N-Phenethyl and N-naphthylmethyl isatins and analogues as in vitro cytotoxic agents

Article abstract of DOI:10.1016/j.bmc.2007.12.026

A range of N-phenethyl, N-phenacyl, and N-(1- and 2-naphthylmethyl) derivatives of 5,7-dibromoisatin 2 were prepared by N-alkylation reactions. Their activity against human monocyte-like histiocytic lymphoma (U937), leukemia (Jurkat), and breast carcinoma

Full text of DOI:10.1016/j.bmc.2007.12.026

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3118–3124
N-Phenethyl and N-naphthylmethyl isatins and analogues
as in vitro cytotoxic agents
Lidia Matesic,a,* Julie M. Locke,a John B. Bremner,a Stephen G. Pyne,a
Danielle Skropeta,a Marie Ransonb and Kara L. Vineb
aSchool of Chemistry, University of Wollongong, Wollongong, NSW 2522, Australia
bSchool of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia
Received 13 November 2007; revised 12 December 2007; accepted 13 December 2007
Available online 7 January 2008
Abstract—ArangeofN-phenethyl, N-phenacyl, andN-(1-and2-naphthylmethyl)derivativesof5,7-dibromoisatin2werepreparedbyN-
alkylation reactions. Their activity against human monocyte-like histiocytic lymphoma (U937), leukemia (Jurkat), and breast carcinoma
(MDA-MB-231) cell lines was assessed. The results allowed further development of structure–activity relationships. The compound 5,7-
dibromo-N-(1-naphthylmethyl)-1H-indole-2,3-dione 5a was the most potent against U937 cells with an IC50 value of 0.19 lM.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Isatin (1H-indole-2,3-dione) 1 is a synthetically versatile
molecule which has led to an array of derivatives dis-
playing a broad spectrum of biological properties
including anti-cancer activities.1–5 Recently, it has been
reported that 5,7-dibromoisatin 2 is significantly more
potent in vitro as a cytotoxin than the parent molecule
against U937 (human monocyte-like histiocytic lym-
phoma) cells.6 Other research within our laboratory
has indicated that N-benzylation of 5,7-dibromoisatin
2 further increased the cytotoxicity toward these lym-
phoma cells and was potent against a range of human
cancer cell lines including a metastatic breast adenocar-
cinoma cell line (MDA-MB-231).7 In this context, it was
of interest to further investigate the cytotoxicity of N-
alkylated 5,7-dibromoisatin analogues by altering the
chain length between N-1 and the aryl group, as well
as increasing the hydrophobicity of the N-substituent
through an extra aromatic ring fusion. Together with
this, in vitro screening against various cancer cell lines
was carried out in order to establish a more comprehen-
sive structure–activity relationship (SAR). The results
are described in this paper.
2. Results and discussion
2.1. Chemistry
In order to determine the structural requirements neces-
sary for cytotoxicity, a simple modification to 2 was
envisaged with a two-atom linker bridge between the
nitrogen and a new aromatic ring, producing N-phen-
ethyl derivatives 4a–e. The phenethyl scaffold was also
modified by introduction of another aromatic ring fu-
sion which subsumed the benzylic methylene group in
the new ring, to afford the 1-naphthylmethyl derivative
5a. The 2-naphthylmethyl analogue 5b was also synthe-
sized for comparative purposes. With a view to assessing
the hydrophobicity requirements, N-phenacyl deriva-
tives 6a–e of 5,7-dibromoisatin 2 were also investigated.
The procedure for the synthesis of the N-phenethyl
derivatives 4a–e was based on a combination of the lit-
erature methods.8–10 Briefly, 2 was treated with a base
such as NaH or K2CO3 which formed an intense purple
colored anion (A) that was subsequently reacted with
the appropriate aryl alkyl halide 3a–e to give the
N-phenethyl derivatives 4a–e in moderate yields
Keywords: Isatins; Cytotoxins; Cancer; Tubulin polymerization
inhibitors.
*
Corresponding author. Tel.: +61 2 4221 4334; fax: +61 2 4221
4287; e-mail: lm66@uow.edu.au
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.12.026

L. Matesic et al. / Bioorg. Med. Chem. 16 (2008) 3118–3124
3119
Table 1. Initial cytotoxicity screening (IC50 lM) of novel N-substi-
tuted isatin derivatives 4a–e, 5a–b, and 6a–ea
(Scheme 1). High temperatures are usually required in
these syntheses to drive the reactions to completion be-
cause the reaction mixtures are prone to crystallization
of the impure product at low temperatures.8 N-Benzyli-
satin derivatives had been obtained previously by heat-
ing at 80 ꢁC,7 however a temperature of 50 ꢁC was
used to produce 4a–e to reduce the risk of substituted
styrenes forming as side products from the base cata-
lyzed elimination of the phenethyl bromides 3a–e. The
isatin intermediate (A) is also an ambidentate anion
which could undergo N- or O-alkylation,11 however
no evidence for O-alkylation was found through 1H
and 13C NMR spectroscopic studies.
