The effect of sulfonate leaving groups on the hypoxia-selective toxicity of nitro analogs of the duocarmycins

Article abstract of DOI:10.1016/j.bmc.2011.06.073

A series of 3-substituted (5-nitro-2,3-dihydro-1H-benzo[e]indol-1-yl)methyl sulfonate (nitroCBI) prodrugs containing sulfonate leaving groups undergo hypoxia-selective metabolism to form potent DNA minor groove alkylating agents. They were evaluated (alon

Full text of DOI:10.1016/j.bmc.2011.06.073

Bioorganic & Medicinal Chemistry 19 (2011) 4851–4860
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The effect of sulfonate leaving groups on the hypoxia-selective toxicity of nitro
analogs of the duocarmycins
Amir Ashoorzadeh, Graham J. Atwell, Frederik B. Pruijn, William R. Wilson, Moana Tercel,
⇑
William A. Denny , Ralph J. Stevenson
Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 3-substituted (5-nitro-2,3-dihydro-1H-benzo[e]indol-1-yl)methyl sulfonate (nitroCBI) pro-
drugs containing sulfonate leaving groups undergo hypoxia-selective metabolism to form potent DNA
minor groove alkylating agents. They were evaluated (along with chloride leaving group analogs for com-
parison) for their cytotoxicity against cultures of SKOV3 and HT29 human tumor cell lines under both
aerobic and hypoxic conditions. Sulfonates with neutral side chains (e.g., 5,6,7-trimethoxyindole; TMI)
show consistently higher hypoxic cytotoxicity ratios (HCRs) (34–246) than the corresponding chloro ana-
logs (2.8–3.1) in SKOV3 cells, but these trends do not hold for compounds with cationic or polar neutral
side chains.
Received 18 May 2011
Revised 22 June 2011
Accepted 26 June 2011
Available online 29 June 2011
Keywords:
Cyclopropylindoline
Hypoxic selectivity
Prodrug
Ó 2011 Elsevier Ltd. All rights reserved.
Sulfonate leaving group
DNA minor groove alkylator
Antitumor
Neutral side chain
1. Introduction
in AT-rich regions of DNA.11 We also showed12 that the corre-
sponding nitro compound 5 was about 3000-fold less toxic than
The class of compounds broadly known as the hydroxyCBIs (e.g.,
1)1, evolved from natural product cyclopropylindolines such as
duocarmycin SA2 (2), have been widely explored as very potent
cytotoxins targeted at the DNA minor groove, where they alkylate
the N3 of adenine in a sequence-selective manner (Fig. 1).1 It has
been shown that the precursor seco analogs such as 1 are equally
cytotoxic, due to their rapid conversion to the cyclopropylindoline
form.4 N-Acyl O-amino- (e.g., 3) and quinone-containing analogs
(e.g., 4) of these have been explored5,6 as bioreductively-activated
prodrugs. We have taken a different approach, using 1-(chloro-
methyl)-5-nitro-2,3-dihydro-1H-benzo[e]indoles as hypoxia-acti-
vated prodrugs.3,7–9
5a in AA8 CHO cells, presumably because it can no longer undergo
spirocyclization and DNA alkylation, but could undergo hypoxia-
selective reduction in cells to the much more potent 5a.8
Nitro compounds such as 5 thus have the potential to be selec-
tively activated in the hypoxic regions that are known to be pres-
ent in a majority of human solid tumors.13 Development of this
class to date has sought to optimize the properties suggested to
be needed for such prodrugs:13,14 (i) high toxicity differential be-
tween prodrug and reduction product(s), (ii) rapid and selective
activation by 1-electron reductases in an oxygen-sensitive process,
(iii) high tissue penetration in order to reach the target hypoxic
cells distant from the vasculature, and (iv) an ability of the active
reduction product(s) to back-diffuse from the site of activation to
kill surrounding cells (the bystander effect).
While nitroCBI 5 showed significantly lower cytotoxicity than
its reduction product 5a, its selective cytotoxicity for hypoxic over
aerobic cell cultures was modest, with an HCR (hypoxic cytotoxic-
ity ratio: IC50aerobic/IC50hypoxic) of only 2.5 in HT29 colon
carcinoma cells after a 4 h exposure.7 The reason for this was
hypothesized to be the relatively low 1-electron reduction poten-
tial of the parent class; for example the more soluble analog 6,
which also has a low HCR, had a one electron reduction potential
(E(1)) of ꢀ512 mV, and that of analog 5 is expected to be similar.
This led to the study of a series of analogs of higher reduction
We previously showed that replacement of the phenol in 1 with
an amino group gave a compound (5a) of similar cytotoxic potency
(IC50s <1 nM in AA8 CHO cells following a 4 h exposure),10 and
with a similar mechanism of action; alkylation at adenine-N3 sites
Abbreviations: CBI, seco-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one;
DIPEA, diisopropylethylamine; DMA, dimethylacetamide; EDCIꢁHCl, N-ethyl-N0-(3-
dimethylaminopropyl)carbodiimide hydrochloride; HCR, hypoxic cytotoxicity
ratio; TMI, 5,6,7-trimethoxyindole.
⇑
Corresponding author. Tel.: +649 923 6144; fax: +64 9 373 7502.
E-mail address: b.denny@auckland.ac.nz (W.A. Denny).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.073

4852
A. Ashoorzadeh et al. / Bioorg. Med. Chem. 19 (2011) 4851–4860
potentials, with electron-withdrawing groups on the A-ring. An
example is the 7-SO2NH2 compound 7, which had an E(1) of
ꢀ390 mV and showed an HCR in the HT29 cell line of 330.7 A later
study on analogs of compound 7 showed that the related 7-
SO2NH(CH2)2OH analog 8 had similarly high hypoxic selectivities
across a range of cell lines.9 The corresponding phosphate 9, pre-
pared as a much more water-soluble pre-prodrug that releases 8
in plasma, showed marked hypoxic cell killing and corresponding
substantial tumor growth delays when used combination with
radiation (to kill the well-oxygenated tumor cells) in mice bearing
SiHa human cervical tumor xenografts.9
To date, while there have been substantial variations in the core
alkylating subunit, the minor groove-binding side chain and the A-
ring substituents of the nitroCBIs, little attention has been paid to
variation of the leaving group which has essentially been restricted
to chloro. A-ring substituents generally diminish the cytotoxicity of
aminoCBIs but were found essential to observe significant HCRs for
nitroCBIs in vitro when the leaving group was chloride. One sulfo-
nate analog has been reported15; the aminoCBI methanesulfonate
10a, which was equipotent with the corresponding chloro com-
pound 5a (IC50s between 0.4 and 1.2 nM in EMT6 and SKOV3 hu-
man tumor cell lines).
boxylic acid20 and (E)-3-(3-hydroxy-4-methoxyphenyl)acrylic acid
using EDCIꢁHCl gave compounds 13 and 15, respectively. Hydroge-
nation of 13 in THF over PtO2 gave the corresponding aminoCBI
13a in excellent yield. AminoCBIs 16a and 18a were prepared from
nitroCBI precursors in the same way.
The cyclohexyl sulfonates 16 and 17 were made in the same
way as the known15 10, from hydroxymethyl-5-nitroCBI interme-
diate 2710 (Scheme 2) via formation of the cyclohexyl sulfonate
28, followed by Boc deprotection and coupling of the resulting
amine 29 with the appropriate side chain acids. Benzyl sulfonates
20 and 21 (Scheme 3), which contain free hydroxyl groups in the
side chains, were prepared the same way, by initially sulfonating
27 to give 33, followed by deprotection to amine 34, to which
the side chains were added as above. Analogs 18 and 19 were made
by an alternative route, initially deprotecting 27 and coupling the
resulting hydroxyl amine 30 with the appropriate side chains to
give 31 and 32, which were then sulfonated to give the end prod-
ucts (Scheme 3).
The synthesis of the tosylates 22–24 (Scheme 4) followed the
former route (previous paragraph), with initial tosylation of 27 to
give 35, followed by Boc deprotection to give common intermedi-
ate 36, to which the appropriate side chains were coupled. The lat-
ter reaction was complicated by the tosylate appearing to be a
better leaving group than the other sulfonates. Thus coupling of
36 with 5-methoxy-1H-indole-2-carboxylic acid using EDCIꢁHCl
mainly afforded the undesired chloromethyl product 14, presum-
ably due to chloride ion scrambling. Use of 1-ethyl-3-(30-dimethyl-
aminopropyl)carbodiimide methiodide (EDCIꢁMeI) gave the
desired product, but the reaction was very slow and the yield
was relatively low. Coupling of 36 with 5,6,7-trimethoxyindole-
2-carboxylic acid using EDCIꢁHCl also gave the chloromethyl prod-
uct 5 together with the desired product 22 in a ratio of 1:3, but
these could be separated by column chromatography. Surprisingly,
reaction of 36 with 5-(2-hydroxyethoxy)-1H-indole-2-carboxylic
acid using EDCIꢁHCl provided the desired product 24 in quantita-
tive yield. Special care, including cooling, had to be taken during
basic workup of the sulfonates as these leaving groups were more
prone to base-promoted elimination (of RSO3H) than their chloride
analogs.
