The effect of a bromide leaving group on the properties of nitro analogs of the duocarmycins as hypoxia-activated prodrugs and phosphate pre-prodrugs for antitumor therapy

Article abstract of DOI:10.1016/j.bmc.2011.08.045

Nitro seco analogs (nitroCBIs) of the antitumor antibiotic duocarmycins are a new class of hypoxia activated prodrugs. These compounds undergo hypoxia-selective metabolism to form potent DNA alkylating agents. A series of four nitroCBI alcohol prodrugs co

Full text of DOI:10.1016/j.bmc.2011.08.045

Bioorganic & Medicinal Chemistry 19 (2011) 5989–5998
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Bioorganic & Medicinal Chemistry
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The effect of a bromide leaving group on the properties of nitro analogs
of the duocarmycins as hypoxia-activated prodrugs and phosphate
pre-prodrugs for antitumor therapy
⇑
Ralph J. Stevenson , William A. Denny, Amir Ashoorzadeh, Frederik B. Pruijn, Wouter F. van Leeuwen,
Moana Tercel
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:
Nitro seco analogs (nitroCBIs) of the antitumor antibiotic duocarmycins are a new class of hypoxia
activated prodrugs. These compounds undergo hypoxia-selective metabolism to form potent DNA alkyl-
ating agents. A series of four nitroCBI alcohol prodrugs containing a bromide rather than chloride or sul-
fonate leaving group was synthesized. In assays for in vitro hypoxia-selective cytotoxicity against human
tumor cell lines the two bromides with DNA minor groove binding basic side chains displayed hypoxic
cytotoxicity ratios (HCRs) of 52–286 in HT29 cells and 41–43 in SiHa cells. These values compare well
with a related previously reported chloride analog. The corresponding more water soluble phosphate
pre-prodrugs of the bromides were synthesized and evaluated for in vivo antitumor activity against SiHa
human tumor xenografts. All four phosphates, with both neutral and basic side chains, demonstrated
activity providing statistically significant hypoxic log10 cell kills of 0.87–2.80 at non-toxic doses, match-
ing or proving superior to those of their chloride analogs.
Received 21 July 2011
Revised 17 August 2011
Accepted 20 August 2011
Available online 25 August 2011
Keywords:
Duocarmycins
Hypoxic selectivity
Phosphate pre-prodrug
Prodrug
Bromide leaving group
DNA minor groove alkylating agent
Antitumor
Ó 2011 Elsevier Ltd. All rights reserved.
Basic side chain
1. Introduction
compounds.13,14
A number of prodrug strategies for tumor-
selective liberation of the cytotoxin have been attempted to address
this issue. Tietze has used a glycosidic prodrug strategy (e.g., 5,
Fig. 2).15,16 Boger17 and Lee18 have described quinone-containing
prodrugs (e.g., 6, Fig. 2). More recently Boger has synthesized N-acyl
O-amino phenol derivatives of CBIs as prodrugs (e.g., 7, Fig. 2).19 Our
laboratory has prepared Co(III) complexes of an aza-hydroxyCBI as
prodrugs (e.g., 8, Fig. 2).20,21 Conjugation to tumor-specific antibod-
ies has also been pursued.22 A recent review10 outlines the chal-
lenges, opportunities and strategies (molecular target inhibitors
or bioreductive prodrugs) in targeting tumor hypoxia.
Some time ago we established that aminoCBIs are very potent
DNA minor groove alkylating agents.23,24 We also showed that
nitroCBIs containing 5,6,7-trimethoxyindole (TMI) or 5-[(dimeth-
ylamino)ethoxy]indole (DEI) DNA minor groove binding side
chains (for structures see Table 1) were up to 300-fold more cyto-
toxic under hypoxic than oxic conditions.1 Unfortunately the effect
was cell line-selective and hypoxic reduction rates were low. In an
attempt to overcome this, we introduced electron-withdrawing
groups (EWGs) into the A-ring (Fig. 1) of a series of nitroCBIs,
thereby raising the one-electron reduction potentials.2 We found
that a primary 7-sulfonamide (e.g., 9, Fig. 2) or 7-carboxamide
(e.g., 10, Fig. 2) A-ring substituent combined with the DEI side
We have recently reported1–6 a class of hypoxia-activated
prodrugs (HAPs), the nitroCBIs (1, Fig. 1), that are selectively cyto-
toxic at low oxygen concentrations. Hypoxia often correlates with
resistance to cancer treatment and negative prognosis;7 thus the
HAP concept may offer a useful strategy to tumor therapy.8–10
The bioreduced ‘effectors’ (aminoCBIs, 2, Fig. 1) of these compounds
are analogs of a small family of antitumor antibiotics including the
duocarmycins (e.g., (+)-duocarmycin SA,11 4, Fig. 2). These natural
products and a number of synthetic analogs monoalkylate in a
sequence-selective manner at N3 of adenine in the minor groove
of DNA and are extremely cytotoxic.12 Myelotoxicity has so far
precluded successful completion of clinical trials of such
Abbreviations: CBI, seco-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one;
DEI, 5-[(dimethylamino)ethoxy]indole; DIPEA, diisopropylethylamine; DMA,
dimethylacetamide; EDCIꢀHBr, N-ethyl-N0-(3-dimethylaminopropyl)carbodiimide
hydrobromide; EWG, electron-withdrawing group; HAP, hypoxia-activated pro-
drug; HCR, hypoxic cytotoxicity ratio; LCK, log10 cell kill; MEI, 5-[(morpholino)eth-
oxy]indole; MS, 4-methoxystyrene; MES, 4-[(morpholino)ethoxy]styrene; TMI,
5,6,7-trimethoxyindole.
⇑
Corresponding author. Tel.: +64 9 3737599; fax: +64 9 3737502.
E-mail address: r.stevenson@auckland.ac.nz (R.J. Stevenson).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.045

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R. J. Stevenson et al. / Bioorg. Med. Chem. 19 (2011) 5989–5998
Y
Y
NCOR
NCOR
NCOR
bioreduction
spirocyclization
A
-HY
7
NH2
NO2
1: nitroCBI
NH
3: adenine N3 alkylator
2: aminoCBI
Figure 1. Suggested mechanism of action of nitroCBI hypoxia-activated prodrugs. R is a DNA minor groove binding side chain. Y is a leaving group (e.g., Cl, Br, sulfonate).
chain provided compounds with high hypoxic cytotoxicity ratios
(HCRs) in SKOV3 (275 and 77 for the two compounds respectively)
and in HT29 (330 and 60) human tumor cell lines. Furthermore,
HCRs of 19–330 were observed for the 7-sulfonamide 9 across an
11-cell line panel. Significant activity against hypoxic cells in a
SiHa human tumor xenograft was observed when 9 was combined
with radiation in vivo.2 In an in vitro comparison with known HAPs
competitive, demonstrating similar hypoxic selectivity and much
greater cytotoxicity (300- to 500-fold) under hypoxic conditions.2
We also found that hypoxic selectivity is reduced by more than
25-fold when the side chain is changed from basic DEI to neutral
TMI.2
In an attempt to improve aqueous solubility, we attached a
tertiary amine to the 7-sulfonamide moiety (11, Fig. 2),3 but this
provided only a moderate increase in solubility.
tirapazamine25 and PR-104A,26 the 7-sulfonamide
9
proved
OMe
OMe
NMe2
O
Cl
N
N
Me
OMe
H
MeO2C
O
N
H
N
N
H
O
O
4
: (+)-duocarmycin SA
5
Br
OMe
O
HO
HO
O
O
OMe
OMe
OH
N
N
MeO2C
Me
HO
Cl
OMe
H
O
N
H
OMe
OMe
OH
N
N
H
4a
Cl
: duocarmycin B2
O
OMe
7
O
OMe
OMe
NHBoc
N
N
H
O
NMe2
O
6
O
Me
Me
Cl
O
Me
O
O
Me
2+
OMe
OMe
OMe
N
N
H
Cl
O
R
NO2
N
N
H
9: R=SO2NH2
10: R=CONH2
O
N
H
11: R=SO2NH(CH2)2NMe2
O
8
N
12: R=SO2NH(CH2)2OP(O)(OH)2
13: R=CONH(CH2)2OP(O)(OH)2
14: R=SO2NH(CH2)2OH
Co
H
N
N
H
Cl
OMe
N
H
15: R=CONH(CH2)2OH
OMe
OMe
N
N
H
O
R
16: R=SO2NH(CH2)2OH
17: R=SO2NH(CH2)2OP(O)(OH)2
NO2
Figure 2. (+)-Duocarmycin SA, duocarmycin B2 and some previously reported prodrug and pre-prodrug analogs of the duocarmycins.