Compound
U937b
Jurkatc
MDA-MB-231d
2
4a
10.5e
0.78
0.78
0.88
1.07
2.35
0.19
0.74
9.97
6.36
9.18
4.70
5.32
6.88g
14.3e
1.52
1.21
1.15
2.00
2.66
0.91
0.41
ntf
42.3e
4.35
2.72
2.01
5.24
4.51
2.49
2.56
nt
4b
4c
4d
4e
5a
5b
6a
6b
nt
nt
6c
6d
nt
nt
nt
nt
6e
Vinblastine
nt
nt
nt
nt
a Values are means of triplicates of at least two independent
experiments.
b Human monocyte-like histiocytic lymphoma cell line.
c Human leukemic T-cell line.
d Human metastatic mammary gland adenocarcinoma cell line.
e Vine et al.6
f Not tested.
g Vine et al.7
The N-naphthylmethyl analogues 5a and 5b were pre-
pared in a similar manner to the N-phenethyl series
4a–e, however, the synthesis of the N-phenacyl deriva-
tives 6a–e proved difficult. Attempts to synthesize these
compounds included various alkylation protocols and
protecting group strategies. A possible competing reac-
tion was a Darzens condensation involving the C3 car-
bonyl group of the isatin 2. While the Darzens
condensation occurs when a ketone or aldehyde reacts
with a haloester to form an epoxy ester, it also proceeds
with halogenomethylsulfones and halogenoketones such
as phenacyl halides.12 It has been reported that phenacyl
halides preferentially yield a Darzens product rather
than the corresponding phenacylisatin,13 although in
the current study this was not observed in the NMR
spectra. The desired compounds 6a–e were obtained in
a pure form but in low yields, which were not optimized
as the required compounds were obtained in sufficient
quantity for cytotoxicity screening.
2.2. Cytotoxic activity and SAR
The N-phenethyl 4a–e and N-naphthylmethyl deriva-
tives 5a–b were initially tested for cytotoxicity using
the MTS cell proliferation assay against three human
cancer cell lines including a lymphoma (U937), leukemia
(Jurkat), and metastatic breast adenocarcinoma cell line
(MDA-MB-231). The results showed that the cytotoxic-
ity of the parent molecule 2 significantly increased
through N-alkylation (Table 1), as reported previously
for the 5,7-dibromo-N-benzylisatin derivatives.7 Com-
pounds 4a–e, which contain a two-carbon linker at the
nitrogen, typically increased the anti-proliferative activ-
ity 10- to 15-fold against the three tumor cell lines com-
pared to that of the parent brominated isatin 2. Three of
the N-phenethyl derivatives, 4a–c, displayed IC50 values
in the sub-micromolar range against U937 cells (e.g., 4a,
IC50 = 0.78 lM, Table 1). Introduction of a hydropho-
bic bromo substituent in the meta (4b) or para (4c) posi-
tion yielded the most active compounds in this series,
while analogous compounds containing methoxy sub-
stituents 4d–e were less cytotoxic. The introduction of
a substituent in the meta position (4b and 4d) enhanced
Scheme 1. Reagents and conditions: (a) NaH or K2CO3, DMF, 4 ꢁC–rt, 20 min to 3 h; (b) KI, 50 ꢁC, 18 h (32–62%).

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L. Matesic et al. / Bioorg. Med. Chem. 16 (2008) 3118–3124
Table 2. Further cytotoxicity screening (IC50 lM) of derivatives 4b–c
and 5a–b against a panel of human adherent tumor cell linesa
Compound
HCT-116b
PC-3c
MCF-7d
A375e
4b
4c
5a
5b
1.41
1.62
1.15
1.51
2.09
2.21
1.44
2.29
4.04
6.02
7.71
3.50
3.44
4.34
3.95
6.56
a Values are means of triplicates of at least two independent
experiments.
b Human colorectal carcinoma cell line.
c Human prostate adenocarcinoma cell line.
d Human non-metastatic mammary gland adenocarcinoma cell line.
e Human malignant melanoma cell line.
cytotoxicity against the U937 and Jurkat cell lines com-
pared to the para position (4c and 4e) although this
trend was not observed against the MDA-MB-231 cell
line. Overall, the U937 lymphoma cell line appeared to
be the most susceptible to treatment with the N-phene-
thylisatins 4a–e, while the metastatic MDA-MB-231 cell
line was the least susceptible.
Figure 1. Elongated U937 cell morphology after treatment with
various isatin derivatives. Cells were incubated for 24 h with (A)
DMSO vehicle control, (B) 5,7-dibromo-N-(2-naphthylmethyl)isatin
5b (0.39 lg/mL), and (C) vinblastine sulfate (0.39 lg/mL). Magnifica-
tion: 1000·.
