Synthesis and antitumor effect in vitro and in vivo of substituted 1,3-dihydroindole-2-ones

Article abstract of DOI:10.1021/jm100763j

Optimization of the anticancer activity for a class of compounds built on a 1,3-dihydroindole-2-one scaffold was performed. In comparison with recently published derivatives of oxyphenisatin the new analogues exhibited an equally potent antiproliferative activity in vitro and improved tolerability and activity in vivo. The best compounds from this series showed low nanomolar antiproliferative activity toward a series of cancer cell lines (compound (S)-38: IC₅₀ of 0.48 and 2 nM in MCF-7 (breast) and PC3 (prostate), respectively) and potent antitumor effects in well tolerated doses in xenograft models. The racemic compound (RS)-38 showed complete tumor regression at a dose of 20 mg/kg administered iv on days 1 and 7 in a PC3 rat xenograft.

Full text of DOI:10.1021/jm100763j

7140 J. Med. Chem. 2010, 53, 7140–7145
DOI: 10.1021/jm100763j
Synthesis and Antitumor Effect in Vitro and in Vivo of Substituted 1,3-Dihydroindole-2-ones
Mette K. Christensen,† Kamille D. Erichsen,† Christina Trojel-Hansen,†,§ Jette Tjørnelund,† Søren J. Nielsen,†
Karla Frydenvang,‡ Tommy N. Johansen,‡ Birgitte Nielsen,‡ Maxwell Sehested,†,§ Peter B. Jensen,† Martins Ikaunieks,
,†,‡
€
Andrei Zaichenko, Einars Loza, Ivars Kalvinsh, and Fredrik Bjorkling*
†TopoTarget A/S, Symbion, Fruebjergvej 3, DK-2100 Copenhagen, Denmark, ‡Department of Medicinal Chemistry, Faculty of Pharmaceutical
Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark, §Experimental Pathology Unit, National University
Hospital, Biocentre, Ole Maaloes Vej 5, 2200 Copenhagen, Denmark, and Latvian Institute of Organic Synthesis, Aizkraules 21,
Riga LV-1006, Latvia
Received June 22, 2010
Optimization of the anticancer activity for a class of compounds built on a 1,3-dihydroindole-2-one
scaffold was performed. In comparison with recently published derivatives of oxyphenisatin the new
analogues exhibited an equally potent antiproliferative activity in vitro and improved tolerability and
activity in vivo. The best compounds from this series showed low nanomolar antiproliferative activity
toward a series of cancer cell lines (compound (S)-38: IC50 of 0.48 and 2 nM in MCF-7 (breast) and PC3
(prostate), respectively) and potent antitumor effects in well tolerated doses in xenograft models. The
racemic compound (RS)-38 showed complete tumor regression at a dose of 20 mg/kg administered iv on
days 1 and 7 in a PC3 rat xenograft.
Introduction
which has been extensively used in man as a laxative. This
nonprescriptiondrug1 was used for morethan 40 years before
it was taken off the market because of transient hepato-
toxicity.3-5 However, the hepatic reaction caused by 1 is pre-
sumably due to a hypersensitivity response rather than a non-
specific toxic effect.3-5
Inhibition of cancer cell proliferation is one of the most
effective principles in the treatment of cancer using chemo-
therapy. Many compounds presently used in cancer treatment
affect proliferation and/or lead to cancer cell death via
apoptosis, necrosis, or alike. A challenge in this approach is
to obtain compounds with sufficient selectivity toward cancer
cells versus normal cells in order to avoid toxicological pro-
blems and side effects commonly seen when using cytotoxics,
such as inhibition of rapidly dividing cells (e.g., bone marrow
and intestinal stem cells). Thus, during any development of
new drugs, an assessment of the toxicological profile is essen-
tial in order to determine a sufficient therapeutic window,
enabling efficient use.
In the present paper further optimization of a recently
described group of compounds based on a 1,3-dihydroin-
dole-2-one scaffold is described.1,2 These compounds exhibit
potent antiproliferative effects in vitro and high efficacy in
mouse xenograft models. However, the first generation of this
compound class showed only a narrow therapeutic window
when tested in rat xenografts and further optimization was
thus warranted.
