Synthesis and SAR study of novel 3,3-diphenyl-1,3-dihydroindol-2-one derivatives as potent eIF2·GTP·Met-tRNAiMet ternary complex inhibitors

Article abstract of DOI:10.1016/j.ejmech.2013.08.030

The growing recognition of inhibition of translation initiation as a new and promising paradigm for mechanism-based anti-cancer therapeutics is driving the development of potent, specific, and druggable inhibitors. The 3,3-diaryloxindoles were recently reported as potential inhibitors of the eIF2·GTP·Met-tRNA_i^Met ternary complex assembly and 3-{5-tert-butyl-2-hydroxyphenyl}-3-phenyl-1,3-dihydro-2H-indol-2- one #1181 was identified as the prototypic agent of this chemotype. Herein, we report our continuous effort to further develop this chemotype by exploring the structural latitude toward different polar and hydrophobic substitutions. Many of the novel compounds are more potent than the parent compound in the dual luciferase ternary complex reporter assay, activate downstream effectors of reduced ternary complex abundance, and inhibit cancer cell proliferation in the low μM range. Moreover, some of these compounds are decorated with substituents that are known to endow favorable physicochemical properties and as such are good candidates for evaluation in animal models of human cancer.

Full text of DOI:10.1016/j.ejmech.2013.08.030

European Journal of Medicinal Chemistry 69 (2013) 537e553
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and SAR study of novel 3,3-diphenyl-1,3-dihydroindol-2-
one derivatives as potent eIF2$GTP$Met-tRNAMi et ternary complex
inhibitors
,
Séverine Denoyelle a 1, Ting Chen a, Hongwei Yang a, Limo Chen a, Yingzhen Zhang a,
,
,
, b,
*
José A. Halperin a b, Bertal H. Aktas a b, Michael Chorev a
a Laboratory for Translational Research, Harvard Medical School, One Kendall Square, Building 600, 3rd Floor, Cambridge, MA 02139, USA
b Department of Medicine, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The growing recognition of inhibition of translation initiation as a new and promising paradigm for
mechanism-based anti-cancer therapeutics is driving the development of potent, specific, and druggable
inhibitors. The 3,3-diaryloxindoles were recently reported as potential inhibitors of the eIF2$GTP$Met-
tRNAMi et ternary complex assembly and 3-{5-tert-butyl-2-hydroxyphenyl}-3-phenyl-1,3-dihydro-2H-
indol-2-one #1181 was identified as the prototypic agent of this chemotype. Herein, we report our
continuous effort to further develop this chemotype by exploring the structural latitude toward different
polar and hydrophobic substitutions. Many of the novel compounds are more potent than the parent
compound in the dual luciferase ternary complex reporter assay, activate downstream effectors of
Received 23 May 2013
Received in revised form
28 August 2013
Accepted 28 August 2013
Available online 17 September 2013
Keywords:
3,3-Diaryloxindole derivatives
Translation initiation TI
Ternary complex TC
Inhibition of cancer cell proliferation
Mechanistic assays
reduced ternary complex abundance, and inhibit cancer cell proliferation in the low mM range. Moreover,
some of these compounds are decorated with substituents that are known to endow favorable physi-
cochemical properties and as such are good candidates for evaluation in animal models of human cancer.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
selective inhibition of weak mRNAs translation will have a detri-
mental effect on cancer cells with negligible impact on normal
Targeting translation initiation (TI) represents a new paradigm
in cancer therapy [1]. Regulation of gene expression at the level of
TI is essential for normal cell growth, proliferation, differentiation,
and apoptosis, and as such it is under very strict control. Conversely,
loss of physiological restraints on TI leads to malignant trans-
formation in vitro [2e5] and plays an important role in the genesis,
progression and maintenance of some cancers. Importantly,
restricting TI by molecular [6] and chemical genetic approaches
reverts the malignant phenotype because it preferentially reduces
translation of weak mRNAs that are characterized by long and
structured 50 untranslated regions (50UTR). These mRNAs pre-
dominantly code for oncogenic proteins, growth factors, and anti-
apoptotic proteins, many of which are upregulated in and are
important for the genesis and progression of cancer. Consequently,
tissues.
In the TI cascade, the ternary complex (TC), which is comprised
of the charged Met-tRNAMi et and the GTP-coupled eukaryotic
initiation factor 2 (eIF2$GTP), is an essential component of the 43S
pre-initiation complex that includes 40S ribosomal subunit and
other translation initiation factors such as eIF1, eIF3 and eIF4F
complexes. 43S pre-initiation complex recruits mRNA to form the
scanning 48S pre-initiation complex. The codon-anticodon pairing
between the AUG start codon in the mRNA and the Met-tRNAMi et in
the ternary complex is coupled to the hydrolysis of GTP. The release
of phosphate and eIF2$GDP from the pre-initiation complex is
coupled to the recruitment of the 60S ribosomal subunit and to
initiation of translation. Due to the much higher affinity of eIF2 to
GDP than GTP, regeneration of the eIF2$GTP depends on catalysis by
eIF2B, a guanine-nucleotide-exchange factor [7]. Activation of one
of the eIF2
and unfolded protein response leads to phosphorylation of S51 in
the subunit of eIF2. Phosphorylated eIF2 binds tighter with eIF2B
and inhibits its guanine nucleotide exchange activity, thus pre-
venting the regeneration of eIF2$GTP that is needed for regenera-
tion of TC, causing reduction in its abundance and inhibition of TI.
a kinases by external stimuli such as starvation, hypoxia,
* Corresponding author. Hematology Laboratory for Translational Research,
Brigham and Women’s Hospital, Harvard Medical School, 20 Shattuck Street, Thorn
7, Boston, MA 02115, USA. Tel.: þ1 617 525 3146; fax: þ1 617 582 6069.
E-mail address: michael_chorev@hms.harvard.edu (M. Chorev).
Institut des Biomolécules Max-Mousseron (UMR5247 CNRS), Faculté de Phar-
macie, 15 Avenue Charles Flahault, 34093 Montpellier, France.
a
1
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.08.030

538
S. Denoyelle et al. / European Journal of Medicinal Chemistry 69 (2013) 537e553
Experimentally, forced expression of eIF2
a
-S51A,
a
non-
androgen and estrogen receptors [27]. Two years beforehand, a
patent had been released in which various N-substituted 3,3-
bisaryloxindole analogs were identified by the same research
group as nuclear receptor modulators for the treatment of
congestive heart failure and other conditions [28]. In cancer ther-
apy, 3,3-diphenyl-indol-2-one derivatives were evaluated as anti-
cancer agents by Felding and coworkers [22,29,30] and lately,
another research group proposed prodrugs of 3,3-diphenyl-1,3-
dihydroindol-2-ones for the treatment of cancer [31]. Neverthe-
less, unlike our series of 3,3-diaryloxindoles, the development of
the oxindole derivatives mentioned above was not targeting TI and
therefore not evaluated by TI-specific bioassays.
In spite of the promising mechanism of action and the proof-of-
principle achieved with #1181, its unfavorable physicochemical
properties and insufficient potency require further optimization
without compromising its in vitro TC inhibitory efficacy. To this end,
we designed, synthesized and tested the biological activity of a
focused library of structurally modified analogs of #1181 in which
we maintained the parental 3-{5-tert-butyl-2-hydroxyphenyl}-3-
phenyl-1,3-dihydro-2H-indol-2-one scaffold and diversified by
decorating aromatic rings A and B, and substituting the hydroxyl
function on ring C (Fig. 1). A wide range of substituents was used,
which included a variety of polar and charged groups that are
commonly employed in medicinal chemistry to enhance aqueous
solubility [32].
phosphorylatable and a constitutively active eIF2
a mutant [8] or of
Met-tRNAMi et, causes transformation of normal cells into malignant
ones [9,10]. In contrast, pharmacological inhibition of TI inhibits
xenograft tumor growth. Importantly, overexpression of eIF2 and
inactivating mutations of eIF2a kinases have been reported in hu-
man cancers [11e14]. Both of these perturbations will increase the
abundance of the TC rendering TI unrestricted.
Taken together, we hypothesize that pharmacological inhibition
of TI by decreasing the availability of the eIF2$GTP$Met-tRNAiMet
(TC) will block preferentially the expression of oncogenes, growth
regulating and anti-apoptotic proteins to which cancer cells are
addicted to, and will result in the elimination of cancer cells. As
such, an inhibitor of TI that targets the TC presents a novel,
mechanism-based, non-toxic, and effective anti-cancer therapy.
Support of this novel paradigm was offered by demonstrating that
molecular probes such as clotrimazole (CLT) [15,16], troglitazone
(TG) [17], and eicosapentanoic acid (EPA) [18] deplete Ca2þ stores
and subsequently phosphorylate eIF2a, inhibit translation initia-
tion, and reduce preferentially the expression of oncoproteins over
“housekeeping” ones. Abrogation of TI accounts at least in part for
the anti-proliferative activity of these agents. In vivo, these agents
demonstrated efficacy in various mouse models [15,18,19] and
inhibited the expression of oncogenes as measured in the excised
tumors [18].
As an extension of our work with CLT, we have developed 3,3-
diarylindolin-2-ones, which share the triarylmethyl as a common
scaffold, and identified #1181 (Fig. 1) that inhibited proliferation of
a large variety of cancer cells with low micromolar potencies
[20,21]. This prototypic 3,3-diarylindolin-2-one inhibits TI by
reducing TC availability. Subsequently, we and others reported anti-
cancer activity of this family of compounds in various mouse
models [1,22e25]. Treatment of mice bearing MCF-7 human breast
cancer cell derived tumors (w150 mm3) with 140 mg/kg/bid of
#1181 sc for 3 weeks resulted in w30% regression in tumor size
[25]. This anti-tumor activity is associated with phosphorylation of
Herein, we report the development of a focused library of newly
substituted 3,3-diphenyl-1,3-dihydroindol-2-ones derived from
#1181 and the characterization of their inhibition of eIF2$GTP$tR-
NAMi et TC formation and cancer cell proliferation.
2. Results and discussion
2.1. Chemistry
The general approach to the synthesis of the library of new
substituted 3,3-diphenyl-1,3-dihydroindol-2-ones is outlined in
Scheme 1. The various isatins used in the condensation with the
phenols were either commercially available or synthesized
following the well-established Sandmeyer’s methodology [33].
Accordingly, anilines such as 2,3-difluoroaniline, methylantranilate,
and 6-aminobenzothiazole were condensed with hydroxylamine
and chloral hydrate generating hydroxyiminoacetanilide in-
termediates that underwent thermal cyclization in concentrated
sulfuric acid to form the corresponding isatins (Fig. 2).
eIF2
findings provide proof-of-principle that #1181-mediated phos-
phorylation of eIF2 and reduction of the TC formation are phar-
a and inhibition of the expression of oncogenic proteins. These
a
macologically relevant and promising targets for anti-cancer
therapy.
Many different 3,3-diarylindolin-2-one analogs have already
been reported as potential drugs. As an example, 3,3-bis(4-amino-
2,5-dimethoxyphenyl)-1,3-dihydroindol-2-one showed
a good
chemical antioxidant activity even if its antioxidative mechanism
was not directly elucidated [26]. A series of N-substituted 3,3-
bisaryloxindoles was identified as potent non-steroidal mineralo-
corticoid receptor (hMR) antagonists, displaying an excellent
selectivity for hMR over human glucocorticoid, progesterone,
Grignard reaction between an appropriately substituted phe-
nylmagnesium bromide and isatin employing standard Grignard
reaction conditions generated 3-hydroxy-3-phenyl-1,3-dihydro-
2H-indol-2-ones 1e17 in moderate to good yield [34]. Yields lower
than 20% were observed when one of the reactants was either 6H-
[1,3]thiazolo[5,4,e]indol-7,8-dione or pyridin-3-ylmagnesium bro-
mide. The second and final step of the general synthetic method-
ology consisted of
a FriedeleCrafts acylation in which the
substituted or non-substituted 4-tert-butylphenol reacted with the
3-hydroxy-3-phenyl-1,3-dihydro-2H-indol-2-ones 1e17 to form
the 3,3-diaryloxindoles 25e49 [35]. The acid used in this final step
was either triflic or p-toluene sulfonic acid depending on the
reactivity of the incoming tert-butyl-substituted ring toward elec-
trophilic aromatic substitution. This synthetic route resulted in
racemic mixtures of the 3,3-diaryloxindoles.
In the absence of commercial sources for some of the 4-tert-
butylphenyl ethers, we developed effective synthetic routes for
their preparation. O-alkylation of 4-tert-butylphenol with 4-(2-
hydroxyethyl)morpholine or the partially protected benzyl 4-(2-
hydroxyethyl)piperazine-1-carboxylate employed a Mitsunobu
reaction in the presence of triphenylphosphine and diethyl
Fig. 1. Clotrimazole (CLT), #1181 and substituted 3,3-diphenylindol-2-one scaffold.

S. Denoyelle et al. / European Journal of Medicinal Chemistry 69 (2013) 537e553
539
Final
comp.
Interm.
R1
H
R2
H
R3
H
R4
H
R5
H
R6
H
X
R7
Modif.a
25
C
C
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
C
1
CH
C
C
H
H
B
2
3
H
H
H
H
H
H
H
H
OMe
H
H
CH
CH
B+C
B
N(CH3)2
B+C
B
4
5
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
CH
CH
N
H
H
H
Cl
H
B
6
H
B
7
H
OMe
OMe
H
OMe
F
CH
CH
CH
CH
B+C
B+C
A
8
H
9
I
H
H
H
10
OCF3
H
H
A
11
12
H
F
H
F
H
H
H
CH
CH
41
H
A
H
H
H
OMe
42
43
44
45
46
47
48
49
H
H
A+B
A+B
13
14
OMe
H
H
H
OMe
OMe
H
H
CH
CH
A+B+C
A+B
H
H
H
CO2CH3
A+B+C
A+B+C
A+B+C
A+B+C
15
16
17
H
H
H
I
OCF3
H
H
H
F
H
H
F
OMe
OMe
H
H
H
CH
CH
CH
N(CH3)2
Scheme 1. General procedure for the synthesis of oxindoles 25e49.aa Modifications on aryl rings A, B and/or C by introduction of different substituents or groups of atoms.
azodicarboxylate (DEAD) in anhydrous THF and generated
the corresponding phenyl ethers 18 and 23, respectively (Scheme
2) [36]. The latter protection strategy prevented the potential
competition between the primary alcohol and the secondary
amine on reacting with 4-tert-butylphenol. The alkylation of the
same phenol with N,N-dimethylaminoethylene bromide, prop-
argylbromide, or tert-butylbromoacetate was carried out in
anhydrous DMF in the presence of NaH yielding the corre-
sponding phenyl ethers 19, 20, and 21 respectively (Scheme 2).
Both methods allowed satisfying yields (46e81%).
Following this general method of two-step synthesis we syn-
thesized 25 new substituted 3,3-diphenyl-1,3-dihydroindol-2-
ones. In some cases, post-assembly modifications such as deme-
thylation of methoxy groups using boron tribromide methyl sulfide
complex in refluxing dichloromethane [37] transformed com-
pounds 30 and 42 into compounds 50 and 51, respectively. Sub-
jecting 3,3-diphenyl-1,3-dihydroindol-2-one 39 to Suzukie
Miyaura cross coupling reaction using a boronic acid pinacol ester
Fig. 2. Sandmeyer methodology for the synthesis of the commercially unavailable
isatins from the corresponding anilines.