The N-naphthylmethylisatins 5a and 5b demonstrated
enhanced cytotoxic activity against the lymphoma
(U937) and leukemia (Jurkat) cell lines compared to
the N-phenethyl derivatives 4a–e. Both 5a and 5b exhib-
ited sub-micromolar IC50 values against these two cell
lines, and were the only compounds to do so against Jur-
kat cells. Compound 5a was the most cytotoxic of all
those tested, with an IC50 value of 0.19 lM against
U937 cells. This compound was four times more potent
against U937 cells than the lead N-phenethyl com-
pounds 4a and 4b.
to undergo elongation. The elongated morphology was
more pronounced when the cells were treated with N-
naphthylmethyl derivatives 5a and 5b (Fig. 1B). This
morphological change was also observed in U937 cells
treated with vinblastine (Fig. 1C), a microtubule desta-
bilizer. This suggested that these isatin analogues inter-
fere with microtubule dynamics in a similar fashion to
previously
derivatives.7
reported
5,7-dibromo-N-benzylisatin
To further investigate the effects of the aforementioned
analogues on microtubule formation, cell-free
The most active analogue in the N-phenacyl series was
6d, which exhibited an IC50 value of 4.70 lM against
U937 cells. The parent phenacyl derivative 6a was the
least active of the series with an IC50 value of 9.97 lM
(Table 1). This suggests that the presence of the polar
carbonyl group is detrimental to activity. Since the N-
phenacylisatins 6a–e were less active than the N-phen-
ethyl 4a–e and N-naphthylmethyl 5a–b series, they were
not tested against any other cell lines.
a
in vitro tubulin polymerization assay was performed
(Fig. 2). The two naphthyl derivatives 5a and 5b were
chosen due to their in vitro potency against U937 and
Jurkat cells, as well as two representative phenethyl
compounds, 4a and 4c. Vinblastine sulfate and paclit-
axel were used as a known microtubule destabilizer
and stabilizer, respectively. Consistent with the litera-
ture reports,14,15 at 10 lM paclitaxel stabilized microtu-
bules, while vinblastine sulfate was a potent microtubule
destabilizer (Fig. 2). The test compounds, in particular
The four most potent derivatives, 4b, 4c, 5a, and 5b,
(Table 1) were selected for further cytotoxic screening
against a panel of other adherent human tumor cell
lines: colorectal (HCT-116), prostate (PC-3), non-meta-
static breast (MCF-7), and melanoma (A375). The re-
sults in Table 2 indicate that activity against the panel
of adherent cell lines varied but all compounds tested
had IC50 values in the low micromolar range. The lower
sensitivity in these adherent cell lines compared to the
non-adherent cell lines may be due to reduced cell sur-
face areas leading to decreased drug uptake as well as
slower proliferation rates in the former.
2.3. Mode of action studies
While performing in vitro cytotoxicity testing, it was ob-
served that the isatin derivatives 4a–e, 5a–b, and 6a–e
caused the U937 and, to a lesser extent, the Jurkat cells
Figure 2. Effect of compounds 4a,4c, 5a, 5b, paclitaxel, and vinblastine
sulfate at 10 lM on the assembly of purified bovine neuronal tubulin.
DMSO was used as a control.

L. Matesic et al. / Bioorg. Med. Chem. 16 (2008) 3118–3124
3121
(A375) tumor cell lines. Cytotoxicity testing showed that
5,7-dibromo-N-(1-naphthylmethyl)isatin 5a was the
most active compound against all of the cell lines tested
and exhibited an IC50 value of 0.19 lM against U937
cells. The N-phenethyl derivatives 4a–c also showed po-
tent sub-micromolar activity against U937 cells. Addi-
tionally, the tumor cells displayed elongated cell
morphology upon treatment with these compounds,
suggesting that these analogues interfere with microtu-
bule dynamics. The results described indicate that these
compounds could serve as the basis for the development
of a new group of cancer chemotherapeutics.
Figure 3. Effect of varying concentrations of the 2-naphthylmethyl
derivative 5b on the assembly of purified bovine neuronal tubulin.
DMSO was used as a control.
4. Experimental
4.1. General
Table 3. Inhibition of tubulin polymerization (IC50 lM) of com-
pounds 4a, 5a, 5b and vinblastine at varying time pointsa
4.1.1. Chemistry. All solvents were of AR grade except
dichloromethane (DCM) which was of LR grade and
distilled before use. The term petroleum spirit (PS) refers
to petroleum spirit with a boiling range of 40–60 ꢁC.
Solvent removal was performed (in vacuo) using tem-
peratures not greater than 60 ꢁC. Organic halides were
purchased from Sigma–Aldrich Chemical Co. and used
as supplied. Sodium hydride was supplied as a 60% dis-
persion in mineral oil and masses used were calculated
appropriately. Melting points were obtained using a
Reichert melting point apparatus and are uncorrected.
Thin-layer chromatography (TLC) on aluminum
backed sheets of Merck Silica Gel 60 F254 plates was em-
ployed to monitor the progress of chemical reactions.
Preparative TLC was performed on 20 · 20 cm plates.
Generally, the isatins were highly colored and visible
on the TLC plate; colorless compounds were detected
by exposure to UV light at k 254 nm. Column chroma-
tography was performed under ‘flash’ conditions21 on
silica gel 60 (230–400 mesh). The solvent used in individ-
ual chromatographic experiments is indicated and all
solvent proportions are given as vol/vol ratios. NMR
spectra were acquired on a Varian Unity 300 MHz spec-
trometer, where proton (1H) and carbon (13C) spectra
were obtained at 300 MHz and 75 MHz, respectively,
Compound
20 min
60 min
4a
5a
4.04
4.71
2.87
0.95
6.60
12.0
6.47
1.66
5b
Vinblastine
a Values are means of duplicates in one experiment.
the 2-naphthyl derivative 5b, appeared to be potent
microtubule destabilizers at 10 lM (Fig. 2), as observed
by the shift of the curve to the right of the control, indi-
cating a decrease in the rate of tubulin polymerization.