Alternative leaving groups have been more extensively used in
the aniline mustard class of DNA-alkylating drugs, where it is
known that cytotoxicity correlates with the nucleofugality of the
leaving group (e.g., bromo mustards are more reactive and more
potent than chloro mustards).16 Mesylate (OSO2Me) and benzyl
sulfonate (OSO2CH2Ph) groups have relative nucleofugalities com-
parable to that of iodide (
x 1.63, 1.34, and 1.33 eV, respectively)
and greater than those of chloride or bromide (
x 0.77 and
0.90 eV, respectively).17,18
In this study we therefore explored structure-activity relation-
ships for a number of nitroCBI prodrugs bearing sulfonate-based
leaving groups and compared their cytotoxicities and HCRs with
those of their chloride analogs and the known sulfonate 10.10,15
Questions to be considered included whether the steric bulk of
the larger sulfonates might hinder binding at the DNA base in
the minor groove and reduce prodrug toxicity, as has been sug-
gested19 with the hydroxyCBI compounds 11 and 12, where the
methyl compound 11 is considerably less potent than 12 (IC50s
0.75 and 0.026 nM, respectively, in A549 human bronchial carci-
noma cells, 24 h exposure).
It should be noted that while several aminoCBIs were able to be
synthesized straightforwardly from their nitroCBI precursors via
hydrogenation (H2/PtO2) and isolated, others (e.g., 18a) appeared
to undergo facile spirocyclization to the resultant imine.7 Those
with cinnamate side chains could not be prepared in this way
due to reduction of the alkene.
In all cases (with the sole exception of 24) the nitroCBI sulfo-
nates with neutral side chains showed higher solubility in culture
medium than the corresponding chloro compounds, although this
gain in aqueous solubility was not seen with nitroCBIs bearing a
basic dimethylaminoethyl side chain (Table 1).
2. Results and discussion
2.1. Chemistry
The new chloromethyl-5-nitroCBI reference analogs were pre-
pared as shown in Scheme 1. Deprotection of the known Boc-pro-
tected intermediate 25,10 followed by the coupling reaction of the
resulting crude amine 26 with 5-(2-hydroxyethoxy)indole-2-car-
Cl
OSO2
OH
NR
NR
NBoc
ii
compounds 13, 15
16, 17
16a
compounds
i
iii
of Table 1
of Table 1
iv
iii
NO2
NO2
NO2
compound
28: R=Boc
27
ii
of Table 2
compound 13a
of Table 2
25: R=Boc
26: R=H
29: R=H
i
Scheme 2. Synthesis of compounds 16, 17, and 16a. Reagents and conditions: (i)
cyclohexanesulfonyl chloride, DIPEA, CH2Cl2, 0 °C; (ii) HCl, dioxane, 10 °C; (iii)
5,6,7-trimethoxyindole-2-carboxylic acid or 5-(2-(dimethylamino)ethoxy)-1H-
indole-2-carboxylic acid, EDCIꢁHCl, DMA; and (iv) H2, PtO2,THF.
Scheme 1. Synthesis of compounds 13, 15, and 13a. Reagents and conditions: (i)
HCl, dioxane, 10 °C; (ii) 5-(2-hydroxyethoxy)indole-2-carboxylic acid or (E)-3-(3-
hydroxy-4-methoxyphenyl)acrylic acid, EDCIꢁHCl, DMA; and (iii) H2, PtO2, THF.

A. Ashoorzadeh et al. / Bioorg. Med. Chem. 19 (2011) 4851–4860
4853
OH
OH
3.1/34 in SKOV3. The higher HCR values for the sulfonates were
mainly due to increased cytotoxicity under hypoxia, although de-
creased aerobic toxicity also contributed. The highest HCRs were
shown by the most bulky sulfonates (16, 18, and 22). This was, sur-
prisingly, not true for the compounds with cationic or polar neutral
side chains, where there was little difference in HCRs between
chlorides and sulfonates in either cell line. Thus for compounds
with cationic side chains the ratios for SKOV3 were: 6/17
(OSO2cyclohexyl) 1.9/1.5; and 6/19 (OSO2CH2Ph) 1.9/1.8. Similarly,
for polar neutral side chains the ratios were: 13/20 (OSO2CH2Ph)
3.8/7.1; 13/23 (OSO2(4-MePh)) 3.8/2.8; and 15/21 (OSO2CH2Ph)
3.3/3.9.
Table 2 records the cytotoxicities and HCRs for two of the chloro
nitroCBIs (5, 13) and their corresponding amine reduction products
(5a, 13a); the active metabolites of these prodrugs.7,8 For com-
pounds with the TMI side chain only, there is comparison of nitro
and amino pairs bearing two different sulfonate leaving groups;
OSO2Me (10, 10a) and OSO2cyclohexyl ( 16, 16a). As previously
shown7,8 for the chloro compounds the nitro group provides a large
degree of deactivation of cytotoxicity, with average nitro/amino
oxic IC50 ratios of about 3000 for 5/5a and 13/13a in the two cell
lines. The amine effectors of two sulfonates (10a, 16a) showed sim-
ilar levels of potency (IC50s 1–5 nM) and degrees of deactivation
(nitro/amino ratios of 2000- to 3000-fold). As expected, all of the
amino analogs showed HCRs close to unity.
NR
NCOR
ii
iii
compounds 18, 19
of Table 1
v
NO2
NO2
OMe
compound 18a
of Table 2
27: R=Boc
i
30
: R=H
R=
for
N
H
31
OMe
O
iii
OMe
NMe2
R=
for
OSO2CH2Ph
NR
N
H
32
iv
compounds 20, 21
of Table 1
NO2
33: R=Boc
34: R=H
i
Scheme 3. Synthesis of compounds 18–21 and 18a. Reagents and conditions: (i)
HCl, dioxane, 10 °C; (ii) 5,6,7-trimethoxyindole-2-carboxylic acid or 5-(2-(dimeth-
ylamino)ethoxy)-1H-indole-2-carboxylic acid hydrochloride, EDCIꢁHCl, DMA; (iii)
a
-toluenesulfonyl chloride, pyridine, 0 °C; (iv) 5-(2-hydroxyethoxy)indole-2-car-
boxylic acid or (E)-3-(3-hydroxy-4-methoxyphenyl)acrylic acid, EDCIꢁHCl, TsOH,
DMA; (v) H2, PtO2,THF.
As noted above, while about half of the nitroCBI sulfonates
studied showed significant hypoxic cell selectivity (>30 in SKOV3
cells), the effect did vary with the nature of the side chain. The
most hypoxia selective compound in SKOV3 and HT29 cultures
was the OSO2CH2Ph/TMI analog 18. Compound 18 was evaluated
in a panel of cell lines, and the results are shown in Table 3. The
HCR values showed large differences between the cell lines, vary-
ing from a low of 3.6 for H1299 cells to a high of 246 for SKOV3
cells, with a median value of 15.6-fold. To evaluate whether this
reflects differences in reductase activity between cell lines, we
compared a transfected A459 line which over-expresses NADPH:
cytochrome P450 reductase (P450R). Forced P450R expression,
to ninefold higher than the parental line22 caused a fourfold
reduction in the hypoxic IC50 but had no significant effect on
the aerobic IC50 (Table 3). This establishes that 18 is a substrate
for P450R, and that redox cycling as a result of its one-electron
reduction in aerobic cells is not responsible for its aerobic cyto-
toxicity in A459 cells. We therefore evaluated whether differences
in P450R activity in these cell lines (measured as NADPH-depen-
dent cyanide-sensitive cytochrome c reductase activity)23 might
account for their differences in sensitivity under hypoxia, but
there was no significant correlation between P450R activity and
hypoxic IC50 of 18 by Pearson product moment correlation (corre-
lation coefficient ꢀ0.159, p = 0.682).
OSO2(4-MePh)
R
NH
iii
NBoc
22-24
compounds
ii
of Table 1
NO2
NO2
36
27
35
: R=OH
i
: R=OSO2(4-MePh)
Scheme 4. Synthesis of compounds 22–24. Reagents and conditions: (i) TsCl,
pyridine, CH2Cl2, 0 °C or TsCl, pyridine, DMAP, CH2Cl2, 0 °C; (ii) TFA, CH2Cl2 or HCl,
dioxane, 10 °C; and (iii) 5,6,7-trimethoxyindole-2-carboxylic acid or 5-methoxy-
1H-indole-2-carboxylic acid or 5-(2-hydroxyethoxy)-1H-indole-2-carboxylic acid,
TsOH, EDCIꢁHCl or EDCIꢁMeI, DMA.