540
S. Denoyelle et al. / European Journal of Medicinal Chemistry 69 (2013) 537e553
Scheme 2. Synthesis of intermediates 18e21 and 24.aa Reagents and reaction conditions: (i) triphenylphosphine, DEAD, anhydrous THF, 0 ꢁC; (ii) NaH, anhydrous DMF; (iii)
benzylchloroformate, 4N NaOH, CH3CN/H2O 1:1; (iv) H2, PdeC, AcOHcat., MeOH, 1 atm.
in the presence of Pd(PPh3)2Cl2 in refluxing THF generated com-
pound 52 in which ring A was decorated with the 6-(morpholin-4-
yl)pyridin-3-yl substituent (Scheme 3) [38].
mechanistic TC availability assay, cell-based dual luciferase reporter
gene assay [39] modified to reflect the abundance of the
eIF2$GTP$Met-tRNAMi et TC [24]. Subsequently, selected represen-
tatives of this oxindole series were subjected to a secondary
mechanistic assay, specifically testing the effect of these com-
pounds on the levels of C/EBP homologous protein (CHOP) mRNA,
which is induced when the TC availability is reduced. We antici-
pated that inhibition of TI by depleting TC formation would
correlate with inhibition of cancer cell proliferation.
The only departure from the canonical scaffold comprised of a
tert-butyl moiety at position 5 of the C ring was its replacement by a
propan-2-yloxy group that maintained most of the bulkiness pre-
sented by tert-butyl but added a polar heteroatom that can act as a
hydrogen bond acceptor (Scheme 4, compound 55). Synthesis of 3-
(4-methoxyphenyl)-3-{2-[2-(morpholin-4-yl)ethoxy]-5-(propan-
2-yloxy)phenyl}-1,3-dihydro-2H-indol-2-one (55) included a non-
discriminating, low yield (19%) double alkylation of the 1,4-
hydroquinone by first 2-iodopropane and then 4-(2-chloroethyl)
morpholine.
All 29 new substituted 3,3-diphenyl-1,3-dihydroindol-2-ones
were purified (purity ꢀ95% determined by analytical reversed
phase HPLC, see Table S1 in “Supporting information”) either by
crystallization, reverse phase (RP) flash chromatography or RP-
preparative HPLC, and their structural integrity was confirmed by
1H and 13C NMR, and HReMS.
2.2.1. Inhibition of cancer cell proliferation
In an effort to modulate the physicochemical properties of
#1181 (see Table S2 in “Supporting information”) we took advan-
tage of O-alkylation of the hydroxyl in position 2 of ring C as a
convenient site for introduction of either polar groups (analogs 25e
27 and 29) or a hydrophobic group (analog 28) (Table 1). Sub-
stitutions with amines such as analogs 25e27 maintained the
inhibitory cell proliferation activity of #1181 while substitution
with either hydrophobic or carboxyl bearing moieties (analogs 28
and 29, respectively) led to substantial increase in the IC50
In general mono- or disubstitutions on ring B are well tolerated
and maintain IC50s in the 2e17 M range in all 3 cell types included
.
2.2. Biological activity
m
in this study (entries 2e5 and 13, Table 2). Likewise, mono-
substitutions on ring A were well tolerated regardless of their
chemical nature (entries 8 and 15, Table 2). In addition, simulta-
neous substitutions on both rings A and B resulted in compounds
The SAR study reported herein aimed at exploring the latitude
for diverse decorations on the phenyl rings in the prototypic #1181,
which has recently been reported as a potent anti-tumor agent
targeting the TC and able to inhibit TI in animal models of human
cancer [25]. In this study we first tested the efficacy of our new
series of oxindoles to inhibit the growth of adherent murine lung
squamous cell carcinoma (KLN 205) and human breast cancer and
melanoma cells (CRL-2351 and CRL-2813, respectively) employing
the sulforhodamine B (SRB) assay [24]. We then carried out the
that had IC50s in the low mM at least in one cell line except for
compound 51 that was weakly potent in KLN-205 and CRL-2813
and inactive in CRL-2351 cells (entries 10e12 and 14, Table 2).
It seems that, out of the three cell lines, the murine KLN-205 is
the least responsive while the human CRL-2351 is the most
responsive to the parent oxindole #1181 and its rings B and C
substituted analogs. Loss of cell growth inhibitory activity
(IC50 > 20 mM) was observed for analogs 36 and 45 in KLN-205 cells
and for analog 51 in CRL-2351 cells (Table 2). The former, analog 36,
was associated with the introduction of 3-pyridyl ring in position B
while analog 45 resulted in the combination of 40-methoxy and 7-
methoxycarbonyl on rings B and A, respectively. The latter was the
trisubstituted analog 51 (6,7-difluoro,40-hydroxy on rings A and B,
respectively).
The most active compounds in CRL-2813 were 40 and 41 and in
Scheme 3. Synthesis of oxindole 52.aa Reagents and reaction conditions: (i) 6-(mor-
pholin-4-yl)pyridin-3-boronic acid pinacol ester, Pd(PPh3)2Cl2, 2M Na2CO3 solution,
degassed THF.
CRL-2351 42 displaying IC50s of w1
mM (Table 2). Relative to the
#1181, all three compounds are substituted on ring A by electron

S. Denoyelle et al. / European Journal of Medicinal Chemistry 69 (2013) 537e553
541
potency was observed with the disubstituted and trisubstituted
analogs 46 and 49, respectively, which were about 2e6-fold less
potent than the parent 25. Interestingly, these losses in potency
were the result of either addition of a 7-CO2Me to 31 or 6,7-diF to
33. Moreover, addition of a 30-OMe on ring B of 33 resulted in the
inactive 30,40-diOMe analog 37 (Table 3).
Evidently, #1181 tolerates a wide range of substitutions as it
relates to inhibition of cancer cell growth, many of these sub-
stitutions being polar and/or charge-bearing ones.
2.2.2. Depletion of the TC
The dual luciferase TC reporter assay that measures the ability of
the small molecule to reduce the availability of the eIF2$GTP$Met-
tRNAMi et (TC) is not only our basic high throughput screening assay
but also the first line assay for gathering structure-TC-mediated TI
inhibitory activity relationship. In general, reduction in TC avail-
ability will result in the inhibition of the translation of most weak
mRNAs except for a small subset of mRNAs that contain multiple
upstream open reading frames (uORFs) in their 50-untranslated
region (50UTRs) such as mRNA coding for activating transcription
factor 4 (ATF-4). Our dual luciferase TC reporter assay has firefly
luciferase mRNA fused to the 50UTR of ATF-4 mRNA and renilla
luciferase fused to the 50UTR that is lacking any uORFs. Validation
and adaptation of this assay to HTS has been previously described
[24]. Therefore, oxindoles that reduce the availability of the TC
would increase firefly luciferase expression and concomitantly
decrease the expression of renilla luciferase, resulting in an
increased firefly/renilla luciferase ratio (F/R).
Scheme 4. Synthesis of oxindole 55.aa Reagents and reaction conditions: (i) 2-
iodopropane, KOH, EtOH, reflux; (ii) 4-(2-chloroethyl)morpholine hydrochloride,
KOH, EtOH, reflux; (iii) intermediate 2, triflic acid, CH2Cl2, 0 ꢁC.
withdrawing substituents and an additional 4-OMe on ring B in
compound 42. Of interest is the remarkable difference in CRL-2351
cell growth inhibitory activity between one of the most active
compounds 42 and the non-active 51, which differ only in the
substituent on ring B (40-OH and 40-OMe, respectively, Table 2). The
same structural difference is presented by the two active com-
pounds 30 and 50 but in this case it results with very similar ac-
tivities in all three cell lines (Table 2).
In an attempt to merge beneficial modifications into a single
structure, we chose oxindole 25 substituted by the polar 2-(mor-
pholino-4-yl)ethoxy group on position 2 of ring C, which was
slightly more potent than #1181 (Table 1), as the basic scaffold and
submitted it to another round of decorations (Table 3). The majority
of analogs modified with either one (31 and 33) or two (38) sub-
stitutions of variable nature on ring B did not change the cell
growth inhibitory activity in a significant way relative to the parent
analog 25. Similarly, disubstitutions on rings A and B (44, 47, and
48) did not affect significantly the IC50s. A notable decrease in
Each compound was tested at 4 different concentrations 10, 20,
40 and 80 mM. Relative to the oxindole #1181, the majority of the
compounds in the current series were at least as potent as the
parent compound in inhibiting TI (Fig. 3). Decorating #1181 with
either 4,5-[1,3]thiazolo fused to positions 4 and 5 of ring A (41), 40-
methoxy on ring B (42), or 5,40-dimethoxy on rings A and B (43) led
to inactive analogs. In general, substitution of ring C on position 20
was more tolerated. Only one compound out of those substituted
by 20-(morpholino-4-yl)ethoxy group on position 20 of ring C was
inactive (46). In addition, substitution of the same position with 20-
piperidino-4-yl)ethoxy (27) or 20-acetoxy (29) led to a loss of ac-
tivity. Relative to the #1181, analogs 28, 30, 38, 48, and 49 were
about 1.2e1.5-fold and analogs 31, 33, and 39 were about 1.5e2-
Table 1
Inhibition of cancer cells proliferation.a
fold more potent at the same concentration (40
analogs 34, 36, and 52 had their maximal increase of ATF-4-50UTR-
mediated expression of firefly luciferase at 80 M (3-, 4.5-, and 4-
mM). Conversely,
m
fold, respectively). These analogs presented the same substitution
pattern on ring C as in #1181, but in addition had 40-morpholino-
methyl on ring B (34), pyridyl-3-yl as ring B (36), or 5-[6-(mor-
pholin-4-yl)pyridin-3-yl] on ring A (52). Importantly, the most
potent analogs in this series displayed higher maximal TI inhibitory
activity in the dual luciferase TC reporter assay but at a higher
concentration compared to the maximal activity of #1181. It is
possible that the for the compounds where the TI inhibitory activity
Entry
Comp.
R7
IC50
KLN
(mM)
2813
2351
1
2
#1181
25
H
14.4 ꢂ 1.5
6.8 ꢂ 3.7
4.8 ꢂ 3.7
2.4 ꢂ 0.3
3.4 ꢂ 0.66
3.35 ꢂ 0.3
at 80
mM is lower than at 40 mM is a result of exceeding the
maximum solubility leading to lower apparent solubility.
3
4
26
27
1.2 ꢂ 0.3
1.2 ꢂ 0.3
2.4 ꢂ 0.4
1.4 ꢂ 0.3
0.9 ꢂ 0.1
12.8 ꢂ 0.8
2.2.3. Expression of CHOP mRNA
Demonstration of the impact of oxindoles as inhibitors of TI
through depletion of the TC on a downstream target, such as
expression of CHOP mRNA, was carried out to provide mechanistic
validation of our above observations. CHOP mRNA levels are tran-
scriptionally controlled by ATF-4, which itself is translationally
upregulated when the TC availability is reduced. Therefore, reduced
TC availability as demonstrated above should result in elevated
CHOP mRNA levels. To this end we selected several active oxindole
derivatives representing a wide range of activity in TC assay and
5
28
29
>20
>20
>20
>20
17.5 ꢂ 2
>20
6
a
To determine IC50 values on murine KLN 205 and human CRL-2813 and CRL-
2351 cell lines, cell proliferation was measured by the sulforhodamine B (SRB)
assay (see Experimental section for assays details). IC50s are indicated as the mean
values of 2 experiments, each done in triplicates.

542
S. Denoyelle et al. / European Journal of Medicinal Chemistry 69 (2013) 537e553
Table 2
Inhibition of cancer cells proliferation.a
Entry
Comp.
R1
R2
R3
R4
R5
R6
X
IC50
KLN
(mM)
2813
2351
1
2
3
#1181
30
32
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH
CH
CH
14.4 ꢂ 1.5
15.7 ꢂ 1.1
10.9 ꢂ 1.3
4.8 ꢂ 3.7
3.4 ꢂ 0.66
2.65 ꢂ 0.1
2.3 ꢂ 0.3
OMe
N(CH3)2
2.75 ꢂ 0.1
11 ꢂ 0.58
4
34
H
H
H
H
H
CH
17.3 ꢂ 2.4
7.8 ꢂ 0.66
7.3 ꢂ 3.6
5
6
7
8
35
36
39
40
H
H
H
H
H
H
I
H
H
H
H
H
H
H
H
Cl
H
H
H
CH3
H
H
CH
N
CH
CH
4.3 ꢂ 1
>20
2.9 ꢂ 0.1
11.5 ꢂ 1.8
2.8 ꢂ 0.2
1 ꢂ 0.1
2.9 ꢂ 0. 1
6.4 ꢂ 3
3.5
2.5 ꢂ 0.9
14.9 ꢂ 3.8
OCF3
H
8 ꢂ 1.7
9
41
H
H
H
H
CH
8 ꢂ 3.3
0.95 ꢂ 0.1
2.2 ꢂ 1
10
11
12
13
14
42
43
45
50
51
H
H
H
H
H
H
OMe
H
H
H
F
F
H
OMe
OMe
OMe
OH
H
H
H
H
H
CH
CH
CH
CH
CH
12.3 ꢂ 2
14.3 ꢂ 3
>20
3 ꢂ 0.9
11.1 ꢂ 1.1
12.2 ꢂ 0.5
4 ꢂ 0.28
0.95 ꢂ 0.08
2.9 ꢂ 0.3
3.4 ꢂ 0.6
2 ꢂ 0.3
H
H
H
F
CO2CH3
H
F
17.3 ꢂ 1.8
OH
15.3 ꢂ 1.9
10.7 ꢂ 3.9
>20
15
52
H
H
H
H
H
CH
13.3 ꢂ 2.4
2.5 ꢂ 1.5
1.8 ꢂ 0.4
a
To determine IC50 values on murine KLN 205 and human CRL-2813 and CRL-2351 cell lines, cell proliferation was measured by the sulforhodamine B (SRB) assay (see
Experimental section for assays details). IC50s are indicated as the mean values of 2 experiments, each done in triplicates.
Table 3
Inhibition of cancer cells proliferation.a
Entry
Comp.
R2
R3
R4
R5
R6
IC50
KLN
(mM)
2813
2351
1
2
3
4
5
6
7
8
9
25
31
33
37
38
44
46
47
48
49
H
H
H
H
H
OMe
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
OMe
F
H
H
H
H
6.8 ꢂ 3.7
4.3 ꢂ 1
2.4 ꢂ 0.3
2.4 ꢂ 0.9
7.5 ꢂ 1
3.35 ꢂ 0.3
2.65 ꢂ 1.5
4.7 ꢂ 0.3
>20
2.5 ꢂ 0.6
4.5 ꢂ 0.7
16.3 ꢂ 1.6
6.1 ꢂ 1.7
2.9 ꢂ 0.27
4 ꢂ 1
OMe
N(CH3)2
OMe
OMe
OMe
OMe
OMe
OMe
N(CH3)2
2.2 ꢂ 0.6
>20
>20
3.4 ꢂ 1.2
2.75 ꢂ 0.3
17 ꢂ 3.5
12 ꢂ 1.2
9.3 ꢂ 2.7
15.7 ꢂ 1.9
2.7 ꢂ 0.4
2.9 ꢂ 0.3
14.6 ꢂ 2.8
2.6 ꢂ 0.4
1.9 ꢂ 0.7
11 ꢂ 1.3
CO2CH3
I
H
H
F
OCF3
H
10
H
a
To determine IC50 values on murine KLN 205 and human CRL-2813 and CRL-2351 cell lines, cell proliferation was measured by the sulforhodamine B (SRB) assay (see
Experimental section for assays details). IC50s are indicated as the mean values of 2 experiments, each done in triplicates.

