Chiral resolution and pharmacological characterization of the enantiomers of the Hsp90 inhibitor 2-amino-7-[4-fluoro-2-(3-pyridyl)phenyl]-4-methyl-7,8- dihydro-6H-quinazolin-5-one oxime

Article abstract of DOI:10.1002/cmdc.201400037

Heat-shock protein 90 (Hsp90) is a molecular chaperone involved in the stabilization of key oncogenic signaling proteins, and therefore, inhibition of Hsp90 represents a new strategy in cancer therapy. 2-Amino-7-[4-fluoro-2-(3- pyridyl)phenyl]-4-methyl-7,8-dihydro-6H-quinazolin-5-one oxime is a racemic Hsp90 inhibitor that targets the N-terminal adenosine triphosphatase site. We developed a method to resolve the enantiomers and evaluated their inhibitory activity on Hsp90 and the consequent antitumor effects. The (S) stereoisomer emerged as a potent Hsp90 inhibitor in biochemical and cellular assays. In addition, this enantiomer exhibited high oral bioavailability in mice and excellent antitumor activity in two different human cancer xenograft models. Problem resolved: A chemical method to resolve the enantiomers of 2-amino-7-[4-fluoro-2-(3-pyridyl)phenyl]-4-methyl-7,8-dihydro-6H-quinazolin-5- one oxime, a Hsp90 inhibitor targeting the N-terminal adenosine triphosphatase site, and the characterization of their inhibitor activity on Hsp90, along with the consequent antiproliferative effect on cancer cells is explored. Pharmacokinetic properties and antitumor activity are also evaluated.

Full text of DOI:10.1002/cmdc.201400037

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DOI: 10.1002/cmdc.201400037
Chiral Resolution and Pharmacological Characterization of
the Enantiomers of the Hsp90 Inhibitor 2-Amino-7-[4-
fluoro-2-(3-pyridyl)phenyl]-4-methyl-7,8-dihydro-6H-
quinazolin-5-one Oxime
Raffaella Amici,*[a, f] Chiara Bigogno,[b, g] Roberto Boggio,[a, h] Andrea Colombo,[b, i]
Stephen M. Courtney,[c] Roberto Dal Zuffo,[d, f] Giulio Dondio,[b, g] Fulvia Fusar,[d, f]
Stefania Gagliardi,[b, j] Saverio Minucci,[e] Marco Molteni,[b, k] Christian A. G. N. Montalbetti,[c, l]
Annalisa Mortoni,[b, m] Mario Varasi,[d, f] Stefania Vultaggio,[d, f] and Ciro Mercurio[d, f]
Heat-shock protein 90 (Hsp90) is a molecular chaperone in-
volved in the stabilization of key oncogenic signaling proteins,
and therefore, inhibition of Hsp90 represents a new strategy in
cancer therapy. 2-Amino-7-[4-fluoro-2-(3-pyridyl)phenyl]-4-
methyl-7,8-dihydro-6H-quinazolin-5-one oxime is a racemic
Hsp90 inhibitor that targets the N-terminal adenosine triphos-
phatase site. We developed a method to resolve the enantio-
mers and evaluated their inhibitory activity on Hsp90 and the
consequent antitumor effects. The (S) stereoisomer emerged
as a potent Hsp90 inhibitor in biochemical and cellular assays.
In addition, this enantiomer exhibited high oral bioavailability
in mice and excellent antitumor activity in two different
human cancer xenograft models.
Introduction
Heat-shock protein 90 (Hsp90) is an adenosine triphosphate
(ATP)-dependent molecular chaperone involved in the confor-
mational maturation of several client proteins implicated in
multiple and diverse cellular functions.[1] The Hsp90 protein
consists of three distinct domains:[2] the N-terminal ATP bind-
ing domain, the middle domain involved in the ATPase cycle
and in the binding with co-chaperone and client proteins, and
the C-terminal domain, with a role in a homodimerization pro-
cess and in the allosteric control over both substrates and N-
terminal pocket.[3]
The function of Hsp90 is regulated by a concerted mecha-
nism, defined as the chaperone cycle; it involves the nucleo-
tide domain pocket in the N-terminal region of the protein, as
well the middle region and the C-terminal nucleotide binding
pocket.[4–6] An additional level of regulation of Hsp90 function
is represented by both the interaction of Hsp90 with diverse
co-chaperones and by several post-translational modifications
such as acetylation, nitrosylation, and phosphorylation.[6] The
occupancy of both the N-terminal ATP binding site and the C-
terminal pocket by high affinity ligands, which inhibit Hsp90,
[a] Dr. R. Amici, Dr. R. Boggio
[g] Dr. C. Bigogno, Dr. G. Dondio
Genextra Group, Congenia s.r.l., Via Adamello 16, 20139 Milan (Italy)
E-mail: raffaella.amici@ieo.eu
Current address: Aphad s.r.l.
Via della Resistenza 65, 20090 Buccinasco (MI) (Italy)
[b] Dr. C. Bigogno, Dr. A. Colombo, Dr. G. Dondio, Dr. S. Gagliardi,
Dr. M. Molteni, Dr. A. Mortoni
[h] Dr. R. Boggio
Current address: IRBM Promidis s.r.l.
Via Pontina km 30, 600, 00040 Pomezia (RM) (Italy)
NiKem Research s.r.l., Via Zambeletti 25, 20021 Baranzate (Italy)
[c] Dr. S. M. Courtney, Dr. C. A. G. N. Montalbetti
[i] Dr. A. Colombo
Evotec, 114 Milton Park, Abingdon, Oxfordshire, OX10 4SA (UK)
Current address: Teva Pharmaceuticals Fine Chemicals
Strada Statale Briantea, 23892 Bulciago (Italy)
[d] Dr. R. Dal Zuffo, Dr. F. Fusar, Dr. M. Varasi, Dr. S. Vultaggio, Dr. C. Mercurio
Genextra Group, DAC s.r.l., Via Adamello 16, 20139 Milan (Italy)
[j] Dr. S. Gagliardi
Current address: Flamma S.p.A.
Via Bedeschi 22, 24040 Chignolo d’Isola (BG) (Italy)
[e] Prof. Dr. S. Minucci
IEO–European Institute of Oncology, Via Adamello 16, 20139 Milan (Italy)
and
[k] Dr. M. Molteni
Department of Life Sciences, University of Milan
Via Celoria 26, 20133 Milan (Italy)
Current address: Chorisis s.r.l.
Via R. Lepetit 34, 21040 Gerenzano (VA) (Italy)
[f] Dr. R. Amici, Dr. R. Dal Zuffo, Dr. F. Fusar, Dr. M. Varasi, Dr. S. Vultaggio,
Dr. C. Mercurio
[l] Dr. C. A. G. N. Montalbetti
Current address: Inventiva, 50 rue de Dijon, 21121 Daix (France)
Current address: Drug Discovery Program
Department of Experimental Oncology
IEO-European Institute of Oncology, Via Adamello 16, 20139 Milan (Italy)
[m] Dr. A. Mortoni
Current address: CISI S.C.R.L.