2.2. Biology
The nitroCBIs bearing sulfonate leaving groups were evaluated
for their cytotoxicity against cultures of SKOV3 (ovarian carci-
noma) and HT29 (colon carcinoma), under both aerobic (20% O2)
and hypoxic (<20 ppm O2) conditions as described previously,21
and the results are given in Table 1, Figures. 2 and 3. Compounds
5, 6, and 13–15 are the comparators with the chloride leaving
group, bearing a range of lipophilic (5, 14), polar neutral (13, 15)
and cationic (6) side chains. Compounds 10, 16–24 are ordered
according to the four different leaving groups studied; OSO2Me
(10), OSO2cyclohexyl (16, 17), OSO2CH2Ph (18–21) and OSO2(4-
MePh) (22–24). Overall, the cytotoxicities of the compounds under
both oxic and hypoxic conditions were broadly similar in SKOV3
and HT29 cell lines, although the HCR values for the more selective
compounds were higher in SKOV3.
The sulfonates with A side chains (TMI) show consistently high-
er HCRs than the corresponding chloride compounds. Thus, com-
pounds 5 (Cl) and 10 (OSO2Me) have HCRs of 2.8 and 39,
respectively, in SKOV3 cells. Similarly, the OSO2cyclohexyl (16),
OSO2CH2Ph (16) and OSO2(4-MePh) (22) leaving groups all pro-
vided much higher HCRs (146, 246 and 72; Table 1) than 5. The
5-OMe-indole pair showed a similar trend: 14/24 (OSO2(4-MePh))
3. Conclusions
The current study was performed to investigate structure–
activity relationships influencing cytotoxicity and hypoxic selectiv-
ity for a series of nitroCBI prodrugs containing sulfonate leaving
groups, with chloride leaving group analogs as comparators. The
general conclusion is that sulfonate leaving groups offer a way of
increasing hypoxic selectivity in nitroCBIs without introducing A-
ring substituents. Furthermore, basic side chains do not appear to
be necessary to produce high HCRs, in contrast to chloro analogs.
This is potentially useful as basic side chains may compromise dis-
tribution (by sequestration in acidic organelles) or metabolism.
The sulfonates without solubilizing side chains showed mod-
estly higher solubility in culture medium than the corresponding
chloro compounds, although additional solubilizing strategies will

4854
A. Ashoorzadeh et al. / Bioorg. Med. Chem. 19 (2011) 4851–4860
OMe
Cl
OMe
OMe
OMe
OMe
N
N
N
N
H
N
OMe
H
MeO2C
O
O
N
H
2
: Duoc
O
N
armycin S
A
1: hydroxyCBI
OH
Cl
OMe
Cl
OMe
OMe
OMe
OMe
OMe
N
H
N
H
O
O
O
O
O
3
4
NHBoc
N
Me
Me
Me
OMe
Y
O
OMe
OMe
N
H
O
O
Me
5: R=NO2; Y=Cl
5a
R
:
R=NH2; Y=Cl
10a: R=NH2; Y=OSO2Me
NMe2
NMe2
O
O
Cl
Cl
H
R
N
N
H
N
N
H
A
O
O
R
6
: R=H
: R=SO2NH2
: R=SO2NH(CH2)2OH
: R=SO2NH(CH2)2OP(O)(OH)2
11: R=Me
12
NO2
7
8
9
OH
: R=H
Figure 1. Structures of some previously reported CBIs.
be required to formulate these prodrugs for in vivo evaluation. The
results demonstrate the utility of sulfonate leaving groups, due to
the equivalent potencies of the amino compounds and the gener-
ally much larger HCRs of the nitro compounds compared with
the chloro series. This study suggests that combining suitable A-
ring substituents, which may be useful (even if not needed for high
HCR) for appending functionality to allow preparation of phos-
phate pre-prodrugs as recently demonstrated in the chloro series,9
and sulfonate leaving groups could provide analogs with higher
hypoxic selectivity and improved water solubility. Regarding the
criteria listed earlier for prodrugs, several of the sulfonates do
show high toxicity differentials between prodrug and effector
and effective oxygen-sensitive activation. The transport issues
have not been addressed here.
product purity was also assessed by combustion analysis carried
out in the Campbell Microanalytical Laboratory, University of Otag-
o, Dunedin, New Zealand. Melting points were determined on an
Electrothermal 2300 Melting Point Apparatus. NMR spectra were
obtained on a Bruker Avance 400 spectrometer at 400 MHz for
1H and 100 MHz for 13C spectra.
4.1.1. (1-(Chloromethyl)-5-nitro-1H-benzo[e]indol-3(2H)-yl)(5-
(2-hydroxyethoxy)-1H-indol-2-yl)methanone (13) (Scheme 1)
A
solution of tert-butyl 1-(chloromethyl)-5-nitro-1H-ben-
zo[e]indole-3(2H)-carboxylate10 (25) (600 mg, 1.65 mmol) in diox-
ane (15 mL) was saturated with HCl gas at 10 °C, stirred at room
temperature for 1 h, and then concentrated under reduced pres-
sure. The resulting crude amine 26 was dissolved in DMA (5 mL)
and 5-(2-hydroxyethoxy)indole-2-carboxylic acid20 (402 mg,
1.82 mmol) and EDCIꢁHCl (793 mg, 4.14 mmol) were added, and
the mixture was stirred at room temperature for 4 h. Addition of
aqueous KHCO3 provided a solid that was further recrystallized
from DMF/EtOAc/petroleum ether (2ꢂ) to give 13 (620 mg, 80%):
mp 249–250 °C; 1H NMR [(CD3)2SO] d 11.71 (s, 1H), 9.16 (s, 1H),
8.35 (dd, J = 7.2, 2.5 Hz, 1H), 8.22 (dd, J = 6.9, 2.3 Hz, 1H), 7.79–
7.70 (m, 2H), 7.42 (d, J = 8.9 Hz, 1H), 7.19 (d, J = 1.3 Hz, 1H), 7.17
(d, J = 2.2 Hz, 1H), 6.96 (dd, J = 8.9, 2.4 Hz, 1H), 4.93 (t, J = 10.2 Hz,
1H), 4.88 (t, J = 5.5 Hz, 1H), 4.70 (dd, J = 10.9, 2.3 Hz, 1H), 4.66–
4.57 (m, 1H), 4.18–4.08 (m, 2H), 4.01 (t, J = 5.0 Hz, 2H), 3.76 (dd,
4. Experimental
4.1. Chemistry
Final products were analyzed by reverse-phase HPLC (Alltima
C18 5
l
m column, 150 ꢂ 3.2 mm; Alltech Associated Inc., Deer-
field, IL) using an Agilent HP1100 equipped with a diode-array
detector. Mobile phases were gradients of 80% CH3CN/20% H2O
(v/v) in 45 mM NH4O2CH at pH 3.5 and 0.5 mL/min. Purity was
determined by monitoring at 330 50 nm and was >95%. Final

A. Ashoorzadeh et al. / Bioorg. Med. Chem. 19 (2011) 4851–4860
4855
Table 1
Structure, solubility and in vitro cytotoxicity data for nitroCBIs with varying leaving groups and side chains
Y
OMe
O
NMe2
R
N
H
A
N
H
OMe
N
OMe
R =
O
B
OMe
OH
OMe
O
NO2
OH
N
H
N
H
D
E
C
b
Compd
Y
R
Sola
(l
M)
IC50 average (lM)
SKOV3
HT29
Oxic
Hypoxic
HCRc
Oxic
3.4 0.4
Hypoxic
HCRc
5d
Cl
Cl
Cl
Cl
A
B
C
D
E
A
A
B
A
B
C
E
5
193
13
38
9
69
70
22
17
91
46
35
89
29
12
4.2 1.3
1.4 0.5
9.3 2.0
4.8 0.2
0.89 0.19
2.2 1.0
16 1.3
1.7 0.22
0.87 0.12
3.3 0.06
2.8 0.9
1.9 0.6
3.8 0.2
3.1 0.1
3.3 0.9
39 29
146 74
1.5 0.28
246 57
1.8 0.2
7.1 0.9
3.9 1.5
72 19
1.6 0.6
0.48 0.11
5.7 2.0
2.5 0.7
1.1 0.13
1.1 0.2
7.4 0.1
4.5 2.3
4.6 1.4
26 11
2.3 0.3
37 7
4 2
2.4 0.5
1.1 0.14
28 2
0.81 0.04
7.9 2.2
6e
0.47 0.05
6.6 0.9
9.7 0.2
1.7 0.1
1.5 0.6
9.9 2.9
0.41 0.13
9.8 1.4
0.24 0.04
1.8 0.8
1.0 0.24
12 0.8
13
14e
15
10d
16
17
18
19
20
21
22
23
24
1.54 0.02
0.40 0.06
0.14 0.04
0.19 0.06
0.30 0.03
0.082 0.02
0.15 0.01
0.21 0.07
0.16 0.04
0.22 0.06
0.77 0.09
0.38 0.09
1.30 0.14
0.59 0 l.14
0.31 0.04
0.44 0.09
0.20 0.03
0.36 0.09
0.08 0.02
0.66 0.18
1.0 0.36
0.47 0.07
2.7 0.15
0.7 0.6
Cl
OSO2Me
OSO2cyclohexyl
OSO2cyclohexyl
OSO2CH2Ph
OSO2CH2Ph
OSO2CH2Ph
OSO2CH2 Ph
OSO2(4-MePh)
OSO2(4-MePh)
OSO2(4-MePh)
0.47 0.04
12 1.5
0.27 0.02
1.2 0.3
0.57 0.08
8.8 1.1
3.0 0.08
11 2
A
C
D
2.8 0.08
34 15
3.3 1.0
6.5 3.5
a
b
c
Solubility in culture medium (aMEM) containing 5% FCS.