S. Denoyelle et al. / European Journal of Medicinal Chemistry 69 (2013) 537e553
543
Fig. 3. Ternary complex assays on KLN-205 cell line (see Experimental section for assays details). Activity of compounds was measured by the firefly/renilla luciferase ratio (F/R)
compared to vehicle treated cells, and expressed for each compound as a function of concentrations (10 M, 20 M, 40 M, 80 M). Compounds were compared to #1181 which
maximal activity (obtained at a concentration of 40 M) is represented by the dashed line.
m
m
m
m
m
tested them for induction of CHOP mRNA (Table 4). We were very
encouraged by the consistent and direct relationship between the
compound’s activity in the TC assay and the fold increase over
control (DMSO) of CHOP mRNA level, both carried out in KLN-205
cells. This concurrence validates our notion that active oxindoles
reduce TC availability and induce ATF-4 expression that leads to
downstream increase in CHOP mRNA transcription.
Overall, these results were very encouraging. Nevertheless, it
should be noted that the correlation between the inhibition of
cancer cell proliferation and activity of compounds in the ternary
complex assay was observed for those compounds that display
significant activity (ꢀ3-fold depletion of TC as compared to the
control DMSO) in the ternary complex assay at concentrations
4. Experimental section
4.1. Chemistry
All reagents and solvents were purchased from commercial
sources and used without further purification. All the solvents were
anhydrous and maintained under nitrogen. Melting points were
determined using a Mel-Temp Electrothermal apparatus and are
uncorrected. Proton and carbon NMR analyses were performed on
Varian 400 MHz and 500 MHz spectrometers using deuterium sol-
vents. Chemical shifts data are reported in parts per million (ppm)
relative to the residual peak of the solvent (
DMSO-d6, and 3.31 and 49.00 ppm for CD3OD) according to Got-
tlieb et al. [40]. All the reactions were monitored by LC-MS analysis
m particle
d 2.50 and 39.52 ppm for
d
below 40
mM. The compounds that in the ternary complex assay
displayed large activity peak at 80
mM without apparent activity at
on reverse phase (column XTerra MS C8, 3 ꢃ 100 mm, 5
m
the lower doses were usually devoid of activity in the cell prolif-
eration assay. Similarly, cell growth inhibitory activity of some
compounds appear to originate at least in part from the off-target
effects that lead to inhibition of many other critical mechanisms
required for cell growth and proliferation and have limited
contribution from the depletion of TC and inhibition of translation
initiation. A good example of such dissociation is displayed by the
high cell inhibitory activity of 40e42 that are devoid of ATF-4 ac-
tivity (Fig. 3). Therefore, in the future we suggest using high po-
tency of analogs in the ternary complex assay obtained at low
concentrations as the best guide during SAR aiming to improve the
activity and specificity of this class of compounds.
size) using a ThermoFisher Finnigan/LCQ Advantage apparatus with
the binary system water/acetonitrile containing 0.1% of trifluoro-
acetic acid (TFA) as eluent. Purifications by flash chromatography
were performed on Biotage SP1 using pre-packed columns filled
with silica gel and were monitored by UV detection at 254 and
280 nm. Purifications by preparative HPLC on reverse phase (column
XTerra Prep MS C8,19 ꢃ150 mm, 5
mm particle size) were carried out
on a Waters 2525 using the binary system water/acetonitrile con-
taining 0.1% of acetic acid as eluent. The HR-MS analyses were per-
formed by the FAS Center for Systems Biology, Harvard University.
The purity of all final compounds was determined by analytical
HPLC on reverse phase (column XBridge BEH130 C18, 4.6 ꢃ 100 mm,
5 mm particle size) using a Waters Alliance 2695 with the binary
3. Conclusion
system water/acetonitrile containing 0.1% of acetic acid as eluent
(see Table S1 in “Supporting information” for more details).
In this study, our iterative lead optimization process has focused
on the introduction of polar groups on ring C that could improve
bioavailability (Table 1), a diverse spectrum of substituents on rings
A and B that may contribute to potency enhancement (Table 2), and
combination of the above to explore possible complementarity
(Table 3). We are very encouraged by the structural insight gained
and the realization of active compounds that display remarkable
enhancement in their activities in the TC reporter assay and CHOP
mRNA induction (Fig. 4). We are confident that our results will
enable further improvement in bioavailability and potency that will
lead to compounds of this chemotype that warrant in vivo
evaluation.
4.1.1. General procedure A for the synthesis of compounds 1e17
4.1.1.1. 3-Hydroxy-3-phenyl-1,3-dihydro-2H-indol-2-one (1). A 1 M
solution of phenylmagnesium bromide in THF (20.40 mL,
20.40 mmol) was added dropwise to a stirred ice-cold solution of
isatin (1 g, 6.80 mmol) in anhydrous THF (20 mL) that was kept
under nitrogen. The reaction was stirred at room temperature for
2 h and then quenched with a saturated aqueous solution of NH4Cl
(20 mL). Water was then added (30 mL) and the aqueous phase was
extracted twice with dichloromethane (2 ꢃ 60 mL). The organic
phase was washed with brine and dried over anhydrous Na2SO4.
The solvent was removed in vacuum and the crude was purified by

544
S. Denoyelle et al. / European Journal of Medicinal Chemistry 69 (2013) 537e553
Table 4
Expression of CHOP mRNA of active oxindole derivatives representing a wide range of increased ATF-4 activity compared to #1181.a
Comp.
Structure
ATF-4
CHOP PCR
Max fold increase of F/R luciferase over control @ [
m
M]
Fold over control
#1181
3.7 ꢂ 0.7 @ 40
2.9 ꢂ 0.6 @ 40
3.77
3.09
51
48
5.11 ꢂ 0.48 @ 40
3.49
30
31
5.6 ꢂ 0.5 @ 40
6.3 ꢂ 0.3 @ 40
4.09
7.1
33
7.15 ꢂ 0.49 @ 40
4.15
39
34
7.2 ꢂ 1.2 @ 40
12.4 ꢂ 3.6 @ 80
4.36
4.68
52
15.6 ꢂ 0.4 @ 80
20.8
a
Ternary complex and CHOP PCR assays were carried out on murine KLN-205 cell line (see Experimental section for assays details). CHOP PCR values represent the fold
increase over control (DMSO).
crystallization from EtOAc/Hexane to afford 1 (1.18 g, 77.1%) as a
white powder. Mp ¼ 215.4e216.7 ꢁC. 1H NMR (500 MHz, DMSO-d6):
DMSO-d6):
6.86 (m, 4H, CHarom.), 6.55 (s, 1H, OH), 3.62 (s, 3H, CH3). 13C NMR
(125.7 MHz, DMSO-d6): 178.74, 158.70, 141.91, 133.78, 133.49,
d 10.35 (s, 1H, NH), 7.26e7.12 (m, 4H, CHarom.), 6.99e
d
10.43 (s, 1H, NH), 7.32e7.24 (m, 6H, CHarom.), 7.12 (d, J ¼ 7 Hz, 1H,
d
CHarom.), 6.98 (m, 1H, CHarom.), 6.93 (d, J ¼ 7.5 Hz, 1H, CHarom.), 6.65
129.19, 126.80 (2C), 124.78, 122.03, 113.50 (2C), 109.77, 76.96, 55.04.
(s, 1H, OH). 13C NMR (125.7 MHz, DMSO-d6):
d
178.54, 141.98,
MS (ESI) m/z 237.96 (M ꢄ OH)þ.
141.58, 133.79, 129.27, 128.11 (2C), 127.44, 125.46 (2C), 124.82,
122.08, 109.90, 77.37. MS (ESI) m/z 225.75 (M þ H)þ.
4.1.1.3. 3-[4-(Dimethylamino)phenyl]-3-hydroxy-1,3-dihydro-2H-
indol-2-one (3). A 0.5 M solution of 4-(dimethylamino)phenyl-
magnesium bromide in THF (20.40 mL, 10.20 mmol) and isatin
(500 mg, 3.40 mmol) were used following the General procedure A
to afford 3 (834 mg, 91.5%) as a white powder. Mp ¼ decomp. 1H
4.1.1.2. 3-Hydroxy-3-(4-methoxyphenyl)-1,3-dihydro-2H-indol-2-
one (2). A 0.5 M solution of 4-methoxyphenylmagnesium bromide
in THF (40.80 mL, 20.40 mmol) and isatin (1 g, 6.80 mmol) were
used following the General procedure A to afford 2 (0.90 g, 51.9%)
as a white powder. Mp ¼ 208.7e210.0 ꢁC. 1H NMR (500 MHz,
NMR (400 MHz, DMSO-d6):
d 10.28 (s, 1H, NH), 7.23 (m, 1H,
CHarom.), 7.14e7.10 (m, 3H, CHarom.), 6.97 (m, 1H, CHarom.), 6.87 (d,

S. Denoyelle et al. / European Journal of Medicinal Chemistry 69 (2013) 537e553
545
Fig. 4. Schematic summary of SAR of the series of 3,3-diphenylindol-2-ones reported herein based on the ternary complex assay (Fig. 3 and Table 4). Blue substituents are
enhancing potency, green ones are most affecting the potency, and the red ones are reducing the potency relative the prototypic #1181. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
J ¼ 8 Hz, 1H, CHarom.), 6.65 (d, J ¼ 8.8 Hz, 2H, CHarom.), 6.39 (s, 1H,
OH), 2.84 (s, 6H, CH3). 13C NMR (100.6 MHz, DMSO-d6):
179.00,
organic phase was washed with brine and dried over anhydrous
Na2SO4. The solvent was removed in vacuum and the crude was
purified by flash chromatography (10e40% of ethyl acetate in
cyclohexane) to afford 6 (151 mg, 18%) as a yellow powder.
d
149.89, 141.87, 134.01, 128.93, 128.87, 126.42 (2C), 124.83, 121.84,
111.93 (2C), 109.68, 76.96, 40.19 (2C). MS (ESI) m/z 269.17 (M þ H)þ.
Mp ¼ 292.5e294.2 ꢁC. 1H NMR (500, MHz, DMSO-d6):
d 8.52 (s,
4.1.1.4. 3-Hydroxy-3-[4-(morpholin-4-ylmethyl)phenyl]-1,3-dihydro-
2H-indol-2-one (4). A 0.25 M solution of 4-(morpholin-4-ylmethyl)
phenylmagnesium bromide in THF (30 mL, 7.48 mmol) and isatin
(550 mg, 3.74 mmol) were used following the General procedure
A. The crude was purified by flash chromatography (0e20% of
methanol in dichloromethane) to afford 4 (1.12 g, 92.4%) as a yellow
1H, NH), 8.47 (s, 1H, CHarom.), 7.84 (m, 1H, CHarom.), 7.70 (d,
J ¼ 8.5 Hz, 1H, CHarom.), 7.40 (m, 1H, CHarom.), 7.35 (d, J ¼ 8.0 Hz,
1H, CHarom.), 7.24e7.19 (m, 2H, CHarom., OH), 7.08 (m, 1H, CHarom.),
6.99 (d, J ¼ 8.0 Hz, 1H, CHarom.). 13C NMR (125.7 MHz, DMSO-d6):
d
179.35, 148.19, 146.53, 141.96, 134.79, 130.12, 128.62, 125.77,
124.92, 123.82, 123.12, 110.52, 84.40. MS (ESI) m/z 227.03
oil. 1H NMR (400 MHz, CDCl3):
d 9.50 (s, 1H, NH), 7.29 (m, 2H,
(M þ H)þ.
CHarom.), 7.18 (m, 2H, CHarom.), 7.07e7.13 (m, 2H, CHarom.), 6.92 (t,
J ¼ 7.6 Hz, 1H, CHarom.), 6.73 (d, J ¼ 8.0 Hz, 1H, CHarom.), 3.56 (m, 4H,
CH2OCH2), 3.37 (s, 2H, CH2F), 2.32 (m, 4H, CH2NCH2). 13C NMR
4.1.1.7. 3-(3,4-Dimethoxyphenyl)-3-hydroxy-1,3-dihydro-2H-indol-
2-one (7). A 0.5 M solution of 3,4-dimethoxyphenylmagnesium
bromide in THF (20.40 mL, 10.20 mmol) and isatin (500 mg,
3.40 mmol) were used following the General procedure A to afford
7 (767 mg, 79.1%) as a white powder. Mp ¼ 219.2e220.3 ꢁC. 1H NMR
(100.6 MHz, CDCl3):
d 180.81, 141.04 (2C), 139.34, 137.26, 132.97,
129.81 (2C), 125.67 (2C), 125.27, 123.48, 111.01, 78.54, 66.84 (2C),
63.02, 53.50 (2C). MS (ESI) m/z 325.10 (M þ H)þ.
(400 MHz, DMSO-d6):
d 10.33 (s, 1H, NH), 7.25e7.10 (m, 3H,
4.1.1.5. 3-(4-Chloro-3-methylphenyl)-3-hydroxy-1,3-dihydro-2H-
indol-2-one (5). A 0.5 M solution of (4-chloro-3-methyl)phenyl-
magnesium bromide in THF (32.70 mL, 16.35 mmol) and isatin
(1.20 g, 8.16 mmol) were used following the General procedure A.
The crude was purified by flash chromatography (5e40% of ethyl
acetate in cyclohexane) to afford HW (1.48 g, 67%) as a yellow oil. 1H
CHarom.), 6.98e6.82 (m, 3H, CHarom.), 6.56 (m, 2H, CHarom., OH), 3.72
(s, 3H, CH3), 3.70 (s, 3H, CH3). 13C NMR (125.7 MHz, DMSO-d6):
d
178.56, 148.46, 148.35, 141.89, 133.97, 133.64, 129.16, 124.83 (2C),
121.95, 117.72, 111.38 (2C), 109.78, 76.97, 55.50 (2C). MS (ESI) m/z
268.21 (M ꢄ OH)þ.
NMR (500 MHz, DMSO-d6):
d
10.48 (s, 1H, NH), 7.32 (d, J ¼ 8.0 Hz,
4.1.1.8. 3-(3-Fluoro-4-methoxyphenyl)-3-hydroxy-1,3-dihydro-2H-
1H, CHarom.), 7.22e7.25 (m, 2H, CHarom.), 7.07 (d, J ¼ 7.0 Hz, 1H,
CHarom.), 7.02 (dd, J ¼ 2.5 Hz, J ¼ 8.0 Hz, 1H, CHarom.), 6.95 (dd,
J ¼ 2.5 Hz, J ¼ 8.0 Hz, 1H, CHarom.), 6.90 (d, J ¼ 8.0 Hz, 1H, CHarom.),
6.69 (s, 1H, OH), 2.27 (s, 3H, CH3). 13C NMR (125.7 MHz, DMSO-d6):
indol-2-one (8). A 0.5
M
solution of 3-fluoro-4-methox
yphenylmagnesium bromide in THF (12.25 mL, 6.12 mmol) and isa-
tin (300 mg, 2.04 mmol) were used following the General procedure
A to afford 8 (443 mg, 79.5%) as a white powder. Mp ¼ 197.8e199 ꢁC.
d
178.75, 142.64, 141.23, 135.72, 133.99, 133.00, 130.07, 129.24,
1H NMR (500 MHz, DMSO-d6):
d 10.40 (s, 1H, NH), 7.26 (m, 1H,
128.84,125.40 (2C),122.80, 110.67, 77.53, 20.43. MS (ESI) m/z 273.85
(M þ H)þ.
CHarom.), 7.19e7.13 (m, 2H, CHarom.), 7.07 (m,1H, CHarom.), 6.98 (m,1H,
CHarom.), 6.91e6.87 (m, 2H, CHarom.), 6.67 (s,1H, OH), 3.79 (s, 3H, CH3).
13C NMR (125.7 MHz, DMSO-d6):
d 178.15, 152.12, 150.18, 146.50,
4.1.1.6. 3-Hydroxy-3-(pyridine-3-yl)-1,3-dihydro-2H-indol-2-one
(6). To a suspension of magnesium (216 mg, 8.88 mmol) in
anhydrous THF (5 mL) was added a few drops of 3-bromopyridine
141.91, 134.45, 133.09, 129.42, 124.84, 122.13, 121.66, 113.39, 109.95,
76.53, 56.03. MS (ESI) m/z 256.30 (M ꢄ OH)þ.
and 1,2-dibromoethane (100
reaction mixture was warmed up for 2 min to initiate the reaction
and 3-bromopyridine (713 L, 7.40 mmol) was added dropwise.
After refluxing under nitrogen for 6 h, the mixture was cooled to
room temperature and added dropwise to a solution of isatin
(544 mg, 3.70 mmol) in anhydrous THF (20 mL) maintained at 0 ꢁC
under nitrogen. After stirring for 2 h at room temperature, the
reaction was quenched with a saturated aqueous solution of NH4Cl
(20 mL). Water was then added (30 mL) and the aqueous phase
was extracted twice with dichloromethane (2 ꢃ 60 mL). The
m
L, 1.16 mmol) under nitrogen. The
4.1.1.9. 3-Hydroxy-5-iodo-3-phenyl-1,3-dihydro-2H-indol-2-one (9).
A 1 M solution of phenylmagnesium bromide in THF (22 mL,
22.00 mmol) and 5-iodoisatin (2 g, 7.33 mmol) were used following
the General procedure A to afford 9 (1.88 g, 73.1%) as a white
powder. Mp ¼ 313.6e314.3 ꢁC. 1H NMR (500 MHz, DMSO-d6):
m
d
10.56 (s,1H, NH), 7.61 (d, J ¼ 8.5 Hz,1H, CHarom.), 7.36e7.26 (m, 6H,
CHarom.), 6.79 (s, 1H, CHarom.), 6.76 (s, 1H, OH). 13C NMR (125.7 MHz,
DMSO-d6): 177.77, 141.70, 140.89, 137.74, 136.42, 132.88, 128.25
d
(2C), 127.64, 125.27 (2C), 112.51, 84.79, 77.17. MS (ESI) m/z 334.15
(M ꢄ OH)þ.