Via G. Fantoli 16/15, 20138 Milan (Italy)
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prevents the Hsp90 client proteins from achieving their mature
functional conformation.
tion have been raised, there are currently 38 clinical trials that
are ongoing with these molecules. One compound, STA-9090,
is in phase III evaluation in patients with non-small-cell lung
cancer treated with docetaxel plus or minus ganetespib. More-
over, the very recent clinical data obtained with BIIB021, which
indicates both good tolerability and responses consistent with
anti-Hsp90 biologic activity, demonstrates that important prog-
ress has been made in overcoming some issues previously ob-
served.[25]
Additional strategies to inhibit Hsp90 have also been identi-
fied[7] and pursued by targeting the co-chaperone/Hsp90 inter-
action and specifically by targeting the Cdc37/Hsp90 interac-
tion[8] and interaction between Hsp90 and the co-chaperone
containing a tetratricopeptide region[9] as well as targeting
client/Hsp90 associations.[10,11]
Many of the Hsp90 client proteins are overexpressed and/or
mutated in cancer and are directly associated with cancer pro-
gression. Among them, we can find ERBB2, CDK4, c-Raf, B-Raf,
c-Met, and h-Tert. Consequently, there is no surprise that
Hsp90 has an important role in maintaining the activity and
stability of key oncogenic signaling proteins.[5] Furthermore,
elevated Hsp90 expression has been documented in different
tumor types, and a correlation exists between high expression
of Hsp90 and poor disease prognosis.[12–19] The inhibition of
Hsp90 and the loss of its chaperone function leads client pro-
teins to be degraded by the ubiquitin proteasome pathways,
which consequently disrupted their oncogenic function. Given
that Hsp90 is involved in the control of a multitude of path-
ways relevant for cancer progression, its inhibition permits
a concerted attack on the unrestricted proliferation and surviv-
al of cancer cells, and this leads to cell growth inhibition and
apoptosis.
With respect to C-terminal inhibitors (Figure 2), clear im-
provement in the prototypic C-terminal inhibitor novobiocin
has been accomplished, and molecules showing interesting in
vivo efficacy data in preclinical studies have been identified
(KU174 and KU363).[38–41]
Importantly, the C-terminal inhibitors represent an interest-
ing and possibly alternative approach, because these mole-
cules, differently from N-terminal inhibitors, do not induce
a prosurvival heat-shock response,[42] and this could increase
the effectiveness of the inhibitors as antitumor agents.[43]
As part of our efforts to find new potent Hsp90 inhibitors,
we performed
a high-throughput biochemical fragment
screening campaign. Further optimization of the hits by using
a combination of in silico commercial analogue selection and
structure-based design led to the identification of compound
1.[44] This compound, which exhibits good biochemical and cel-
The relevance of Hsp90 as target for cancer therapy has
been extensively established and reviewed in the last
years.[20–23] Up to now, the vast majority of drug-development
efforts have focused on inhibitors binding to the N-terminal
ATP binding site, but a second druggable site was identified in
the C-terminal domain of Hsp90.[24] To date, no C-terminal in-
hibitor has advanced into clinical trial. Indeed, all of the 17 clin-
ically evaluated Hsp90 inhibitors belong to the so called N-ter-
minal inhibitors.[25]
With regard to these inhibitors, the natural product geldana-
mycin was the first reported inhibitor of Hsp90. Since then,
several geldanamycin derivatives such as 17-AAG, 17-DMAG,
and IPI-504 (Figure 1)[26] have been investigated in preclinical
and clinical studies. Furthermore, synthetic small-molecule in-
hibitors from unrelated chemical classes have been discovered,
some of which are currently in clinical trials for the treatment
of cancer. The structures of these new agents belong to three
main classes: ATP mimetics through an amino-substituted
fused heteroaromatic bicyclic ring system, resorcinol ana-
logues, and 2-aminobenzamides.[26] All of them target the N-
terminal ATP pocket of Hsp90. Representative examples
(Figure 1) are the oral-drug candidates XL-888 (Exelixis,
lular activity, has one stereogenic carbon atom; consequently,
it corresponds to a racemic mixture of two possible enantio-
mers. Our further efforts were therefore directed toward the
separation of racemic compound 1 and biological characteriza-
tion of the two enantiomers to investigate the influence of the
stereochemistry on the identified Hsp90 inhibitory activity.
In this study, we report a method to produce the pure enan-
tiomers of compound 1 on both small and large scales. The
paper further reports their biological and absorption, distribu-
tion, metabolism, and excretion (ADME) characterization, their
in vivo pharmacokinetic profile, and in vivo efficacy in xeno-
graft models.
phase I);[27]
DEBIO-0932/CUDC-305
(Debiopharm/Curis,
phase I);[28] MPC-3100 (Myrexis Inc., phase I);[29] PF-4929113/
SNX-5422 (Pfizer, phase I), which is a glycine prodrug of PF-
4928473/SNX-2112;[30] BIIB021 (Biogen Idec, phase II);[31]and Results and Discussion
NVP-HSP990 (Novartis, phase I).[32] Compounds pursued as in-
Enantiomeric separation and in vitro characterization
travenous agents include NVP-AUY922 (Novartis, phase II),[33]
AT13387 (Astex, phase II),[34] STA-9090 (Synta, phase II/III),[35]
KW-2478 (Kyowa Hakko Kirin, phase I/II),[36] and PU-H71 (Memo-
rial Sloan-Kettering Cancer Center, phase I).[37] Although some
concerns about the safety profile of N-terminal Hsp90 inhibi-
For the initial characterization of the enantiomers, we conduct-
ed the resolution of racemic mixture 1 by chiral chromatogra-
phy by using a Daicel Chiralpak AD semipreparative column
with a heptane/2-propanol mixture (75:25) containing 0.1%
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Figure 1. Hsp90 inhibitors in clinical development.
triethylamine as the eluent. The obtained enantiomers were
evaluated for their Hsp90 inhibitory activity as well as for their
antiproliferative effects in comparison with the racemic mix-
ture. Interestingly, a clear difference in the activity profiles for
the distinct enantiomers was observed. As shown in Table 1,
stereoisomer 2 exhibited very potent Hsp90 inhibitory activity
(IC50 =19 nm), which was clearly superior to that of enantiomer
3 and racemate 1. Similarly and congruently, enantiomer 2 was
more potent than enantiomer 3 and racemic mixture 1 in the
antiproliferative assay on the two human cancer cell lines A549
and HCT-116.
We subsequently analyzed the molecular signature of Hsp90
inhibition on HCT-116 cells by following the induction of
Hsp70 and the depletion of the well-known Hsp90 client pro-
tein c-Raf.[45] Racemate 1 and enantiomers 2 and 3 were tested
at concentrations that were 0.2-, 1-, and 5-fold the antiprolifer-
ative IC50 value on HCT-116 cells. The objective was to find
a correlation between the observed antiproliferation effect and
Hsp90 inhibition. As reported in Figure 3, all three compounds
were able to determine a dose-dependent induction of Hsp70,
whereas only racemate 1 and enantiomer 2, but not enantio-
mer 3, were able to induce a down-regulation of c-Raf under
Table 1. Hsp90 enzyme and antiproliferative activity data for racemate
1 and enantiomers.[a]
Compound
Activity [mm]
Enzyme
A549
HCT-116
racemate 1
enantiomer 2
enantiomer 3
0.030ꢀ0.001
0.019ꢀ0.001
1.10ꢀ0.04
0.85ꢀ0.01
0.29ꢀ0.038
>2.5
0.85ꢀ0.09
0.42ꢀ0.079
2.3ꢀ0.779
[a] Assays done in replicates (nꢁ2); mean values and standard deviations
are shown.