R. J. Stevenson et al. / Bioorg. Med. Chem. 19 (2011) 5989–5998
5991
Table 1
Cytotoxicity data for nitroCBI alcohols 18–21 (bromides) compared with known 14 and 16 (chlorides)
OMe
O
OMe
Y
NMe2
N
H
N
H
OMe
O
R
N
OMe
TMI
MS
O
DEI
R=
H
N
O
O
HO
S
N
N
O
O
NO2
O
N
H
MES
MEI
a
Compd
R
Y
IC50 (lM)
HT29
Hypoxic
SiHa
Oxic
HCRb
Oxic
Hypoxic
HCRb
18
19
20
TMI
MS
MEI
MES
TMI
DEI
Br
Br
Br
Br
Cl
Cl
12.3 2.9
2.45 0.1
22.4 2.8
11.2 1.2
1.35 0.13
<0.1c
0.44 0.14
0.08 0.042
10
>25
52 16
286 124
2.1 0.1
160 40
2
2.61 0.36
1.44 0.11
11.7 1.3
2.72 0.24
0.27 0.05
0.12 0.03
0.28 0.03
0.09 0.014
3.5 2.5
10 1.6
17 4.8
43
41 11
16
6
21
16d
14d
28
2
11
2
13
1
4
9.3
1
0.09 0.02
2.0 0.2
0.07 0.01
46 10
a
b
c
Drug concentration to reduce cell density to 50% of that of controls, 5 days after 4 h exposure (mean SEM for 3–6 experiments).
Hypoxic cytotoxicity ratio = IC50(oxic)/IC50(hypoxic). Values are means of intraexperiment ratios ( SEM for 2–6 experiments).
Single determination.
d
Synthesis and cytotoxicity data (2–14 experiments) of nitroCBIs with chloride leaving groups (Fig. 2) previously reported.4
Table 2
Antitumor activity for nitroCBI phosphates 22–25 (bromides) compared with known 12 and 17 (chlorides) in SiHa human tumor xenografts
Y
R
N
H
N
O
O
(HO)2PO
S
O
O
NO2
Compd
Ra
Y
Controlb
NitroCBIc
pd
Radiatione
Radiation + nitroCBIf
Hypoxic LCKg
ph
22
23
24
25
17j
12j
TMI
MS
MEI
MES
TMI
DEI
Br
Br
Br
Br
Cl
Cl
7.15 0.18
7.15 0.20
7.37 0.01
7.46 0.10
7.62 0.10
7.52 0.12
6.51 0.16
7.07 0.03
6.05 0.08
7.01 0.13
7.29 0.31
6.89 0.26
NS
NS
<0.001
0.038
NS
5.46 0.04
5.37 0.04
5.40 0.05
5.34 0.12
5.50 0.15
5.73 0.05
3.62 0.26
4.50 0.23
2.60 0.21
3.78 0.06
4.96 0.14
3.90 0.18
1.84 0.21
0.87 0.18
2.80 0.17i
1.56 0.10
0.55 0.17
1.83 0.15
<0.001
0.005
<0.001
<0.001
NS
NS
<0.001
NS: not significant.
a
See Table 1.
b
Values are mean of log10 clonogens/g tumor SEM for a control group of 3 mice assayed by excision and clonogenic assay 18 h after iv dosing.
Values are mean of log10 clonogens/g tumor SEM for groups of 3 mice treated with nitroCBI alone. Dose was 42 lmol/kg accept for 25 (30 lmol/kg).
p-values for nitroCBI alone vs control. Calculated using intraexperiment controls.
c
d
e
f
Values are mean of log10 clonogens/g tumor SEM for groups of 5 mice for
Values are mean of log10 clonogens/g tumor SEM for groups of 5 mice for
(30 mol/kg).
Hypoxic log10 cell kill: radiation + nitroCBI (vs radiation alone).
c
irradiation (15 Gy).
c
irradiation (15 Gy) plus nitroCBI. Dose was 42 lmol/kg except for compound 25
l
g
h
i
p-values for radiation + nitroCBI versus radiation alone. Calculated using intraexperiment controls
No clonogens detected in 1 of 5 treated tumors.
Synthesis and in vivo data for chlorides 12 (included highest LCK of four reported experiments: LCK 1.36–1.82) and 17 previously reported.4
j
A
modified series of 7-sulfonamide and 7-carboxamide
provided low HCRs (2.1–16) in vitro and that was matched by
phosphate 17 having less activity (LCK 0.55) than phosphate 12
(1.36–1.82) in vivo.4
nitroCBIs were prepared by attaching a phosphate-containing moi-
ety to the sulfonamide (e.g., 12, Fig. 2) or carboxamide (e.g., 13,
Fig. 2).4 It has been shown that phosphate 12 rapidly hydrolyzes
to alcohol 14 in plasma,4 and thus the corresponding alcohols
(i.e., 14 and 15, Fig. 2) were prepared. As expected, the phosphates
of these had much greater aqueous solubility (>2 mM) than the
alcohols.4 Furthermore, the phosphate 12 was highly selective for
and active against hypoxic cells in SiHa human tumor xenografts
in vivo (Table 2).4 The corresponding alcohol 14 exhibited HCRs
of 9.4–250 in a panel of 14 human tumor cell lines (examples in
Table 1).4 Alcohol 16 containing the neutral TMI side chain
We have recently reported the effect of sulfonate leaving groups
on the hypoxia-selective toxicity of A-ring unsubstituted nitroCBIs
and observed that unlike their chloride counterparts, compounds
with neutral DNA minor groove binding side chains (e.g., TMI) pro-
vided the greatest hypoxic selectivity.5 Duocarmycin B2 (4a, Fig. 2),
a seco hydroxyCBI containing a bromide leaving group, is among
the antitumor antibiotics isolated from the culture broth of Stepto-
myces sp.27 Nagamura and co-workers have reported the syntheses
and antitumor activity of duocarmycin derivatives and prodrugs

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R. J. Stevenson et al. / Bioorg. Med. Chem. 19 (2011) 5989–5998
containing a bromide leaving group.28–35 We now report the syn-
theses of nitroCBIs containing a bromide leaving group36,37 and
the effects of the bromide leaving group in combination with
A-ring one-electron reduction potential-modifying substituents
and DNA minor groove binding side chains on cytotoxicity and
antitumor activity.
tography prior to the coupling reaction. The known 5-[(morpho-
lino)ethoxy]indole38 (MEI) and 4-[(morpholino)ethoxy]styrene37
(MES) side chains were introduced in this way. As previously re-
ported2 the EDCI-mediated reactions were most successful under
acidic conditions (catalytic anhydrous TsOH). Thus, EDCIꢀHBr-
mediated couplings of aniline 32 were carried out with four
carboxylic acids to introduce two neutral DNA minor groove
binding side chains, 5,6,7-trimethoxyindole (TMI) (18, 87%) and
4-methoxystyrene (MS) (19, 67%), and two weakly basic minor
groove binding side chains, MEI (20, 57%) and MES (21, 57%) shown
in Table 1. Although there was no advantage, 19 was also alterna-
tively prepared from the TBDMS-protected alcohol 33 (Scheme 1).
Reduction of bromide-containing nitroCBIs to aminoCBIs was
not attempted due to the presumed instability via facile ring-clo-
sure to an imino version (3, Fig. 1) of a spirocyclopropylcyclohexa-
dienone. We have previously reported on the difficulty of obtaining
aminoCBIs with sulfonate leaving groups.5
The corresponding phosphates (Scheme 2) were prepared via
tert-butyl phosphate esters, as previously,4 in a synthetic strategy
designed to minimize handling of the very polar phosphates them-
selves. The intermediate sulfonamide phosphate ester 35 was
formed (81%) by reaction of sulfonyl chloride 31 with 2-amin-
oethyldi-tert-butyl phosphate and deprotection of the trifluoroace-
tamide-protected aniline with aqueous Cs2CO3. EDCIꢀHBr-
mediated couplings using 35 were carried out with the same four
carboxylic acids as above, introducing the TMI (36a, 55%), MS (36b,
38%), MEI (36c, 38%) and MES (37d, 50%) minor groove binding
side chains. The final step required phosphate ester cleavage with
TFA, to produce compounds 22–25 (Scheme 2, 100%).