In vitro, compound 2 is found to potently inhibit prolifera-
tion in the breast cancer cell line MDA-MB-468 (IC50=3 nM).1
Interestingly, this class of compounds has a broad but distinct
selectivity for inhibition of cancer cells, some being very sen-
sitive, others almost resistant as further discussed below. Also,
compound 2 is found to be well tolerated and shows efficient
effects in vivo when tested in breast (MCF-7) and prostate
cancer (PC3) xenograft models in mice.2 To further explore the
therapeutic potential of this compound, the maximum toler-
ated dose (MTDa) of 2 was determined in rat, where the
compound was found to be much more toxic than in mice.
Thus, potent effects in rat xenografts with compounds closely
related to 2 (e.g., compound 4) were only observed at doses
close to or above the MTD. Therefore, it was decided to
optimize these compounds further with respect to the in vivo
effects and the therapeutic window using the rat in the toxico-
logical assessment.
The starting point for this investigation was the recently
described 3,3-substituted oxindole compounds such as 6,7-
difluoro-3,3-bis(4-hydroxyphenyl)-2-oxindole (2, TOP216)
(Figure1, Table1)whichwas showntobe a potent inhibitor of
cell proliferation. This class of compounds was discovered in a
screen for compounds with antiproliferative activity and was
later optimized. Compound 2 is a substituted derivative of
oxyphenisatin (1, 3,3-bis(4-hydroxyphenyl)-2-oxindole) (Figure 1),
Since the precise mode of action and the biological target of
the compounds are not known at present, the optimization
was performed using standard cancer cell proliferation assays,
followed by pharmacokinetic and toxicological assessment in
a Abbreviations: AUC, area under the curve; DCM, dichloro-
methane; DMEM, Dulbecco’s modified Eagle medium; DMSO, di-
methylsulfoxide; HP-β-CD, (2-hydroxypropyl)-β-cyclodextrin; HRMS,
highresolutionmassspectrometry;IC50, concentration of a test compound
that produces half maximal inhibition; LC-MS, liquid chromatography-
mass spectrometry; MTD, maximum tolerated dose; NMR, nuclear
magnetic resonance; p-TSA, p-toluenesulfonic acid; SAR, structure-
activity relationship; SD, standard deviation; SDS-PAGE sodium dode-
cyl sulfate-polyacrylamide gel electrophoresis.
*To whomcorrespondence should beaddressed. Address:Department
of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University
of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
Phone: þ45 35 33 62 31. Fax: þ45 35 33 60 41. E-mail: fb@farma.ku.dk.
r
pubs.acs.org/jmc
Published on Web 09/16/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7141
Results and Discussion
As earlier reported, the SAR studies for substituted analo-
gues of compound 1 have only been explored for the sym-
metrically substituted 3,3-diphenyl-substituted scaffold. In
summary, the most potent compounds are 3,3-bis(4-hydro-
xyphenyl) moieties with small substituents in the 6 and 7
positions of the oxindole ring.1 Alternative or further sub-
stitution of the phenyl rings and larger substituents in the
oxindole do not improve in vitro activity.
Figure 1. Oxyphenisatin (1) and the analogue compound TOP216 (2).
Table 1. SAR: Determination of Antiproliferative Activity of
Compounds 1-14
Lead Optimization. In the initial optimization approach,
aromatic substituents different from 4-hydroxyphenyl were
introduced in place of one of the 4-hydroxyphenyl substitu-
ents in the 3-position of the lead compound 2. The effects of
these substitutions were determined by the antiproliferative
activities of the compounds in MCF-7 (breast) and PC3
(prostate) cancer cell lines (Table 1). A simple removal of one
of the hydroxyl groups from the 4-hydroxyphenyl moieties
gave a compound 3, which retained high antiproliferative
potency (Table 1). Removal of both hydroxyl groups gave
inactive compounds (data not shown), and this illustrates
that only one phenolic group is needed for high activity.