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Figure 4. The S enantiomer (grey) and R enantiomer (black) docked into the
N-terminal ATP binding site of Hsp90.
tions with the aromatic cage constituted by Phe138, Tyr139,
and Trp162, whereas the R equatorial enantiomer was sterically
hindered (Figure 4). As shown in the figure, the fluorine atom
of the R enantiomer in particular shows an unfavorable interac-
tion with the protein.
Figure 2. Structures of C-terminal inhibitors.
As the next step, enantiomers 2 and 3 were evaluated in
a series of ADME experiments. The compounds were incubated
with mouse and human microsomes at 378C for 30 min, and
the percentage of unmetabolized product was assessed by
LC–MS/MS. As illustrated in Table 2, enantiomers 2 and 3 had
similar stability in mouse and human microsomes. The deriva-
tives showed low aqueous solubility at neutral pH and accept-
able solubility at pH 4. The protein binding was 97 and 95%
for enantiomers 2 and 3, respectively. Moreover, inhibition of
key drug-metabolizing cytochromes P450 (CYPs) was assessed,
which is relevant both in the pharmacokinetics of xenobiotics
and in adverse drug–drug interactions.[46] For this purpose, the
compounds were incubated at final concentrations of 1 mm
with the cytochrome isozymes CYP1A2, CYP2C9, CYP2D6, and
CYP3A4 and the specific substrates for 15–45 min. The percen-
tages of inhibition of the cytochrome isoenzymes are summar-
ized in Table 2. Similar inhibition (ꢂ24%) on CYP2C9 was ob-
served for both enantiomers. Interestingly, enantiomer 2
showed only minor interference (<5%) on the CYP1A2,
CYP2D6, and CYP3A4 cytochromes. In contrast, enantiomer 3
inhibited the enzyme activity of cytochromes CYP3A4 and
CYP2D6 by ~30%.
Figure 3. Western blot of racemate 1 and enantiomers 2 and 3 on HCT-116
cell lines. Target modulation at 24 h tested at 0, 0.2, 1, and 5 times the re-
spective cellular IC50 value. The expression levels of Erk were used as loading
control.
the experimental conditions; this suggested that the antiproli-
ferative effect determined by enantiomer 3 was not directly re-
lated to Hsp90 inhibition.
Co-crystallization experiments were conducted on both
enantiomers, but the only crystal structure that was obtained
in our hands was that of enantiomer 2, to which an S configu-
ration was assigned (PDB code: 3FT8).[44] On the basis of these
results, redocking experiments were performed on all four pos-
sible conformations of the two enantiomers (i.e., S with axial 3-
pyridylphenyl, S with equatorial 3-pyridylphenyl, and the corre-
sponding R conformations). In agreement with the X-ray data,
only the S equatorial configuration showed favorable interac-
On the basis of the promising in vitro data, we decided to
evaluate the compounds in vivo. It was therefore necessary to
establish a method to produce quantities of the chiral deriva-
tives sufficient for the in vivo evaluations. The use of a variety
Table 2. In vitro ADME parameters of enantiomers 2 and 3.[a]
Compd
Microsomal stability [%]
Solubility [mm]
PPB [%]
Cytochrome P450 inhibition at 1 mm [%]
Mouse
Human
pH 7.4
pH 4
1A2
2C9
2D6
3A4
2
3
23
34
38
38
34
29
208
181
97
95
<5
7.6
24
24
<5
32
<5
32
[a] Assays done in replicates (nꢁ2); mean values are shown, and standard deviations are <30% of the mean.
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Scheme 1. Reagents and conditions: DMF, EDC, HOBT, TEA, R*COOH at room temperature or with microwave heating. Boc=tert-butoxycarbonyl.
of chiral auxiliaries in the synthe-
sis of racemate 1 did not provide
satisfactory results. Thus, the
oxime group of compound
1 was selected as a functionaliza-
tion site, and various commercial
chiral acids were used for the
synthesis of the corresponding
oxime esters (Scheme 1). The
syntheses were performed with
hydroxybenzotriazole (HOBt), 1-
ethyl-3-(3-dimethylaminopropyl)-
carbodiimide (EDC), and triethyl-
amine (TEA) in DMF at room
Scheme 2. Reagents and conditions: Enzyme, EtOH, room temperature.
temperature or by heating
under microwave irradiation if
necessary. With the exception of
acids 4, 9, 12, and 15, all other acids afforded the desired
esters; however, separation of the diastereomers was not pos-
sible.
The most promising results were obtained by using lipase
immobilized from Candida antarctica, which led to low conver-
sion into the hydrolyzed free oxime (20% by UPLC analysis)
with S/R=76:24 (HPLC analysis on a chiral stationary phase).
Optimal conditions were found with the lipase acrylic resin
from C. antarctica in THF with butanol at 308C over 36 h
(Scheme 3). Under these conditions, hydrolysis of the O-acetyl
oxime moiety of compound 1 resulted in 50% conversion into
the free oxime. The enantiomeric ratio was S/R=9:1, and the
remaining O-acetylated oxime had an enantiomeric ratio of S/
R=7:93. The free oxime was subsequently re-acetylated and
triturated with methanol. Both the filtered solid and the
mother liquors were analyzed by HPLC on a chiral stationary
phase. Surprisingly, the mother liquors contained 99% of the S
enantiomer, and the solid contained the acetyl oxime with S/
R=8:2. It is well known that nonracemic enantiomeric mix-
tures can form homochiral and heterochiral aggregates in melt
or suspension during adsorption or crystallization. These dia-
stereomeric associations determine the distribution of the
The use of enzymatic methods was then evaluated for re-
solving racemate 1. Lipases are extensively used to catalyze
stereoselective esterifications, transesterifications, and hydroly-
ses in non-aqueous media.[47,48] Oxime esters show intermedi-
ate reactivity as irreversible acyl-transfer agents for lipase catal-
ysis and, therefore, can be used under mild conditions in enzy-
matic transesterification reactions.[49–52] As a first attempt, enan-
tioselective acetylation of compound 1 was undertaken. The
reaction was performed with vinyl acetate in ethanol by using
amano lipase AK from Pseudomonas fluorescens; however, no
desired products were obtained. Enantioselective hydrolysis of
acetylated derivative 1 was then considered. Racemate 1 was
treated with acetyl chloride and TEA in DMF at room tempera-
ture overnight. The resultant acetate was then treated with
several enzymes in ethanol at room temperature (Scheme 2).
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tissue distribution. Following
oral administration, enantiomer
2
was rapidly absorbed with
t
max =15 min and showed high
oral bioavailability (95%). Enan-
tiomer 3, however, exhibited
a higher clearance rate (56.4 vs.
32 mLminꢃ1 kgꢃ1
)
and signifi-
cantly lower oral exposure and
bioavailability (33.7 vs. 95%)
than enantiomer 2.