Drug concentration to reduce cell density to 50% of that of controls, 5 days after 4 h exposure (mean SEM for two to seven experiments).
Hypoxic cytotoxicity ratio = IC50(oxic)/IC50(hypoxic). Values are means of intraexperiment ratios ( SEM for two to six experiments).
d
e
Previously reported.10,15
Previously reported.15,20
J = 5.1, 2.5 Hz, 2H). Anal. Calcd for C24H20ClN3O5: C, 61.87; H, 4.33;
N, 9.02. Found: C, 61.61; H, 4.54; N, 9.31.
4.1.4. (5-Nitro-3-(5,6,7-trimethoxy-1H-indole-2-carbonyl)-2,3-
dihydro-1H-benzo[e]indol-1-yl)methyl cyclohexanesulfonate
(16) (Scheme 2)
4.1.2. (E)-1-(1-(Chloromethyl)-5-nitro-1H-benzo[e]indol-3(2H)-
yl)-3-(3-hydroxy-4-methoxyphenyl)prop-2-en-1-one (15)
Similar deprotection of 25 (352 mg, 0.97 mmol) with HCl/diox-
ane, followed by reaction with (E)-3-(3-hydroxy-4-methoxy-
phenyl)acrylic acid (207 mg, 1.07 mmol) and EDCIꢁHCl (558 mg,
2.91 mmol) in DMA (3 mL) gave 15 (308 mg, 72%): mp 258–
259 °C; 1H NMR [(CD3)2SO] d 9.21 (br s, 1H), 9.16 (s, 1H), 8.32 (d,
J = 8.7 Hz, 1H), 8.17 (d, J = 8.1 Hz, 1H), 7.77–7.66 (m, 2H), 7.62 (d,
J = 15.3 Hz, 1H), 7.28 (d, J = 1.9 Hz, 1H), 7.22 (dd, J = 8.4, 2.0 Hz,
1H), 7.02–6.94 (m, 2H), 4.69–4.52 (m, 3H), 4.07 (d, J = 4.4 Hz,
2H), 3.84 (s, 3H). Anal. Calcd for C23H19ClN2O5: C, 62.94; H, 4.36;
N, 6.38. Found: C, 62.71; H.4.33; N, 6.36.
Cyclohexanesulfonyl chloride (350 mg, 1.92 mmol) was added
dropwise to a stirred solution of tert-butyl 1-(hydroxymethyl)-5-
nitro-1H-benzo[e]indole-3(2H)-carboxylate
(27)10
(330 mg,
0.96 mmol) and DIPEA (310 mg, 2.40 mmol) in CH2Cl2 (25 mL) at
0 °C. The mixture was stirred at 0 °C for 1 h and at room tempera-
ture for further 16 h, and then diluted with CH2Cl2, washed with
aqueous KHCO3 and water, dried and concentrated under reduced
pressure. The residue was purified by column chromatography
eluting with CH2Cl2 followed by trituration of the resulting oil with
i-Pr2O to give tert-butyl 1-(((cyclohexylsulfonyl)oxy)methyl)-5-ni-
tro-1H-benzo[e]indole-3(2H)-carboxylate (28) as a yellow solid
(349 mg, 74%): mp (CH2Cl2/i-Pr2O) 201–204 °C; 1H NMR [(CD3)2SO]
d 8.74 (br s, 1H), 8.31 (d, J = 8.6 Hz, 1H), 8.14 (d, J = 8.2 Hz, 1H),
7.78–7.60 (m, 2H), 4.56–4.44 (m, 2H), 4.42–4.32 (m, 1H), 4.26 (t,
J = 10.4 Hz, 1H), 4.14 (dd, J = 11.4, 2.4 Hz, 1H) 3.08 (br, s, 1H),
1.88–1.80 (m, 1H), 1.73–1.43 (m, 13H), 1.24–0.90 (m, 5H). Anal.
Calcd for C24H30N2O7S: C, 58.76; H, 6.16; N, 5.71. Found: C,
58.49; H, 6.33; N, 5.62.
A solution of 28 (112 mg, 0.23 mmol) in dioxane (2 mL) was sat-
urated with HCl gas at 10 °C, stirred at room temperature for 1 h,
and then concentrated under reduced pressure. The resulting crude
amine 29 was dissolved in DMA (2 mL) and 5,6,7-trimethoxyin-
dole-2-carboxylic acid (63 mg, 0.25 mmol) and EDCIꢁHCl (131 mg,
0.68 mmol) were added, and the mixture was stirred at room tem-
perature for 2 h. Addition of aqueous KHCO3 precipitated a solid
that was purified by column chromatography eluting with
CH2Cl2/EtOAc (9:1) followed by trituration of the resulting oil with
i-Pr2O to give 16 as a yellow solid (74 mg, 52%): mp (EtOAc) 191–
4.1.3. (1-(Chloromethyl)-5-amino-1H-benzo[e]indol-3(2H)-
yl)(5-(2-hydroxyethoxy)-1H-indol-2-yl)methanone (13a)
A solution of 13 (127 mg, 0.27 mmol) in THF (100 mL) was
hydrogenated over PtO2 (30 mg) at 45 psi for 2 h. The catalyst
was removed by filtration and the solution was concentrated under
reduced pressure to a small volume and diluted with i-Pr2O to give
13a (114 mg, 96%): mp >250 °C; 1H NMR [(CD3)2SO] d 11.55 (d,
J = 1.6 Hz, 1H), 8.08 (d, J = 8.5 Hz, 1H), 7.76 (d, J = 8.3 Hz, 1H),
7.71 (s, 1H), 7.46 (t, J = 7.6 Hz, 1H), 7.40 (d, J = 8.9 Hz, 1H), 7.28
(t, J = 7.5 Hz, 1H), 7.15 (d, J = 2.3 Hz, 1H), 7.07 (d, J = 1.3 Hz, 1H),
6.92 (dd, J = 8.9, 2.4 Hz, 1H), 5.97 and 5.95 (2s, 2H), 4.85 (t,
J = 5.6 Hz, 1H), 4.73 (dd, J = 10.7, 9.0 Hz, 1H), 4.51 (dd, J = 10.9,
1.6 Hz, 1H), 4.16–4.08 (m, 1H), 4.03–3.93 (m, 3H), 3.80–3.70 (m,
3H). Anal. Calcd for C24H22ClN3O3: C, 66.13; H, 5.09; N, 9.64; Cl,
8.13. Found: C, 66.15; H, 5.10; N, 9.50; Cl, 7.88.

4856
A. Ashoorzadeh et al. / Bioorg. Med. Chem. 19 (2011) 4851–4860
100
10
nitroCBI oxic
nitroCBI hypoxic
SKOV3 cell line
1
0.1
0.01
5
10 16
23
18 22
6
17 19
13
20
14 24
15 21
l
)
l
l
)
l
)
l
l
l
e
h
y
h
y
h
C
C
C
C
C
Ph
Ph
P
Ph
2
M
P
e
P
2
2
2
2
ex
ex
ePh
H
H
H
H
h
h
M
-
M
Me
-
C
C
C
C
o
o
-
SO
l
2
2
2
2
4
4
4
cl
O
(
(
(
O
O
yc
2
2
2
cy
2
SO
c
S
S
SO
O
O
2
O
O
O
O
S
SO
S
O
O
O
SO
SO
O
O
D
E
C
B
A
Figure 2. Cytotoxicity of nitroCBI compounds under oxic and hypoxic conditions in the SKOV3 cell line. The compounds are grouped according to side chains A–E of Table 1.
100
nitroCBI oxic
nitroCBI hypoxic
HT29 cell line
10
1
0.1
0.01
10 16 18
22
6
19
23
20
13
14 24
15
21
5
17
D
E
C
B
A
Figure 3. Cytotoxicity of nitroCBI compounds under oxic and hypoxic conditions in the HT29 cell line. The compounds are grouped according to side chains A–E of Table 1.

A. Ashoorzadeh et al. / Bioorg. Med. Chem. 19 (2011) 4851–4860
4857
Table 2
Cytotoxicity of corresponding aminoCBI analogs
Y
OMe
OMe
O
R
OH
N
R
=
N
H
A
N
H
O
OMe
C
X
b
Compd
X
Y
R
Sola
(lM)
IC50 (SKOV3) average (
lM)
Oxic
Hypoxic
HCRc
Oxic NO2/NH2
5ad
13a
10ad
16a
1
NH2
NH2
NH2
NH2
OH
Cl
Cl
A
C
A
A
A
197
22
15
0.0013 0.00014
0.0033 0.00018
0.0012 0.00012
0.0049 0.0019
0.0017 0.00020
0.0042 0.0001
1.0 0.1
0.8 0.04
3230
2820
1830
3265
OSO2Me
OSO2cyclo hexyl
Cl
37
0.0065 0.0029
1.0 0.3
0.00061 0.00017
a
b
c
Solubility in culture medium (aMEM) containing 5% FCS.