These results are consistent with reports that structur-
ally similar indole16–18 and indolinone7,15,19 compounds
inhibit tubulin polymerization.
The three most potent destabilizers, 4a, 5a, and 5b, were
chosen for further studies on tubulin polymerization. All
of these compounds inhibited the rate of tubulin poly-
merization in a dose-dependent manner (representative
result shown in Fig. 3) and the IC50 values are reported
in Table 3. Vinblastine displayed similar inhibition in
this assay to that reported previously.20
1
It was of interest to note that in Figure 3, the test com-
pound 5b displayed potent destabilizing effects at higher
concentrations (as seen by the shift in the curves to the
right of the DMSO control) and stabilized microtubules
at lower concentrations (as seen by the shift in the curves
to the left of the DMSO control). This phenomenon has
also been reported in the case of vinblastine14 and sug-
gests that these compounds may bind to more than
one binding site on tubulin with different affinities.
or on a Varian Inova 500 spectrometer, where the H
and 13C were obtained at 500 MHz and 126 MHz,
respectively. All spectra were obtained with a probe tem-
perature of 298 K. Spectra were recorded in CDCl3 (un-
less otherwise indicated) and were referenced to the
residual non-deuterated solvent signal or TMS. Hydro-
gen and carbon assignments were also made using gradi-
ent correlation spectroscopy (gCOSY), gradient
heteronuclear single quantum correlation (gHSQC),
and gradient heteronuclear multiple bond correlation
(gHMBC) spectroscopic techniques. The superscript let-
a
3. Conclusions
ter denotes coincident peaks. Low resolution electron
ionization mass spectra (LREI-MS) were obtained using
a Shimadzu QP5050 spectrometer. High resolution elec-
tron ionization mass spectra (HREI-MS) were obtained
using a Fisons/VG Autospec spectrometer operating
with an electron beam of 70 eV, with a source tempera-
ture of 250 ꢁC and perfluorokerosene (PFK) as the inter-
nal standard. Compounds for testing were >95% pure
A number of N-phenethyl, N-phenacyl, and N-naphthyl-
methyl derivatives of 5,7-dibromoisatin 2 were synthe-
sized and tested against a range of human cancer cell
lines. These compounds displayed greater cytotoxicity
against non-adherent U937 and Jurkat cells compared
to adherent human breast (MDA-MB-231 and MCF-
7), colon (HCT-116), prostate (PC-3), and melanoma
1
on the basis of TLC and H NMR analysis.

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L. Matesic et al. / Bioorg. Med. Chem. 16 (2008) 3118–3124
4.1.2. Cell biology. All cancer cell lines were obtained
from the American Type Culture Collection (ATCC,
VA, USA). Cells were routinely maintained in RPMI-
1640 medium, containing 2 mM L-glutamine, 5.6%
(2 g/L) sodium bicarbonate, and 5% fetal calf serum
(FCS) (at 37 ꢁC 95% humidified atmosphere and 5%
CO2). Adherent cells were detached with sterile tryp-
sin–EDTA, washed with culture media, and reseeded
following centrifugation for 5 min at 1600 rpm. The
number of viable cells was counted with the aid of a
hemocytometer and Trypan blue staining. Cytotoxicity
of the isatin derivatives was determined using the CellT-
iter 96ꢂ AQueous One Solution Cell Proliferation Assay,
utilizing [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymeth-
H600), 7.69 (d, J = 1.5 Hz, 1H, H4), 7.89 (d, J = 1.5 Hz,
1H, H6). 13C NMR (126 MHz): d 35.0, 42.4, 104.5,
116.6, 121.1, 126.7, 127.2, 128.4a, 128.6a, 136.7, 144.8,
146.4, 157.5, 181.1. HREI-MS: m/z calcd for
C16H11NO279Br79Br [M+]: 406.9157; found: 406.9166.
4.2.3. 5,7-Dibromo-1-[2-(3-bromophenyl)ethyl]-1H-indole-
2,3-dione (4b). The compound was prepared according to
the method for 4a using 5,7-dibromoisatin 2 (100 mg,
0.33 mmol) and 3-bromophenethyl bromide 3b (193 mg,
0.11 mL, 0.73 mmol) as starting materials. The resulting
solid was purified by flash chromatography on silica gel
(DCM) to yield 4b (67.1 mg, 42%) as bright red/orange
crystals, mp 188–190 ꢁC, Rf 0.54 (silica, DCM). 1H NMR
(acetone-d6, 500 MHz): d 3.09 (t, J = 8 Hz, 2H, H20),
4.38 (t, J = 8 Hz, 2H, H10), 7.27 (t, J = 8 Hz, 1H, H500),
7.34 (d, J = 8 Hz, 1H, H600), 7.42 (d, J = 8 Hz, 1H, H400),
7.51 (s, 1H, H200), 7.72 (d, J = 1.5 Hz, 1H, H4), 8.06 (d,
J = 1.5 Hz, 1H, H6). 13C NMR (acetone-d6, 126 MHz): d
34.9, 42.4, 104.7, 110.0, 116.0, 122.3, 126.8, 128.2, 130.0,
130.8, 132.0, 140.9, 144.4, 147.5, 158.4, 181.6. HREI-MS:
m/z calcd for C16H10NO279Br81Br81Br [M+]: 488.8221;
found: 488.8207.
oxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner
salt] (MTS) (Promega Co., Madison, WI, USA) as de-
scribed previously.6,7 The inhibitory concentration re-
quired to inhibit 50% of the metabolic activity of the
cell population (IC50) was calculated from sigmoidal
dose–response curves using GraphPad Prism 5.00
(GraphPad Software Inc.). Cell images were obtained
by brightfield microscopy on an inverted light micro-
scope using a Leica DC500 12-megapixel high-perfor-
mance FireWire camera system. The in vitro tubulin
polymerization assay was obtained from Cytoskeleton
Inc. (Jomar Diagnostics, Australia) and the assay was
conducted with reagents as described by the manufac-
turer. Briefly, 5 lL of vehicle control, paclitaxel, vinblas-
tine sulfate, and compounds 4a, 4c, 5a, and 5b at the
desired concentrations were incubated with 50 lL of
purified bovine neuronal tubulin reaction mix. The rate
of polymerization was monitored kinetically for 1 h at
37 ꢁC using an excitation wavelength of 360 10 nm
and fluorescence emission at 440 10 nm.
4.2.4. 5,7-Dibromo-1-[2-(4-bromophenyl)ethyl]-1H-indole-
2,3-dione (4c). The compound was prepared according to
the method for 4a using 5,7-dibromoisatin 2 (100 mg,
0.33 mmol) and 4-bromophenethyl bromide 3c (193 mg,
0.11 mL, 0.73 mmol) as starting materials. The resulting
solid was purified by flash chromatography on silica gel
(CHCl3) to yield 4c (81.1 mg, 50%) as bright red crystals,
mp 189–190 ꢁC, Rf 0.48 (silica, DCM). 1H NMR
(300 MHz): d 2.99 (t, J = 8.4 Hz, 2H, H20), 4.35 (t,
J = 8.4 Hz, 2H, H10), 7.13 (d, J = 8.7 Hz, 2H, H200, H600),
7.42 (d, J = 8.7 Hz, 2H, H300, H500), 7.69 (d, J = 1.8 Hz,
1H, H4), 7.89 (d, J = 1.8 Hz, 1H, H6). 13C NMR
(126 MHz): d 35.1, 42.6, 104.9, 117.3, 121.2, 121.6, 127.9,
130.8a, 132.1a, 136.2, 145.4, 146.8, 158.0, 181.4. HREI-
MS: m/z calcd for C16H10NO279Br81Br81Br [M+]:
488.8221; found: 488.8214.
4.2. Chemistry
4.2.1. 5,7-Dibromoisatin (2)6. Yield: 2.90 g, (28%) as
bright red/orange needles, mp 251–253 ꢁC (lit.22 248–
1
250 ꢁC), Rf 0.63 (silica, DCM/MeOH, 9:1). H NMR
(DMSO-d6, 500 MHz): d 7.66 (d, J = 1.5 Hz, 1 H, H4),
8.02 (d, J = 1.5 Hz, 1H, H6), 11.43 (br s, 1H, NH). 13C
NMR (DMSO-d6, 126 MHz): d 105.7, 114.5, 121.3,
125.9, 141.1, 148.6, 159.4, 182.5. LREI-MS: m/z 303;
305; 307 [M+]79Br79Br; 79Br81Br; 81Br81Br.
4.2.5. 5,7-Dibromo-1-[2-(3-methoxyphenyl)ethyl]-1H-indole-
2,3-dione (4d). The compound was prepared according to
the method for 4a using 5,7-dibromoisatin 2 (100 mg,
0.33 mmol) and 3-methoxyphenethyl bromide 3d
(157 mg, 0.11 mL, 0.73 mmol) as starting materials. The
resulting solid was purified by flash chromatography on
silica gel (DCM) to yield 4d (65.0 mg, 45%) as bright
red/orange crystals, mp 179–180 ꢁC, Rf 0.56 (silica,
4.2.2. 5,7-Dibromo-1-phenethyl-1H-indole-2,3-dione (4a).
A mixture of 5,7-dibromoisatin 2 (200 mg, 0.66 mmol)
and K2CO3 (128 mg, 0.92 mmol) or NaH (36.8 mg,
0.92 mmol) was dissolved in anhydrous DMF (4 mL)
and stirred under nitrogen at 4 ꢁC for 3 h (or 20 min
at rt for NaH) before the addition of KI (22.0 mg,
0.13 mmol) and (2-bromoethyl)benzene 3a (270 mg,
0.2 mL, 1.46 mmol). The reaction mixture was heated
at 50 ꢁC and stirred at this temperature for 18 h. To
the resulting solution were added water (80 mL) and
1 M HCl (2 mL) to acidify to pH 1. The suspension
was filtered and the precipitate washed with water.