546
S. Denoyelle et al. / European Journal of Medicinal Chemistry 69 (2013) 537e553
4.1.1.10. 3-Hydroxy-3-phenyl-5-(trifluoromethoxy)-1,3-dihydro-2H-
indol-2-one (10). A 1 M solution of phenylmagnesium bromide in
THF (6.50 mL, 6.50 mmol) and 5-(trifluoromethoxy)isatin (500 mg,
2.16 mmol) were used following the General procedure A to afford
10 (357 mg, 53.4%) as a white powder. Mp ¼ 241.5e243.0 ꢁC. 1H
4.1.1.15. 3-Hydroxy-5-iodo-3-(4-methoxyphenyl)-1,3-dihydro-2H-
indol-2-one (15). A 0.5 M solution of 4-methoxyphenylmagnesium
bromide in THF (11 mL, 5.50 mmol) and 5-iodoisatin (500 mg,
1.83 mmol) were used following the General procedure A to afford
15 (533 mg, 76.4%) as a white powder. Mp ¼ 256.8e258.1 ꢁC. 1H
NMR (500 MHz, DMSO-d6):
d
10.62 (s, 1H, NH), 7.44e7.26 (m, 6H,
NMR (500 MHz, DMSO-d6):
d
10.49 (s, 1H, NH), 7.58 (d, J ¼ 8 Hz, 1H,
CHarom.), 7.08 (s,1H, CHarom.), 6.99 (d, J ¼ 8.5 Hz,1H, CHarom.), 6.83 (s,
CHarom.), 7.36 (s, 1H, CHarom.), 7.19 (m, 2H, CHarom.), 6.88 (m, 2H,
1H, OH). 13C NMR (125.7 MHz, DMSO-d6):
d
178.35, 143.39, 141.15,
CHarom.), 6.75 (d, J ¼ 8 Hz, 1H, CHarom.), 6.65 (s, 1H, OH), 3.71 (s, 3H,
140.70, 135.33, 128.25 (2C), 127.74, 125.32 (2C), 122.55, 119.23,
118.26, 110.92, 77.42. MS (ESI) m/z 309.87 (M þ H)þ and 292.23
(M ꢄ OH)þ.
CH3). 13C NMR (125.7 MHz, DMSO-d6):
d 177.95, 158.80, 141.62,
137.63, 136.47, 132.91, 132.82, 126.67 (2C), 113.63 (2C), 112.44, 84.71,
76.77, 55.10. MS (ESI) m/z 364.18 (M ꢄ OH)þ.
4.1.1.11. 8-Hydroxy-8-phenyl-6,8-dihydro-7H-[1,3]thiazolo[5,4,e]
indol-7-one (11). A 1 M solution of phenylmagnesium bromide in
THF (7.35 mL, 7.35 mmol) and 6H-[1,3]thiazolo[5,4,e]indol-7,8-
dione (500 mg, 2.45 mmol) were used following the General
procedure A. The crude was purified by flash chromatography on
reverse phase (0e60% of acetonitrile in water) and the pure frac-
tions were lyophilized to afford 11 (128 mg, 18.5%) as a white
4.1.1.16. 3-Hydroxy-3-(4-methoxyphenyl)-5-(trifluoromethoxy)-1,3-
dihydro-2H-indol-2-one (16). A 0.5
M
solution of 4-
methoxyphenylmagnesium bromide in THF (13 mL, 6.50 mmol)
and 5-(trifluoromethoxy)isatin (500 mg, 2.16 mmol) were used
following the General procedure A to afford 16 (584 mg, 79.6%) as a
white powder. Mp ¼ 226.9e228.4 ꢁC. 1H NMR (400 MHz, DMSO-
d6):
d 10.55 (s, 1H, NH), 7.30e7.15 (m, 3H, CHarom.), 7.08 (s, 1H,
powder. Mp ¼ decomp. 1H NMR (400 MHz, CD3OD):
d
8.99 (s, 1H,
CHarom.), 7.00e6.83 (m, 3H, CHarom.), 6.73 (s, 1H, OH), 3.70 (s, 3H,
N]NHeS), 8.02 (d, J ¼ 8 Hz, 1H, CHarom.), 7.40e7.24 (s, 6H,
CH3). 13C NMR (100.6 MHz, DMSO-d6):
d
178.59, 158.92, 143.39,
CHarom.). 13C NMR (100.6 MHz, CD3OD):
d
181.93, 155.14 (2C),
141.10, 135.40, 132.65, 126.78 (2C), 122.50, 118.93, 118.29, 113.66
151.71, 140.99, 139.45, 130.91, 129.51 (2C), 129.27, 126.61 (2C),
125.16, 111.16, 79.74. MS (ESI) m/z 283.16 (M þ H)þ and 265.26
(M ꢄ OH)þ.
(2C), 110.88, 77.06, 55.10. MS (ESI) m/z 322.24 (M ꢄ OH)þ.
4.1.1.17. 3-[4-(Dimethylamino)phenyl]-6,7-difluoro-3-hydroxy-1,3-
dihydro-2H-indol-2-one (17). A 0.5 M solution of 4-(dimethyla-
mino)phenylmagnesium bromide in THF (9.85 mL, 4.92 mmol) and
6,7-difluoro-1H-indole-2,3-dione (300 mg, 1.64 mmol) were used
following the General procedure A to afford 17 (387 mg, 77.6%) as a
white powder. Mp ¼ decomp. 1H NMR (400 MHz, DMSO-d6):
4.1.1.12. 6,7-Difluoro-3-hydroxy-3-(4-methoxyphenyl)-1,3-dihydro-
2H-indol-2-one (12). A 0.5
M
solution of 4-methoxy
phenylmagnesium bromide in THF (9.85 mL, 4.92 mmol) and 6,7-
difluoro-1H-indole-2,3-dione (300 mg, 1.64 mmol) were used
following the General procedure A to afford 12 (304 mg, 63.7%) as a
white powder. Mp ¼ 169.2e170.1 ꢁC. 1H NMR (400 MHz, DMSO-d6):
d
11.04 (s, 1H, NH), 7.07 (m, 2H, CHarom.), 6.98e6.94 (m, 2H, CHarom.),
6.65 (m, 2H, CHarom.), 6.56 (s, 1H, OH), 2.85 (s, 6H, CH3). 13C NMR
(100.6 MHz, DMSO-d6): 178.78, 150.04, 136.34, 133.89, 131.69,
d
11.14 (s, 1H, NH), 7.21 (m, 2H, CHarom.), 6.98e6.96 (m, 2H, CHarom.),
d
6.89 (m, 2H, CHarom.), 6.73 (s, 1H, OH), 3.72 (s, 3H, CH3). 13C NMR
(100.5 MHz, DMSO-d6): 178.53,158.93,151.35,149.40,136.24,134.15,
130.67, 127.79, 126.34 (2C), 120.90, 111.94 (2C), 109.63, 76.71, 40.15
d
(2C). MS (ESI) m/z 304.94 (M þ H)þ.
132.57, 131.46, 130.90, 126.78, 120.99, 113.62, 109.80, 76.73, 55.12. MS
(ESI) m/z 273.99 (M ꢄ OH)þ.
4.1.2. 4-[2-(4-tert-Butylphenoxy)ethyl]morpholine (18)
A ice-cold solution of 4-tert-butylphenol (1 g, 6.67 mmol), 4-(2-
hydroxyethyl)morpholine (0.87 g, 6.67 mmol) and triphenylphos-
phine (2.28 g, 8.67 mmol) in anhydrous THF (50 mL) was stirred
under nitrogen for 20 min. After a dropwise addition of 40% DEAD
in toluene (3.77 mL, 8.67 mmol), the reaction mixture was allowed
to react for 2 more hours at room temperature. The solvent was
then removed in vacuum and the residue was dissolved in
dichloromethane. Addition of n-Hexane formed a precipitate of
triphenylphosphine oxide that was filtered off. After removal of the
solvents under reduced pressure, the crude was purified by flash
chromatography (10e50% of ethyl acetate in cyclohexane) to afford
18 (0.96 g, 54.7%) as a colorless oil. 1H NMR (400 MHz, CDCl3):
4.1.1.13. 3-Hydroxy-5-methoxy-3-(4-methoxyphenyl)-1,3-dihydro-
2H-indol-2-one (13). A 0.5
M
solution of 4-methoxy
phenylmagnesium bromide in THF (17 mL, 8.50 mmol) and 5-
methoxyisatin (500 mg, 2.82 mmol) were used following the Gen-
eral procedure A to afford 13 (690 mg, 85.7%) as a white powder.
Mp ¼ 189.8e191.2 ꢁC. 1H NMR (500 MHz, DMSO-d6):
d 10.17 (s, 1H,
NH), 7.22 (m, 2H, CHarom.), 6.89e6.82 (m, 4H, CHarom.), 6.73 (s, 1H,
CHarom.), 6.54 (s, 1H, OH), 3.72 (s, 3H, CH3), 3.67 (s, 3H, CH3). 13C NMR
(125.7 MHz, DMSO-d6): d 178.64,158.71,155.15,135.07,134.99,133.53,
126.85 (2C), 113.91, 113.49 (2C), 111.46, 110.31, 77.38, 55.48, 55.10. MS
(ESI) m/z 268.28 (M ꢄ OH)þ.
d
7.31e7.28 (m, 2H, CHarom.), 6.86e6.83 (m, 2H, CHarom.), 4.09 (m,
4.1.1.14. Methyl 3-hydroxy-3-(4-methoxyphenyl)-2-oxindoline-7-
2H, OCH2), 3.73 (m, 4H, CH2OCH2), 2.79 (m, 2H, CH2N), 2.57 (m, 4H,
carboxylate (14). A 0.5
M
solution of 4-methoxyphe
CH2NCH2), 1.29 (s, 9H, C(CH3)3). 13C NMR (100.6 MHz, CDCl3):
nylmagnesium bromide in THF (14.65 mL, 7.32 mmol) and
methyl 2,3-dioxoindoline-7-carboxylate (1 g, 4.88 mmol) were
used following the General procedure A. The crude was purified
by flash chromatography on reverse phase (30e80% of acetonitrile
in water) and the pure fractions were lyophilized to afford 14
(1.06 g, 69.4%) as a white powder. Mp ¼ 173.6e175.2 ꢁC. 1H NMR
d 156.38, 143.41, 126.16 (2C), 113.98 (2C), 66.84 (2C), 65.63, 57.65,
54.03 (2C), 34.03, 31.51 (3C). MS (ESI) m/z 264.19 (M þ H)þ.
4.1.3. General procedure B for the synthesis of compounds 19e21
4.1.3.1. 2-(4-tert-Butylphenoxy)-N,N-dimethylethanamine
(19).
A solution of 4-tert-butylphenol (2 g, 13.33 mmol) in anhydrous
DMF (25 mL) was added dropwise to a stirred mixture of NaH 60%
(0.71 g,17.75 mmol) in anhydrous DMF (50 mL) that was kept under
nitrogen. After 30 min, a solution of N,N-dimethylaminoethyl-
bromide (1.35 g, 8.89 mmol) in anhydrous DMF (25 mL) was added
dropwise using a cannula and the reaction mixture was stirred
under nitrogen for 16 h at 80 ꢁC. After removal of DMF under
reduced pressure, the crude was dissolved in dichloromethane
(500 MHz, DMSO-d6):
d
10.25 (s, 1H, NH), 7.79 (d, J ¼ 8 Hz, 1H,
CHarom.), 7.37 (d, J ¼ 7.5 Hz, 1H, CHarom.), 7.20 (m, 2H, CHarom.), 7.12
(m, 1H, CHarom.), 6.89 (m, 2H, CHarom.), 6.71 (s, 1H, OH), 3.89 (s, 3H,
CH3), 3.72 (s, 3H, CH3). 13C NMR (125.7 MHz, DMSO-d6):
d 179.65,
166.48, 159.66, 143.27, 135.72, 133.05, 130.77, 130.21, 127.52 (2C),
123.24, 114.48 (2C), 112.48, 76.69, 55.88, 53.11. MS (ESI) m/z 296.20
(M ꢄ OH)þ.