Substitution of the 3-phenyl group showed that small sub-
stituents in the 4-position of the phenyl moiety were well
tolerated (compounds 4-7). Compounds 4 and 5 with a
4-fluorophenyl and a 4-Me-phenyl group were the most
potent with IC50 values of 2.6 and 1.7 nM, respectively, in
the MCF-7 WST-1 screening assay. Larger alkoxy substitu-
ents, such as propoxy (8) and pentoxy (9), in the 4-position
gave compounds with low or no activity, suggesting a
hydrophobic interaction with some limitation in size. Com-
pound 10 showed that the 4-hydroxyphenyl as one of the
substituents in the 3-position of the oxindole is crucial for
high activity. Further substitution in the second phenyl ring
is allowed, as exemplified by compound 11. However, this
did not improve in vitro activity substantially, and com-
pounds that are more structurally distinct from the starting
compound 2 were therefore synthesized to tentatively in-
crease the chances for altered activity and toxicological
profile.
IC50 (nM)b
compd
Ar
R
WST-1a MCF-7
PC3
1
73% at 0.5 μM >1000
2
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
4-OH
20 ( 4.4
7.1 ( 0.24
2.6 ( 0.09
1.7 ( 0.01
6.3 ( 0.42
11 ( 1.5
356
699
3
H
4-F
8.9 ( 0.35
8.6 ( 0.14
0.8 ( 0.7
2.2 ( 3.4
24 ( 13
>1000
4
5
4-Me
4-OMe
4-OEt
4-O-n-Pr
6
7
8
9
4-O-n-Pent >1000
(3-OH-Ph),c Ph 4-OMe
>1000
10
11
12
13
14
196 ( 18
7.8 ( 3.1
82 ( 2.0
37 ( 3.7
63 ( 0.63
145 ( 25
19 ( 1.5
0.3 ( 0.1
110 ( 184
>1000
Ph
3,4-di-F
2-thiophene
4-pyridyl
3-pyridyl
H
H
H
a Cancer cells were incubated for the designated time and the number
of viable cells assessed using cell proliferation reagent WST-1. b IC50
values are the mean ( SD of at least two experiments performed in
triplicate. c 3-OH-Ph in place of 4-OH-Ph.
Scheme 1. Synthesis of 3,3-Diaryl- or 3-Alkyl-3-aryloxindoles
IIIa
Replacement of one of the 4-hydroxyphenyl groups by
heterocycles, such as compounds 12-14, did not improve
activity in the MCF-7 cell lines (Table 1) and was not further
explored, mainly because of sluggish chemistry that gener-
ally gave low yields and complicated purifications. Further
replacement of one 4-hydroxyphenyl group with alkyl and
cycloalkyl groups gave a new series of interesting compounds
(Table 2). Compounds with small n-alkyl substituents (15,
16) showed only low activity in the MCF-7 cell lines
(Table 2). However, compounds with cyclic substituents
(17-19) were found to be active with an optimum for the
cycloheptyl derivative 19 (Table 2). Larger alkyl and alkyl-
aryl groups (20-22) led to compounds with low activity or
inactive compounds (Table 2).
a Reagents and conditions: (a) R2MgX (3 equiv), THF, -78 °C;
(b) phenol (5 equiv), p-TSA (7.5 equiv), dichloroethane.
vivo and efficacy determination in xenograft models. Ongoing
studies of the mode of action are also presented.
Chemistry
The general synthetic approach to the 3,3-diaryl- or 3-alkyl-
3-aryloxindoles III is outlined in Scheme 1. The first step
involved a Grignard addition to the isatin derivative I, using
an aryl- or alkylmagnesium halide, and the second step
involved an elimination-addition reaction.6,7 This was ac-
complished by an acid-catalyzed Friedel-Crafts type con-
densation of compound II with phenol to generate 3,3-diaryl-
or 3-alkyl-3-aryloxindoles III. The isatin derivatives I were
either commercially available or synthesized according to
literature procedures.8-14
The antiproliferative activities were analyzed for substi-
tuted analogues of the most interesting compounds from
Tables 1 and 2, such as compounds carrying different
substituents in position 4, 5, 6, or 7 of the oxindol (Table 3).