2. Results and discussion
2.1. Chemistry
Syntheses of the nitroCBI bromides of Table 1 began by bromide
displacement of the known chloride 26b,2 or the mesylates 26a5 or
295 (Scheme 1). Starting with 26a rather than 26b proved a better
option, as chloride starting material was difficult to remove from
the product bromide. However, chlorosulfonylation of bromide
27 was more successful than converting the mesylate 29 into bro-
mide 28. During the formation of 7-sulfonyl chloride 28 from bro-
mide 27, a considerable amount of the 7-sulfonic acid formed as a
precipitate which could be converted to 28 by dissolving the acid
in DMF and treating the solution with oxalyl chloride, ultimately
giving a 93% yield of 28 from 27. Nitration of 28 (KNO3/H2SO4) gave
a mixture of the 9-nitro and 5-nitro regioisomers (30 and 31,
respectively, determined by 1H NMR). The desired major isomer
31 was separated by either flash chromatography (59%) or precip-
itation from CH2Cl2/i-Pr2O (75%). This intermediate and all of the
following products had to be handled with extra care—rings A
and B both contain EWGs that enhance elimination of bromide
(producing HBr), particularly under basic conditions, and of halide
ion scrambling from any chloride ions present. Thus whenever
reactions were subjected to basic conditions or reagents, or when-
ever basic workup was required, the system was routinely kept
cold to minimize these potential side reactions. The intermediate
sulfonamide alcohol 32 was formed quantitatively by reaction of
31 with ethanolamine, followed by trifluoroacetamide cleavage
with aqueous Cs2CO3. EDCI-mediated couplings of aniline 32 with
carboxylic acids using EDCIꢀHCl produced a considerable amount
(up to 40%) of chloride ion scrambling, which was circumvented
by the use of EDCIꢀHBr (requiring CH2Cl2 as a co-solvent to dissolve
it). In addition, where salts of basic side chains were used, these
were converted from HCl to HBr forms by ion exchange chroma-
2.2. In vitro cytotoxicity
In accordance with our previous protocol,4 hypoxia-selective
cytotoxicity in vitro was assessed with the cell-permeable alcohols
rather than the highly polar phosphates. Thus alcohols 18–21 were
compared by measuring IC50 values under oxic and hypoxic condi-
tions and deriving a hypoxic cytotoxicity ratio [HCR = IC50(oxic)/
IC50(hypoxic)] (Table 1, Fig. 3). Data for previously reported4 alco-
hols containing a chloride leaving group (14 and 16) are presented
for comparison. Compound 14 is suitable for comparison as it pro-
vided the highest HCRs of all sulfonamides reported and was the
Br
Br
Y
NO2
NCOCF3
+
NCOCF3
NCOCF3
d
ClO2S
ClO2S
Z
NO2
26a or 26b: Y=OMs or Cl, Z=H
27: Y=Br, Z=H
28: Y=Br, Z=SO2Cl
29: Y=OMs, Z=SO2Cl
30
a
31
b
c
Br
Br
e
NCOR
NR
f or h
H
N
H
N
HO
S
Z
S
O
O
O
O
NO2
NO2
32: Z=OH, R=H
18: R=TMI of Table 1
19: R=MS of Table 1
20: R=MEI of Table 1
g
f
33: Z=OTBDMS, R=H
34: Z=OTBDMS, R=COMS
(MS of Table 1)
21: R=MES of Table 1
Scheme 1. Synthesis of nitroCBI alcohols 18–21 of Table 1. Reagents and conditions: (a) LiBr, THF or LiBr, 2-butanone, 85 °C; (b) (i) ClSO3H, CH2Cl2, ꢁ78 °C to 5 °C, (ii) oxalyl
chloride, DMF, 0 °C; (c) LiBr, THF; (d) (from 28) KNO3, 98% H2SO4, ꢁ12 °C; (e) (i) ethanolamine, CH2Cl2, 0 °C, (ii) Cs2CO3, MeOH, H2O, 0 °C; (f) (from 32, for 18–21) RCO2H,
EDCIꢀHBr, TsOH, DMA, CH2Cl2; (g) TBDMSCl, DIPEA, DMF, 0 °C; (h) (from 34, for 19) TFA, CH2Cl2.

R. J. Stevenson et al. / Bioorg. Med. Chem. 19 (2011) 5989–5998
5993
Br
Br
b
a
31
NH
NCOR
H
N
H
N
O
O
(t-BuO)2PO
S
(ZO)2PO
S
O
O
O
O
35 NO2
NO2
36a-36d: Z=t-Bu, R=TMI, MS, MEI, MES of Table 1
c
22: Z=H, R=TMI of Table 1
23: Z=H, R=MS of Table 1
24: Z=H, R=MEI of Table 1
25: Z=H, R=MES of Table 1
Scheme 2. Synthesis of nitroCBI phosphates 22–25 of Table 2. Reagents and conditions: (a) (i) H2N(CH2)2OP(O)(Ot-Bu)2, DIPEA, CH2Cl2, ꢁ50 °C, (ii) Cs2CO3, MeOH, H2O,
ꢁ10 °C; (b) RCO2H, EDCIꢀHBr, TsOH, DMA, CH2Cl2; (c) TFA, CH2Cl2.
only compound in this series with a basic side chain. We have pre-
viously demonstrated that nitroCBIs (chlorides) containing a basic
side chain (e.g., DEI in 14) provide significantly greater HCRs than
those with neutral side chains (e.g., TMI in 16).4 We now show that
this is also true for nitroCBIs containing a bromide leaving group.
Bromide 21 containing the MES basic side chain has an HCR of
286 in HT29 human colon carcinoma cells and an HCR of 41 in SiHa
human cervical carcinoma. The ratios were less but still highly sig-
nificant for bromide 20 containing the MEI basic side chain with an
HCR of 52 in HT29 cells and an HCR of 43 in SiHa cells. This com-
pares well with the HCRs of their chloride analog 14 containing the
DEI basic side chain (160 in HT29 cells and 46 in SiHa cells). As
with their chloride analogs, the bromides with neutral side chains
provided lower HCRs. Bromide 18 containing the TMI side chain
has an HCR of 10 in both HT29 cells and SiHa cells while bromide
19 containing the MS side chain has an HCR >25 in HT29 cells and
17 in SiHa cells. These lower ratios follow the trends of the chloride
analog 16 containing the TMI side chain with HCR 2.1 in HT29 and
HCR 16 in SiHa. Although we did not synthesize the corresponding
bromide aminoCBIs, cytotoxicity values under hypoxia for bromide
nitroCBIs are comparable to those of their chloride nitroCBI coun-
terparts (Table 1). From these we infer that bromide aminoCBIs
would have comparable cytotoxicity values to chloride aminoCBIs.
2.3. In vivo activity
We have previously shown that the phosphate moiety of nitro-
CBI 12 is rapidly hydrolyzed in plasma to the corresponding alco-
hol, and that a dose of 42
tumor cell kill.4 Higher doses (>100
icity in some animals and a maximum tolerated dose was difficult
to define.4 Thus, in the present study, an iv dose of 30 or 42
mol/
lmol/kg provides significant hypoxic
lmol/kg) promoted acute tox-
l
kg of phosphates was administered to SiHa tumor-bearing mice
either alone or 5 min after a single 15 Gy dose of ionizing radiation.
100
HT29
SiHa
10
1
0.1
0.01
18 19 20 21 16 14
R = TMI MS MEI MES TMI DEI
18 19 20 21 16 14
R = TMI MS MEI MES TMI DEI
Br
Cl
Br
Cl
nitroCBI Oxic
nitroCBI Hypoxic
Figure 3. Comparative cytotoxicity of bromides 18–21 versus known4 chlorides 14 and 16 under oxic and hypoxic conditions in HT29 and SiHa human tumor cell lines. R
refers to the DNA minor groove binding side chains shown in Table 1.

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R. J. Stevenson et al. / Bioorg. Med. Chem. 19 (2011) 5989–5998
8
7
6
5
4
3
2
Control
nitroCBI
Radiation
Radiation + nitroCBI
1/5
17 12 22 23 24 25
17 12 22 23 24 25
+ Radiation
Figure 4. Antitumor activity of bromides 22–25 versus known4 chlorides 12 and 17 in SiHa human tumor xenografts assayed by tumor excision and clonogenic assay 18 h
after iv dosing with nitroCBI (30 or 42 mol/kg) only or irradiation (15 Gy) plus nitroCBI. Values are mean SEM for groups of 3 mice (control or nitroCBI alone) or 5 mice
l
c
(radiation alone or radiation + nitroCBI). Values for control and radiation alone are averages SEM from six independent experiments. Downward arrow: the number of
tumors for which no surviving clonogens could be detected.
At these doses no acute toxicity was observed for any of the ana-
logs. Eighteen hours later the tumors were excised and surviving
clonogens assessed. An irradiation dose of 15 Gy is sufficient to
sterilize aerobic tumor cells and provided 1.69–2.12 logs of cell kill
compared to control in 6 independent experiments (Table 2).