S. Denoyelle et al. / European Journal of Medicinal Chemistry 69 (2013) 537e553
547
(200 ml) and the resulting solution was washed 3 times with brine
and dried over anhydrous Na2SO4. After removal of the solvent in
vacuum, the crude was purified by flash chromatography (0e10% of
methanol in dichloromethane) to afford 19 (0.90 g, 45.6%) as a
under reduced pressure, the crude was purified by flash chroma-
tography (0e50% of ethyl acetate in cyclohexane) to afford 23
(1.62 g, 72.2%) as a colorless oil. 1H NMR (500 MHz, CDCl3):
d 7.39e
7.32 (m, 7H, CHarom.), 6.89e6.87 (m, 2H, CHarom.), 5.18 (s, 2H, CH2F),
4.12 (m, 2H, OCH2), 3.58 (m, 4H, (CH2)2NCO), 2.83 (m, 2H, CH2N),
2.57 (m, 4H, CH2NCH2), 1.34 (s, 9H, C(CH3)3). 13C NMR (125.7 MHz,
colorless oil. 1H NMR (400 MHz, CDCl3):
d 7.30e7.26 (m, 2H,
CHarom.), 6.87e6.84 (m, 2H, CHarom.), 4.05 (m, 2H, OCH2), 2.89 (m,
2H, CH2N), 2.45 (s, 6H, N(CH3)2), 1.28 (s, 9H, C(CH3)3). 13C NMR
CDCl3): d 156.63, 155.44, 143.79, 137.00, 128.76 (2C), 128.27, 128.16
(100.6 CDCl3):
d
156.49, 143.51, 126.22 (2C), 113.99 (2C), 65.83,
(2C), 126.49 (2C), 114.29 (2C), 67.38, 66.02, 57.52, 53.48 (2C), 44.03
57.85, 45.45 (2C), 34.08, 31.56 (2C). MS (ESI) m/z 222.13 (M þ H)þ.
(2C), 34.32, 31.82 (3C). MS (ESI) m/z 397.10 (M þ H)þ.
4.1.3.2. 1-tert-Butyl-4-(prop-2-yn-1-yloxy)benzene
(20).
4.1.6. 1-[2-(4-tert-Butylphenoxy)ethyl]piperazine (24)
4-tert-butylphenol (1 g, 6.67 mmol), NaH 60% (0.53 g, 13.33 mmol)
and propargylbromide (0.95 g, 7.98 mmol) in anhydrous DMF were
used following the General procedure B. The crude was purified by
flash chromatography (0e10% of ethyl acetate in cyclohexane) to
afford 20 (0.72 g, 57.5%) as a yellow oil. 1H NMR (500 MHz, CDCl3):
Palladium on carbon 10% (75.7 mg, 60 mg per mmol) was
carefully added to a solution of compound 23 (500 mg, 1.26 mmol)
in methanol (10 mL). A catalytic amount of acetic acid was added.
The reaction mixture was flushed under hydrogen at atmospheric
pressure for 1.5 h and then filtered over celite. The solvent was then
removed in vacuum to afford 24 (320 mg, 96.7%) as a yellow oil
which was taken to the next step without further purification. MS
(ESI) m/z 263.19 (M þ H)þ.
d
7.34 (m, 2H, CHarom.), 6.94 (m, 2H, CHarom.), 4.69 (s, 2H, CH2), 2.53
(s, 1H, CH), 1.30 (s, 9H, C(CH3)3). 13C NMR (125.7 MHz, CDCl3):
155.46, 144.41, 126.39 (2C), 114.45 (2C), 79.02, 75.45, 55.96, 34.26,
d
31.61 (3C). MS (ESI) no ionization.
4.1.7. General procedure C for the synthesis of compounds 25e33,
37e49 and 55
4.1.3.3. tert-Butyl-(4-tert-butylphenoxy)acetate (21).
4-tert-butylphenol (2 g, 13.33 mmol), NaH 60% (1.07 g, 26.67 mmol)
and tert-butylbromoacetate (1.73 g, 8.89 mmol) in anhydrous DMF
were used following the General procedure B. The crude was pu-
rified by flash chromatography (0e10% of ethyl acetate in cyclo-
hexane) to afford 21 (1.89 g, 80.7%) as a yellow oil. 1H NMR
4.1.7.1. 3-{5-tert-Butyl-2-[2-(morpholin-4-yl)ethoxy]phenyl}-3-
phenyl-1,3-dihydro-2H-indol-2-one (25). Triflic acid (790
mL,
8.89 mmol) was added to a stirred ice-cold solution of compound 1
(200 mg, 0.89 mmol) and compound 18 (280 mg, 1.07 mmol) in
dichloromethane (10 mL) that was kept under nitrogen. After 1.5 h,
the reaction mixture was quenched over several grams of ice. The
aqueous phase was then extracted twice with dichloromethane and
the combined organic phases were washed with brine and dried
over anhydrous Na2SO4. After removal of the solvent in vacuum, the
crude was purified by crystallization from EtOAc/Hexane to afford
25 (250 mg, 59.8%) as a white powder. Mp ¼ 288.8e289.4 ꢁC. 1H
(400 MHz, CDCl3):
(s, 2H, OCH2), 1.51 (s, 9H, C(CH3)3), 1.31 (s, 9H, C(CH3)3). 13C NMR
(100.6 MHz, CDCl3): 168.19, 155.69, 144.04, 126.20 (2C), 114.04
d 7.31 (m, 2H, CHarom.), 6.84 (m, 2H, CHarom.), 4.50
d
(2C), 82.05, 65.73, 34.04, 31.48 (3C), 28.01 (3C). MS (ESI) no
ionization.
4.1.4. Benzyl 4-(2-hydroxyethyl)piperazine-1-carboxylate (22)
Benzylchloroformate (4.72 g, 27.69 mmol) in acetonitrile
(30 mL) was added dropwise over 30 min to a solution of 1-(2-
hydroxyethyl)piperazine (3 g, 23.08 mmol) in water (30 mL) via
an isobar cylindrical funnel. The pH was maintained around 8e9 by
addition of 4 N NaOH. The reaction was stirred overnight at room
temperature. The mixture was first extracted with dichloro-
methane (100 mL) in order to remove the fully protected com-
pound and then acidified with 4 N HCl. The acidic aqueous phase
was extracted twice with dichloromethane (2 ꢃ 100 mL). The
organic phase was washed with brine and dried over anhydrous
Na2SO4. The solvent was removed in vacuum and the crude was
purified by flash chromatography (0e8% of methanol in dichloro-
methane) to afford 22 (5.41 g, 88.8%) as a colorless oil. 1H NMR
NMR (500 MHz, DMSO-d6): d 10.39 (s, 1H, NH), 7.31e7.09 (m, 8H,
CHarom.), 6.96e6.18 (m, 4H, CHarom.), 3.90 (m, 1H, CH2O), 3.66 (m,
1H, CH2O), 3.50 (m, 4H, CH2OCH2), 2.32e2.24 (m, 6H, CH2N,
CH2NCH2), 1.11 (s, 9H, C(CH3)3). 13C NMR (100.6 MHz, DMSO-d6):
d
179.89, 154.84, 143.00, 142.74, 140.19, 133.40, 131.21, 129.03 (2C),
128.58 (2C), 127.94, 127.58 (2C), 126.27, 125.63, 121.88, 113.11, 110.17,
66.93, 66.83 (2C), 60.62, 57.02, 54.06 (2C), 34.43, 31.85 (3C). HRMS
(ESI) for C30H34N2O3: calculated (M þ H)þ ¼ 471.26422, found
(M þ H)þ ¼ 471.26413.
4.1.7.2. 3-{5-tert-Butyl-2-[2-(dimethylamino)ethoxy]phenyl}-3-
phenyl-1,3-dihydro-2H-indol-2-one (26). Triflic acid (790
mL,
8.89 mmol), compound 1 (200 mg, 0.89 mmol) and compound 19
(236 mg, 1.07 mmol) in dichloromethane (10 mL) were used
following the General procedure C to afford 26 (260 mg, 68.3%) as a
white powder. Mp ¼ 161.7e162.7 ꢁC. 1H NMR (500 MHz, DMSO-d6):
(400 MHz, DMSO-d6):
d 7.37e7.29 (m, 5H, CHarom.), 5.05 (s, 2H,
CH2F), 4.41 (s, 1H, OH), 3.47 (m, 2H, HOCH2), 3.35 (m, 4H,
(CH2)2NCO), 2.38e2.35 (m, 6H, CH2N, CH2NCH2). 13C NMR
d
10.38 (s, 1H, NH), 7.40e7.32 (m, 5H, CHarom.), 7.23e7.21 (m, 2H,
(100.6 MHz, DMSO-d6):
d
155.04, 137.59, 129.09 (2C), 128.49, 128.22
CHarom.), 7.06 (m, 1H, CHarom.), 6.95e6.83 (m, 3H, CHarom.), 6.80 (s,
(2C), 66.81, 60.82, 59.14, 53.51 (2C), 44.16 (3C). MS (ESI) m/z 265.02
1H, CHarom.), 3.85 (m, 1H, OCH2), 3.60 (m, 1H, OCH2), 2.29 (m, 1H,
(M þ H)þ.
CH2N), 2.13e2.09 (m, 7H, CH2N, N(CH3)2), 1.11 (s, 9H, C(CH3)3). 13
C
NMR (125.7 MHz, CDCl3):
d 179.39, 154.27, 142.49, 142.18, 139.41,
4.1.5. Benzyl 4-[2-(4-tert-butylphenoxy)ethyl]piperazine-1-
carboxylate (23)
132.81, 130.86, 128.53 (2C), 127.95 (3C), 127.39, 127.00, 125.51,
125.01, 121.23, 112.67, 109.51, 66.74, 59.94, 56.98, 45.44 (2C), 33.84,
A ice-cold solution of 4-tert-butylphenol (0.85 g, 5.67 mmol),
compound 22 (1.50 g, 5.67 mmol) and triphenylphosphine (1.93 g,
7.37 mmol) in anhydrous THF (50 mL) was stirred and kept under
nitrogen for 20 min. After a dropwise addition of 40% DEAD in
toluene (3.2 mL, 7.37 mmol), the reaction mixture was allowed to
react for 2 more hours at room temperature. The solvent was then
removed in vacuum and the residue was dissolved in dichloro-
methane. Addition of hexane formed a precipitate of triphenyl-
phosphine oxide that was filtered off. After removal of the solvents
31.25
(3C).
HRMS
(ESI)
for
C
28H32N2O2:
calculated
(M þ H)þ ¼ 429.25365, found (M þ H)þ ¼ 429.25347.
4.1.7.3. 3-{5-tert-Butyl-2-[2-(piperazin-1-yl)ethoxy]phenyl}-3-
phenyl-1,3-dihydro-2H-indol-2-one (27). Triflic acid (690 L,
7.77 mmol), compound 1 (175 mg, 0.78 mmol) and compound 24
(244 mg, 0.93 mmol) in dichloromethane (10 mL) were used
following the General procedure C to afford 27 (146 mg, 40%) as a
white powder. Mp ¼ 228.1e229.3 ꢁC. 1H NMR (500 MHz, DMSO-
m