The resulting solid was purified by flash chromatogra-
phy on silica gel (CHCl3) to yield 4a (86.3 mg, 32%) as
bright red crystals, mp 165–168 ꢁC, Rf 0.71 (silica,
1
DCM). H NMR (500 MHz): d 3.00 (t, J = 8 Hz, 2H,
H20), 3.79 (s, 3H, OCH3), 4.38 (t, J = 8 Hz, 2H, H10),
6.76 (d, J = 7.5 Hz, 1H, H400), 6.80 (s, 1H, H200), 6.84 (d,
J = 7.5 Hz, 1H, H600), 7.22 (t, J = 7.5 Hz, 1H, H500), 7.69
(d, J = 2 Hz, 1H, H4), 7.89 (d, J = 2 Hz, 1H, H6). 13C
NMR (126 MHz): d 35.3, 42.6, 55.2, 104.8, 112.2, 114.8,
116.9, 121.2, 121.4, 127.5, 129.8, 138.5, 145.1, 146.7,
157.8, 159.8, 181.3. HREI-MS: m/z calcd for
C17H13NO3 79Br81Br [M+]: 438.9242; found: 438.9244.
4.2.6. 5,7-Dibromo-1-[2-(4-methoxyphenyl)ethyl]-1H-indole-
2,3-dione (4e). The compound was prepared according to
the method for 4a using 5,7-dibromoisatin 2 (100 mg,
0.33 mmol) and 4-methoxyphenethyl bromide 3e
1
DCM). H NMR (500 MHz): d 3.03 (t, J = 8 Hz, 2H,
H20), 4.39 (t, J = 8 Hz, 2H, H10), 7.30 (m, 5H, H200–

L. Matesic et al. / Bioorg. Med. Chem. 16 (2008) 3118–3124
3123
(157 mg, 0.11 mL, 0.73 mmol) as starting materials. The
resulting solid was purified by flash chromatography on
silica gel (CHCl3) to yield 4e (89.5 mg, 62%) as bright
red/orange crystals, mp 176–178 ꢁC, Rf 0.53 (silica,
freezer at ꢀ18 ꢁC for 20 h.23 A mixture of the 5,7-dibro-
moisatin 2 (103 mg, 0.34 mmol) and K2CO3 (47.0 mg,
0.34 mmol) or NaH (13.7 mg, 0.34 mmol) was dissolved
in anhydrous DMF (5 mL) and stirred under nitrogen at
4 ꢁC for 3 h. This anion solution was added in portions
(0.5 mL) to the phenacyl iodide maintained at ꢀ2 ꢁC
such that each portion had reacted before the addition
of the next portion (monitored by TLC). The yellow/
brown reaction mixture was stirred at rt for 25 h but
no change in color intensity was observed after 2 h. To
the resulting solution were added water (60 mL) and
1 M HCl (2 mL) to acidify to pH 1. The suspension
was filtered and the precipitate washed with water.
The resulting solid was purified by flash chromatogra-
phy on silica gel (DCM) to yield 6a (13.5 mg, 9%) as
bright red/orange crystals, mp 173–175 ꢁC, Rf 0.58 (sil-
1
DCM). H NMR (500 MHz): d 2.96 (t, J = 8 Hz, 2H,
H20), 3.78 (s, 3H, OCH3), 4.34 (t, J = 8 Hz, 2H, H10),
6.83 (d, J = 8.5 Hz, 2H, H300, H500), 7.16 (d, J = 8.5 Hz,
2H, H200, H600), 7.68 (d, J = 2 Hz, 1H, H4), 7.89 (d,
J = 2 Hz, 1H, H6). 13C NMR (126 MHz): d 34.7, 43.1,
55.5, 105.0, 114.4a, 117.1, 121.6, 127.8, 129.2, 130.1a,
145.3, 147.0, 158.1, 158.8, 181.6. HREI-MS: m/z calcd
for C17H13NO3 79Br81Br [M+]: 438.9242; found:
438.9241.
4.2.7. 5,7-Dibromo-1-(1-naphthylmethyl)-1H-indole-2,3-
dione (5a). A mixture of 5,7-dibromoisatin 2 (101 mg,
0.33 mmol) and NaH (18.0 mg, 0.46 mmol) was dis-
solved in anhydrous DMF (2.5 mL) and stirred under
nitrogen at rt for 20 min before the addition of KI
(11.0 mg, 0.066 mmol) and 1-chloromethylnaphthalene
(128 mg, 0.11 mL, 0.73 mmol). The reaction mixture
was heated at 60 ꢁC and stirred at this temperature for
19 h. After cooling, ethyl acetate (50 mL) was added
and the resulting solution was extracted with 0.5 M
HCl (50 mL) followed by brine (50 mL). The orange or-
ganic layer was dried over MgSO4 and the solvent was
removed to yield a sticky red/orange residue. The result-
ing solid was purified by flash chromatography on silica
gel [DCM/PS (3:2)] to yield 5a (90.8 mg, 62%) as dark
1
ica, DCM). H NMR (500 MHz): d 5.64 (s, 2H, H10),
7.54 (t, J = 7.5 Hz, 2H, H300, H500), 7.67 (t, J = 7.5 Hz,
1H, H400), 7.75 (d, J = 2 Hz, 1H, H4), 7.80 (d,
J = 2 Hz, 1H, H6), 8.00 (d, J = 7.5 Hz, 2H, H200, H600).
13C NMR (126 MHz): d 48.2, 105.4, 117.1, 121.3,
126.5, 127.6, 128.3a, 129.1a, 134.4, 144.8, 146.9, 158.2,
180.8, 191.3. HREI-MS: m/z calcd for C16H9NO3
79Br81Br [M+]: 422.8929; found: 422.8928.