When bromides 22–25 were combined with radiation, a greater
kill was achieved (2.65–4.77 log), indicating elimination of radiore-
sistant hypoxic cells within the tumors by the nitroCBIs. This com-
pares favorably with the two chloride comparators 17 (2.66 log)
and 12 (3.62 log) (Table 2, Fig. 4). Phosphate 17 contains the TMI
side chain while 12 contains the DEI basic side chain and was
the most active sulfonamide previously reported.4 The hypoxic
log10 cell kill (LCK) of radiation plus drug (vs radiation alone) for
22–25 ranged from 0.87–2.80. The most active compound was 24
containing the MEI basic side chain, for which no clonogens could
be detected in 1 of 5 treated tumors. All four bromides were much
more active (p-values 60.005) in combination with radiation (vs
radiation alone). As single agents (nitroCBI alone) 24 and 25, the
two bromides with basic side chains, exhibited statistically signif-
icant activity with p-values of <0.001 and 0.038 versus control,
respectively. Single agent activity may be due to hypoxic cell kill,
oxic cell kill or aminoCBI diffusion from hypoxic regions (bystander
effect). Bromides 22 and 23 with neutral side chains exhibited
weak but not statistically significant single agent activity, as did
the chlorides 12 and 17.
with a bromide leaving group offer an alternative and promising
approach in antitumor therapy, especially targeting radioresistant
tumor cells. It would be useful to understand toxicity differentials
between bromide prodrug and effector, made difficult by the
intrinsic instability of the latter due to the powerful leaving group
effect of the bromide. The superior activity of 24 in vivo could be
due to bromide instead of chloride or the MEI side chain versus
the DEI side chain. Future investigations will involve in vivo studies
of dose-dependent tumor hypoxic cell kill, tumor growth delay as-
says and an in depth study incorporating morpholine side chains
with different leaving groups. Bromide is slightly more lipophilic
and bulky than chloride and the issues of plasma pharmacokinet-
ics, tissue penetration and bystander effect also need to be
investigated.
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 P95% for all fi-
nal products. Final product purity was also assessed by combustion
analysis carried out in the Campbell Microanalytical Laboratory,
University of Otago, 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.
3. Conclusions
The current study was performed to investigate structure–
activity relationships influencing cytotoxicity and hypoxic selectiv-
ity for a small series of nitroCBI prodrugs and more water soluble
phosphate pre-prodrugs containing a bromide leaving group, and
to compare the results with those of previously reported chloride
analogs. Undoubtedly the bromides are synthetically more chal-
lenging than their chloride counterparts. However, in both
in vitro and in vivo the bromides matched or proved superior to
their chloride analogs in the small series examined. Thus nitroCBIs
4.1.1. 1-(1-(Bromomethyl)-1H-benzo[e]indol-3(2H)-yl)-2,2,
2-trifluoroethanone (27)
4.1.1.1. Method A.
A solution of (3-(2,2,2-trifluoroacetyl)-2,3-
dihydro-1H-benzo[e]indol-1-yl)methyl methanesulfonate5 (26a)
(8.9 g, 23.9 mmol) and LiBr (41.5 g, 477 mmol) in THF (100 mL)

R. J. Stevenson et al. / Bioorg. Med. Chem. 19 (2011) 5989–5998
5995
was stirred in the dark for 36 h. EtOAc was added and the solution
was washed with water, brine and dried (Na2SO4). The organic
layer was evaporated and the crude product was recrystallized
(MeOH/H2O) to give 27 (8.1 g, 95%) as colorless crystals: mp
152–154 °C; 1H NMR (CDCl3) d 8.43 (d, J = 9.0 Hz, 1H), 7.92 (d,
J = 9.1 Hz, 1H), 7.88 (d, J = 9.3 Hz, 1H), 7.79 (d, J = 8.3 Hz, 1H),
7.58 (td, J = 8.1, 1.2 Hz, 1H), 7.49 (td, J = 8.1, 1.1 Hz, 1H), 4.62 (dt,
J = 11.5, 1.4 Hz, 1H), 4.43 (dd, J = 11.4, 8.5 Hz, 1H), 4.25 (t,
J = 9.1 Hz, 1H), 3.84 (ddd, J = 10.7, 3.2, 0.8 Hz, 1H), 3.39 (t,
J = 10.2 Hz, 1H). Anal. Calcd for C15H11BrF3NO: C, 50.30; H, 3.10;
N, 3.91. Found: C, 50.58; H, 3.07; N, 3.92.
1H), 4.72 (br d, J = 11.5 Hz, 1H), 4.61 (dd, J = 11.5, 8.9 Hz, 1H),
4.43 (tt, J = 8.6, 3.0 Hz, 1H), 3.81 (dd, J = 11.0, 3.2 Hz, 1H), 3.58
(dd, J = 11.0, 7.8 Hz, 1H). Anal. Calcd for C15H9BrClF3N2O5S: C,
35.91; H, 1.81; N, 5.58. Found: C, 36.00; H, 1.77; N, 5.27. A por-
tion of 30 was recrystallized (CH2Cl2/i-Pr2O) to give colorless
crystals: mp 238–243 °C; 1H NMR (CDCl3) d 8.91 (d, J = 9.1 Hz,
1H), 8.81 (d, J = 1.9 Hz, 1H), 8.36 (d, J = 2.0 Hz, 1H), 8.26 (d,
J = 9.1 Hz, 1H), 4.58–4.45 (m, 2H), 4.18–4.11 (m, 1H), 3.47 (dd,
J = 8.1, 3.2 Hz, 1H), 3.18 (dd, J = 11.0, 7.3 Hz, 1H). Anal. Calcd
for C15H9BrClF3N2O5Sꢀ0.2i-Pr2O: C, 37.27; H, 2.28; N, 5.37. Found:
C, 37.10; H, 2.11; N, 5.18.
A
repeat reaction with 28 (870 mg, 1.91 mmol) gave 31
4.1.1.2. Method B.
A solution of 1-(1-(chloromethyl)-1H-
(724 mg, 75%) after re-precipitation (CH2Cl2/i-Pr2O) of the crude
product. Compound 30 could not be detected in the precipitated
material.
benzo[e]indol-3(2H)-yl)-2,2,2-trifluoroethanone2 (26b) (500 mg,
1.60 mmol) and LiBr (2.19 g, 26.39 mmol) in 2-butanone (15 mL)
was heated (85 °C) for 9 days. EtOAc and water were added and
the organic portion was washed with water, brine and dried
(Na2SO4). Flash chromatography (petroleum ether/EtOAc; 100:0
then 99.5:0.5) gave 27 (333 mg, 55%) contaminated with 26b
(37 mg, 10%), which could not be removed by recrystallization
(EtOAc/petroleum ether).
4.1.4. 1-(Bromomethyl)-N-(2-hydroxyethyl)-5-nitro-2,
3-dihydro-1H-benzo[e]indole-7-sulfonamide (32)
A solution of ethanolamine (18 mg, 0.30 mmol) in CH2Cl2
(0.5 mL) was added dropwise to a cooled (0 °C) solution of 31
(50 mg, 0.10 mmol) in CH2Cl2 (10 mL). After 15 min Cs2CO3
(67 mg, 0.20 mmol), MeOH (3 mL) and water (2 drops) were added.
After a further 15 min ice was added and the mixture was poured
into cold water/cold EtOAc. The organic portion was washed with
cold water, cold brine and dried (Na2SO4). Recrystallization
(EtOAc/i-Pr2O) gave 32 (43 mg, 100%) as red crystals: mp 144–
147 °C; 1H NMR [(CD3)2SO] d 8.59 (d, J = 1.7 Hz, 1H), 8.02 (d,
J = 8.9 Hz, 1H), 7.79 (dd, J = 8.9, 1.8 Hz, 1H), 7.69 (t, J = 5.9 Hz,
1H), 6.73 (s, 1H), 4.63 (t, J = 5.6 Hz, 1H), 4.33–4.25 (m, 1H), 3.89
(td, J = 10.4, 1.9 Hz, 1H), 3.82 (dd, J = 10.2, 3.4 Hz, 1H), 3.74–3.66
(m, 2H), 3.43–3.30 (m, 3H), 2.81 (q, J = 6.1 Hz, 2H). Anal. Calcd
for C15H16BrN3O5Sꢀ0.1i-Pr2O: C, 42.54; H, 3.98; N, 9.54. Found: C,
42.73; H, 3.92; N, 9.30.