In vivo efficacy
The two enantiomers were then
submitted to an in vivo efficacy
experiment in an established
Scheme 3. Reagents and conditions: a) Lipase acrylic resin from C. antarctica, THF/nBuOH, 308C; b) separation by
flash chromatography; c) CH3COCl, TEA, DMF, room temperature; d) trituration in MeOH; e) NaOH, MeOH, room
temperature.
human
HCT-116
xenograft
model. Human cancer cells HCT-
116 (5 ꢁ 106) were injected sub-
cutaneously in the flank of
enantiomers between the solid phase and other phases.[53,54]
female CD-1 nude mice, and treatment was initiated once the
tumors reached a volume of 100 mm3. Enantiomer 2 was orally
administered at doses of 30, 50, and 100 mgkgꢃ1 for five days
per week for 16 days, whereas enantiomer 3 was orally admin-
istered only at the highest dose of 100 mgkgꢃ1. As shown in
Figure 5a, oral administration of enantiomer 2 resulted in a sig-
nificant dose-dependent decrease in the tumor volume relative
to the control group. Specifically, the calculated tumor/control
ratio (T/C) at doses of 30, 50, and 100 mgkgꢃ1 were 0.42, 0.37,
and 0.3, respectively, with p values of 0.01, 0.004, and 0.0003.
Importantly, enantiomer 3 was less active than enantiomer 2
at the dose of 100 mgkgꢃ1 with a calculated T/C of 0.69. No
significant body weight differences among the groups of mice
and no signs of evident toxicity were observed during treat-
ment.
The distribution depends on the stability order of the homo-
and heterochiral aggregates (conglomerates or racemate for-
mation). These aggregates are diastereomeric, and they can be
differentiated under achiral conditions. The (S)-acetyl oxime
from the mother liquors was then treated with NaOH to obtain
free enantiomer 2 (enantiomeric purity S>98%). The originally
obtained O-acetylated oxime with S/R= 7:93 was converted
following the same procedure into the (R)-acetylated enantio-
mer (enantiomeric purity of 100%). Similar results in terms of
enantiomeric purity and recovered yield were obtained by
treating the O-acetyl oximes in a second run with lipase acrylic
resin from C. antarctica under the previously described condi-
tions. However, it is evident that this second enzymatic run is
more expensive and time consuming than simple trituration
with methanol.
Four hours after the end of the treatment, three tumors
were randomly taken both from the group of control mice and
from the group of mice treated with compound 2 at doses of
30, 50, and 100 mgkgꢃ1. Western blotting of the lysates of the
recovered tumors showed that enantiomer 2 effectively in-
duced the degradation of the Hsp90 client protein as c-Raf in
the majority of the analyzed samples (Figure 5b). Concurrently,
a significant inhibition of mitogen-activated protein kinase
(MAPK), as measured by decreased phosphorylation levels of
Following isolation of sufficient quantities of the enantio-
mers, the in vivo pharmacokinetic behavior was determined.
The compounds were administrated to CD-1 mice in a single
intravenous (IV) dose of 5 mgkgꢃ1 or an oral dose of
15 mgkgꢃ1. The main plasma pharmacokinetic parameters are
reported in the Table 3. Following IV administration, enantio-
mer
2
showed
a
systemic plasma clearance of
32 mLminꢃ1 kgꢃ1, which is lower than hepatic blood flow in
mice (86 mLminꢃ1 kgꢃ1),[55] an estimated elimination half-life of
71 min, and a volume of distribution that was three times
higher than mice total body water; this is suggestive of good
Erk, was observed mainly at doses of 50 and 100 mgkgꢃ1
.
Enantiomer 2 was then evaluated in a non-small-cell lung
(NSCL) xenograft model as a single agent and in combination
Table 3. Pharmacokinetic data of enantiomers 2 and 3 in mice.[a]
Compd
Cmax(IV)
[ngmLꢃ1
AUC(IV)0–1
t1/2
[min]
Cl
Vss
[Lkgꢃ1
C
max(OS)
tmax
[min]
AUC(OS)0–1
F
[%]
]
[minngmLꢃ1
]
[mLminꢃ1 kgꢃ1
]
]
[ngmLꢃ1
]
[minngmLꢃ1
]
2
3
3979
4756
156429
88680
71
163
32
56.4
2.1
2.45
1964
858
15
15
447063
89836
95
33.7
[a] Cmax: maximum concentration; AUC: area under curve; t1/2: half-life; Cl: clearance; Vss: steady-state volume of distribution; F: bioavailability.
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Table 4. Tumor weight change ratio treated versus control (T/C) of single
agent and combined treatment of paclitaxel and enantiomer 2.
Compound
Dose [mgkgꢃ1
]
T/C
enantiomer 2
50
0.3
enantiomer 2
100
0.1
paclitaxel
paclitaxel and enantiomer 2
paclitaxel and enantiomer 2
25
0.18
0.05
0.02
25 and 50
25 and 100
paclitaxel was well tolerated and no significant loss of body
weight was observed after three weeks of dosing.
Conclusions
In conclusion, a method was developed to resolve the enantio-
mers of racemate 1. Once obtained, the Hsp90 inhibitory activ-
ity of the enantiomers as well as their antiproliferative effects
were investigated in comparison with those of racemic com-
pound 1. Enantiomer 2 was found to be a potent Hsp90 inhibi-
tor in enzyme and cell-based assays. Enantiomer 2 had good
ADME characteristics and a high oral bioavailability in mice
pharmacokinetic studies. In addition, the compound demon-
strated excellent antitumor activity in two different human
cancer xenograft models as a single agent. Enantiomer 2 also
demonstrated a synergistic effect in combination with paclitax-
el in a NSCL xenograft model. We can therefore conclude that
all the data obtained are supportive of the therapeutic poten-
tial of (S)-2 in the treatment of cancer.
Figure 5. a) Antitumor activity of 2 and 3 against HCT-116 human tumor
xenografts implanted in mice, expressed as the mean tumor volume (ex-
pressed as mm3)ꢀstandard error of the mean (SEM). Enantiomer 2 was
orally administered at doses of 30, 50, and 100 mgkgꢃ1 for five days per
week for 16 days, whereas enantiomer 3 was orally administered only at the
highest dose of 100 mgkgꢃ1 (os die: per os daily). b) Analysis by western
blot of c-Raf, phosphorylated Erk (Erk-p), and Erk of the lysate of HCT-116
xenograft tumor at the end of treatment.
Experimental Section
Chemistry: General methods
Unless otherwise indicated, all the starting reagents and solvents
(HPLC purity) were commercially available and were used without
any further purification. The reactions were monitored by thin-
layer chromatography (TLC) by using Merck plates 0.2 mm (60F-
254) and spotting the reaction products with UV light at 254 nm.
Flash chromatography purifications were performed with Merck
with paclitaxel. NSCL human cancer cells H1975 (5ꢁ106) were
injected subcutaneously in the flank of female CD-1 nude
mice, and treatment was initiated once the tumors reached
a volume of 100 mm3. Compound 2 was orally administered at
doses of 50 and 100 mgkgꢃ1 for five days per week for three
weeks, whereas paclitaxel was injected by IV once a week at
the optimal dose of 25 mgkgꢃ1. For the combined treatment,
paclitaxel was administered by IV at day 1, 8, and 15, whereas
inhibitor 2 was dosed orally from day 2 to 5, day 9 to 12, and
day 16 to 19.
1
silica gel 60 (0.04–0.063 mm). H NMR spectra were recorded with
a Bruker 300 MHz spectrometer, and 13C NMR spectra were record-
ed with a Varian 500 MHz spectrometer. Splitting patterns describe
apparent multiplicities and are designated as s (singlet), d (dou-
blet), t (triplet), q (quartet), quint. (quintet), sext. (sextet), m (mul-
tiplet), and br. s (broad signal). HPLC–MS analyses were recorded
by using one of the following methods.