Compounds with a substituent at the 4- and 5-position of
the oxindole did not show any significant activity (data not
shown). With the toxicological effects observed for com-
pound 2 in mind we wanted to substitute the 6,7-difluoro
with alternative groups in order to structurally discriminate
these compounds from the starting compound but with

7142 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Christensen et al.
Table 2. SAR: Determination of Antiproliferative Activity of
Compounds 15-22
Table 4. SAR: Determination of Antiproliferative Activity of Enantio-
mers of Compounds 6, 18, and 38
IC50 (nM)b
compd
enantiomer
WST-1a MCF-7
PC3
6
1
2
1
2
1
2
2.1 ( 0.12
>1000
2 ( 0.42
>1000
9.2 ( 1.8
>1000
2 ( 1.1
>1000
6
18
18 ( 11.2
>1000
18
(S)-38
(R)-38
0.48 ( 0.19
>1000
IC50 (nM)b
a Cancer cells were incubated for the designated time and the number
of viable cells assessed using cell proliferation reagent WST-1. b IC50
values are mean values ( SD of at least two experiments performed in
triplicate.
compd
R
WST-1a MCF-7
PC3
15
16
17
18
19
20
21
22
n-propyl
n-pentyl
>1000
>1000
96 ( 7.8
12 ( 1.6
13 ( 2.4
1.2 ( 1.9
>1000
1.1 ( 1.2
8.2 ( 6.9
32 ( 17
12 ( 1.2
>1000
cyclopentyl
cyclohexyl
35-38, and the cycloheptyl analogue 38 was found to be
one of the most potent derivatives in this series in both cell
lines, structurally distinct from the starting compound 2
and exhibiting very high antiproliferative activity (IC50 of
4.7 and 12 nM in WST-1 MCF-7 and PC3, respectively).
Stereochemistry. The new compounds discussed above
were all racemates with a chiral center at the 3-position of
the oxindole. To explore the stereochemical preference of the
target, selected potent compounds 6, 18, and 38 were re-
solved by chiral chromatography (Chiracel OD) and the
antiproliferative activity was determined for the pure enan-
tiomers. Interestingly, a high eudismic ratio was observed for
all three compounds, the first eluting enantiomer being the
eutomer (Table 4). The great difference in activity was
further confirmed in a clonogenic assay using MCF-7 and
A2780 cell lines (Figure 2). The high activity difference for
the stereoisomers suggested a specific biological interaction
with a receptor or an enzyme. The absolute configuration of
the active stereoisomer of compound 38 was determined to
be S by X-ray crystallography of the corresponding Mosher
ester. The diastereoisomeric Mosher esters of 38 were sepa-
rated by chromatography followed by crystallization and
determination of the absolute configuration by X-ray dif-
fraction. The absolute configuration of the Mosher ester
could then be correlated back to 38 by hydrolysis of the
Mosher ester and analysis using chiral chromatography.
Because of the close structural similarity between 6, 18,
and 38 and because of the similar elution order on Chiralcel
OD-H, it is most likely that the active enantiomers possess
the same structural configuration, which would be (R)-6 and
(S)-18, respectively.
cycloheptyl
cyclooctyl
methylcyclohexyl
methylphenyl
102 ( 70
>1000
>1000
>1000
a Cancer cells were incubated for the designated time and the number
of viable cells assessed using cell proliferation reagent WST-1. b IC50
values are the mean ( SD of at least two experiments performed in
triplicate.