4.1.2. 1-(Bromomethyl)-3-(2,2,2-trifluoroacetyl)-2,3-dihydro-
1H-benzo[e]indole-7-sulfonyl chloride (28)
4.1.2.1. Method A.
A solution of 27 (7.50 g, 21.0 mmol) in
CH2Cl2 (40 mL) was added dropwise to a cooled (ꢁ78 °C) solution
of ClSO3H (9.72 g, 83.8 mmol) in CH2Cl2 (40 mL). The stirred mix-
ture was allowed to warm to 5 °C over 7 h producing a gray precip-
itate. The mixture was kept at 5 °C overnight and then cooled
(0 °C). DMF was slowly added (the minimum amount required to
dissolve the precipitate) and oxalyl chloride (4 mL) was added
dropwise. After 1 h the mixture was poured into ice water. The sys-
tem was extracted with cold EtOAc and the organic portion was
washed with cold water, cold brine and dried (Na2SO4). After filtra-
tion through a plug of Celite/silica gel and removal of solvent, the
crude product was dissolved in CH2Cl2/i-Pr2O. Slow removal of
CH2Cl2 under vacuum gave 28 (8.88 g, 93%) as a white powder:
mp 194–196 °C; 1H NMR (CDCl3) d 8.65 (d, J = 7.5 Hz, 1H), 8.63
(d, J = 2.0 Hz, 1H), 8.11 (d, J = 9.0 Hz, 1H), 8.09 (dd, J = 9.0, 2.0 Hz,
1H), 7.99 (d, J = 9.0 Hz, 1H), 4.67 (dt, J = 11.7, 1.6 Hz, 1H), 4.52
(dd, J = 11.4, 8.7 Hz, 1H), 4.32 (tt, J = 8.7, 2.6 Hz, 1H), 3.80 (dd,
J = 10.8, 3.3 Hz, 1H), 3.47 (dd, J = 10.8, 9.1 Hz, 1H). Anal. Calcd for
4.1.5. 1-(Bromomethyl)-N-(2-hydroxyethyl)-5-nitro-3-(5,6,
7-trimethoxy-1H-indole-2-carbonyl)-2,3-dihydro-1H-
benzo[e]indole-7-sulfonamide (18)
A solution of EDCIꢀHCl (9.0 g, 58.1 mmol) in CH2Cl2 (20 mL) was
washed with a solution of 40% aq K2CO3. Addition of pyridini-
umꢀHBr (7.5 g, 46.5 mmol) to the organic portion followed by addi-
tion of Et2O produced a precipitate. The precipitate was filtered off
and the solid was washed with Et2O to give EDCIꢀHBr (8.63 g, 79%)
as a white powder.
A solution of EDCIꢀHBr (165 mg, 0.69 mmol), TsOH (4 mg,
0.02 mmol), 5,6,7-trimethoxyindole-2-carboxylic acid (58 mg,
0.23 mmol) and 32 (50 mg, 0.12 mmol) in CH2Cl2 (6 mL) and
DMA (0.5 mL) was stirred for 1 h and the mixture was poured into
cold EtOAc/cold water. The organic layer was washed with cold
water, cold brine and then dried (Na2SO4) and evaporated. The res-
idue was purified by re-precipitation (CH2Cl2/MeOH) followed by
trituration (EtOAc) to give 18 (67 mg, 87%) as a yellow powder:
mp 248–250 °C; 1H NMR [(CD3)2SO] d 11.61 (s, 1H), 9.24 (s, 1H),
8.86 (d, J = 1.4 Hz, 1H), 8.42 (d, J = 8.9 Hz, 1H), 8.02 (dd, J = 8.9,
1.6 Hz, 1H), 7.91 (t, J = 5.8 Hz, 1H), 7.20 (d, J = 2.1 Hz, 1H), 7.00 (s,
1H), 4.93 (t, J = 10.3 Hz, 1H), 4.67 (t, J = 5.5 Hz, 2H), 4.61 (dd,
J = 10.9, 2.1 Hz, 1H), 4.07–3.97 (m, 2H), 3.94 (s, 3H), 3.83 (s, 3H),
3.81 (s, 3H), 3.38 (q, J = 5.8 Hz, 2H), 2.87 (q, J = 6.1 Hz, 2H). Anal.
Calcd for C27H27BrN4O9SꢀEtOAc: C, 49.54; H, 4.69; N, 7.45. Found:
C, 49.71; H, 4.58; N, 7.46.
C
15H10BrClF3NO3Sꢀ0.1i-Pr2O: C, 40.13; H, 2.46; N, 3.00. Found: C,
40.17; H, 2.27; N, 2.97.
4.1.2.2. Method B.
A solution of 295 (80 mg, 0.17 mmol) and
LiBr (400 mg, 4.60 mmol) in THF (8 mL) was stirred for 4 days.
EtOAc and water were added and the organic portion was washed
with water, brine and dried (Na2SO4). Flash chromatography
(petroleum ether/EtOAc; 100:0 to 4:1) gave 28 (36 mg, 47%).
4.1.3. 1-(Bromomethyl)-5-nitro-3-(2,2,2-trifluoroacetyl)-2,
3-dihydro-1H-benzo[e]indole-7-sulfonyl chloride (31) and
1-(bromomethyl)-9-nitro-3-(2,2,2-trifluoroacetyl)-2,3-dihydro-
1H-benzo[e]indole-7-sulfonyl chloride (30)
A cooled (0 °C) solution of KNO3 (117 mg, 1.16 mmol) in 98%
H2SO4 (1 mL) was added dropwise to a cooled (ꢁ12 °C) solution
of 28 (440 mg, 0.97 mmol) in 98% H2SO4 (40 mL). After 15 min,
the mixture was poured into ice water and extracted with cold
EtOAc. The organic portion was washed with cold water, cold
brine and dried (Na2SO4). Flash chromatography (petroleum
ether/EtOAc; 100:0 to 7:3) gave 31 (287 mg, 59%) as a white
powder and 30 (10 mg, 2%) as a white powder. A portion of
31 was recrystallized (CH2Cl2/i-Pr2O) to give colorless crystals:
4.1.6. (E)-1-(Bromomethyl)-N-(2-hydroxyethyl)-3-(3-(4-
methoxyphenyl)acryloyl)-5-nitro-2,3-dihydro-1H-
benzo[e]indole-7-sulfonamide (19)
4.1.6.1. Method A.
1.81 mmol), TsOH (10 mg, 0.06 mmol), (E)-3-(4-methoxy-
phenyl)acrylic acid (65 mg, 0.36 mmol) and 32 (130 mg,
A
solution of EDCIꢀHBr (430 mg,
mp 217–219 °C; 1H NMR (CDCl3)
J = 1.7 Hz, 1H), 8.23 (dd, J = 9.0, 1.9 Hz, 1H), 8.11 (d, J = 9.1 Hz,
d
9.35 (s, 1H), 9.29 (d,

5996
R. J. Stevenson et al. / Bioorg. Med. Chem. 19 (2011) 5989–5998
0.30 mmol) in CH2Cl2 (8 mL) was stirred for 1 h. Further portions of
EDCIꢀHBr (100 mg, 0.42 mmol), TsOH (5 mg, 0.03 mmol) and (E)-3-
(4-methoxyphenyl)acrylic acid (50 mg, 0.28 mmol) were added
and the solution was stirred for 3 h and the mixture was poured
into cold EtOAc/cold water. The organic layer was washed with
cold water, cold brine and then dried (Na2SO4) and evaporated.
The residue was purified by precipitation (CH2Cl2/MeOH) to give
19 (120 mg, 67%). Further purification by preparative HPLC
(Synergi-Max RP column; flow rate 15 mL/min; pump 1: H2O/
TFA, pH 2.5, gradient 80%-5%-80%; pump 2: CH3CN/H2O, 9:1, gradi-
ent 20%–95%–20%) gave a yellow powder (106 mg, 99.4% purity):
mp 246 °C (dec); 1H NMR [(CD3)2SO] d 9.35 (s, 1H), 8.83 (d,
J = 1.4 Hz, 1H), 8.37 (d, J = 8.9 Hz, 1H), 8.01 (dd, J = 8.9, 1.6 Hz,
1H), 7.88 (t, J = 5.9 Hz, 1H), 7.81 (d, J = 8.8 Hz, 2H), 7.73 (d,
J = 15.3 Hz, 1H), 7.10 (d, J = 15.3 Hz, 1H), 7.02 (d, J = 8.7 Hz, 2H),
4.72–4.63 (m, 3H), 4.56 (t, J = 7.7 Hz, 1H), 4.03–3.94 (m, 2H), 3.83
(s, 3H), 3.39 (q, J = 5.8 Hz, 2H), 2.87 (q, J = 6.1 Hz, 2H). Anal. Calcd
for C25H24BrN3O7S: C, 50.86; H, 4.10; N, 7.12. Found: C, 50.97; H,
4.13; N, 6.95.
4.1.7. 1-(Bromomethyl)-N-(2-hydroxyethyl)-3-(5-(2-
morpholinoethoxy)-1H-indole-2-carbonyl)-5-nitro-2,3-
dihydro-1H-benzo[e]indole-7-sulfonamide (20)
Biorad AG 1-X4 (Clꢁ form) resin (45 g) was converted into the
Brꢁ form by eluting with a solution of NaBr (45 g) in water
(450 mL). After washing the resin with water (450 mL) and MeOH
(450 mL), a solution of the HCl salt of 5-(2-morpholinoethoxy)-1H-
indole-2-carboxylic acid38 (1.5 g) in MeOH (20 mL) was passed
through the resin to give 5-(2-morpholinoethoxy)-1H-indole-2-
carboxylic acid hydrobromide (1.33 g, 78%) as a cream powder
after removal of MeOH.