Method 1: Waters Acquity UPLC, Micromass ZQ 2000 single quad-
rupole (Waters). Column: Phenomenex Kinetex UPLC C18 (50ꢁ
2.1 mm, 1.7 mm). Mobile phase: phase A: H2O/CH3CN=95:5+0.1%
trifluoroacetic acid (TFA); phase B: H2O/CH3CN=5:95+0.1% TFA;
flow rate: 0.5 mLminꢃ1. Detection: UV (diode array) among 210 e
400 nm; ESI+; full scan from m/z=100 to 2000. Gradient: 0–0.3 (A:
95%, B: 5%), 0.3–1.5 (A: 0%, B: 100%), 1.5–2.0 (A: 0%, B: 100%),
2.0–2.4 min (A: 95%, B: 5%).
As reported in Figure 6, the administration of both 50 and
100 mgkgꢃ1 doses of enantiomer 2 as well as the optimal dose
of paclitaxel strongly decreased tumor growth with a T/C of
0.3, 0.1, and 0.18, respectively (Table 4). Interestingly, the com-
bined treatment at the 50 mgkgꢃ1 dose of enantiomer 2 and
the optimal dose of paclitaxel resulted in a significant en-
hancement in antitumor activity (T/C of 0.05 vs. 0.3 and 0.18).
Importantly, the combined treatment of the 100 mgkgꢃ1 dose
of enantiomer 2 and the optimal dose of paclitaxel led to an
almost complete disappearance of the tumors with a T/C of
0.02. Moreover, the combined treatment of enantiomer 2 and
Method 2: Waters Acquity HPLC, Micromass ZQ 2000 Single quad-
rupole (Waters). Column: Acquity Atlantis C18 (50ꢁ2.1 mm, 3 mm).
Mobile phase: phase A: H2O+0.1% TFA; phase B: CH3CN+0.1%
TFA; flow rate: 0.3 mLminꢃ1. Detection: UV at 254 nm or base peak
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(2.88 mL, 20.64 mmol) maintaining
the temperature in the 0–58C
range. The reaction was stirred at
room
temperature
overnight.
Three further additions of acetyl
chloride (0.3 mL, 4.22 mmol) and
TEA (0.6 mL, 4.30 mmol) were
made, and the reaction mixture
was stirred at room temperature
for 1 h between each addition. The
solvent was removed under re-
duced pressure, and the crude ma-
terial was washed with water and
filtered. The solid thus obtained
was washed with methanol, fil-
tered, and dried under vacuum to
obtain the desired compound as
a white solid (5.18 g, 12.78 mmol,
93%).
1H NMR
(300 MHz,
[D6]DMSO): d=8.58 (dd, J=4.7,
1.8 Hz, 1H), 8.55 (dd, J=2.3,
0.9 Hz, 1H), 7.79 (dt, J=7.9, 2.3 Hz,
1H), 7.71 (dd, J=8.8, 5.9 Hz, 1H),
7.46 (ddd, J=7.8, 4.8, 0.9 Hz, 1H),
7.33 (td, 1H), 7.13 (dd, J=9.5,
2.8 Hz, 1H), 7.01 (s, 2H), 2.55–3.26
(m, 5H), 2.52 (brs, 3H), 2.16 ppm
(s, 3H); LC–MS (ESI): m/z: 406
[M+H]+; LC–MS: tR =0.98 min
(method 1).
Figure 6. Antitumor activity of 2 and paclitaxel against NCI-H1975 human tumor xenografts implanted in mice, ex-
pressed as the mean tumor volume (expressed as mm3)ꢀSEM.
Racemate 1[44] was obtained by functionalization of the intermedi-
ate ketone as a single isomer. Likely, steric hindrance of the methyl
group at the 4-position prevented formation of one isomer, which
led to a single derivative, the (E)-oxime.
intensity (BPI) with ESI+ at 3.2 KV, 25 V, 3508C. Gradient: 0–0.2 (A:
95%, B: 5%), 0.2–5.0 (A: 0%, B: 100%), 5.0–6.0 (A: 0%, B: 100%),
6.0–6.1 (A: 95%, B: 5%), 6.1–7.0 min (A: 95%, B: 5%).
Method 3: (Chiral Column) Waters Alliance HPLC. Column: Chiralcel
OD-H (150ꢁ4.6 mm, 5 mm). Mobile phase: phase A: n-hexane+
0.1% TEA; phase B: iPrOH+0.1% TEA; flow rate: 0.4 mLminꢃ1. De-
tection: UV at 260 nm. Isocratic gradient: A: 45%, B: 55% for
25 min.
(S,E)-2-Amino-7-[4-fluoro-2-(pyridin-3-yl)phenyl]-4-methyl-7,8-dihy-
droquinazolin-5(6H)-one oxime [ꢂ80% enantiomeric excess (ee)]
and (R,E)-2-amino-7-[4-fluoro-2-(pyridin-3-yl)phenyl]-4-methyl-7,8-
dihydroquinazolin-5(6H)-one O-acetyl oxime (ꢂ80%ee): Lipase
acrylic resin from C. antarctica (0.3 g) was added to a solution of
(E)-2-amino-7-[4-fluoro-2-(pyridin-3-yl)phenyl]-4-methyl-7,8-dihydro-
quinazolin-5(6H)-one O-acetyl oxime (0.2 g, 0.493 mmol) in THF
(20 mL) and nBuOH (0.034 mL, 0.37 mmol). The suspension was
stirred at 308C for 36 h. The enzyme was removed by filtration,
and a mixture of CH2Cl2 and acetone (4:1, 200 mL) was added to
the solution. The solution thus obtained was concentrated under
reduced pressure, and the crude was purified by flash column
chromatography (CH2Cl2/MeOH 99:1 to 95:5). Two fractions were
obtained.
Care and husbandry of animals were in conformity with the institu-
tional guidelines in compliance with the Italian Law (D. L.vo 116/
92).
Enantiomer separation by chiral chromatography: Compound
1 was solubilized in methanol/2-propanol=2:1 v/v (ꢂ2 mgmLꢃ1).
Enantiomeric separation was achieved by using a chiral column
(Daicel Chiralpak AD Semi-preparative 250ꢁ20 mm i.d., 10 mm)
and an isocratic mobile phase consisting of 75% heptane/25% 2-
propanol (0.1% triethylamine). Separation conditions: flow rate
18 mLminꢃ1; injection volume 1000 mL (ꢂ2 mg column loading);
run time 60 min; UV detection at 261 nm. tR ꢂ25 (enantiomer 3),
40 min (enantiomer 2). Owing to considerable tailing of enantio-
mer 3, enantiomer 2 was recycled to achieve the desired enantio-
meric purity. The enantiomers were obtained as white/cream-col-
ored powders.
Fraction 1:
(R,E)-2-Amino-7-[4-fluoro-2-(pyridin-3-yl)phenyl]-4-
methyl-7,8-dihydroquinazolin-5(6H)-one O-acetyl oxime (0.073 g,
0.180 mmol, 73%) as a pale yellow solid. LC–MS (ESI): m/z: 406
[M+H]+; LC–MS: tR =1.00 min (method 1); HPLC: tR =16.695 min,
enantiomeric ratio: S/R=7:93 (method 3).