Table 3. SAR: Determination of Antiproliferative Activity of
Compounds 23-38
IC50 (nM)b
compd
R1
R2
WST-1a MCF-7
PC3
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
H
4-F-Ph
28 ( 6.7
247 ( 121
>1000
9.7
>1000
>1000
>1000
91
H
7-Me
cycloheptyl
cycloheptyl
cycloheptyl
imidazolyl
4-F-Ph
7-CF3
7-CF3
>1000
40 ( 51
>1000
>1000
19
>1000
209 ( 94
>1000
>1000
>1000
30 ( 21
150
5,7-di-Me
5,7-di-Me
5-F,7-Me
6,7-di-Me
6-F,7-Me
6-Cl,7-Me
6-Me,7-Cl
cycloheptyl
cycloheptyl
cycloheptyl
cycloheptyl
cycloheptyl
cycloheptyl
20 ( 0.21
51 ( 27
2.8 ( 1
6.3
17
6-OMe,7-Me 4-F-Ph
9 ( 2.4
111 ( 179
28 ( 3.9
12 ( 15
6-OMe,7-Me cyclopentyl
6-OMe,7-Me cyclohexyl
6-OMe,7-Me cycloheptyl
1 ( 0.69
14 ( 2.5
4.7 ( 0.05
In Vitro Activity and Mode of Action. As reported, com-
pound 2 has a similar selectivity profile as the mTOR
inhibitor CCI-779 with high cytotoxic activity at low nano-
molar concentrations toward a subset of human cancer cell
lines and with a striking selectivity (>1000-fold) against
a Cancer cells were incubated for 72 h and the number of viable cells
assessed using cell proliferation reagent WST-1. b IC50 values are the
mean ( SD of at least two experiments performed in triplicate.
naturally resistant cell lines, e.g., MDA-MB-468 IC50
=
retained activity. The structure-activity relationship (SAR)
was carefully investigated for the two very active compounds
4 and 19. The unsubstituted derivatives (23, 24) lost much of
their activity, but some of the activity could be retained by
monosubstitution, preferably at the 7-position (e.g., com-
pound 26, Table 3). On the basis of earlier work, larger or
polar substituents reduce the potency.1 However, as it evident
from Table 3, the data indicate that halogens and small
lipophilic substituents in the 6 and 7 positions not only are
allowed but are important to achieve high antiproliferative
activity (compounds 31-38). The preferred substitution
pattern was the 6-methoxy and 7-methyl in compounds
20 nM vs MDA-MB-231 IC50 > 3 μM.1 The molecular basis
for the very high selectivity of compound 2 and analogues
toward specific cancer cell lines has not yet been elucidated,
but it is currently being investigated. In an attempt to learn
more about the mode of action of 2 and the new analogues
with the same profile, two resistant cell lines, MCF-7/TOP216
and A2780/TOP216, were generated by culturing the pa-
rental cell lines with increasing concentrations of 2 (Trojel-
Hansen et al., manuscript in preparation). The compound 2
resistant cell lines showed cross-resistance with the new
analogues prepared, indicating a similar mode of action.
Furthermore, it has been demonstrated that treatment of

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7143
Figure 2. Clonogenic assay for racemate and pure enantiomers (1 or 2) of compound 6 and compound 2. In vitro colony forming assays were
performed in MCF-7 (breast carcinoma) and A2780 (ovarian carcinoma) cancer cells essentially as previous published.16
Table 5. In Vivo PC3 Xenograft Activities in Mouse and Rata
mouse tumor
regressionb
compd dose (mg/kg) dose (mg/kg)
rat tumor
regressionb,c
rat MTD rat therapeutic
indexd
(mg/kg)
2
5
ND
<2.5
<10
ND
1
0.4
2
4
2.5
10
10
20
10
20
2.5
10
18
19
26
35
38
2
>40
40
8
>20
<10
10
4
15
70
3
7
a The antitumor effect in vivo was tested in a subcutaneous (sc)
xenograft model in nude mice or rat. ND: not determined. b Schedule:
ꢀ3/week iv, PC3 mouse or rat xenograft model. c The effective doses in
rats were estimated from mouse xenograft models (mouse to rat
conversion factor: 0.5). d Therapeutic index: (toxic dose in rats)/(effec-
tive dose in rats).
tested induced tumor stasis and regression (including cures)
at low concentrations (5-20 mg/kg) comparable to or better
than those of compound 2 (Table 5). Interestingly, in mice no
signs of toxicity were observed at concentrations, where the
compound was highly active. To further select the best com-
pounds, the MTD in rat was determined, followed by mea-
surements of efficacy in a prostate cancer (PC3) rat xenograft
model. The MTD of 2 was determined to be 1 mg/kg at a single
dose after iv administration, leading to a therapeutic index
below 1 (Table 5). At doses above the MTD, the main toxico-
logical findings were respiratory distress immediately after
treatment followed by a mucous-containing diarrhea. In some
cases the rats died with vascular shocklike symptoms shortly
after dosing. Compounds with only one phenol group and an
aromatic or cycloalkyl substituent in the 3-position (such as 4,
18, and 19) showed a weak but significant improvement in the
MTD. Further substitution in the oxindole group also gave
compounds with improved toxicological profile, e.g., com-
pounds 26 and 35. In this series, compound 38 was observed
with the highest MTD (70 mg/kg) and a therapeutic index of 7.