4.2.10. 5,7-Dibromo-1-[2-(3-bromophenyl)-2-oxo-ethyl]-
1H-indole-2,3-dione (6b). This compound was prepared
according to the method for 6a using 5,7-dibromoisatin
2 (305 mg, 1.00 mmol) and 3-bromophenacyl bromide
(278 mg, 1.00 mmol) as starting materials. The reaction
mixture was stirred at 100 ꢁC for 16 h.24 The resulting
solid was purified by flash chromatography on silica
gel (DCM) and subsequent preparative TLC (silica,
DCM) to yield 6b (10.6 mg, 2%) as bright red/orange
crystals, mp 160–162 ꢁC, Rf 0.54 (silica, DCM). 1H
NMR (500 MHz): d 5.60 (s, 2H, H10), 7.44 (t, J = 8 Hz,
1H, H500), 7.77 (d, J = 2 Hz, 1H, H4), 7.80 (d, J = 8.5 Hz,
1H, H400), 7.81 (d, J = 2 Hz, 1H, H6), 7.93 (d, J = 8.5 Hz,
1H, H600), 8.13 (s, 1H, H200). 13C NMR (126 MHz): d
48.0, 105.3, 117.3, 121.3, 123.5, 126.6, 127.7, 130.7, 131.2,
135.4, 137.3, 144.8, 146.6, 158.0, 180.6, 190.2. HREI-MS:
m/z calcd for C16H8NO3 79Br79Br81Br [M+]: 500.8034;
found: 500.8037.
1
red crystals, mp 218–219 ꢁC, Rf 0.35 (silica, DCM). H
NMR (500 MHz): d 5.81 (s, 2H, H10), 7.06 (d, J = 7 Hz,
1H, H200), 7.34 (t, J = 7.5 Hz, 1H, H300), 7.53 (t, J = 7 Hz,
1H, H600), 7.58 (t, J = 7 Hz, 1H, H700), 7.76 (m, 3H, H4,
H6, H400) 7.88 (d, J = 8.5 Hz, 1H, H500), 7.93 (d,
J = 8.5 Hz, 1H, H800). 13C NMR (126 MHz): d 42.8,
105.5, 117.1, 121.3, 121.4, 122.1, 125.3, 126.0, 126.6,
127.5, 128.0, 129.0, 129.8, 130.7, 133.8, 145.3, 146.8,
158.1, 181.2. HREI-MS: m/z calcd forC19H11NO279Br81Br
[M+]: 444.9136; found: 444.9131.
4.2.8. 5,7-Dibromo-1-(2-naphthylmethyl)-1H-indole-2,3-
dione (5b). The compound was prepared according to
the method for 5a using 5,7-dibromoisatin 2 (50.5 mg,
0.16 mmol) and 2-bromomethylnaphthalene (80.1 mg,
0.36 mmol) as starting materials. The resulting red solid
was purified by flash chromatography on silica gel
[DCM/PS (3:2)] to yield 5b (46.6 mg, 64%) as dark red
crystals, mp 140–142 ꢁC, Rf 0.58 (silica, DCM). 1H
NMR (500 MHz): d 5.56 (s, 2H, H10), 7.36 (dd,
J = 2 Hz, 8 Hz, 1H, H300), 7.47 (m, 2H, ArH · 2), 7.63
(s, 1H, H100), 7.74 (d, J = 2 Hz, 1H, H4), 7.76 (m, 1H,
ArH), 7.79 (d, J = 2 Hz, 1H, H6), 7.81 (m, 2H,
ArH · 2). 13C NMR (126 MHz): d 45.1, 105.5, 117.5,
121.7, 124.6, 125.3, 126.4, 126.7, 127.8, 128.0, 128.0,
129.1, 133.0, 133.3, 133.5, 145.6, 147.0, 158.6, 181.5.
HRMS: m/z calcd for C19H11NO279Br81Br [M+]:
444.9136; found: 444.9135.
4.2.11. 5,7-Dibromo-1-[2-(4-bromophenyl)-2-oxo-ethyl]-
1H-indole-2,3-dione (6c). 5,7-Dibromoisatin 2(153 mg,
0.5 mmol) and NaH (20.0 mg, 0.5 mmol) were dissolved
in anhydrous DMF (1.25 mL) and stirred at rt under
nitrogen for 20 min before the addition of freshly distilled
trimethylsilyl chloride (81.0 mg, 0.095 mL, 1.5 mmol).25
The reaction mixture was heated at 50 ꢁC and stirred at
this temperature for 1 h before the addition of 4-brom-
ophenacyl bromide (139 mg, 0.5 mmol) and further heat-
ing at 100 ꢁC for 1.5 h. Upon cooling, water (15 mL) was
added, the suspension was filtered, and the precipitate
washed with hot water (90 ꢁC) to yield a rust colored com-
pound. The product was recrystallized from glacial
AcOH, filtered, and washed with ice cold water to yield
6c (11.2 mg, 5%) as a light yellow powder, mp 184–
4.2.9. 5,7-Dibromo-1-(2-oxo-2-phenylethyl)-1H-indole-
2,3-dione (6a). A mixture of KI (113 mg, 0.68 mmol)
and phenacyl bromide (68.0 mg, 0.34 mmol) was dis-
solved in anhydrous DMF (0.5 mL) and stirred at
ꢀ5 ꢁC under nitrogen for 5 h, followed by cooling in a
1
186 ꢁC, Rf 0.65 (silica, DCM). H NMR (500 MHz): d
5.60 (s, 2H, H10), 7.70 (d, J = 8.5 Hz, 2H, H300, H500),
7.76 (d, J = 2 Hz, 1H, H4), 7.80 (d, J = 2 Hz, 1H, H6),
7.87 (d, J = 8.5 Hz, 2H, H200, H600). 13C NMR

3124
L. Matesic et al. / Bioorg. Med. Chem. 16 (2008) 3118–3124
(126 MHz): d 47.9, 105.3, 117.2, 121.3, 127.6, 129.6a,
129.8, 132.5a, 132.5, 144.7, 146.7, 158.1, 180.7, 190.4.