Fraction 2:
methyl-7,8-dihydroquinazolin-5(6H)-one
0.165 mmol, 67%) as a white solid. LC–MS (ESI): m/z: 364 [M+H]+;
LC–MS: tR =0.92 min (method 1); HPLC: tR =10.646 min, enantio-
meric ratio: S/R=9:1 (method 3).
(S,E)-2-Amino-7-[4-fluoro-2-(pyridin-3-yl)phenyl]-4-
oxime (0.060 g,
Procedures and characterization
(E)-2-Amino-7-[4-fluoro-2-(pyridin-3-yl)phenyl]-4-methyl-7,8-dihy-
droquinazolin-5(6H)-one O-acetyl oxime: Acetyl chloride (0.982 mL,
13.76 mmol) was added dropwise to a solution of (E)-2-amino-7-[4-
fluoro-2-(pyridin-3-yl)phenyl]-4-methyl-7,8-dihydroquinazolin-5(6H)-
one oxime (1; 5 g, 13.76 mmol) in dry DMF (100 mL) and TEA
(R,E)-2-Amino-7-(4-fluoro-2-pyridin-3-yl-phenyl)-4-methyl-7,8-dihy-
dro-6H-quinazolin-5-one oxime (enantiomer 3): Step A, method 1:
(R,E)-2-Amino-7-[4-fluoro-2-(pyridin-3-yl)phenyl]-4-methyl-7,8-dihy-
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droquinazolin-5(6H)-one O-acetyl oxime (S/R=7:93; 0.073 g,
0.180 mmol) was triturated with a minimal amount of methanol. A
white solid was obtained that was separated from the mother
liquor by filtration. HPLC of the mother liquor: R>99%
(method 3). HPLC of the solid: S/R=4:6 (method 3). The mother
liquor was concentrated under reduced pressure to give (R,E)-2-
amino-7-[4-fluoro-2-(pyridin-3-yl)phenyl]-4-methyl-7,8-dihydroqui-
nazolin-5(6H)-one O-acetyl oxime (0.043 g, 0.108 mmol, 60%). LC–
MS (ESI): m/z: 406 [M+H]+; LC–MS: tR =0.98 min (method 1); HPLC:
tR =16.09 min, enantiomeric ratio: R>99% (method 3).
mother liquor were concentrated under reduced pressure to give
(S,E)-2-amino-7-[4-fluoro-2-(pyridin-3-yl)phenyl]-4-methyl-7,8-dihy-
droquinazolin-5(6H)-one O-acetyl oxime (0.940 g, 2.319 mmol,
60%). LC–MS (ESI): m/z: 406 [M+H]+; LC–MS: tR =0.98 min
(method 1); HPLC: tR =15.919 min, enantiomeric ratio: S>98%
(method 3).
Method 2: Lipase immobilized from C. antarctica (0.8 g) was added
to a solution of (E)-2-amino-7-[4-fluoro-2-(pyridin-3-yl)phenyl]-4-
methyl-7,8-dihydroquinazolin-5(6H)-one O-acetyl oxime (S/R=9:1;
0.433 g, 1.069 mmol) in dry THF (50 mL) and nBuOH (0.093 mL,
1.018 mmol). The mixture was stirred at 308C for two days. HPLC
analysis on a chiral stationary phase showed an enantiomeric ratio
of S/R=99:1. The enzyme was removed by filtration, and a mixture
of CH2Cl2 and acetone (4:1, 100 mL) was added to the filtered solu-
tion. The obtained solution was concentrated under reduced pres-
sure. The crude material was purified by silica gel flash chromatog-
raphy (CH2Cl2 100% to CH2Cl2/MeOH=98:2) to give the desired
product (0.202 g, 1.764 mmol, 52%) as a white solid. LC–MS (ESI):
m/z: 406 [M+H]+; LC–MS: tR =0.99 min (method 1); HPLC: tR =
16.09 min, enantiomeric ratio: S/R=99:1 (method 3).
Step A, method 2: Lipase immobilized from C. antarctica (1.5 g)
was added to a solution of (E)-2-amino-7-[4-fluoro-2-(pyridin-3-yl)-
phenyl]-4-methyl-7,8-dihydroquinazolin-5(6H)-one O-acetyl oxime
(S/R=5:95; 1.26 g, 3.11 mmol) in dry THF (100 mL) and nBuOH
(0.142 mL, 1.554 mmol). The mixture was stirred at 308C for 40 h.
HPLC analysis on a chiral stationary phase showed an enantiomeric
ratio of S/R=1:99. The enzyme was removed by filtration, and
a mixture of CH2Cl2 and acetone (4:1, 200 mL) was added to the fil-
tered solution. The obtained solution was concentrated under re-
duced pressure. The crude was purified by silica gel flash chroma-
tography (CH2Cl2 100% to CH2Cl2/MeOH=98:2) to give the desired
product (0.715 g, 1.764 mmol, 56.7%) as a pale yellow solid. LC–MS
(ESI): m/z: 406 [M+H]+; LC–MS: tR =0.99 min (method 1); HPLC:
tR =16.09 min, enantiomeric ratio: S/R=1:99 (method 3).
Step B: 1m NaOH (10 mL, 10 mmol) was added to a solution of
(S,E)-2-amino-7-[4-fluoro-2-(pyridin-3-yl)phenyl]-4-methyl-7,8-dihy-
droquinazolin-5(6H)-one O-acetyl oxime (S>98%; 0.940 g,
2.319 mmol) in MeOH (60 mL). The mixture was stirred at room
temperature for 15 min. The solvent was removed under reduced
pressure, and the crude material was washed with water, filtered,
and dried under vacuum to give (S,E)-2-amino-7-[4-fluoro-2-(pyri-
din-3-yl)phenyl]-4-methyl-7,8-dihydroquinazolin-5(6H)-one oxime
(0.710 g, 1.954 mmol, 84%) as a white solid: [a]2D0 = +39.6 (c=0.80,
acetic acid); 1H NMR (300 MHz, [D6]DMSO): d=10.91 (br. s, 1H),
8.57 (dd, J=4.8, 1.6 Hz, 1H), 8.53 (dd, J=2.3, 0.9 Hz, 1H), 7.77 (dt,
J=7.9, 2.1 Hz, 1H), 7.68 (dd, J=8.9, 6.0 Hz, 1H), 7.45 (ddd, J=7.8,
4.8, 0.9 Hz, 1H), 7.31 (td, J=8.7, 2.9 Hz, 1H), 7.10 (dd, J=9.7,
2.9 Hz, 1H), 6.65 (s, 2H), 3.01–3.12 (m, 1H), 2.99 (dd, J=15.3,
12.0 Hz, 1H), 2.79–2.92 (m, 1H), 2.54–2.68 (m, 2H), 2.45 ppm (s,
3H); 13C NMR (126 MHz, [D6]DMSO): d=167.8, 165.9, 161.4, 160.7
Step B: 1m NaOH (3.53 mL, 3.53 mmol) was added to a solution of
(R,E)-2-amino-7-[4-fluoro-2-(pyridin-3-yl)phenyl]-4-methyl-7,8-dihy-
droquinazolin-5(6H)-one O-acetyl oxime (0.715 g, 1.764 mmol) in
MeOH (45 mL). The mixture was stirred at room temperature for
15 min. The solvent was removed under reduced pressure, and the
crude material was washed with water, filtered, and dried under
vacuum overnight to obtain the desired product (0.590 g,
1.624 mmol, 92%) as a white solid. [a]2D0 =ꢃ42.2 (c=0.74, acetic
1
acid); H NMR (300 MHz, [D6]DMSO): d=10.91 (br. s, 1H), 8.57 (dd,
J=4.8, 1.6 Hz, 1H), 8.53 (dd, J=2.3, 0.9 Hz, 1H), 7.77 (dt, J=7.9,
2.1 Hz, 1H), 7.68 (dd, J=8.9, 6.0 Hz, 1H), 7.45 (ddd, J=7.8, 4.8,
0.9 Hz, 1H), 7.31 (td, J=8.7, 2.9 Hz, 1H), 7.10 (dd, J=9.7, 2.9 Hz,
1H), 6.65 (s, 2H), 3.01–3.12 (m, 1H), 2.99 (dd, J=15.3, 12.0 Hz, 1H),
1
3
(d, JC,F =240.1 Hz), 152.6, 149.5, 149.2, 140.1 (d, JC,F =7.5 Hz), 138.6
(d, 4JC,F =2.9 Hz), 136.9, 135.8, 129.2 (d, 3JC,F =8.5 Hz), 123.9, 117.1
(d, 2JC,F =20.5 Hz), 115.9 (d, 2JC,F =20.5 Hz), 112.5, 40.1, 33.6, 32.2,
26.8 ppm; LC–MS (ESI): m/z: 364.08 [M+H]+; LC–MS: purity=99%,
tR =1.71 min (method 2); HPLC: tR =10.83 min, enantiomeric ratio:
S/R=98:2 (method 3).