This compound showed a very high antitumor activity in
the rat PC3 xenograft model with no signs of toxicity when
administered iv on days 0 and 7 at a dose of 20 mg/kg (Figure 4).
In this experiment the PC3 tumors disappeared and no
regrowth was observed until the end of study at day 26. The
observed increase in body weight was normal. The difference
in toxicology between the selected compounds does not seem
to be explained by simple pharmacokinetic data, since the T1/2
and AUC for the compounds were similar in rat (Table 6). The
more dramatic difference in toxicological sensitivity found
between species (mouse vs rat) could not be easily explained.
However, some distribution differences in the two species were
observed with higher concentrations of the drug in the rat lung
compared to concentrations in the mouse lung. Also, a more
Figure 3. Treatment with the active compound 38 but not the
inactive analogue 27 led to an increased p-eIF2R phosphorylation.
MCF-7 and A2780 including their 2-resistant sublines were treated
with 100 mM 38 and 27 for 4 and 24 h, respectively. The resistant
sublines showed cross-resistance toward 38 as indicated by lack of
p-eIF2R induction.
cells with 3,3-diaryloxindoles, compounds with a structural
similarity to the compounds in this paper, leads to phosphor-
ylation of the translational initiation factor eIF2R at the
regulatory site, serine 51.15 To investigate whether this
mechanism is conserved among the present compounds,
human breast cancer MCF-7 cells and human ovary cancer
A2780 cells were incubated in the presence of the active
compound 38 or the inactive analogue 27. Treatment with
38 for 24 h leads to a robust induction of the phosphorylation
of eIF2R, while 27 had no effect on eIF2R phosphorylation
(Figure 3). In the MCF-7 and A2780 subcell lines with induced
resistance to 2 (MCF-7/TOP216 and A2780/TOP216, re-
spectively) 38 had no effect on eIF2R phosphorylation, sug-
gesting that the induction of eIF2R phosphorylation is an
integral part of the mechanism of compound 38 mediating
inhibition of cell proliferation.
In Vivo Antitumor Activity and Toxicology. Selected com-
pounds with potent (nanomolar) activity in the in vitro
screen (Tables 1-4) were tested in vivo in a PC3 (human
prostate cancer) mouse xenograft model. The PC3 cells were
grown on nude NMRI mice, and treatment was initiated at
large tumor size (800-1000 mm3) to observe for tumor
regression. The compounds were formulated in 2% DMSO/
20% HP-β-CD and isotonic sterile saline and administered iv
at 5-20 mL/kg three times weekly. Several of the compounds

7144 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Christensen et al.
1H spectra (CDCl3, 7.26; CD3OD, 3.31; (CD3)2SO, 2.50) and 13
C
spectra (CDCl3, 77.23; CD3OD, 49.00; (CD3)2SO, 39.52). The
value of a multiplet, defined doublet (d), triplet (t), double
doublet (dd), double triplet (dt), quartet (q), or not (m) at the
approximate midpoint is given unless a range is quoted. bs
indicates a broad singlet. The purity of the compounds was
determined using an LC-MS (Bruker Esquire 3000þ ESI ion
trap with an Agilent 1200 HPLC system) and was confirmed to be
g95% for all compounds. HRMS was carried out on a Micro-
mass Q-Tof micro mass spectrometer. The HPLC system for the
semipreparative resolution of the Mosher ester of compound 38
consisted of a Jasco 880 pump, a Rheodyne 7125 injector
equipped with a 5 mL loop, and a Shimadzu SPD-6A UV
detector connected to a Hitachi-D2000 Chromato integrator.