548
S. Denoyelle et al. / European Journal of Medicinal Chemistry 69 (2013) 537e553
d6):
d
10.40 (s, 1H, NH), 7.30e7.21 (m, 7H, CHarom.), 7.11 (d, J ¼ 7 Hz,
7.00e6.86 (m, 6H, CHarom.), 6.83 (s, 1H, CHarom.), 3.87 (m, 1H, OCH2),
3.73 (m, 3H, OCH3), 3.69 (m,1H, OCH2), 3.49 (m, 4H, CH2OCH2), 2.35
(m, 1H, CH2N), 2.28e2.24 (m, 5H, CH2N, CH2NCH2), 1.12 (s, 9H,
1H, CHarom.), 6.95e6.86 (m, 3H, CHarom.), 6.82 (s, 1H, CHarom.), 3.87
(m, 1H, OCH2), 3.60 (m, 1H, OCH2), 2.60 (m, 4H, CH2NHCH2), 2.28
(m, 1H, CH2N), 2.17e2.08 (m, 5H, CH2N, CH2NCH2), 1.11 (s, 9H,
C(CH3)3). 13C NMR (100.6 MHz, DMSO-d6):
d 179.63, 158.47, 154.15
C(CH3)3). 13C NMR (100.6 MHz, CDCl3):
d
179.81, 154.86, 143.00,
(2C), 142.42, 142.05, 133.02, 131.02, 130.81, 129.66, 127.76, 126.98,
125.36, 124.86, 121.08, 113.33 (2C), 112.38, 109.43, 66.43, 66.18 (2C),
59.14, 56.36, 55.15, 53.45 (2C), 33.79, 31.20 (3C). HRMS (ESI) for
142.61, 140.21, 133.41, 131.21, 128.96 (2C), 128.52 (3C), 127.84,
127.44, 126.33, 125.58, 121.83, 113.05, 110.08, 66.82, 60.61, 57.28,
54.97 (2C), 46.21 (2C), 34.41, 31.84 (3C). HRMS (ESI) for C30H35N3O2:
calculated (M þ Na)þ ¼ 492.26215, found (M þ Na)þ ¼ 492.26211.
C
31H36N2O4: calculated (M
þ
H)þ
¼
501.27478, found
(M þ H)þ ¼ 501.27420.
4.1.7.4. 3-{5-tert-Butyl-2-[(prop-2-yn-1-yl)oxy]phenyl}-3-phenyl-
4.1.7.8. 3-[5-tert-Butyl-2hydroxyphenyl)-3-[4-(dimethylamino)
phenyl]-1,3-dihydro-2H-indol-2-one (32). Triflic acid (330 L,
1,3-dihydro-2H-indol-2-one (28). Triflic acid (197
mL, 2.21 mmol),
m
compound 1 (100 mg, 0.44 mmol) and compound 20 (100 mg,
0.53 mmol) in dichloromethane (5 mL) were used following the
General procedure C. The crude was purified by flash chroma-
tography (10e40% of ethyl acetate in cyclohexane) to afford 28
(98 mg, 55.8%) as a white powder. Mp ¼ 252.4e253.0 ꢁC. 1H NMR
3.71 mmol), compound 3 (200 mg, 0.75 mmol) and tert-butylphe-
nol (134 mg, 0.89 mmol) in dichloromethane (10 mL) were used
following the General procedure C. The crude was purified by flash
chromatography (2e4% of methanol in dichloromethane) to afford
32 (124 mg, 41.5%) as a white powder. Mp ¼ 149.9e151.2 ꢁC. 1H
(500 MHz, DMSO-d6):
d
10.42 (s, 1H, NH), 7.33e7.19 (m, 7H,
NMR (500 MHz, DMSO-d6): d 10.24 (s, 1H, NH), 9.15 (s, 1H, OH), 7.16
CHarom.), 7.02e6.89 (m, 4H, CHarom.), 6.83 (s, 1H, CHarom.), 4.42 (d,
J ¼ 16 Hz, 1H, OCH2), 4.29 (d, J ¼ 16 Hz, 1H, OCH2), 3.37 (s, 1H, CH),
(m, 1H, CHarom.), 7.09e7.05 (m, 3H, CHarom.), 6.91e6.84 (m, 4H,
CHarom.), 6.66 (m, 2H, CHarom.), 6.57 (d, J ¼ 8.5 Hz, 1H, CHarom.), 2.86
(s, 6H, N(CH3)2), 1.11 (s, 9H, C(CH3)3). 13C NMR (100.6 MHz, DMSO-
1.11 (s, 9H, C(CH3)3). 13C NMR (100.6 MHz, DMSO-d6):
d 179.17,
153.27, 143.04, 142.44, 139.20, 132.43, 131.26, 128.52 (2C), 128.04
(3C), 127.42, 127.08, 125.49, 124.96, 121.21, 113.26, 109.56, 78.90,
77.88, 59.81, 56.19, 33.89, 31.18 (3C). HRMS (ESI) for C27H25NO2:
calculated (M þ H)þ ¼ 396.19581, found (M þ H)þ ¼ 396.19537.
d6): d 180.27, 152.90, 149.50, 142.51, 140.18, 133.51, 129.16 (2C),
128.68, 127.46, 127.00, 126.25, 125.33, 124.49, 120.85, 114.86, 111.79
(2C), 109.16, 59.16, 40.15 (2C), 33.71, 31.42 (3C). HRMS (ESI) for
C
26H28N2O2: calculated (M
þ
H)þ
¼
401.22235, found
(M þ H)þ ¼ 401.22205.
4.1.7.5. [4-tert-Butyl-2-(2-oxo-3-phenyl-2,3-dihydro-1H-indol-3-yl)
phenoxy]acetic acid (29). Triflic acid (395
m
L, 4.44 mmol), com-
4.1.7.9. 3-{5-tert-Butyl-2-[2-(morpholin-4-yl)ethoxy]phenyl}-3-[4-
(dimethylamino)phenyl]-1,3-dihydro-2H-indol-2-one (33).
Triflic acid (200 L, 2.24 mmol), 3 (120 mg, 0.45 mmol) and
compound 18 (141 mg, 0.54 mmol) in dichloromethane (5 mL)
were used following the General procedure C. The crude was
purified by flash chromatography (2e5% of methanol in
dichloromethane) to afford 33 (176 mg, 76.6%) as a white powder.
pound 1 (200 mg, 0.89 mmol) and compound 21 (282 mg,
1.07 mmol) in dichloromethane (10 mL) were used following the
General procedure C to afford 29 (166 mg, 45%) as a white powder.
m
Mp ¼ 303.5e304.8 ꢁC. 1H NMR (500 MHz, DMSO-d6):
d 12.77 (s, 1H,
OH), 10.38 (s, 1H, NH), 7.33e7.15 (m, 8H, CHarom.), 6.96e6.83 (m, 4H,
CHarom.), 4.26 (d, J ¼ 16 Hz, 1H, OCH2), 4.99 (d, J ¼ 16 Hz, 1H, OCH2),
1.12 (s, 9H, C(CH3)3). 13C NMR (100.6 MHz, DMSO-d6):
d
178.97,
Mp ¼ 245.1e245.9 ꢁC. 1H NMR (500 MHz, DMSO-d6):
d 10.21 (s,
169.82, 153.95, 143.26, 142.31, 139.54, 132.47, 131.53, 128.26e127.87
(5C), 127.19, 126.81, 125.72, 125.04, 121.17, 114.19, 109.51, 66.36,
59.89, 33.84, 31.12 (3C). HRMS (ESI) for C26H25NO4: calculated
(M þ H)þ ¼ 416.18563, found (M þ H)þ ¼ 416.18509.
1H, NH), 7.20e7.16 (m, 3H, CHarom.), 7.08 (m, 1H, CHarom.), 6.90e
6.84 (m, 5H, CHarom.), 6.66 (m, 2H, CHarom.), 3.84 (m, 1H, OCH2),
3.68 (m, 1H, OCH2), 3.48 (m, 4H, CH2OCH2), 2.86 (s, 6H, N(CH3)2),
2.36 (m, 1H, CH2N), 2.28e2.21 (m, 5H, CH2N, CH2NCH2), 1.12 (s, 9H,
C(CH3)3). 13C NMR (100.6 MHz, DMSO-d6):
d 179.95, 154.20, 149.56,
4.1.7.6. 3-(5-tert-Butyl-2-hydroxyphenyl)-3-(4-methoxyphenyl)-1,3-
142.49, 141.99, 133.39, 131.16, 129.27, 127.48, 127.25, 125.89, 125.12,
124.63, 120.81 (2C), 112.26, 111.73 (2C), 109.24, 66.51, 66.17 (2C),
58.92, 56.36, 53.45 (2C), 40.10 (2C), 33.78, 31.27 (3C). HRMS (ESI)
dihydro-2H-indol-2-one (30). Triflic acid (348
mL, 3.92 mmol),
compound 2 (200 mg, 0.78 mmol) and tert-butylphenol (141 mg,
0.94 mmol) in dichloromethane (10 mL) were used following the
General procedure C. The crude was purified by flash chroma-
tography (10e50% of ethyl acetate in cyclohexane) to afford 30
(238 mg, 78.4%) as a white powder. Mp ¼ 132.6e134.4 ꢁC. 1H NMR
for
C
32H39N3O3: calculated (M
þ
H)þ
¼
514.30642, found
(M þ H)þ ¼ 514.30572.
4.1.8. General procedure D for the synthesis of compounds 34e36
4.1.8.1. 3-(5-tert-Butyl-2-hydroxyphenyl)-3-[4-(morpholin-4-
ylmethyl)phenyl]-1,3-dihydro-2H-indol-2-one (34).
4-tert-Butylphenol (225 mg, 1.50 mmol) and compound 4 (324 mg,
1 mmol) were dissolved in 1,2-dichloroethane (10 mL). The reaction
mixture was heated to 85 ꢁC and p-TsOH (323 mg, 1.70 mmol) was
added. After stirring at 85 ꢁC for 6 h, the solvent was removed in
vacuum and the crude was purified by flash chromatography (0e
20% of ethyl acetate in cyclohexane) to afford 34 (430 mg, 94.3%) as
a white powder. Mp > 314 ꢁC (decomp). 1H NMR (400 MHz, DMSO-
(500 MHz, DMSO-d6):
d 10.33 (s, 1H, NH), 9.21 (s, 1H, OH), 7.20 (m,
3H, CHarom.), 7.09 (m, 1H, CHarom.), 6.97e6.87 (m, 5H, CHarom.), 6.79
(m, 1H, CHarom.), 6.60 (d, J ¼ 8.5 Hz, 1H, CHarom.), 3.73 (s, 3H, OCH3),
1.10 (s, 9H, C(CH3)3). 13C NMR (125.7 MHz, DMSO-d6):
d 180.52,
159.01, 153.46, 143.07, 140.88, 133.73, 131.88, 130.21 (2C), 129.06,
128.31, 127.39, 126.04, 125.29, 121.67, 115.58, 113.92 (2C), 119.92,
59.91, 55.74, 34.30, 31.97 (3C). HRMS (ESI) for C25H25NO3: calcu-
lated (M þ H)þ ¼ 388.19072, found (M þ H)þ ¼ 388.19170.
4.1.7.7. 3-{5-tert-Butyl-2-[2-(morpholin-4-yl)ethoxy]phenyl}-3-(4-
d6): d 10.40 (s, 1H, NH), 9.25 (s, 1H, OH), 7.46 (m, 2H, CHarom.), 7.19
methoxyphenyl)-1,3-dihydro-2H-indol-2-one
(31). Triflic
acid
(m,1H, CHarom.), 7.09 (m, 2H, CHarom.), 7.06 (dd, J ¼ 2.0 Hz, J ¼ 8.0 Hz,
1H, CHarom.), 7.00 (m, 1H, CHarom.), 6.93 (m, 1H, CHarom.), 6.87 (d,
J ¼ 8.0 Hz, 1H, CHarom.), 6.67 (d, J ¼ 2.0 Hz, 1H, CHarom.), 6.58 (d,
J ¼ 8.4 Hz, 1H, CHarom.), 3.56 (m, 4H, CH2OCH2), 3.34 (s, 2H, CH2),
2.27 (m, 4H, CH2NCH2), 1.06 (s, 9H, C(CH3)3). 13C NMR (100.6 MHz,
(350 L, 3.94 mmol) compound 18 (247 mg, 0.94 mmol) and
m
compound 2 (200 mg, 0.78 mmol) in dichloromethane (10 mL)
were used following the General procedure C. The crude was pu-
rified by preparative HPLC on reverse phase (20e55% of acetonitrile
in water) and the pure fractions were lyophilized to afford 31
(165 mg, 42.1%) as a white powder. Mp ¼ 222.4e223.7 ꢁC. 1H NMR
DMSO-d6):
d 180.08, 153.51, 145.51, 143.06, 140.90, 138.89, 133.36,
130.03, 129.06 (2C), 128.91, 128.48, 127.58, 126.22 (2C), 125.45,
121.81, 115.78, 110.15, 65.82 (2C), 61.58, 60.49, 52.99 (2C), 34.90,
(500 MHz, DMSO-d6): d 10.32 (s,1H, NH), 7.21e7.19 (m, 4H, CHarom.),

S. Denoyelle et al. / European Journal of Medicinal Chemistry 69 (2013) 537e553
549
31.90
(3C).
HRMS
(ESI)
for
C29H32N2O3:
calculated
(m, 4H, CH2NCH2), 1.12 (s, 9H, C(CH3)3). 13C NMR (75.47 MHz,
DMSO-d6): 179.14, 153.76, 152.46, 149.25, 146.40, 142.17, 132.23,
130.22, 128.07, 126.71, 125.41, 125.06, 124.45, 121.32, 115.98, 115.72,
113.17, 112.62, 109.58, 65.55 (3C), 58.91, 56.02 (2C), 52.95 (2C),
(M þ H)þ ¼ 457.24857, found (M þ H)þ ¼ 457.24982.
d
4.1.8.2. 3-(5-tert-Butyl-2-hydroxyphenyl)-3-(4-chloro-3-
methylphenyl)-1,3-dihydro-2H-indol-2-one (35).
33.84, 30.99 (3C). HRMS (ESI) for
C31H35FN2O4: calculated
4-tert-Butylphenol (225 mg, 1.50 mmol), intermediate 5 (273 mg,
1 mmol) and p-TsOH (323 mg, 1.70 mmol) in 1,2-dichloroethane
(10 mL) at 85 ꢁC were used following the General procedure D.
The crude was purified by flash chromatography (0e20% of ethyl
acetate in cyclohexane) to afford 35 (300 mg, 74%) as a white
(M þ H)þ ¼ 519.26536, found (M þ H)þ ¼ 519.26711.
4.1.11. 3-(5-tert-Butyl-2-hydroxyphenyl)-5-iodo-3-phenyl)-1,3-
dihydro-2H-indol-2-one (39)
Triflic acid (2.15 mL, 24.22 mmol), compound 9 (1.70 g,
4.84 mmol) and 4-tert-butylphenol (0.87 g, 5.81 mmol) in
dichloromethane (100 mL) were used following the General pro-
cedure C. The crude was purified by flash chromatography (10e70%
of ethyl acetate in cyclohexane) to afford 39 (1.44 g, 61.5%) as a
white powder. Mp ¼ 264.2e265.6 ꢁC. 1H NMR (400 MHz, DMSO-
powder. Mp ¼ 140.4e141.6 ꢁC. 1H NMR (400 MHz, CD3OD):
d 7.23e
7.20 (m, 3H, CHarom.), 7.15 (dd, J ¼ 2.0 Hz, J ¼ 8.4 Hz, 1H, CHarom.),
7.06 (m, 2H, CHarom.), 7.00e6.95 (m, 3H, CHarom.), 6.69 (d, J ¼ 8.4 Hz,
1H, CHarom.), 2.23 (s, 3H, CH3), 1.14 (s, 9H, C(CH3)3). 13C NMR
(100.6 MHz, CD3OD): d 182.34, 153.04, 141.86, 141.81, 139.20, 135.57,
133.26, 133.12, 130.84 (2C), 128.52, 128.22, 127.18, 126.87, 126.05,
125.57, 122.16, 116.14, 110.12, 61.24, 33.83, 30.85 (3C), 19.15. HRMS
(ESI) for C25H24ClNO2: calculated (M þ H)þ ¼ 406.15683, found
(M þ H)þ ¼ 406.15685.
d6):
d
10.56 (s, 1H, NH), 9.38 (s, 1H, OH), 7.56 (d, J ¼ 7.2 Hz, 1H,
CHarom.), 7.33e7.29 (m, 6H, CHarom.), 7.12 (d, J ¼ 7.2 Hz, 1H, CHarom.),
6.77 (m, 2H, CHarom.), 6.66 (d, J ¼ 8 Hz, 1H, CHarom.), 1.11 (s, 9H,
C(CH3)3). 13C NMR (100.6 MHz, DMSO-d6):
d 178.73, 152.76, 142.35,
140.39, 138.79, 136.42, 135.70, 133.38, 128.25 (2C), 128.05 (2C),
127.62, 127.37, 126.71, 125.17, 115.09, 111.88, 83.87, 60.04, 33.69,
4.1.8.3. 3-(5-tert-Butyl-2-hydroxyphenyl)-3-(pyridin-3-yl)-1,3-
dihydro-2H-indol-2-one
(36). 4-tert-Butylphenol
(135
mg,
31.28
(3C).
HRMS
(ESI)
for
C
24H22INO2:
calculated
0.90 mmol), intermediate 6 (140 mg, 0.62 mmol) and p-TsOH
(190 mg, 1 mmol) in 1,2-dichloroethane (10 mL) at 85 ꢁC were used
following the General procedure D. The crude was purified by flash
chromatography (0e20% of methanol in dichloromethane) to
afford 36 (30 mg, 14%) as a white powder. Mp ¼ 134.9e136.1 ꢁC. 1H
(M þ H)þ ¼ 484.07680, found (M þ H)þ ¼ 484.07523.
4.1.12. 3-(5-tert-Butyl-2-hydroxyphenyl)-3-phenyl-5-
(trifluoromethoxy)-1,3-dihydro-2H-indol-2-one (40)
Triflic acid (430
mL, 4.84 mmol), compound 10 (300 mg,
NMR (400 MHz, CDCl3):
d
8.58 (m, 1H, CHarom.), 8.46 (s, 1H, CHarom.),
0.97 mmol) and 4-tert-butylphenol (175 mg, 1.17 mmol) in
dichloromethane (10 mL) were used following the General pro-
cedure C. The crude was purified by flash chromatography (15e35%
of ethyl acetate in cyclohexane) to afford 40 (175 mg, 40.9%) as a
white powder. Mp ¼ 278.5e279.1 ꢁC. 1H NMR (500 MHz, DMSO-
8.00 (m, 1H, CHarom.), 7.64 (m, 1H, CHarom.), 7.42 (d, J ¼ 7.6 Hz, 1H,
CHarom.), 7.31 (dd, J ¼ 1.6 Hz, J ¼ 8.0 Hz, 1H, CHarom.), 7.21 (dd,
J ¼ 2.0 Hz, J ¼ 8.8 Hz, 1H, CHarom.), 7.11e7.02 (m, 3H, CHarom.), 6.73
(d, J ¼ 8.0 Hz, 1H, CHarom.), 1.20 (s, 9H, C(CH3)3). 13C NMR
(100.6 MHz, CDCl3):
d
179.85, 154.30, 153.01, 144.95, 144.46, 142.20,
d6): d 10.64 (s, 1H, NH), 9.38 (s, 1H, OH), 7.36e7.31 (m, 3H, CHarom.),
141.34, 140.38 (2C), 131.56, 128.96, 126.42, 126.06 (2C), 122.70 (2C),
116.10, 110.38, 59.95, 33.80, 30.68 (3C). HRMS (ESI) for C23H22N2O2:
calculated (M þ H)þ ¼ 358.16813, found (M þ H)þ ¼ 359.17612.
7.26e7.23 (m, 3H, CHarom.), 7.13 (d, J ¼ 8.5 Hz, 1H, CHarom.), 7.09 (s,
1H, CHarom.), 6.97 (d, J ¼ 8.5 Hz, 1H, CHarom.), 6.80 (s, 1H, CHarom.),
6.64 (d, J ¼ 8.5 Hz, 1H, CHarom.), 1.11 (s, 9H, C(CH3)3). 13C NMR
(100.6 MHz, DMSO-d6):
d 179.27, 152.82, 142.83, 141.68, 140.43,
4.1.9. 3-{5-tert-Butyl-2-[2-(morpholin-4-yl)ethoxy]phenyl}-3-(3,4-
dimethoxyphenyl)-1,3-dihydro-2H-indol-2-one (37)
138.81, 134.67, 128.10 (2C), 128.08 (2C), 127.39, 127.31, 126.66,
125.19,121.93,121.02,119.04,115.14,110.06, 60.45, 33.69, 31.25 (3C).
HRMS (ESI) for C25H22F3NO3: calculated (M þ H)þ ¼ 442.16245,
found (M þ H)þ ¼ 442.16175.
Triflic acid (234 mL, 2.63 mmol), compound 7 (150 mg, 0.53 mmol)
and compound 18 (166 mg, 0.63 mmol) in dichloromethane (5 mL)
were usedfollowingtheGeneralprocedure C. The crudewaspurified
by flash chromatography (2e8% of methanol in dichloromethane) to
afford 37 (187 mg, 66.9%) as awhite powder. Mp ¼ 279.1e280.0 ꢁC.1H
4.1.13. 8-(5-tert-Butyl-2-hydroxyphenyl)-8-phenyl-6,8-dihydro-
7H-[1,3]thiazolo[5,4-e]indol-7-one (41)
NMR (500 MHz, DMSO-d6):
d
10.29 (s, 1H, NH), 7.21e7.19 (m, 2H,
Triflic acid (66 mL, 0.74 mmol), compound 11 (42 mg, 0.15 mmol)
CHarom.), 7.03e6.85 (m, 7H, CHarom.), 6.61 (s,1H, CHarom.), 3.87 (m,1H,
CH2O), 3.73e3.69 (m, 4H, CH2O, OCH3), 3.59 (s, 3H, OCH3), 3.48 (m,
4H, CH2OCH2), 2.37 (m, 1H, CH2N), 2.30e2.23 (m, 5H, CH2N,
CH2NCH2), 1.12 (s, 9H, C(CH3)3). 13C NMR (125.7 MHz, DMSO-d6):
and 4-tert-butylphenol (27 mg, 0.18 mmol) in dichloromethane
(5 mL) were used following the General Procedure C. The crude
was purified by preparative HPLC on reverse phase (20e65% of
acetonitrile in water) to afford 41 (21 mg, 34%) as a white powder.
d
180.23, 154.80, 148.95, 148.76, 143.15, 142.69, 133.47, 131.83, 131.38,
Mp > 300 ꢁC (decomp). 1H NMR (400 MHz, MeOD):
d 8.91 (s, 1H,
128.41, 127.88, 125.89, 125.89, 125.45, 121.62, 121.32, 113.64, 112.93,
112.01, 110.08, 67.18, 66.81 (2C), 59.89, 56.97, 56.12 (2C), 54.08 (2C),
N]CHeS), 8.02 (d, J ¼ 8.8 Hz, 1H, CHarom.), 7.32e7.30 (m, 5H,
CHarom.), 7.26 (d, J ¼ 8.8 Hz, 1H, CHarom.), 7.16 (dd, J ¼ 2.8 Hz,
J ¼ 8.4 Hz, 1H, CHarom.), 7.00 (s, 1H, CHarom.), 6.63 (d, J ¼ 8 Hz, 1H,
CHarom.), 1.17 (s, 9H, C(CH3)3). 13C NMR (100.6 MHz, MeOD):
34.42, 31.87 (3C). HRMS (ESI) for
C32H38N2O5: calculated
(M þ H)þ ¼ 531.28535, found (M þ H)þ ¼ 531.28571.
d
182.65, 153.49, 153.40, 149.79, 141.46, 140.46, 137.63, 130.66,
4.1.10. 3-{5-tert-Butyl-2-[2-(morpholin-4-yl)ethoxy]phenyl}-3-(3-
fluoro-4-methoxyphenyl)-1,3-dihydro-2H-indol-2-one (38)
128.59, 128.18 (2C), 127.89 (2C), 125.57e125.28 (4C), 122.65, 115.19,
109.55, 61.37, 33.77, 30.66 (3C). HRMS (ESI) for C25H22N2O2S:
calculated (M þ H)þ ¼ 415.14748, found (M þ H)þ ¼ 415.14738.
Triflic acid (244
mL, 2.75 mmol), compound 8 (150 mg,
0.55 mmol) and compound 18 (173 mg, 0.66 mmol) in dichloro-
methane (5 mL) were used following the General procedure C to
afford 38 (98 mg, 34.4%) as a white powder. Mp ¼ 196.9e198.2 ꢁC.
4.1.14. 3-(5-tert-Butyl-2-hydroxyphenyl)-6,7-difluoro-3-(4-
methoxyphenyl)-1,3-dihydro-2H-indol-2-one (42)
1H NMR (500 MHz, DMSO-d6):
d
10.45 (s,1H, NH), 7.26e7.23 (m, 2H,
Triflic acid (190 mL, 2.14 mmol), compound 12 (125 mg,
CHarom.), 7.13 (m, 1H, CHarom.), 7.05 (m, 2H, CHarom.), 6.98e6.89 (m,
4H, CHarom.), 6.83 (s, 1H, CHarom.), 3.96 (m, 1H, OCH2), 3.82e3.77 (m,
4H, OCH2, OCH3), 3.55 (m, 4H, CH2OCH2), 3.34 (m, 2H, CH2N), 2.35
0.43 mmol) and 4-tert-butylphenol (77 mg, 0.51 mmol) in
dichloromethane (5 mL) were used following the General proce-
dure C to afford 42 (86 mg, 47.3%) as a white powder. Mp ¼ 182.8e