EDCIꢀHBr (230 mg, 0.98 mmol), TsOH (6 mg, 0.033 mmol) and
5-(2-morpholinoethoxy)-1H-indole-2-carboxylic acid hydrobro-
mide (121 mg, 0.33 mmol) were added to
a solution of 32
(70 mg, 0.16 mmol) in CH2Cl2 (15 mL) and DMA (2 mL). After
30 min further portions of EDCIꢀHBr (230 mg, 0.98 mmol) and
TsOH (20 mg, 0.12 mmol) were added. After 1.5 h a further portion
of 5-(2-morpholinoethoxy)-1H-indole-2-carboxylic acid hydrobro-
mide (60 mg, 0.16 mmol) was added. After a further 5 h the mix-
ture was poured into cold water/cold EtOAc. The organic portion
was washed with cold water, cold brine and dried (Na2SO4). After
removal of solvent the solid was dissolved in CH2Cl2/MeOH and
slow removal of CH2Cl2 under vacuum gave 20 (65 mg, 57%) as a
yellow powder: mp 210–216 °C (dec); 1H NMR [(CD3)2SO] d
11.74 (s, 1H), 9.29 (s, 1H), 8.85 (d, J = 1.5 Hz, 1H), 8.43 (d,
J = 8.9 Hz, 1H), 8.02 (dd, J = 8.9, 1.6 Hz, 1H), 7.91 (t, J = 5.9 Hz,
1H), 7.42 (d, J = 8.9 Hz, 1H), 7.22–7.15 (m, 2H), 6.96 (dd, J = 8.9,
2.3 Hz, 1H), 4.97 (t, J = 10.5 Hz, 1H), 4.73–4.62 (m, 3H), 4.13 (t,
J = 5.8 Hz, 2H), 4.08–3.98 (m, 2H), 3.63–3.56 (m, 4H), 3.39 (q,
J = 6.1 Hz, 2H), 2.88 (q, J = 6.1 Hz, 2H), 2.73 (t, J = 5.7 Hz, 2H), 4 ali-
4.1.6.2. Method B.
A solution of TBDMSCl (21 mg, 0.14 mmol)
and DIPEA (30 mg, 0.23 mmol) in DMF (0.5 mL) was added drop-
wise to a cooled (0 °C) solution of 32 (50 mg, 0.12 mmol) in DMF
(2 mL) and the mixture was allowed to warm to 20 °C over 1 h. Fur-
ther portions of TBDMSCl (42 mg, 0.28 mmol) and DIPEA (30 mg,
0.23 mmol) were added and after 15 min the mixture was poured
into cold water/cold EtOAc. The organic portion was washed with
cold water, cold brine and dried (Na2SO4). Trituration with petro-
leum ether and Et2O gave 1-(bromomethyl)-N-(2-((tert-butyldi-
methylsilyl)oxy)ethyl)-5-nitro-2,3-dihydro-1H-benzo[e]indole-7-
sulfonamide (33) (60 mg, 95%) as a red powder: mp 69–72 °C
(dec); 1H NMR [(CD3)2SO] d 8.58 (s, 1H), 8.02 (d, J = 9.0 Hz, 1H),
7.80 (dd, J = 8.9, 1.6 Hz, 1H), 7.78–7.72 (m, 2H), 6.74 (s, 1H),
4.34–4.24 (m, 1H), 3.88 (t, J = 10.4 Hz, 1H), 3.81 (dd, J = 10.3,
3.4 Hz, 1H), 3.74–3.65 (m, 2H), 3.52 (t, J = 6.3 Hz, 2H), 2.85 (q,
J = 6.0 Hz, 2H), 0.79 (s, 9H), ꢁ0.05 (s, 6H). Anal. Calcd for
phatic
30H32BrN5O8Sꢀ0.5H2O: C, 50.64; H, 4.67; N, 9.84. Found: C,
50.47; H, 4.60; N, 9.67.
protons
not
observed.
Anal.
Calcd
for
C
4.1.8. (E)-1-(Bromomethyl)-N-(2-hydroxyethyl)-3-(3-(4-(2-
morpholinoethoxy)phenyl)acryloyl)-5-nitro-2,3-dihydro-1H-
benzo[e]indole-7-sulfonamide (21)
C
21H30BrN3O5SSiꢀ0.3H2O: C, 45.87; H, 5.61; N, 7.64. Found: C,
45.55; H, 5.28; N, 8.04.
Biorad AG 1-X4 (Clꢁ form) resin (45 g) was converted into Brꢁ
form by eluting with a solution of NaBr (45 g) in water (450 mL).
After washing the resin with water (450 mL) and MeOH
(450 mL), a solution of the HCl salt of (E)-3-(4-(2-morpholinoeth-
oxy)phenyl)acrylic acid37 (1.0 g) in MeOH (20 mL) was passed
through the resin to give (E)-3-(4-(2-morpholinoethoxy)
phenyl)acrylic acid hydrobromide (1.0 g, 88%) as a cream powder
after removal of MeOH.
EDCIꢀHBr (329 mg, 1.40 mmol), TsOH (8 mg, 0.047 mmol) and
(E)-3-(4-(2-morpholinoethoxy)phenyl)acrylic acid hydrobromide
(166 mg, 0.47 mmol) were added to a solution of 32 (100 mg,
0.23 mmol) in CH2Cl2 (8 mL) and DMA (1 mL). After 2 h further
portions of (E)-3-(4-(2-morpholinoethoxy)phenyl)acrylic acid
A solution of EDCIꢀHBr (143 mg, 0.61 mmol), TsOH (4 mg,
0.02 mmol),
(E)-3-(4-methoxyphenyl)acrylic
acid
(27 mg,
0.15 mmol) and 33 (55 mg, 0.10 mmol) in CH2Cl2 (2 mL) was stirred
for 3 h. Further portions of EDCIꢀHBr (143 mg, 0.61 mmol), TsOH
(4 mg, 0.02 mmol) and (E)-3-(4-methoxyphenyl)acrylic acid
(27 mg, 0.15 mmol) were added and the solution was stirred for
1 h and the mixture was poured into cold EtOAc/cold water. The or-
ganic layer was washed with cold water, cold brine and then dried
(Na2SO4) and evaporated. The residue was purified by precipitation
(CH2Cl2/MeOH) followed by trituration (petroleum ether) to give
(E)-1-(bromomethyl)-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)-3-
(3-(4-methoxyphenyl)acryloyl)-5-nitro-2,3-dihydro-1H-ben-
zo[e]indole-7-sulfonamide (34) (50 mg, 70%) as a yellow powder:
mp 128–132 °C; 1H NMR [(CD3)2SO] d 9.39 (s, 1H), 8.89 (d,
J = 1.3 Hz, 1H), 8.42 (d, J = 8.9 Hz, 1H), 8.05 (dd, J = 8.9, 1.6 Hz, 1H),
8.03 (t, J = 5.9 Hz, 1H), 7.86 (d, J = 8.7 Hz, 2H), 7.78 (d, J = 15.3 Hz,
1H), 7.15 (d, J = 15.3 Hz, 1H), 7.07 (d, J = 8.7 Hz, 2H), 4.77–4.67 (m,
2H), 4.65–4.58 (m, 1H), 4.08–3.97 (m, 2H), 3.88 (s, 3H), 3.59 (t,
J = 6.1 Hz, 2H), 2.97 (q, J = 6.0 Hz, 2H), 0.83 (s, 9H), 0.00 (s, 6H). Anal.
Calcd for C31H38BrN3O7SSiꢀ0.4petroleum ether: C, 54.27; H, 5.95; N,
5.69. Found: C, 54.54; H, 5.65; N, 6.03.
hydrobromide
(166 mg,
0.47 mmol),
EDCIꢀHBr
(329 mg,
1.40 mmol) and TsOH (8 mg, 0.047 mmol) were added. After a fur-
ther 2 h the mixture was poured into cold water/cold EtOAc. The
organic portion was washed with cold water, cold brine and dried
(Na2SO4). After removal of solvent the solid was dissolved in
CH2Cl2/MeOH and slow removal of CH2Cl2 under vacuum gave 21
(91 mg, 57%) as a yellow powder: mp 185–190 °C (dec); 1H NMR
[(CD3)2SO] d 9.34 (s, 1H), 8.83 (s, 1H), 8.35 (d, J = 8.8 Hz, 1H),
8.01 (d, J = 8.9 Hz, 1H), 7.90 (t, J = 5.8 Hz, 1H), 7.79 (d, J = 8.7 Hz,
2H), 7.73 (d, J = 15.3 Hz, 1H), 7.09 (d, J = 15.4 Hz, 1H), 7.03 (d,
J = 8.7 Hz, 2H), 4.73–4.63 (m, 3H), 4.59–4.51 (m, 1H), 4.16 (t,
J = 5.7 Hz, 3H), 4.05–3.92 (m, 3H), 3.63–3.55 (m, 4H), 3.54–3.47
(m, 3H), 2.86 (q, J = 6.0 Hz, 3H), 2.72 (t, J = 5.7 Hz, 2H). Anal. Calcd
for C30H33BrN4O8SꢀMeOH: C, 51.60; H, 5.17; N, 7.76. Found: C,
51.89; H, 5.07; N, 7.60.