2.79–2.92 (m, 1H), 2.54–2.68 (m, 2H), 2.45 ppm (s, 3H); 13C NMR
1
(126 MHz, [D6]DMSO): d=167.8, 165.9, 161.4, 160.7 (d, JC,F
=
240.1 Hz), 152.6, 149.5, 149.2, 140.1 (d, 3JC,F =7.5 Hz), 138.6 (d,
4JC,F =2.9 Hz), 136.9, 135.8, 129.2 (d, 3JC,F =8.5 Hz), 123.9, 117.1 (d,
2JC,F =20.5 Hz), 115.9 (d, 2JC,F =20.5 Hz), 112.5, 40.1, 33.6, 32.2,
26.8 ppm; LC–MS (ESI): m/z: 364.18 [M+H]+; LC–MS: purity=98%,
tR =1.71 min (method 2); HPLC: tR =13.55 min, enantiomeric ratio:
S/R=1:99 (method 3).
Biological assays
(S,E)-2-Amino-7-[4-fluoro-2-(pyridin-3-yl)phenyl]-4-methyl-7,8-dihy-
dro-6H-quinazolin-5-one oxime (enantiomer 2): Step A: Acetyl chlo-
ride (0.357 mL, 5.00 mmol) was added to a solution of (S,E)-2-
amino-7-[4-fluoro-2-(pyridin-3-yl)phenyl]-4-methyl-7,8-dihydroqui-
nazolin-5(6H)-one oxime (S/R=9:1; 1.397 g, 3.84 mmol) in dry DMF
(20 mL) and TEA (0.804 mL, 5.77 mmol) maintaining the tempera-
ture in the 0–58C range. The mixture was stirred at room tempera-
ture. After 1 h, additional aliquots of acetyl chloride (0.273 mL,
3.84 mmol) and TEA (0.536 mL, 3.84 mmol) were added, and the
mixture was stirred at room temperature overnight. The solvent
was removed, and the crude material was washed with water, fil-
tered, and dried under vacuum.
Hsp90 inhibitory activity: Hsp90 inhibitory activity was determined
as described by Schilb et al. and Kim et al.[56,57] A Tamra–geldanami-
cin ligand was used as a fluorescent tracer for the Hsp90 ATPase
domain. Its displacement by small-molecule inhibitors was mea-
sured by fluorescence changes.
Cell growth assay: The antiproliferative effect of the compounds
was evaluated in A549 (non-small-cell lung cancer) and in HCT-116
(human colon cancer) tumor cell lines with the CellTiter-Glo Lumi-
nescent Cell Viability Assay (Promega, Madison, WI) according to
the manufacturer’s instructions. A549 and HCT-116 cells, in expo-
nential growth, were incubated for 72 h at different concentrations
of the inhibitors. Then, the CellTiter-Glo reagent (1 equiv) was
added, the solution was mixed for 2 min to induce cell lysis, and
the luminescence was recorded after an additional 10 min. The IC50
was calculated by using GraphPad Software.
Method 1: The product thus obtained was triturated with a minimal
amount of methanol. A white solid was obtained that was separat-
ed from the mother liquor by filtration. HPLC of the mother liquor:
S>98% (method 3). HPLC of the solid: S/R=63:37 (method 3). The
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Client protein degradation: Cells both from in vitro and in vivo ex-
terface in MRM mode. The percentage of compound bound to the
plasma protein (PPB) was determined according to Equation (2), in
which fu is given by Equation (3), and NSB is given by Equation (4):
*
periments were lysed in lysis buffer composed of 50 mm Tris HCl,
pH 6.8, 2% SDS, and 10% glycerol. Equal amounts of protein were
separated by 10% SDS-PAGE and transferred onto a nitrocellulose
membrane. The membranes were incubated with 5% milk in TBST
PPB ¼ ð1ꢃfuÞ ꢄ 100
ð2Þ
ð3Þ
*
[20 mm Tris HCl (pH 7.5), 500 mm NaCl, and 0.5% Tween 20] at
areafiltrate ꢄ ð1 þ NSBÞ
room temperature for 1 h and probed with primary antibodies in
5% nonfat dried milk in TBST overnight at 48C. After washing
three times with TBST for 5 min, the membrane was incubated
with secondary anti-rabbit or anti-mouse antibody for 1 h at room
temperature and visualized by enhanced chemiluminescence de-
tection (Amersham, Bioscience Little Chalfont Buckinghamshire
HP7 9NA England).
fu
¼
areatotal
areafiltrate in buffer
areatotal in buffer
NSB ¼ 1ꢃð
Þ
ð4Þ
in which areatotal and areatotalinbuffer refer to the samples of the test
compound in a mixture composed of the initial plasma solution or
buffer solution (40 mL) diluted with CH3CN/MeOH (1:1, 160 mL) and
then centrifuged.
For the experiments, the following antibodies were used: Hsp70
(Hsp72) Monoclonal Antibody (C92F3A-5) form Assay Design-
Stressgen (Ann Arbor USA) used at 1:1000 dilution in TBST over-
night at 48C. Raf-1 Rabbit antibody (C-12) from Santa Cruz Biotech-
nologies Inc. (Dallas, TX, USA) used at 1:200 dilution in TBST over-
night at 48C.