General Procedure A: Grignard Reaction to Form Tertiary
Alcohols of General Formula II, Followed by Friedel-Crafts
Reaction with Phenol. To a stirred solution of isatin derivative
I in dry THF under nitrogen at -78 °C was added 3 equiv of
Grignard reagent. After 30 min, the dry ice bath was removed
and the mixture was left to reach room temperature over
4-14 h.7 Excess Grignard reagent was quenched with water,
and the reaction mixture was acidified with 1 N HCl or saturated
NH4Cl solution, extracted with EtOAc (ꢀ2), dried over MgSO4,
filtered, and concentrated. The residue was purified by chroma-
tography (1% MeOH in DCM or mixtures of petroleum ether
and EtOAc) to afford racemic tertiary alcohol II.
Figure 4. Treatment with compound 38 in rat xenograft PC3 tumor
model. Compound 38, 20 mg/kg iv on days 0 and 7, resulted in
tumor regression of PC3 human prostate cancer cell growth on nude
rats. Each of two rats (R1 and R2) had four tumor nodes (a-d). The
tumor volume at start was from 50 to 450 mm3. Regression was
observed after the first treatment. Rat received an extra treatment
on day 7, and regrowth was not observed at day 26 when the
experiment was terminated. Treatment with 38 had no effect on
body weights or clinical condition of the rats.
Table 6. Pharmacokinetic Parameters for Selected Compounds in
Mouse and Rata
To a solution of tertiary alcohol of general formula II in
dichloroethane was added phenol (5 equiv) and p-TSA (7.5
equiv). The reaction mixture was heated to 90 °C for 2-4 h and
then cooled to room temperature. The solid (mainly p-TSA) was
filtered off and washed with dichloroethane or DCM. The
solution was concentrated and the residue was purified by
chromatography (1% MeOH in DCM or mixtures of petroleum
ether and EtOAc) to afford racemic compounds of general
formula III.
(RS)-3-Cycloheptyl-3-(4-hydroxyphenyl)-6-methoxy-7-methy-
lindolin-2-one (38). Preparation of 38 was performed according
to general procedure A using 6-methoxy-7-methylindoline-2,3-
dione and cycloheptylmagnesium bromide.
mouse
rat
dose iv
compd (mg/kg)
AUC
dose iv
AUC
T1/2 (h) (h ng/mL) (mg/kg) T1/2 (h) (h ng/mL)
3
3
2
5
1.7
2.1
0.4
3.7
1.6
1.3
ND
ND
6769
1913
395
ND
2.5
2.5
2.5
5
ND
0.8
0.7
1.1
1.1
2.1
1.5
1.5
ND
4
5
2534
2634
2396
1353
2288
3949
5760
12
17
18
19
35
38
5
5
1849
3554
694
5
5
5
ND
ND
ND
ND
10
10
a ND: not determined.
(RS)-3-Cycloheptyl-3-hydroxy-6-methoxy-7-methylindolin-2-
one: 1H NMR ((CD3)2SO) δ 10.21 (bs, 1H), 7.00 (d, J = 8.3 Hz,
1H), 6.49 (d, J = 8.3 Hz, 1H), 5.58 (s, 1H), 3.75 (s, 3H), 2.08 (m,
1H), 2.01 (s, 3H), 1.88 (m, 1H), 1.73 (m, 1H), 1.55 (m, 1H),
1.50-1.20 (m, 8H), 0.75 (m, 1 H). Yield 35%.
rapid uptake was found for compound 2 in the lung compared
to the less toxic compound 38. This may be one reason for the
acute toxicological reactions in the rat. Further pharmaco-
kinetic studies may clarify this possibility and suggest ways to
further improve the toxicological profile of these interesting
drug leads.
Product 38: 1H NMR ((CD3)2SO) δ 10.38 (bs, 1H), 9.28 (bs,
1H), 7.13 (m, 2H), 7.07 (d, J = 8.2 Hz, 1 H), 6.67 (m, 2H), 6.61
(d, J = 8.2 Hz, 1H), 3.79 (s, 3H), 2.27 (m, 1H), 2.05 (s, 3H), 1.58
(m, 3H), 1.41 (m, 2H), 1.13 (m, 3H), 0.93 (m, 1H), 0.68 (m, 1H).