TFA (0.5 mL) was added dropwise to a solution of 34 (47 mg, 0.
07 mmol) in CH2Cl2 (2 mL) and the mixture was stirred for 4 h. Sol-
vents were removed and the residue was triturated (Et2O) to give
19 (39 mg, 100%). Further purification by preparative HPLC (Syner-
gi-Max RP column; flow rate 15 mL/min; pump 1: H2O/TFA, pH 2.5,
gradient 80%–5%–80%; pump 2: CH3CN/H2O, 9:1, gradient 20%–
95%–20%) gave a yellow powder (29 mg, 99.4% purity).

R. J. Stevenson et al. / Bioorg. Med. Chem. 19 (2011) 5989–5998
5997
4.1.9. 2-(1-(Bromomethyl)-5-nitro-3-(5,6,7-trimethoxy-1H-
indole-2-carbonyl)-2,3-dihydro-1H-benzo[e]indole-7-
sulfonamido)ethyl dihydrogen phosphate (22)
precipitation (CH2Cl2/MeOH) to give (E)-2-(1-(bromomethyl)-3-
(3-(4-methoxyphenyl)acryloyl)-5-nitro-2,3-dihydro-1H-benzo[e]
indole-7-sulfonamido)ethyl di-tert-butyl phosphate (36b) (72 mg,
38%) as a yellow powder: mp 186–189 °C; 1H NMR [(CD3)2SO] d
9.35 (s, 1H), 8.84 (d, J = 1.6 Hz, 1H), 8.38 (d, J = 8.9 Hz, 1H), 8.15
(t, J = 5.8 Hz, 1H), 7.99 (dd, J = 8.9, 1.6 Hz, 1H), 7.81 (d, J = 8.8 Hz,
2H), 7.74 (d, J = 15.3 Hz, 1H), 7.10 (d, J = 15.3 Hz, 1H), 7.02 (d,
J = 8.8 Hz, 2H), 4.71–4.64 (m, 2H), 4.57 (t, J = 7.8 Hz, 1H), 4.02–
3.94 (m, 2H), 3.87–3.80 (m, 2H), 3.83 (s, 3H), 3.05 (q, J = 5.9 Hz,
2H), 1.36 (s, 18H). Anal. Calcd for C33H41BrN3O10PSꢀH2O: C, 49.51;
H, 5.41; N, 5.25. Found: C, 49.13; H, 5.04; N, 5.56.
TFA (98 mg, 0.86 mmol) was added dropwise to a solution of
36b (67 mg, 0.09 mmol) in CH2Cl2 (1.5 mL) and stirred for 15 h.
Solvents were removed and the residue was triturated (Et2O) to
give 23 (57 mg, 100%) as a yellow powder: mp 191 °C (dec); 1H
NMR [(CD3)2SO] d 9.35 (s, 1H), 8.85 (d, J = 1.6 Hz, 1H), 8.37 (d,
J = 8.9 Hz, 1H), 8.12 (t, J = 5.8 Hz, 1H), 8.01 (dd, J = 8.9, 1.7 Hz,
1H), 7.81 (d, J = 8.8 Hz, 2H), 7.72 (d, J = 15.3 Hz, 1H), 7.10 (d,
J = 15.3 Hz, 1H), 7.03 (d, J = 8.8 Hz, 2H), 4.72–4.62 (m, 2H), 4.56
(t, J = 7.8 Hz, 1H), 4.03–3.94 (m, 2H), 3.83 (s, 3H), 3.82–3.76 (m,
2H), 3.03 (q, J = 5.6 Hz, 2H), 2 protons not observed. Anal. Calcd
for C25H25BrN3O10PSꢀH2O: C, 43.61; H, 3.95; N, 6.10. Found: C,
43.83; H, 3.82; N, 5.77.
A solution of DIPEA (155 mg, 1.20 mmol) and H2N(CH2)2O-
4
P(O)(Ot-Bu)2 (300 mg, 1.20 mmol) in CH2Cl2 (1 mL) was added
dropwise to a cooled (ꢁ50 °C) solution of 31 (500 mg, 1.00 mmol)
in CH2Cl2 (10 mL). After 30 min Cs2CO3 (650 mg, 2.0 mmol), MeOH
(5 mL) and water (2 mL) were added. The cooled (ꢁ10 °C) mixture
was stirred for 15 min and poured into cold water/cold EtOAc. The
organic portion was washed with cold water, cold brine, dried
(Na2SO4) and filtered through a plug of silica gel (EtOAc/petroleum
ether: 9:1) to give 2-(1-(bromomethyl)-5-nitro-2,3-dihydro-1H-
benzo[e]indole-7-sulfonamido)ethyl di-tert-butyl phosphate (35)
(500 mg, 81%). A small portion was recrystallized (CH2Cl2/i-Pr2O)
to give red crystals: mp 76 °C (dec); 1H NMR [(CD3)2SO] d 8.59
(d, J = 1.6 Hz, 1H), 8.04 (d, J = 8.9 Hz, 1H), 7.94 (t, J = 5.6 Hz, 1H),
7.80 (dd, J = 9.0, 1.8 Hz, 1H), 7.75 (s, 1H), 6.75 (s, 1H), 4.33–4.25
(m, 1H), 3.89 (td, J = 10.5, 1.9 Hz, 1H), 3.84–3.76 (m, 3H), 3.74–
3.65 (m, 2H), 3.01 (q, J = 5.6 Hz, 2H), 1.35 (s, 18H). Anal. Calcd for
C23H33BrN3O8PS: C, 44.38; H, 5.34; N, 6.75. Found: C, 44.48; H,
5.20; N, 6.65.
A solution of EDCIꢀHBr (273 mg, 1.16 mmol), TsOH (7 mg,
0.04 mmol), 5,6,7-trimethoxyindole-2-carboxylic acid (97 mg,
0.39 mmol) and 35 (120 mg, 0.19 mmol) in CH2Cl2 (5 mL) was
stirred for 1 h and the mixture was poured into cold EtOAc/cold
water. The organic layer was washed with cold water, cold
brine and then dried (Na2SO4) and evaporated. The residue was
purified by trituration with EtOAc followed by Et2O to give 2-
(1-(bromomethyl)-5-nitro-3-(5,6,7-trimethoxy-1H-indole-2-carbonyl)-
2,3-dihydro-1H-benzo[e]indole-7-sulfonamido)ethyl di-tert-butyl
phosphate (36a) (90 mg, 55%) as a yellow powder: mp 206–
210 °C (dec); 1H NMR [(CD3)2SO] d 11.61 (d, J = 1.7 Hz, 1H),
9.24 (s, 1H), 8.87 (d, J = 1.6 Hz, 1H), 8.44 (d, J = 8.9 Hz, 1H),
8.17 (t, J = 5.9 Hz, 1H), 8.02 (dd, J = 8.9, 1.7 Hz, 1H), 7.19 (d,
J = 2.1 Hz, 1H), 6.09 (s, 1H), 4.93 (t, J = 9.5 Hz, 1H), 4.70–4.63
(m, 1H), 4.61 (dd, J = 10.9, 2.3 Hz, 1H), 4.08–3.96 (m, 2H), 3.94
(s, 3H), 3.87–.81 (m, 2H), 3.84 (s, 3H), 3.81 (s, 3H), 3.06 (q,
J = 5.9 Hz, 2H), 1.36 (s, 18H), 2 protons not observed. Anal. Calcd
for C35H44BrN4O12PS: C, 49.13; H, 5.18; N, 6.55. Found: C, 49.37;
H, 5.21; N, 6.43.
TFA (66 mg, 0.58 mmol) was added dropwise to a solution of
36a (50 mg, 0.06 mmol) in CH2Cl2 (2 mL) and stirred for 15 h. Sol-
vents were removed and the residue was triturated (MeOH) to give
22 (43 mg, 100%) as a yellow powder: mp 215–219 °C (dec); 1H
NMR [(CD3)2SO] d 11.60 (s, 1H), 9.24 (s, 1H), 8.87 (d, J = 1.6 Hz,
1H), 8.42 (d, J = 8.9 Hz, 1H), 8.23 (br s, 1H), 8.03 (dd, J = 8.9,
1.6 Hz, 1H), 7.19 (d, J = 2.1 Hz, 1H), 6.99 (s, 1H), 4.92 (t,
J = 10.4 Hz, 1H), 4.70–4.63 (m, 1H), 4.61 (dd, J = 10.9, 2.2 Hz, 1H),
4.07–3.98 (m, 2H), 3.94 (s, 3H), 3.84 (s, 3H), 3.82–3.75 (m, 2H),
3.81 (s, 3H), 3.03 (t, J = 5.7 Hz, 2H). Anal. Calcd for
4.1.11. 2-(1-(Bromomethyl)-3-(5-(2-morpholinoethoxy)-1H-
indole-2-carbonyl)-5-nitro-2,3-dihydro-1H-benzo[e]indole-7-
sulfonamido)ethyl dihydrogen phosphate trifluoroacetate (24)
EDCIꢀHBr (3.74 g, 15.8 mmol), TsOH (109 mg, 0.63 mmol) and
5-(2-morpholinoethoxy)-1H-indole-2-carboxylic acid hydrobro-
mide (1.30 g, 3.48 mmol) were added to
a solution of 35
(1.97 g, 3.17 mmol) in CH2Cl2 (55 mL) and DMSO (5 mL). After
1 h further portions of EDCIꢀHBr (3.0 g, 12.7 mmol), TsOH
(200 mg, 1.16 mmol) and 5-(2-morpholinoethoxy)-1H-indole-2-
carboxylic acid hydrobromide (600 mg, 1.61 mmol) were added.