Drug concentration to reduce cell density to 50% of that of controls following 4 h exposure (mean SEM for two to seven experiments).
Hypoxic cytotoxicity ratio = IC50(oxic)/IC50(hypoxic). Values are means of intraexperiment ratios ( SEM for two to six experiments).
Previously reported.10,15
d
8.35 (d, J = 8.7 Hz, 1H), 8.25 (d, J = 8.1 Hz, 1H), 7.82–7.70 (m, 2H),
7.42 (d, J = 8.9 Hz, 1H), 7.22–7.13 (m, 2H), 6.94 (dd, J = 8.9,
2.4 Hz, 1H), 4.94 (t, J = 10.0 Hz, 1H), 4.77–4.70 (m, 1H), 4.63–4.50
(m, 3H), 4.07 (t, J = 5.9 Hz, 2H), 3.04–2.95 (m, 1H), 2.65 (t,
J = 5.8 Hz, 2H), 2.24 (s, 6H), 1.78–1.68 (m, 1H), 1.60–1.37 (m, 4H),
1.17–0.81 (m, 5H). Treatment of the free base with CH2Cl2/petro-
leum ether/HCl gave the hydrochloride salt, mp 150–154 °C. Anal.
Calcd for C32H37ClN4O7 Sꢁ½H2O: C, 57.69; H, 5.75; N, 8.41. Found:
C, 57.67; H, 5.50; N, 8.47.
Table 3
Cytotoxicity of 18 in a panel of human tumor cell lines.
b
Cell linea
IC50
(lM)
Oxic
Hypoxic
HCRc
A549
A549-P450R
H460
H1299
C33A
SiHa
HCT116
HT29
A375
PC3
SKOV3
1.08 0.48
0.93 0.39
0.25 0.05
0.51 0.04
0.43 0.14
3.00 2.15
0.53 0.08
9.8 1.4
0.40 0.18
0.095 0.008
0.10 0.07
0.16 0.03
0.033 0.005
0.45 0.42
0.05 0.02
0.36 0.09
0.41 0.22
0.21 0.04
0.082 0.02
3.73 0.36
16.5 3.2
7.19 4.6
3.60 0.93
15.6 0.26
30.1 16.8
12.1 3.4
37.1 6.9
4.12 2.08
10.2 3.9
246 57
4.1.6. (5-Amino-3-(5,6,7-trimethoxy-1H-indole-2-carbonyl)-2,3-
dihydro-1H-benzo[e]indol-1-yl)methyl cyclohexanesulfonate
(16a)
1.23 0.07
1.9 0.47
12 1.5
A solution of 16 (95 mg, 0.15 mmol) in THF (20 mL) was hydro-
genated over PtO2 (25 mg) at 45 psi for 30 min. The catalyst was
removed by filtration and the solution was concentrated under re-
duced pressure and diluted with i-Pr2O to give 16a as a yellow so-
lid (79 mg, 87%): mp (CH2Cl2/i-Pr2O) 214–217 °C; 1H NMR
[(CD3)2SO] d 11.38 (s, 1H), 8.08 (d, J = 8.5 Hz, 1H), 7.77 (d,
J = 8.1 Hz, 1H), 7.66 (br s, 1H), 7.46 (t, J = 7.7 Hz, 1H), 7.28 (t,
J = 7.7 Hz, 1H), 7.03 (d, J = 2.1 Hz, 1H), 6.95 (s, 1H), 5.97 (s, 2H),
4.67 (dd, J = 10.8, 9.1 Hz, 1H), 4.47–4.36 (m, 2H), 4.25 (dd,
J = 10.2, 7.1 Hz, 1H), 4.12–4.03 (m, 1H), 3.94 (s, 3H), 3.82 (s, 3H),
3.80 (s, 3H), 3.05 (dd, J = 11.8, 3.3 Hz, 1H) 1.88–1.79 (m, 1H),
1.71–1.40 (m, 4H), 1.28–0.90 (m, 5H). Anal. Calcd for
a
A549: nonsmall-cell lung carcinoma; A549-P450R: stable transfectant of A549
overexpressing human NADPH/cytochrome P450 oxidoreductase (ninefold higher
rate of cytochrome c reduction than parent cell line; H460: large-cell lung carci-
noma; H1299: lung carcinoma; C33A, SiHa: cervical carcinoma; HCT116: colorectal
carcinoma; HT29: colon carcinoma; A375: melanoma; PC3: prostate carcinoma;
SKOV3: ovarian carcinoma.
b
Drug concentration to reduce cell density to 50% of that of controls following
4 h exposure (mean SEM for two to seven experiments).
c
Hypoxic cytotoxicity ratio = IC50(oxic)/IC50(hypoxic). Values are means of
intraexperiment ratios ( SEM for two to six experiments).
C
31H35N3O7Sꢁ½H2O: C, 61.77; H, 6.02; N, 6.97. Found: C, 61.75;
192 °C; 1H NMR [(CD3)2SO] d 11.59 (s, 1H), 9.13 (s, 1H), 8.35 (d,
J = 8.7 Hz, 1H), 8.24 (d, J = 8.2 Hz, 1H), 7.81–7.70 (m, 2H), 7.16 (d,
J = 2.1 Hz, 1H), 6.97 (s, 1H), 4.90 (t, J = 10.1 Hz, 1H), 4.65 (dd,
J = 12.4, 1.7 Hz, 1H), 4.61–4.46 (m, 3H), 3.94 (s, 3H), 3.83 (s, 3H),
3.81 (s, 3H), 3.06–2.94 (m, 1H), 1.97 (d, J = 11.5 Hz, 1H), 1.61–
1.34 (m, 4H), 1.20–0.79 (m, 5H). Anal. Calcd for C31H33N3O9S: C,
59.70; H, 5.33; N, 6.74. Found: C, 59.67; H, 5.48; N, 6.70.
H, 6.04, N, 6.83.
4.1.7. (5-Nitro-3-(5,6,7-trimethoxy-1H-indole-2-carbonyl)-2,
3-dihydro-1H-benzo[e]indol-1-yl)methyl
phenylmethanesulfonate (18) (Scheme 3)
A solution of 27 (1.00 g, 2.90 mmol) in dioxane (20 mL) was sat-
urated with HCl gas at 10 °C, stirred at room temperature for
30 min, and then evaporated under reduced pressure below
30 °C. The resulting crude amine 30 was dissolved in DMA
(10 mL) and 5,6,7-trimethoxyindole-2-carboxylic acid (0.77 g,
3.06 mmol) and EDCIꢁHCl (1.95 g, 10.2 mmol) were added, and
the mixture was stirred at room temperature for 3 h. Addition of
aqueous KHCO3 precipitated a solid that was dissolved in EtOAc/
CH2Cl2 and the solution was filtered through a silica gel plug,
concentrated to a small volume and diluted with i-Pr2O to give
(1-(hydroxymethyl)-5-nitro-1H-benzo[e]indol-3(2H)-yl)(5,6,7-tri-
methoxy-1H-indol-2-yl)methanone (31) as a yellow solid (0.92 g,
4.1.5. (3-(5-(2-(Dimethylamino)ethoxy)-1H-indole-2-carbonyl)-
5-nitro-2,3-dihydro-1H-benzo[e]indol-1-yl)methyl
cyclohexanesulfonate (17)
Similar deprotection of 28 (200 mg, 0.41 mmol) with HCl/diox-
ane, followed by reaction with 5-(2-(dimethylamino)ethoxy)-1H-
indole-2-carboxylic acid hydrochloride21 (139 mg, 0.49 mmol)
and EDCIꢁHCl (313 mg, 1.63 mmol) in DMA (6 mL) gave a solid that
was recrystallized from CH2CI2/i-Pr2O/petroleum ether (2ꢂ) to give
17 (120 mg, 47%): 1H NMR [(CD3)2SO] d 11.68 (s, 1H), 9.16 (s, 1H),

4858
A. Ashoorzadeh et al. / Bioorg. Med. Chem. 19 (2011) 4851–4860
66%): mp (EtOAc/CH2Cl2/i-Pr2O) 211–212 °C; 1H NMR [(CD3)2SO] d
11.55 (s, 1H), 9.14 (s, 1H), 8.40–8.31 (m, 1H), 8.20–8.10 (m, 1H),
7.77–7.65 (m, 2H), 7.17 (s, 1H), 6.98 (s, 1H), 5.09 (t, J = 5.2 Hz,
1H), 4.78 (t, J = 9.8 Hz, 1H), 4.65 (dd, J = 10.6, 2.0 Hz, 1H), 4.17–
4.09 (m, 1H), 3.94 (s, 3H), 3.86–3.74 (m, 7H), 3.65–3.56 (m, 1H).