550
S. Denoyelle et al. / European Journal of Medicinal Chemistry 69 (2013) 537e553
183.4 ꢁC. 1H NMR (500 MHz, DMSO-d6):
d
11.06 (s, 1H, NH), 9.35 (s,
dichloromethane (10 mL) were used following the General pro-
cedure C. The crude was purified by preparative HPLC on reverse
phase (50e90% of acetonitrile in water) and the pure fractions were
lyophilized to afford 46 (180 mg, 33.6%) as a white powder.
1H, OH), 7.21 (m, 2H, CHarom.), 7.10 (dd, J ¼ 2 Hz, J ¼ 8 Hz, 1H,
CHarom.), 6.94e6.92 (m, 3H, CHarom.), 6.75 (s, 2H, CHarom.), 6.60 (d,
J ¼ 8 Hz, 1H, CHarom.), 3.75 (s, 3H, OCH3), 1.10 (s, 9H, C(CH3)3). 13
C
NMR (125.7 MHz, DMSO-d6):
d
179.73, 158.69, 152.69, 148.59,
Mp ¼ 207.2e208.1 ꢁC. 1H NMR (300 MHz, DMSO-d6):
d 10.17 (s, 1H,
140.44, 134.14, 131.51, 130.88, 129.97, 129.68, 128.00, 126.73, 125.06,
121.26, 114.82, 113.50 (2C), 108.55, 108.40, 59.06, 54.98, 33.57, 31.20
NH), 7.76 (d, J ¼ 7.8 Hz, 1H, CHarom.), 7.25e7.04 (m, 5H, CHarom.),
6.95e6.82 (m, 4H, CHarom.), 3.87e3.84 (m, 4H, OCH2, OCH3), 3.74e
3.69 (m, 4H, OCH2, OCH3), 3.46 (m, 4H, CH2OCH2), 2.35e2.22 (m,
6H, CH2N, CH2NCH2), 1.12 (s, 9H, C(CH3)3). 13C NMR (75.47 MHz,
(3C).
HRMS
(ESI)
for
C
25H23F2NO3:
calculated
(M þ H)þ ¼ 424.17188, found (M þ H)þ ¼ 424.17208.
DMSO-d6):
d 179.61, 165.67, 158.69, 153.88, 143.40, 142.13, 134.58,
4.1.15. 3-(5-tert-Butyl-2-hydroxyphenyl)-5-methoxy-3-(4-
methoxyphenyl)-1,3-dihydro-2H-indol-2-one (43)
130.07, 129.70 (2C), 129.61, 128.46, 126.96, 125.09, 120.99, 113.49
(2C), 112.07, 111.08, 66.07, 65.99 (3C), 57.95, 56.32, 55.11, 53.29 (2C),
51.95, 33.72, 31.13 (3C). HRMS (ESI) for C33H38N2O6: calculated
(M þ H)þ ¼ 559.28026, found (M þ H)þ ¼ 559.27958.
Triflic acid (310
mL, 3.49 mmol), compound 13 (200 mg,
0.70 mmol) and 4-tert-butylphenol (126 mg, 0.84 mmol) in
dichloromethane (10 mL) were used following the General proce-
dure C. The crude was purified by flash chromatography (10e45% of
ethyl acetate in cyclohexane) to afford 43 (181 mg, 61.8%) as a white
powder. Mp ¼ 140.3e151.9 ꢁC (decomp). 1H NMR (500 MHz, DMSO-
4.1.19. 3-{5-tert-Butyl-2-[2-(morpholin-4-yl)ethoxy]phenyl}-5-
iodo-3-(4-methoxyphenyl)-1,3-dihydro-2H-indol-2-one (47)
Triflic acid (175
mL, 1.97 mmol), compound 15 (150 mg,
d6):
d
10.22 (s,1H, NH), 9.23 (s,1H, OH), 7.18 (m, 2H, CHarom.), 7.10 (dd,
0.39 mmol) and compound 18 (124 mg, 0.47 mmol) in dichloro-
methane (5 mL) were used following the General procedure C to
afford 47 (122 mg, 49.5%) as a white powder. Mp ¼ 274.2e275.8 ꢁC.
J ¼ 2.5 Hz, J ¼ 8.5 Hz,1H, CHarom.), 6.90e6.79 (m, 5H, CHarom.), 6.63e
6.60 (m, 2H, CHarom.), 3.72 (s, 3H, OCH3), 3.65 (s, 3H, OCH3), 1.11 (s,
9H, C(CH3)3). 13C NMR (125.7 MHz, DMSO-d6):
d
179.63, 158.28,
1H NMR (500 MHz, DMSO-d6):
d
10.48 (s,1H, NH), 7.56 (d, J ¼ 7.5 Hz,
154.42, 152.88, 140.26, 135.81, 134.60, 131.38, 129.35 (2C), 128.16,
126.61,124.70,115.16,113.28 (2C),112.91,111.76,109.47, 59.90, 55.38,
55.05, 33.66, 31.31 (3C). HRMS (ESI) for C26H27NO4: calculated
(M þ H)þ ¼ 418.20128, found (M þ H)þ ¼ 418.20064.
1H, CHarom.), 7.25e7.15 (m, 4H, CHarom.), 6.93e6.90 (m, 3H, CHarom.),
6.80 (s, 1H, CHarom.), 6.75 (d, J ¼ 7.5 Hz, 1H, CHarom.), 3.87 (m, 1H,
CH2O), 3.77e3.63 (m, 4H, CH2O, OCH3), 3.50 (m, 4H, CH2OCH2),
2.31e2.23 (m, 6H, CH2N, CH2NCH2), 1.12 (s, 9H, C(CH3)3). 13C NMR
(125.7 MHz, DMSO-d6):
d 178.96, 158.62, 154.03, 142.23, 142.22,
4.1.16. 3-{5-tert-Butyl-2-[2-(morpholin-4-yl)ethoxy]phenyl}-5-
methoxy-3-(4-methoxyphenyl)-1,3-dihydro-2H-indol-2-one (44)
136.49, 135.82, 133.08, 130.23, 129.96, 129.50 (2C), 127.01, 125.25,
113.53 (2C), 112.51, 112.03, 83.76, 66.44, 66.13 (2C), 59.20, 56.36,
55.15, 53.41 (2C), 33.80, 31.20 (3C). HRMS (ESI) for C31H35IN2O4:
calculated (M þ H)þ ¼ 627.17143, found (M þ H)þ ¼ 627.17127.
Triflic acid (235
mL, 2.64 mmol), compound 13 (150 mg,
0.53 mmol) and compound 18 (166 mg, 0.63 mmol) in dichloro-
methane (5 mL) were used following the General procedure C. The
crude was purified by flash chromatography (0e10% of methanol in
ethyl acetate) to afford 44 (108 mg, 37.2%) as a white powder.
4.1.20. 3-{5-tert-Butyl-2-[2-(morpholin-4-yl)ethoxy]phenyl}-3-(4-
methoxyphenyl)-5-(trifluoromethoxy)-1,3-dihydro-2H-indol-2-one
(48)
Mp ¼ 255.8e256.9 ꢁC. 1H NMR (500 MHz, DMSO-d6):
d 10.17 (s, 1H,
NH), 7.22e7.16 (m, 3H, CHarom.), 6.90e6.79 (m, 6H, CHarom.), 6.60 (s,
1H, CHarom.), 3.88 (m, 1H, OCH2), 3.72 (s, 3H, OCH3), 3.70 (m, 1H,
OCH2), 3.65 (s, 3H, OCH3), 3.32 (m, 4H, CH2OCH2), 2.34 (m, 1H,
Triflic acid (393 mL, 4.39 mmol), compound 16 (300 mg,
0.88 mmol) and compound 18 (279 mg, 1.06 mmol) in dichloro-
methane (10 mL) were used following the General procedure C to
afford 48 (160 mg, 31%) as a white powder. Mp ¼ decomp. 1H NMR
CH2N), 2.27e2.22 (m, 5H, CH2N, CH2NCH2), 1.12 (s, 9H, C(CH3)3). 13
C
NMR (125.7 MHz, DMSO-d6):
d
179.34,158.42,154.47,154.22,142.08,
(400 MHz, DMSO-d6): d 10.82 (s, 1H, NH), 7.41e6.80 (m, 10H,
135.85,134.47,131.21,130.66,129.51 (2C),126.91,124.92,113.32 (2C),
112.93, 112.51, 111.72, 109.63, 66.44, 66.13 (2C), 59.72, 56.43, 55.43,
55.12, 53.47 (2C), 33.80, 31.23 (3C). HRMS (ESI) for C32H38N2O5:
calculated (M þ H)þ ¼ 553.26729, found (M þ H)þ ¼ 553.26739.
CHarom.), 4.20 (m, 2H, OCH2), 3.85e3.59 (m, 7H, CH2OCH2, OCH3),
3.23e2.78 (m, 6H, CH2N, CH2NCH2), 1.10 (s, 9H, C(CH3)3). 13C NMR
(125.7 MHz, DMSO-d6):
d 180.63, 159.64, 153.80, 144.22, 143.64,
142.11, 135.23, 131.15e130.64 (4C), 128.09, 126.16, 122.07 (2C),
119.55, 114.33 (2C), 114.16, 111.46, 64.27, 63.82 (2C), 59.77, 55.73
(2C), 55.46, 52.13, 34.16, 31.46 (3C). HRMS (ESI) for C32H35F3N2O5:
calculated (M þ H)þ ¼ 585.25708, found (M þ H)þ ¼ 585.25694.
4.1.17. Methyl 3-(5-tert-butyl-2-hydroxyphenyl)-3-(4-
methoxyphenyl)-2-oxoindoline-7-carboxylate (45)
Triflic acid (430
mL, 4.84 mmol), compound 14 (300 mg,
0.96 mmol) and 4-tert-butylphenol (172 mg, 1.15 mmol) in
dichloromethane (10 mL) were used following the General pro-
cedure C to afford 45 (280 mg, 65.6%) as a white powder.
4.1.21. 3-{5-tert-Butyl-2-[2-(morpholin-4-yl)ethoxy]phenyl}-3-[4-
(dimethylamino)phenyl]-6,7-difluoro-1,3-dihydro-2H-indol-2-one
(49)
Mp ¼ 279.1e280.0 ꢁC. 1H NMR (400 MHz, DMSO-d6):
d
10.18 (s, 1H,
Triflic acid (150 mL, 1.69 mmol), compound 17 (100 mg,
NH), 9.32 (s, 1H, OH), 7.74 (d, J ¼ 8 Hz, 1H, CHarom.), 7.23e7.04 (m,
5H, CHarom.), 6.94e6.92 (m, 2H, CHarom.), 6.78 (s, 1H, CHarom.), 6.59
(d, J ¼ 8 Hz, 1H, CHarom.), 3.86 (s, 3H, OCH3), 3.73 (s, 3H, OCH3), 1.10
0.33 mmol) and compound 18 (104 mg, 0.39 mmol) in dichloro-
methane (5 mL) were used following the General procedure C. The
crude was purified by preparative HPLC on reverse phase (50e70%
of acetonitrile in water) and the pure fractions were lyophilized to
afford 49 (145 mg, 80.3%) as a white powder. Mp ¼ 207.8e208.9 ꢁC.
(s, 9H, C(CH3)3). 13C NMR (100.6 MHz, DMSO-d6):
d 179.74, 165.73,
158.62, 152.62, 143.50, 140.35, 134.82, 129.97e129.74 (4C), 128.45,
128.01, 126.83, 124.92, 120.96, 114.71, 113.46 (2C), 110.87, 58.09,
55.12, 52.03, 33.68, 31.31 (3C). HRMS (ESI) for C27H27NO5: calcu-
lated (M þ H)þ ¼ 446.19620, found (M þ H)þ ¼ 446.19531.
1H NMR (400 MHz, DMSO-d6):
d 10.99 (s, 1H, NH), 7.20 (m, 1H,
CHarom.), 7.06 (m, 1H, CHarom.), 6.92e6.94 (m, 4H, CHarom.), 6.68e
6.64 (m, 3H, CHarom.), 3.81 (m, 2H, OCH2), 3.47 (m, 4H, CH2OCH2),
2.86 (s, 6H, N(CH3)2), 2.36 (m, 2H, CH2N), 2.22 (m, 4H, CH2NCH2),
4.1.18. Methyl 3-{5-tert-butyl-2-[2-(morpholin-4-yl)ethoxy]
1.09 (s, 9H, C(CH3)3). 13C NMR (125.7 MHz, DMSO-d6):
d 179.87,
phenyl}-3-(4-methoxyphenyl)-2-oxoindoline-7-carboxylate (46)
153.95, 149.73, 148.43, 142.08, 134.35, 132.07e131.04 (3C), 130.20
(2C), 129.18, 128.76, 127.16, 125.02, 124.43, 120.92 (2C), 111.99,
111.78, 108.30, 108.15, 66.01 (2C), 58.72, 56.34 (2C), 53.34 (2C),
Triflic acid (430
mL, 4.84 mmol), compound 14 (300 mg,
0.96 mmol) and compound 18 (302 mg, 1.15 mmol) in