HRMS m/z calcd for C23H27NO3 [M þ Na], 388.1889; found,
388.1936. Yield 90%.
Conclusion
Cell Culture. Human breast carcinoma MCF-7, ovarian
carcinoma A2780, and prostate PC3 cell lines were grown
according to American Type Culture Collection guidelines. Cell
culture media were from Invitrogen unless otherwise stated.
MCF-7 was maintained in DMEM and A2780 in RPMI 1640
with GlutaMax. Media were supplemented with 10% (v/v) FCS
(Perbio, Thermo Fischer Scientific), penicillin (100 U/mL),
streptomycin (0.1 mg/mL) and cells incubated at 37 °C in an
atmosphere containing 5% CO2.
WST-1 Proliferation Assay. Cells were seeded in 96-well
plates at 3ꢀ103 cells/well in 100 μL of culture medium. The
following day compounds were serially diluted in culture medium
and an amount of 100 μL of each dilution was added per well in
triplicate to the cell culture plates. Plates were incubated for 72 h
at 37 °C in a 5% CO2 atmosphere and the number of viable
cells assessed using cell proliferation reagent WST-1 (Roche,
Mannheim, Germany). An amount of 10 μL of reagent was added
to each well, and after a 1 h incubation period, absorbance was
measured at 450 nm, subtracting absorbance at 690 nm as a
reference. Data were analyzed using GraphPad Prism (GraphPad
The present study describes the successful optimization of
the therapeutic and toxicological effects of 1,3-dihydroindole-
2-ones as anticancer agents in vivo. Modification of the
substitution patternon thisscaffold gavecompound38, which
showed very potent anticancer effects in a rat PC3 xenograft
model at well tolerated doses, suggesting this compound to be
an interesting candidate for further preclinical evaluation.
Present activities aim for improved pharmacokinetics and
possibly distribution to diminish toxicological side effects
through the preparation of prodrugs of the most active
compounds. Future work is also pursued to elucidate the
mode of action of this unique active group of compounds.
Experimental Section
Reaction conditions and yields were not optimized. 1H and 13
C
NMR spectra were recorded on a Bruker Avance 300 spectro-
meter (300 MHz). Chemical shifts are reported in parts per
million (δ) and referenced according to deuterated solvent for

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7145
Software, CA) and Calcusyn (Biosoft, Cambridge, U.K.) as
appropriate.
Vissing Holst, Department of Chemistry, University of
Copenhagen, Denmark, for collecting X-ray data.
Western Blotting. Cells were lysed in ELB buffer on ice
for 15 min, sonicated for 5-10 s, and centrifuged at 20000g
for 15 min at 4 °C. Protein extracts (20 μg, as determined by
Bio-Rad protein assay (Bio-Rad)) were diluted in sample buffer
(4 ꢀ Novex Nupage sample buffer), heated at 95 °C for 5 min,
and separated by SDS-PAGE followed by blotting onto a nitro-
cellulose membrane using the NuPAGE Novex BisTris (XCell
SureLock) system (Invitrogen). Membranes were blocked with
5% nonfat milk in Tris-buffered saline/0.1% Tween (TBS-T) for
1 h, incubated with primary antibody overnight at 4 °C, washed
3 times in TBS-T, and incubated with horseradish peroxidase
labeled secondary antibodies for 1 h at room temperature. The
membranes were then washed 3ꢀ10 min in TBS-T. Detection was
achieved using ECL SuperSignal West Femto maximum sensi-
tivity substrate(Pierce) together witha ChemiDoc XRS/Quantity
One documentation system (Bio-Rad).
Supporting Information Available: Experimental procedures,
analytical and spectral data for all intermediate and final
compounds, and X-ray crystallographic analysis results. This
material is available free of charge via the Internet at http://
pubs.acs.org. X-ray crystallographic information of frac-
tional atomic coordinates, list of anisotropic displacement
parameters, and a complete list of geometrical data have been
deposited in Cambridge Crystallographic Data Centre (No.
CCDC 779154).
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Acknowledgment. We thank Annette Nielsen and Anita
Johansen for invaluable technical assistance. We thank Niels