Anal. Calcd for C25H23N3O7: C, 62.88; H, 4.85; N, 8.80. Found: C,
62.64; H, 4.99; N, 8.62.
4.1.10. (3-(5-(2-Hydroxyethoxy)-1H-indole-2-carbonyl)-5-nitro-
2,3-dihydro-1H-benzo[e]indol-1-yl)methyl
phenylmethanesulfonate (20)
Et3N (91 lL, 0.65 mmol) and then a-toluenesulfonyl chloride
(115 mg, 0.60 mmol) were added to a stirred solution of 30
(173 mg, 0.50 mmol) in CH2Cl2 (15 mL) at 0 °C. After 15 min fur-
ther Et3N (91 lL, 0.65 mmol) and then a-toluenesulfonyl chloride
A solution of 31 (155 mg, 0.32 mmol) in pyridine (2 mL) was
(115 mg, 0.60 mmol) were added and the mixture was stirred at
0 °C for a further 5 min. Water was added and the CH2Cl2 layer
was separated. The aqueous phase was extracted with CH2Cl2
and the combined organic layers were dried with MgSO4 and con-
centrated under reduced pressure. The residue was purified by col-
umn chromatography eluting with EtOAc/petroleum ether (1:4) to
give tert-butyl 1-((benzylsulfonyloxy)methyl)-5-nitro-1H-ben-
zo[e]indole-3(2H)-carboxylate (33) as a yellow foam (251 mg,
100%). A sample was crystallized from benzene/petroleum ether:
mp 148–151 °C; 1H NMR (CDCl3) d 8.79 (br s, 1H), 8.39 (d,
J = 7.8 Hz, 1H), 7.68 (d, J = 7.8 Hz, 1H), 7.62–7.53 (m, 2H), 7.38–
7.30 (m, 5H), 4.36 (s, 2H), 4.35–4.30 (m, 1H), 4.21–4.16 (m, 1H),
4.09–3.98 (m, 2H), 3.95–3.89 (m, 1H), 1.61 (s, 9H); 13C NMR
(CDCl3) (one C not observed) d 152.0, 148.1, 140.3, 130.5, 130.4,
129.3, 129.0, 128.5, 127.4, 127.2, 124.1, 122.8, 121.8, 113.3, 82.4,
70.2, 57.2, 51.6, 39.6, 28.4. Anal. Calcd for C25H26N2O7S: C, 60.23;
H, 5.26; N, 5.62. Found: C, 60.33; H, 5.28; N, 5.57.
treated with
a-toluenesulfonyl chloride (74 mg, 0.39 mmol) at
0 °C, and then stirred at this temperature for a further 15 min.
The mixture was diluted with water and the resulting solid was
purified by column chromatography eluting with CH2Cl2/EtOAc
(9:1) to give 18 as a yellow solid (171 mg, 83%): mp (EtOAc/CH2Cl2)
185 °C; 1H NMR [(CD3)2SO] d 11.61 (s, 1H), 9.12 (s, 1H), 8.35 (dd,
J = 7.9, 1.7 Hz, 1H), 8.15 (dd, J = 7.3, 1.5 Hz, 1H), 7.81–7.69 (m,
2H), 7.22–7.08 (m, 6H), 6.98 (s, 1H), 4.90–4.80 (m, 1H), 4.67–4.43
(m, 6H), 3.94 (s, 3H), 3.83 (s, 3H), 3.81 (s, 3H). Anal. Calcd for
C
32H29N3O9S: C, 60.84; H, 4.63; N, 6.65. Found: C, 60.85; H, 4.76;
N, 6.64.
4.1.8. (3-(5-(2-(Dimethylamino)ethoxy)-1H-indole-2-carbonyl)-
5-nitro-2,3-dihydro-1H-benzo[e]indol-1yl)methyl
phenylmethanesulfonate (19)
Similar reaction of 30 (250 mg, 0.73 mmol) in dry DMA (5 mL)
with 5-(2-(dimethylamino)ethoxy)-1H-indole-2-carboxylic acid
hydrochloride (227 mg, 0.80 mmol) and EDCIꢁHCl (418 mg,
2.18 mmol), and crystallization of the product from EtOAc (2ꢂ)
gave (5-(2-(dimethylamino)ethoxy)-1H-indol-2-yl)(1-(hydroxy-
methyl)-5-nitro-1H-benzo[e]indol-3(2H)-yl)methanone (32) as a
yellow solid (202 mg, 59%): mp 135–137 °C; 1H NMR [(CD3)2SO]
d 11.65 (s, 1H), 9.17 (s, 1H), 8.39–8.31 (m, 1H), 8.20–8.12 (m,
1H), 7.77–7.67 (m, 2H), 7.41 (d, J = 8.9 Hz, 1H), 7.18 (s, 2H),
6.94 (dd, J = 8.9, 2.4 Hz, 1H), 5.13–5.03 (m, 1H), 4.81 (t,
J = 9.8 Hz, 1H), 4.72 (dd, J = 10.5, 2.3 Hz, 1H), 4.21–4.12 (m,
1H), 4.07 (t, J = 5.9 Hz, 2H), 3.85–3.76 (m, 1H), 3.69–3.58 (m,
1H), 2.65 (t, J = 5.8 Hz, 2H), 2.24 (s, 6H). Anal. Calcd for
Trifluoroacetic acid (1.0 mL, 13 mmol) was added to a solution
of 33 (122 mg, 0.24 mmol) and anisole (40 lL, 0.36 mmol) in
CH2Cl2 (10 mL). The mixture was stirred at room temperature for
2.5 h and then evaporated to dryness. The residue was suspended
in water and extracted with CH2Cl2, and the extract was dried with
MgSO4 and concentrated under reduced pressure. The residue was
purified by column chromatography eluting with EtOAc/petroleum
ether (1:4 then 2:3) to give (5-nitro-2,3-dihydro-1H-benzo[e]in-
dol-1-yl)methyl phenylmethanesulfonate (34) as a red-orange oil
(89 mg, 91%) that was used directly. 1H NMR (CDCl3) d 8.35 (d,
J = 8.7 Hz, 1H), 7.63 (s, 1H), 7.62–7.59 (m, 1H), 7.54–7.50 (m, 1H),
7.45–7.40 (m, 1H), 7.38–7.32 (m, 5H), 4.39–4.31 (m, 2H), 4.26–
4.20 (m, 1H), 4.07–3.97 (m, 3H), 3.81–3.71 (m, 2H).
C26H26N4O5: C, 65.81; H, 5.52; N, 11.81. Found: C, 65.43; H,
Amine 34 (89 mg, 0.22 mmol) was dissolved in dry DMA (3 mL)
and 5-(2-hydroxyethoxy)-1H-indole-2-carboxylic acid20 (64 mg,
0.29 mmol), anhydrous toluenesulfonic acid (38 mg, 0.22 mmol),
and EDCIꢁHCl (0.17 g, 0.88 mmol) were added. The mixture was stir-
red at room temperature for 1 h, then cooled to 0 °C and diluted with
cold dilute aqueous NaHCO3. The mixture was extracted with EtOAc
(2ꢂ) and the extracts were washed with cold dilute aqueous NaH-
CO3 and then dried with MgSO4 and concentrated under reduced
pressure. The residue was recrystallized from CH2Cl2/MeOH to give
20 as a yellow solid (119 mg, 81%): mp 211–213 °C; 1H NMR
[(CD3)2SO] d 11.70 (s, 1H), 9.17 (s, 1H), 8.37–8.33 (m, 1H), 8.17–
8.14 (m, 1H), 7.81–7.72 (m, 2H), 7.44 (d, J = 8.9 Hz, 1H), 7.22–7.11
(m, 7H), 6.97 (dd, J = 8.9, 2.4 Hz, 1H), 4.94–4.84 (m, 2H), 4.67–4.50
(m, 6H), 4.02 (t, J = 5.1 Hz, 2H), 3.76 (q, J = 5.1 Hz, 2H). HRMS (FAB)
calcd for C31H28N3O8S (MH+) m/z 602.15971, found: 602.15998.
HPLC analysis showed this material to have a purity of 98.8%.
5.82; N, 11.67.
A solution of 32 (96 mg, 0.20 mmol) in pyridine (2 mL) at 0 °C
was treated portionwise with
a-toluenesulfonyl chloride
(115 mg, 0.60 mmol), and the mixture was stirred at 0 °C for 1 h
and at room temperature for a further 6 h, then basified with aque-
ous NH3. The resulting solid was recrystallized from EtOAc/i-Pr2O
to give 19 (64 mg, 50%): mp 136–139 °C; 1H NMR [(CD3)2SO] d
11.69 (s, 1H), 9.15 (s, 1H), 8.35 (dd, J = 7.6, 2.0 Hz, 1H), 8.15 (dd,
J = 7.2, 1.9 Hz, 1H), 7.81–7.70 (m, 2H), 7.43 (d, J = 8.9 Hz, 1H),
7.22–7.09 (m, 7H), 6.95 (dd, J = 8.9, 2.4 Hz, 1H), 4.94–4.84 (m,
1H), 4.67–4.47 (m, 6H), 4.07 (t, J = 5.9 Hz, 2H), 2.66 (t, J = 5.8 Hz,
2H), 2.24 (s, 6H). Anal. Calcd for C33H32N4O7S: C, 63.04; H, 5.13;
N, 8.91. Found: C, 62.67; H, 5.02; N, 8.71.