S. Denoyelle et al. / European Journal of Medicinal Chemistry 69 (2013) 537e553
551
33.76, 31.20 (3C). HRMS (ESI) for C32H37F2N3O3: calculated
d 179.42, 158.11, 152.87, 144.94, 141.52, 140.33, 139.45, 135.48,
(M þ H)þ ¼ 550.28757, found (M þ H)þ ¼ 550.28718.
133.91, 130.87, 128.38 (2C), 128.09, 127.99 (2C), 127.14, 126.76,
125.85, 125.53, 124.86, 123.03, 115.20, 109.83, 107.02, 65.94 (2C),
60.37, 45.24 (2C), 33.68, 31.30 (3C). HRMS (ESI) for C33H33N3O3:
calculated (M þ H)þ ¼ 520.25947, found (M þ H)þ ¼ 520.25935.
4.1.22. General procedure E for the synthesis of compounds 50 and
51
4.1.22.1. 3-(5-tert-Butyl-2-hydroxyphenyl)-3-(4-hydroxyphenyl)-1,3-
dihydro-2H-indol-2-one (50). To a solution of boron tribromide
methyl sulfide complex (323 mg, 1.03 mmol) in dichloroethane
(10 mL) kept under nitrogen was added compound 30 (100 mg,
0.26 mmol). The reaction mixture was stirred at reflux under ni-
trogen overnight. When the starting material was totally
consumed, the reaction mixture was cooled to room temperature,
hydrolyzed by adding approx.10 ml of water, and stirred for 20 min.
The aqueous phase was extracted twice by dichloromethane
(2 ꢃ 30 ml). The combined extracts were washed with 1 M NaHCO3,
dried over Na2SO4 and concentrated in vacuum. The resulting res-
idue was purified by flash chromatography (5e55% of ethyl acetate
in cyclohexane) to afford 50 (54 mg, 56% yield) as a white powder.
4.1.24. 4-(Propan-2-yloxy)phenol (53)
A solution of KOH (2.64 g, 47.06 mmol) in water (40 mL) was
added to a mixture of hydroquinone (2.85 g, 25.88 mmol) and 2-
iodopropane (4 g, 23.53 mmol) in ethanol (50 mL). After 16 h at
reflux, the dark brown reaction mixture was cooled down and
filtered. After removal of ethanol in vacuum, the resulting aqueous
phase was acidified with 2 N HCl and then extracted twice with
ethyl acetate (2 ꢃ 60 mL). The organic phase was washed with brine
and dried over anhydrous Na2SO4. The solvent was removed in
vacuum and the crude was purified by flash chromatography (0e
20% of ethyl acetate in cyclohexane) to afford 53 (1.25 g, 35%) as a
brown oil. 1H NMR (400 MHz, CDCl3):
6.60 (s, 1H, OH), 4.41 (m, 1H, CH(CH3)2), 1.35e1.30 (m, 6H,
d 6.84e6.75 (m, 4H, CHarom.),
Mp ¼ 178.9e181.6 ꢁC. 1H NMR (500 MHz, DMSO-d6):
d 10.28 (s, 1H,
NH), 9.39 (s,1H, OH), 9.17 (s,1H, OH), 7.17 (m,1H, CHarom.), 7.11e7.06
(m, 3H, CHarom.), 6.93 (m, 2H, CHarom.), 6.85 (d, J ¼ 8 Hz,1H, CHarom.),
6.78 (s, 1H, CHarom.), 6.70 (m, 2H, CHarom.), 6.57 (d, J ¼ 8 Hz, 1H,
CHarom.), 1.11 (s, 9H, C(CH3)3). 13C NMR (100.6 MHz, DMSO-d6):
CH(CH3)2). 13C NMR (100.6 MHz, CDCl3):
d 151.32, 150.03, 118.25
(2C), 116.27 (2C), 72.00, 22.13 (2C). MS (ESI) 153.13 (M þ H)þ.
4.1.25. 4-{2-[4-(Propan-2-yloxy)phenoxy]ethyl}morpholine (54)
A solution of KOH (0.74 g, 13.16 mmol) in water (10 mL) was
added to a mixture of compound 53 (1 g, 6.58 mmol) and 4-(2-
chloroethyl)morpholine hydrochloride (1.47 g, 7.89 mmol) in
ethanol (20 mL). After 16 h at reflux, the reaction mixture was
cooled down, ethanol was removed in vacuum and the resulting
aqueous phase was extracted twice with ethyl acetate (2 ꢃ 30 mL).
The organic phase was washed with brine and dried over anhy-
drous Na2SO4. The solvent was removed in vacuum and the crude
was purified by flash chromatography (20e80% of ethyl acetate in
cyclohexane) to afford 54 (0.94 g, 54%) as a yellow oil. 1H NMR
d
180.16, 156.53, 152.86, 142.54, 140.26, 133.33, 129.71 (2C), 129.37,
128.74, 127.62, 126.98, 125.45, 124.61, 121.00, 114.90, 114.77 (2C),
109.28, 59.22, 33.75, 31.42 (3C). HRMS (ESI) for C24H23NO3: calcu-
lated (M þ H)þ ¼ 374.17507, found (M þ H)þ ¼ 374.17507.
4.1.22.2. 3-(5-tert-Butyl-2-hydroxyphenyl)-6,7-difluoro-3-(4-
hydroxyphenyl)-1,3-dihydro-2H-indol-2-one (51). Boron tribromide
methyl sulfide complex (250 mg, 0.80 mmol) and compound 42
(85 mg, 0.20 mmol) in dichloroethane (10 mL) were used following
the General procedure E. The crude was purified by flash chro-
matography (10e30% of ethyl acetate in cyclohexane) to afford 51
(40 mg, 48.7%) as a white powder. Mp ¼ 208.3e210.2 ꢁC. 1H NMR
(400 MHz, CDCl3):
d 6.77e6.75 (m, 4H, CHarom.), 4.34 (m, 1H,
CH(CH3)2), 3.98 (t, J ¼ 6 Hz, 2H, OCH2), 3.66 (m, 4H, CH2OCH2), 2.70
(500 MHz, DMSO-d6):
d 11.02 (s, 1H, NH), 9.52 (s, 1H, OH), 9.34 (s,
(t, J ¼ 6 Hz, 2H, CH2N), 2.49 (m, 4H, CH2NCH2), 1.25e1.21 (m, 6H,
1H, OH), 7.11e7.07 (m, 3H, CHarom.), 6.90 (m, 1H, CHarom.), 6.75e6.68
(m, 4H, CHarom.), 6.58 (d, J ¼ 8.5 Hz, 1H, CHarom.), 1.09 (s, 9H,
CH(CH3)2). 13C NMR (100.6 MHz, CDCl3):
d 152.74, 151.88, 117.18
(2C), 115.34 (2C), 70.59, 66.72 (2C), 66.10, 57.62, 53.93 (2C), 21.99
C(CH3)3). 13C NMR (125.7 MHz, DMSO-d6):
d
179.90, 156.80, 152.65,
(2C). MS (ESI) 266.19 (M þ H)þ.
148.52, 140.38, 134.11, 131.05, 131.03, 129.72, 128.23, 128.07, 126.87,
124.90, 121.18, 114.91 (2C), 114.71, 108.43, 108.29, 58.95, 33.71, 31.34
(3C). HRMS (ESI) for C24H21F2NO3: calculated (M þ H)þ ¼ 41
0.15623, found (M þ H)þ ¼ 410.15582.
4.1.26. 3-(4-Methoxyphenyl)-3-{2-[2-(morpholin-4-yl)ethoxy]-5-
(propan-2-yloxy)phenyl}-1,3-dihydro-2H-indol-2-one (55)
Triflic acid (260
mL, 2.93 mmol), compound 2 (150 mg,
0.59 mmol) and compound 54 (187 mg, 0.71 mmol) in dichloro-
methane (5 mL) were used following the General procedure C. The
crude was purified by flash chromatography on reverse phase (20e
60% of acetonitrile in water) and the pure fractions were lyophilized
to afford 55 (108 mg, 36.6%) as a white powder. Mp ¼ 139.3e
4.1.23. 3-(5-tert-Butyl-2-hydroxyphenyl)-5-[6-(morpholin-4-yl)
pyridin-3-yl]-3-phenyl-1,3-dihydro-2H-indol-2-one (52)
To a degassed solution of 39 (300 mg, 0.62 mmol), 6-(mor-
pholin-4-yl)pyridin-3-boronic acid pinacol ester (217 mg,
0.75 mmol), and Pd(PPh3)2Cl2 (72 mg, 0.06 mmol) in THF (6 mL)
was added 2 M Na2CO3 solution (6.2 mL). The reaction mixture was
refluxed under nitrogen for 30 min, cooled to room temperature,
and concentrated in vacuum to remove most of the solvent. The
formed residue was partitioned between Et2O (30 mL) and H2O
(15 mL), and the whole was stirred for 30 min. The organic layer
was separated, and the aqueous layer was extracted with Et2O
(2 ꢃ 20 mL). The combined extracts were dried over Na2SO4 and
concentrated in vacuum. The resulting residue was purified by flash
chromatography (50e60% of ethyl acetate in cyclohexane) to afford
52 (83 mg, 25.7% yield) as a white powder. Mp > 205 ꢁC(decomp).
149.9 ꢁC (decomp). 1H NMR (500 MHz, MeOD):
d 7.27e7.24 (m, 2H,
CHarom.), 7.00e6.80 (m, 8H, CHarom.), 6.53 (m, 1H, CHarom.), 4.42 (m,
1H, CH(CH3)2), 4.16 (m, 2H, OCH2), 3.87 (m, 4H, CH2OCH2), 3.78 (s,
3H, OCH3), 3.42 (m, 2H, CH2N), 3.24 (m, 4H, CH2NCH2), 1.19 (d,
J ¼ 6 Hz, 3H, CHCH3), 0.60 (d, J ¼ 6 Hz, 3H, CHCH3). 13C NMR
(100.6 MHz, MeOD): d 183.32, 160.75, 152.16, 151.36, 143.92, 134.73,
131.32, 129.06 (2C), 126.57 (2C), 122.67, 119.74, 114.33e113.93 (4C),
110.81, 70.78, 65.12 (2C), 63.4ziegeler1, 60.97, 57.68, 55.73 (2C),
53.62 (2C), 21.75, 20.75. HRMS (ESI) for C30H34N2O5: calculated
(M þ H)þ ¼ 503.25405, found (M þ H)þ ¼ 503.25309.
1H NMR (400 MHz, DMSO-d6):
d
10.53 (s, 1H, NH), 9.33 (s, 1H, OH),
4.2. Biological evaluation
8.32 (s,1H, CHarom.), 7.73 (d, J ¼ 8.4 Hz,1H, CHarom.), 7.46 (d, J ¼ 8 Hz,
1H, CHarom.), 7.35e7.28 (m, 6H, CHarom.), 7.09 (d, J ¼ 8.4 Hz, 1H,
CHarom.), 6.96 (d, J ¼ 8 Hz, 1H, CHarom.), 6.86e6.84 (m, 2H, CHarom.),
6.62 (d, J ¼ 8.4 Hz, 1H, CHarom.), 3.69 (m, 4H, CH2OCH2), 3.43 (m, 4H,
CH2NCH2), 1.10 (s, 9H, C(CH3)3). 13C NMR (100.6 MHz, DMSO-d6):
4.2.1. Cell growth assay
Adherent mouse (KLN) and human solid tumor cells (CRL-2351
and CRL-2813) were plated in 96-well plates, allowed to settle
overnight, and maintained for 5 days in the presence of 0.54e

552
S. Denoyelle et al. / European Journal of Medicinal Chemistry 69 (2013) 537e553
20
m
M of individual compound, and cell proliferation was measured
manufacturer specifications. The thermal cycler conditions and the
primers used are detailed under Supplementary Methods and in
Ref. [41]. All PCRs were performed in triplicate in at least two in-
dependent PCR runs. Mean values of these repeated measurements
were used for calculation. To calibrate the results, all the transcript
quantities were normalized to 18S rRNA.
by the sulforhodamine B (SRB) assay as described [18]: briefly, cells
were fixed in 10% cold trichloroacetic acid at 4 ꢁC for 1 h, exten-
sively washed with double-distilled H2O and air-dried. Plates were
then incubated with 0.4% SRB in 1% acetic acid for 1 h, washed with
1% acetic acid to remove the unbound dye, and air-dried. The bound
dye was solubilized by addition of 10 mM Tris (pH 10), and the
absorbance was determined in a Titertek Multiscan plate reader at
490 nm. The data calculations were carried out as described (the
values for mean ꢂ SD of data from replicate wells are calculated):
data are expressed in terms of %T/C [(OD of treated cells/OD of
control cells) ꢃ 100], as a measure of cell viability and survival in
the presence of test materials. Calculations are also made for the
concentration of test agents giving a T/C value of 50%, or 50%
growth inhibition (IC50). With the SRB assay, a measure is made of
the cell population density at time 0 (the time at which drugs are
added) from two extra reference plates of inoculated cells fixed
with TCA at the time of drug addition to the test plates. Thus, we
have three measurements: control optical density (C), test optical
density (T), and optical density at time zero (T0). The calculation is
100 ꢃ [(T ꢄ T0)/(C ꢄ T0)]. If T is less than T0, cell killing has occurred
and can be calculated from 100 ꢃ [(T ꢄ T0)/T0]. Thus, for each drug-
cell line combination, a doseeresponse curve is generated and
three levels of effect are calculated.
Acknowledgments
This work was supported by a Sponsored Research Agreement
with Egenix.
Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2013.08.030. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
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