HREI-MS: m/z calcd for C16H8NO3 79Br81Br81Br [M+]:
502.8013; found: 502.8007.
2. Sharma, V. M.; Prasanna, P.; Seshu, V. A.; Renuka, B.;
Rao, V. L.; Kumar, G. S.; Narasimhulu, C. P.; Babu, P.
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4.2.12. 5,7-Dibromo-1-[2-(3-methoxyphenyl)-2-oxo-ethyl]-
1H-indole 2,3-dione (6d). This compound was prepared
according to the method for 6a using 5,7-dibromoisatin 2
(306 mg, 1.00 mmol) and 3-methoxyphenacyl bromide
(230 mg, 1.00 mmol) as starting materials.26 The resulting
solid was purified by flash chromatography on silica gel
(DCM)toyield6d(43.5 mg, 10%)asbrightorangecrystals,
m.p. 155–157 ꢁC, Rf 0.50 (silica, DCM). 1H NMR
(500 MHz): d 3.88 (s, 3H, OCH3), 5.62 (s, 2H, H10), 7.20
(d, J = 8 Hz, 1H, H400), 7.45 (t, J = 8 Hz, 1H, H500), 7.51
(s, 1H, H200), 7.58 (d, J = 8 Hz, 1H, H600), 7.75 (d,
J = 2 Hz, 1H, H4), 7.81 (d, J = 2 Hz, 1H, H6). 13C NMR
(75 MHz): d 48.1, 55.5, 105.4, 112.5, 117.1, 120.5, 120.7,
121.3, 127.5, 130.1, 135.0, 144.7, 146.9, 158.1, 160.1,
180.8, 191.2. HREI-MS: m/z calcd for C17H11NO4
79Br79Br [M+]: 450.9055; found: 450.9048.
10. Maynard, G. D.; Cheng, H. C.; Kane, J. M.; Staeger, M.
A. Bioorg. Med. Chem. Lett. 1993, 3, 753–756.
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4.2.13. 5,7-Dibromo-1-[2-(4-methoxyphenyl)-2-oxo-ethyl]-
1H-indole-2,3-dione (6e). This compound was prepared
according to the method for 6d using 5,7-dibromoisatin
2 (111 mg, 0.36 mmol) and 4-methoxyphenacyl bromide
(82 mg, 0.36 mmol) as starting materials. The reaction
mixture was then stirred at rt for 51 h. The suspension
was filtered and the precipitate washed with water. The
resulting solid was purified by flash chromatography on
silica gel (DCM) to yield 6e (18.2 mg, 11%) as bright or-
1
ange crystals, mp 205–207 ꢁC, Rf 0.50 (silica, DCM). H
NMR (500 MHz): d 3.91 (s, 3H, OCH3), 5.59 (s, 2H,
H10), 7.00 (d, J = 8 Hz, 2H, H300, H500), 7.74 (d,
J = 2 Hz, 1H, H4), 7.80 (d, J = 2 Hz, 1H, H6), 7.98 (d,
J = 8 Hz, 2H, H200, H600). 13C NMR (126 MHz): d 47.9,
55.9, 105.7, 114.6a, 117.2, 121.6, 126.9, 127.7, 130.7a,
145.0, 147.3, 158.5, 164.7, 181.2, 186.9. HREI-MS: m/z
calcd for C17H11NO4 79Br81Br [M+]: 452.9034; found:
452.9048.
20. Goldbrunner, M.; Loidl, G.; Polossek, T.; Mannschreck,
A.; von Angerer, E. J. Med. Chem. 1997, 40, 3524–3533.
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1120.
24. Jones, A. W.; Purwono, B.; Bowyer, P. K.; Mitchell, P. S.
R.; Kumar, N.; Nugent, S. J.; Joliffe, K. A.; Black, D. C.
Tetrahedron 2004, 60, 10779–10786.
Acknowledgments
We are grateful to the University of Wollongong for
financial support and an Australian Postgraduate
Award for L. Matesic. A University of Wollongong
Post-doctoral Fellowship to K.L. Vine and a Cancer
Institute New South Wales Fellowship to M. Ranson
are gratefully acknowledged.
25. Rekhter, M. A. Chem. Het. Compounds 1999, 35, 1165–
1166.
References and notes
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