4.1.9. (5-Amino-3-(5,6,7-trimethoxy-1H-indole-2-carbonyl)-2,
3-dihydro-1H-benzo[e]indol-1-yl)methyl phenylmethane-
sulfonate (18a)
4.1.11. (E)-(3-(3-(3-Hydroxy-4-methoxyphenyl)acryloyl)-5-
nitro-2,3-dihydro-1H-benzo[e]indol-1-yl)methyl
phenylmethanesulfonate (21)
Crude amine 34 (76 mg, 0.20 mmol) was obtained as above
from 31 (107 mg, 0.21 mmol), TFA (1.0 mL, 13 mmol), and anisole
A solution of 18 (55 mg, 0.09 mmol) in THF (10 mL) was hydro-
genated over PtO2 at 45 psi for 45 min. The mixture was filtered
through a Celite pad, washed with CH2Cl2, and the filtrate was con-
centrated under reduced pressure and diluted with petroleum
ether to give 18a (42 mg, 80%): mp 215–220 °C; 1H NMR
[(CD3)2SO] d 11.42 (s, 1H), 8.08 (d, J = 8.9 Hz, 1H), 7.73–7.61 (m,
2H), 7.48 (t, J = 7.4 Hz, 1H), 7.34–7.15 (m, 6H), 7.00 (d, J = 2.0 Hz,
1H), 6.96 (s, 1H), 4.70–4.58 (m, 3H), 4.44–4.31 (m, 2H), 4.17–
3.99 (m, 2H), 3.94 (s, 3H), 3.83 (s, 3H), 3.81 (s, 3H). Anal. Calcd
for C32H31N3O7SꢁH2O: C, 62.02; H, 5.37; N, 6.78. Found: C, 62.15;
H, 5.64; N, 6.80.
(35 lL, 0.31 mmol) in CH2Cl2 (8 mL). The amine was then dissolved
in dry DMA (2 mL) and (E)-3-(3-hydroxy-4-methoxyphenyl)acrylic
acid (49 mg, 0.25 mmol), anhydrous toluenesulfonic acid (37 mg,
0.22 mmol), and EDCIꢁHCl (150 mg, 0.78 mmol) were added. The
mixture was stirred at room temperature for 2 h, then cooled to
0 °C and diluted with cold dilute aqueous KHCO3. The precipitated
solid was filtered off and washed with dilute aqueous KHCO3 and

A. Ashoorzadeh et al. / Bioorg. Med. Chem. 19 (2011) 4851–4860
4859
water. The solid was dissolved in THF and the solution was dried,
concentrated to a small volume and then diluted with MeOH.
The precipitated solid was filtered off and dried to give 21
(mono-THF solvate) as a yellow solid (79 mg, 57%): mp 190–
192 °C; 1H NMR [(CD3)2SO] d 9.21 (s, 1H), 9.14 (s, 1H), 8.33 (d,
J = 8.0 Hz, 1H), 8.11 (d, J = 7.7 Hz, 1H), 7.79–7.70 (m, 2H), 7.64 (d,
J = 15.3 Hz, 1H), 7.29–7.20 (m, 7H), 7.01 (d, J = 8.4 Hz, 1H), 6.93
(d, J = 15.3 Hz, 1H), 4.66 (s, 2H), 4.62–4.56 (m, 1H), 4.54–4.46 (m,
4H), 3.84 (s, 3H), 3.63–3.58 (m, 4H, THF), 1.79–1.74 (m, 4H, THF).
Anal. Calcd for C30H26N2O8SꢁTHF: C, 63.15; H, 5.30; N, 4.33. Found:
C, 62.90; H, 5.08; N, 4.40.
collected by filtration, washed with cold water and i-Pr2O and then
dried. The residue was recrystallized from CHCl3/i-Pr2O to give 22
as a yellow solid (16 mg, 28%): mp (CHCl3/i-Pr2O) 212–216 °C; 1H
NMR [(CD3)2SO] d 11.71 (s, 1H), 9.07 (s, 1H), 8.28 (d, J = 8.5 Hz,
1H), 7.99 (d, J = 8.1 Hz, 1H), 7.71–7.61 (m, 2H), 7.45 (d, J = 8.9 Hz,
1H), 7.29–7.27 (m, 2H), 7.16 (d, J = 2.3 Hz, 1H), 7.09 (d, J = 1.7 Hz,
1H), 6.99–6.94 (m, 3H), 4.85–4.80 (m, 1H), 4.52–4.49 (m, 1H),
4.37–4.47 (m, 3H), 3.81 (s, 3H), 2.16 (s, 3H). HRMS (FAB) calcd
for C30H26N3O7S (MH+) m/z 572.1492, found: 572.1489. HPLC anal-
ysis showed this material to have a purity of 100%.
4.1.14. (3-(5-(2-Hydroxyethoxy)-1H-indole-2-carbonyl)-5-nitro-
2,3-dihydro-1H-benzo[e]indol-1yl)methyl 4-
methylbenzenesulfonate (24)
4.1.12. (5-Nitro-3-(5,6,7-trimethoxy-1H-indole-2-carbonyl)-2,3-
dihydro-1H-benzo[e]indol-1-yl)methyl 4-
methylbenzenesulfonate (22) (Scheme 4)
A solution of 28 (120 mg, 0.35 mmol) in CH2Cl2 (10 mL) was
treated with pyridine (340 lL, 4.20 mmol) and tosyl chloride
A solution of 36 (40 mg, 0.10 mmol) in DMA (1 mL) was treated
with anhydrous toluenesulfonic acid (19 mg, 0.11 mmol), 5-(2-
hydroxyethoxy)-1H-indole-2-carboxylic acid (29 mg, 0.13 mmol)
and EDCIꢁHCl (77 mg, 0.40 mmol). The mixture was stirred at room
temperature overnight, then cooled to 0 °C, treated with 5% aque-
ous NaHCO3 (3 mL) and stirred for a further 10 min. The precipitate
was collected by filtration, washed with cold water and dried to
give 24 as a yellow solid (59 mg, 98%): mp (CHCl3/i-Pr2O) 127–
129 °C; 1H NMR [(CD3)2SO] d 11.70 (d, J = 1.4 Hz, 1H), 9.07 (s, 1H),
8.28 (d, J = 8.3 Hz, 1H), 7.99 (d, J = 8.1 Hz, 1H), 7.60–7.71 (m, 2H),
7.45 (d, J = 8.9 Hz, 1H), 7.27–7.29 (m, 2H), 7.16 (d, J = 2.3 Hz, 1H),
7.08 (d, J = 1.7 Hz, 1H), 6.94–7.00 (m, 3H), 4.80–4.86 (m, 1H),
4.50–4.53 (m, 1H), 4.39–4.47 (m, 3H), 4.02 (t, J = 5.1 Hz, 2H), 3.76
(dd, J = 10.3, 5.3 Hz, 2H), 2.16 (s, 3H). Anal. Calcd for C31H27N3O8S:
C, 61.89; H, 4.52; N, 6.98. Found: C, 61.75; H, 4.53; N, 7.05.
(667 mg, 3.50 mmol) at 0 °C. The mixture was stirred at room tem-
perature for 48 h, diluted with CH2Cl2, washed with water and
brine, dried with Na2SO4, and concentrated under reduced pres-
sure. The residue was purified by column chromatography eluting
with petroleum ether/EtOAc (4:1 then 3:2) followed by trituration
of the product with petroleum ether/i-Pr2O to give tert-butyl
1-(tosyloxymethyl)-5-nitro-1H-benzo[e]indole-3(2H)-carboxylate
(35) as a yellow solid (174 mg, 100%): mp 131–132 °C; 1H NMR
[(CD3)2SO] d 8.65 (br s, 1H), 8.24 (d, J = 9.3 Hz, 1H), 7.86 (d,
J = 8.2 Hz, 1H), 7.54–7.63 (m, 2H), 7.32–7.34 (m, 2H), 7.10–7.12
(m, 2H), 4.33–4.41 (m, 2H), 4.14–4.23 (m, 2H), 4.00–4.06 (m,
1H), 2.30 (s, 3H), 1.55 (s, 9H). Anal. Calcd for C25H26N2O7S: C,
60.23; H, 5.26; N, 5.62. Found: C, 60.48; H, 5.27; N, 5.61.