Cytochrome P450 inhibition: Experiments were performed by
adapting protocols described previously[59,60] and according to the
manufacturer’s instructions (BD Biosciences, Franklin Lakes, NJ,
USA). The compounds were dissolved in a 96-well plate at 1 mm
final concentration in potassium phosphate buffer (pH 7.4) contain-
ing an NADPH regenerating system. For all enzyme/substrate pairs,
the final cofactor concentration was 1.3 mm NADP+, 3.3 mm glu-
cose-6-phosphate, and 0.4 UmLꢃ1 glucose-6-phosphate dehydro-
genase. The reaction was initiated by the addition of specific isoen-
zymes (Supersomes, Gentest) and substrates at 378C. Furafylline
(for CYP1A2, 100 mm), sulfaphenazole (for CYP2C9, 10 mm), tranylcy-
promine (for CYP2C19, 500 mm), quinidine (for CYP2D6, 0.5 mm),
and ketoconazole (for CYP3A4, 1.66 mm) were employed as control
inhibitors in one-third serial dilution. Incubations were performed
for 15 [0.5 pmol CYP1A2, 5 mm 3-cyano-7-ethoxycoumarin (CEC)],
30 {0.5 pmol CYP2C19, 25 mm CEC; 1 pmol CYP3A4, 50 mm 7-benzy-
loxy-4-(trifluoromethyl)coumarin; 1.5 pmol CYP2D6, 1.5 mm 3-[2-
(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin},
or 45 min [1 pmol CYP2C9, 75 mm 7-methoxy-4-(trifluoromethyl)-
coumarin]. The reaction was then quenched by adding a mixture
containing 80% CH3CN and 20% Tris base (0.5m, 75 mL), and the
plates were read by using a fluorimeter at the appropriate emis-
sion/excitation wavelengths.[60] The percentage of inhibition was
calculated relative to the enzyme samples without inhibitors.
Microsomal stability assay: By adaptation of the protocols de-
scribed by Di et al.,[58] the compounds at 1 mm were pre-incubated
for 10 min at 378C in potassium phosphate buffer (pH 7.4) togeth-
er with 0.5 mgmLꢃ1 mouse or human hepatic microsomes (Xeno-
tech, Kansas City). The cofactor mixture comprising NADP, G6P, and
G6P-DH was added, and aliquots were taken after 0 and 30 min.
Samples were analyzed with an Acquity UPLC, coupled with
a sample organizer, and interfaced with a triple quadrupole Pre-
miere XE (Waters, Milford, MA, USA). Mobile phases consisted of
a phase A [0.1% formic acid in a mixture of water and acetonitrile
(95:5 v/v)] and phase B [0.1% formic acid in a mixture of water and
acetonitrile (5:95 v/v)]. Separations were achieved at 408C with
Acquity BEH C18 columns (50 mmꢁ2.1 mmꢁ1.7 mm with a flow
rate of 0.45 mLminꢃ1 or 50 mmꢁ1 mmꢁ1.7 mm with a flow rate of
0.2 mLminꢃ1). The column was conditioned with 2% of phase B for
0.2 min, then brought to 100% of phase B within 0.01 min and
maintained under these conditions for 1.3 min. The operating pa-
rameters of the mass spectrometer were set as follows: capillary
voltage 3.4 kV, source temperature 1158C, desolvation temperature
4508C, desolvation gas flow 900 Lhꢃ1, cell pressure 0.33 Pa. Cone
voltage and collision energy were optimized for each compound.
LC–MS/MS analyses were performed by using a positive electro-
spray ionization (ESI+) interface in MRM (multiple reaction moni-
toring) mode with verapamil as internal standard. The percentage
of the compound remaining after a 30 min incubation period was
calculated according to Equation (1):
In vivo pharmacokinetic studies: Pharmacokinetic experiments
were performed by using four-week-old male nude CD-1 mice
(Charles River Laboratories, Calco, Italy). Animals were quarantined
for approximately one week prior to the study. They were housed
under standard conditions and had free access to water and stan-
dard laboratory rodent diet.
area at time 30 min
area at time 0 min
Compound 2 was dissolved in a mixture of stoichiometric 0.1n
HCl and 0.9% NaCl at a concentration of 1 mgmLꢃ1 for the IV ad-
ministration or stoichiometric 0.1n HCl in water for the oral dose
at a concentration of 3 mgmLꢃ1. Compound 3 was dissolved in
a mixture of stoichiometric 0.1n HCl, 3% DMSO, and 0.9% NaCl at
a concentration of 1 mgmLꢃ1 for the IV administration or stoichio-
metric 0.1n HCl in water for the oral dose at a concentration of
3 mgmLꢃ1. Each experimental group contained 27 animals. The
compounds were administered to mice either by IV or oral route,
and blood samples were collected at different time points after
dosing. Plasma was separated immediately after blood sampling
by centrifugation, plasma proteins were precipitated by using Si-
rocco filtration plates, and the plasma samples were kept frozen
(ꢃ808C) until submission to LC–MS/MS analysis. Sample analysis
was performed with an Acquity UPLC by using an Acquity HSS T3
column (50 mmꢁ2.1 mmꢁ1.8 mm), coupled with a sample organiz-
er and interfaced to a triple quadrupole Premiere XE (Waters, Mil-
ð1Þ
compound remaining ¼
ꢄ 100 %
Plasma protein binding: Compounds at a final concentration of
5 mm were incubated in mouse plasma at 378C for 1 h. Each
sample (300 mL) was then transferred to a Centrifree vial (Millipore,
Billerica, MA, USA) and centrifuged at 2700 rpm (g value) at 208C
for 25 min. The filtrated portion and the initial plasma solution
(40 mL) were then diluted/extracted with a CH3CN/MeOH mixture
(1:1, 160 mL) and centrifuged at 13000 rpm (g value) for 10 min.
The solutions were analyzed by LC–MS/MS by using an ESI+ inter-
face in multiple reaction monitoring (MRM) mode. Nonspecific
binding (NSB) was determined by transferring a spiked buffer solu-
tion (300 mL) to a Centrifree vial. After centrifugation at 2700 rpm
(g value) at 208C for 25 min, the filtrated portion and the initial
buffer solution (40 mL) were then diluted with a CH3CN/MeOH mix-
ture (1:1, 160 mL) and analyzed by LC–MS/MS by using an ESI+ in-
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a capillary voltage of 3–4 kV, cone voltage of 25–52 V, source tem-
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In vivo xenograft study (HCT-116 xenograft and NSCL xenograft):
For in vivo antitumor efficacy studies, 5 million human cancer cells
(HCT-116 or NCI-H1975) were injected subcutaneously in the flank
of female CD-1 nude mice (aged six weeks, Charles River Laborato-
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of ~100 mm3 were reached. Mice bearing a tumor xenograft were
randomized into treated and control groups of seven animals per
group. Hsp90 inhibitors were dissolved in a vehicle corresponding
to a mixture of stoichiometric 0.1n HCl, 0.5% methylcellulose, and
1% Tween 80 in water. Paclitaxel (LC laboratories Woburn, USA)
was dissolved in a mixture containing 50% Cremophor EL (Sigma–
Aldrich, Steinheim, Germany) and 50% ethanol and further diluted
10-fold in a solution of 0.9% NaCl.
In the HCT-116 xenograft experiment, Hsp90 inhibitors were ad-
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at the optimal dose of 25 mgkgꢃ1. For the combined treatment,
paclitaxel was IV administered at day 1, 8, and 15, whereas inhibi-
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Keywords: antitumor agents · chiral resolution · enantiomers ·
Hsp90 · oximes
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Received: January 14, 2014
Published online on April 17, 2014
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ChemMedChem 2014, 9, 1574 – 1585 1585