A solution of 35 (340 mg, 0.68 mmol) in dioxane (3 mL) was
saturated with HCl gas at 10 °C, stirred at room temperature for
30 min, and then concentrated under reduced pressure. The
residue was purified by column chromatography eluting with
petroleum ether/EtOAc (3:2) to give (5-nitro-2,3-dihydro-1H-ben-
zo[e]indol-1-yl)methyl-4-methylbenzenesulfonate (36) as a red
solid (241 mg, 89%): mp 121–123 °C; 1H NMR [(CD3)2SO] d 8.06
(d, J = 8.6 Hz, 1H), 7.63 (d, J = 8.3 Hz, 1H), 7.58 (s, 1H), 7.48–7.80
(m, 2H), 7.42–7.47 (m, 1H), 7.32–7.38 (m, 1H), 7.23–7.25 (m,
2H), 6.20 (d, J = 2.4 Hz, 1H), 4.07–4.19 (m, 3H), 3.72 (ddd, J = 13.0,
10.2, 2.9 Hz, 1H), 3.51 (dd, J = 10.2, 2.1 Hz, 1H), 2.34 (s, 3H). Anal.
Calcd for C20H18N2O5S: C, 60.29; H, 4.55; N, 7.03. Found: C,
59.93; H, 4.65; N, 6.71.
4.2. Solubility in culture medium
The solubility of the compounds in
aMEM containing 5% FCS
(equilibrated with 5% CO2/air) at 22 °C was determined as previ-
ously described.3
4.3. In vitro cytotoxicity
Inhibition of proliferation of log-phase monolayers was as-
sessed in 96-well plates as previously described.24 The drug expo-
sure time was 4 h under aerobic (20% O2) or anoxic (<20 ppm O2)
conditions followed by sulforhodamine B staining 5 days later.
The IC50 was determined by interpolation as the drug concentra-
tion required to inhibit cell density to 50% of that of the controls
on the same plate.
A solution of 36 (241 mg, 0.60 mmol) in DMA (2 mL) was trea-
ted with 5,6,7-trimethoxyindole-2-carboxylic acid (188 mg,
0.75 mmol) and EDCIꢁHCl (522 mg, 2.72 mmol), and the mixture
was stirred at room temperature for 2 h. Addition of dilute aqueous
KHCO3 gave a solid that was purified by column chromatography
eluting with CH2Cl2/EtOAc (19:1) firstly to give compound 5
(80 mg, 27%) and then a product that was recrystallized from
CH2Cl2/i-Pr2O (2ꢂ) to provide 22 (268 mg, 71%): mp 212–214 °C;
Acknowledgments
The authors are grateful for the assistance of Sisira Kumara for
HPLC and solubility determinations, Maruta Boyd for NMR spec-
trometry, and Jane Botting and Wouter van Leeuwen for cytotoxic-
ity studies. This work was funded by the Foundation for Research,
Science and Technology (NERF grant), the Auckland Division of the
Cancer Society of New Zealand, and the Health Research Council of
New Zealand.
1H NMR [(CD3)2SO]
d 11.62 (s, 1H), 9.05 (s, 1H), 8.28 (d,
J = 8.6 Hz, 1H), 7.98 (d, J = 8.3 Hz, 1H), 7.72–7.58 (m, 2H), 7.28 (d,
J = 8.3 Hz, 2H), 7.06 (d, J = 1.6 Hz, 1H), 6.99–6.89 (m, 3H), 4.79 (t,
J = 10.1 Hz, 1H), 4.52–4.48 (m, 4H), 3.97 (s, 3H), 3.84 (s, 3H), 3.83
(s, 3H). Anal. Calcd for C32H29N3O9Sꢁ1½H2O: C, 58.25; H, 4.90; N,
6.38. Found: C, 58.34; H, 4.58; N, 6.35.
References and notes
1. Boger, D. L.; Johnson, D. S. Angew. Chem., Int. Ed. 1996, 35, 1438.
2. Yasuzawa, T.; Saitoh, Y.; Ichimura, M.; Takahashi, I.; Sano, H. J. Antibiot. 1991,
44, 445.
3. Tercel, M.; Yang, S.; Atwell, G. J.; Smith, E.; Gu, Y.; Anderson, R. F.; Denny, W. A.;
Wilson, W. R.; Pruijn, F. B. Bioorg. Med. Chem. 2010, 18, 4997.
4. Boger, D. L.; Ishizaki, T.; Kitos, P. A.; Suntornwat, O. J. Org. Chem. 1990, 55, 5823.
5. Wei, J.; Trzupek, J. D.; Rayl, T. J.; Broward, M. A.; Vielhauer, G. A.; Weir, S. J.;
Hwang, I.; Boger, D. L. J. Am. Chem. Soc. 2007, 129, 15391.
4.1.13. (3-(5-Methoxy-1H-indole-2-carbonyl)-2,3-dihydro-1H-
benzo[e]indol-1-yl)methyl 4-methylbenzenesulfonate (23)
A solution of 36 (40 mg, 0.10 mmol) in DMA (1 mL) was treated
with anhydrous toluenesulfonic acid (19 mg, 0.11 mmol), 5-meth-
oxy-1H-indole-2-carboxylic acid (25 mg, 0.13 mmol) and EDCIꢁMeI
(119 mg, 0.40 mmol). The mixture was stirred at room tempera-
ture overnight, then cooled to 0 °C, treated with 5% aqueous NaH-
CO3 (3 mL) and stirred for a further 10 min. The precipitate was
6. Townes, H.; Summerville, K.; Purnell, B.; Hooker, M.; Madsen, E.; Hudson, S.;
Lee, M. Med. Chem. Res. 2002, 11, 248.

4860
A. Ashoorzadeh et al. / Bioorg. Med. Chem. 19 (2011) 4851–4860
7. Tercel, M.; Atwell, G. J.; Yang, S.; Stevenson, R. J.; Botting, K. J.; Boyd, M.; Smith,
E.; Anderson, R. F.; Denny, W. A.; Wilson, W. R.; Pruijn, F. B. J. Med. Chem. 2009,
52, 7258.
8. Wilson, W. R.; Stribbling, S. M.; Pruijn, F. B.; Syddall, S. P.; Patterson, A. V.;
Liyanage, H. D. S.; Smith, E.; Botting, K. J.; Tercel, M. Mol. Cancer Ther. 2009, 8,
2903.
9. Tercel, M.; Atwell, G. J.; Yang, S.; Ashoorzadeh, A.; Stevenson, R. J.; Botting, K. J.;
Gu, Y.; Mehta, S. Y.; Denny, W. A.; Wilson, W. R.; Pruijn, F. B. Angew. Chem., Int.
Ed. 2011, 50, 2606.
10. Atwell, G. J.; Tercel, M.; Boyd, M.; Wilson, W. R.; Denny, W. A. J. Org. Chem.
1998, 63, 9414.
15. Atwell, G. J.; Milbank, J. B. J.; Wilson, W. R.; Hogg, A.; Denny, W. A. J. Med. Chem.
1999, 42, 3400.
16. Panthananickal, A.; Hansch, C.; Leo, A. J. Med. Chem. 1978, 21, 16.
17. Jaramillo, P.; Domingo, L. R.; Perez, P. Chem. Phys. Lett. 2006, 420, 95.
18. Pacheco, D. Y.; Stratton, N. K.; Gibson, N. W. Cancer Res. 1989, 49, 5108.
19. Tietze, L. F.; Birgit Krewer, B. Anti-Cancer Agents Med. Chem. 2009, 9, 304.
20. Denny, W. A.; Wilson, W. R.; Stevenson, R. J.; Tercel, M.; Atwell, G. J.; Yang, S.;
Patterson, A. V.; Pruijn, F. B. PCT Int. Appl. WO 2006043839A1, 2006.
21. Milbank, J. B. J.; Tercel, M.; Atwell, G. J.; Wilson, W. R.; Hogg, A.; Denny, W. A. J.
Med. Chem. 1999, 42, 649.
22. Ahn, G.-O.; Botting, K. J.; Patterson, A. V.; Ware, D. C.; Tercel, M.; Wilson, W. R.
Biochem. Pharmacol. 2006, 71, 1683.
23. Su, J.; Wang, J.; Wilson, W. A. Personal communication.
11. Tercel, M.; Gieseg, M. A.; Milbank, J. B.; Boyd, M.; Fan, J. Y.; Tan, L. K.; Wilson,
W. R.; Denny, W. A. Chem. Res. Toxicol. 1999, 12, 700.
12. Atwell, G. J.; Wilson, W. R.; Denny, W. A. Bioorg. Med. Chem. Lett. 1997, 7, 1493.
13. Wilson, W. R.; Hay, M. P. Nat. Rev. Cancer 2011, 11, 393.
14. Chen, Y.; Hu, L. Med. Res. Rev. 2009, 29, 29.
24. Hay, M. P.; Gamage, S. A.; Kovacs, M. S.; Pruijn, F. B.; Anderson, R. F.; Patterson,
A. V.; Wilson, W. R.; Brown, J. M.; Denny, W. A. J. Med. Chem. 2003, 46, 169.