After a further 3 h the mixture was poured into cold water/cold
EtOAc. The organic portion was washed with cold water, cold
brine and dried (Na2SO4). Precipitation (EtOAc) followed by trit-
uration (acetone) gave 2-(1-(bromomethyl)-3-(5-(2-morpholino-
ethoxy)-1H-indole-2-carbonyl)-5-nitro-2,3-dihydro-1H-benzo[e]
indole-7-sulfonamido)ethyl di-tert-butyl phosphate (36c) (1.08 g,
38%) as
a
yellow powder: mp 228–233 °C (dec); 1H NMR
[(CD3)2SO] d 11.73 (s, 1H), 9.30 (s, 1H), 8.87 (d, J = 1.7 Hz, 1H),
8.45 (d, J = 8.9 Hz, 1H), 8.18 (t, J = 5.9 Hz, 1H), 8.02 (dd, J = 8.9,
1.7 Hz, 1H), 7.42 (d, J = 8.9 Hz, 1H), 7.23–7.17 (m, 2H), 6.96
(dd, J = 8.9, 2.4 Hz, 1H), 4.97 (t, J = 10.4 Hz, 1H), 4.73–4.63 (m,
2H), 4.13 (t, J = 5.7 Hz, 2H), 4.08–3.99 (m, 2H), 3.83 (q,
J = 6.0 Hz, 2H), 3.60 (t, J = 4.5 Hz, 4H), 3.07 (q, J = 5.8 Hz, 2H),
2.74 (t, J = 5.4 Hz, 2H), 1.36 (s, 18H), 4 protons not observed.
Anal. Calcd for C38H49BrN5O11PSꢀ0.5H2O: C, 50.50; H, 5.58; N,
7.75. Found: C, 50.68; H, 5.57; N, 7.82.
C27H28BrN4O12PS: C, 43.62; H, 3.80; N, 7.54. Found: C, 43.86; H,
A solution of 36c (140 mg, 0.16 mmol) and TFA (178 mg,
1.56 mmol) in CH2Cl2 (5 mL) was stirred for 20 h. Removal of
solvents gave 24 (146 mg, 100%) as a yellow powder: mp 206–
210 °C (dec); 1H NMR [(CD3)2SO] d 11.78 (s, 1H), 9.28 (s, 1H),
8.87 (d, J = 1.5 Hz, 1H), 8.42 (d, J = 8.9 Hz, 1H), 8.19 (br s, 1H),
8.01 (dd, J = 8.9, 1.5 Hz, 1H), 7.43 (d, J = 8.9 Hz, 1H), 7.24 (d,
J = 2.0 Hz, 1H), 7.21 (d, J = 1.4 Hz, 1H), 6.99 (dd, J = 8.9, 2.3 Hz,
1H), 4.97 (t, J = 10.5 Hz, 1H), 4.72–4.63 (m, 2H), 4.27 (t,
J = 5.9 Hz, 2H), 4.08–3.97 (m, 2H), 3.82 (q, J = 6.1 Hz, 2H), 3.87–
3.70 (m, 2H), 3.03 (t, J = 5.7 Hz, 2H), 9 aliphatic protons not ob-
served (obscured by large water peak). After D2O addition the
following peaks became visible: d 3.56 (t, J = 5.6 Hz, 2H), 3.52–
3.41 (m, 2H), 3.30–3.15 (br m, 2H). Anal. Calcd for
3.88; N, 7.37.
4.1.10. (E)-2-(1-(Bromomethyl)-3-(3-(4-
methoxyphenyl)acryloyl)-5-nitro-2,3-dihydro-1H-
benzo[e]indole-7-sulfonamido)ethyl dihydrogen phosphate
(23)
A solution of EDCIꢀHBr (340 mg, 1.45 mmol), TsOH (8 mg,
0.05 mmol),
(E)-3-(4-methoxyphenyl)acrylic
acid
(56 mg,
0.31 mmol) and 35 (150 mg, 0.24 mmol) in CH2Cl2 (6 mL) was stir-
red for 2 h. Further portions of EDCIꢀHBr (114 mg, 0.48 mmol),
TsOH (8 mg, 0.05 mmol) and (E)-3-(4-methoxyphenyl)acrylic acid
(30 mg, 0.17 mmol) were added and the solution was stirred for
2 h and the mixture was poured into cold EtOAc/cold water. The
organic layer was washed with cold water, cold brine and then
dried (Na2SO4) and evaporated. The residue was purified by
C32H34BrF3N5O13PS: C, 42.87; H, 3.82; N, 7.81. Found: C, 43.21;
H, 4.11; N, 7.73.

5998
R. J. Stevenson et al. / Bioorg. Med. Chem. 19 (2011) 5989–5998
4.1.12. (E)-2-(1-(Bromomethyl)-3-(3-(4-(2-
Acknowledgments
morpholinoethoxy)phenyl)acryloyl)-5-nitro-2,3-dihydro-1H-
benzo[e]indole-7-sulfonamido)ethyl dihydrogen phosphate
trifluoroacetate (25)
The authors are grateful for the assistance of Sisira Kumara and
Karin Tan for HPLC, Dr. Maruta Boyd and Dr. Shannon Black for
NMR spectrometry, and Sunali Mehta and Caroline McCulloch for
in vivo antitumor assays. 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 Re-
search Council of New Zealand.
EDCIꢀHBr (820 mg, 3.47 mmol), TsOH (20 mg, 0.12 mmol) and
(E)-3-(4-(2-morpholinoethoxy)phenyl)acrylic acid hydrobromide
(410 mg, 1.16 mmol) were added to a solution of 35 (360 mg,
0.58 mmol) in CH2Cl2 (15 mL) and DMA (1 mL). After 3 h the
mixture was poured into cold water/cold EtOAc. The organic por-
tion was washed with cold water, cold brine and dried (Na2SO4).
Precipitation (CH2Cl2/MeOH) followed by trituration (MeOH)
gave (E)-2-(1-(bromomethyl)-3-(3-(4-(2-morpholinoethoxy)
phenyl)acryloyl)-5-nitro-2,3-dihydro-1H-benzo[e]indole-7-sulfon-
amido)ethyl di-tert-butyl phosphate (36d) (250 mg, 50%) as a
yellow powder: mp 177–182 °C (dec); 1H NMR [(CD3)2SO] d
9.35 (s, 1H), 8.86 (d, J = 1.7 Hz, 1H), 8.39 (d, J = 8.9 Hz, 1H),
8.16 (t, J = 5.9 Hz, 1H), 8.00 (dd, J = 8.9, 1.7 Hz, 1H), 7.79 (d,
J = 8.8 Hz, 2H), 7.73 (d, J = 15.3 Hz, 1H), 7.10 (d, J = 15.3 Hz, 1H),
7.03 (d, J = 8.8 Hz, 2H), 4.71–4.63 (m, 2H), 4.61–4.53 (m, 1H),
4.17 (t, J = 5.7 Hz, 2H), 4.04–3.93 (m, 2H), 3.82 (q, J = 6.0 Hz,
2H), 3.59 (t, J = 4.6 Hz, 4H), 3.06 (q, J = 5.8 Hz, 2H), 2.72 (t,
J = 5.7 Hz, 2H), 1.35 (s, 18H), 4 protons not observed. Anal. Calcd
for C38H50BrN4O11PS: C, 51.76; H, 5.72; N, 6.35. Found: C, 51.80;
H, 5.91; N, 6.21.
A solution of 36d (250 mg, 0.28 mmol) and TFA (320 mg,
2.84 mmol) in CH2Cl2 (12 mL) was stirred for 20 h. Removal of sol-
vents gave 25 (250 mg, 100%) as a yellow powder: mp 178–182 °C
(dec); 1H NMR [(CD3)2SO] d 9.35 (s, 1H), 8.85 (s, 1H), 8.38 (d,
J = 8.8 Hz, 1H), 8.18–8.08 (m, 1H), 8.01 (d, J = 8.4 Hz, 1H), 7.86 (d,
J = 8.4 Hz, 2H), 7.76 (d, J = 15.2 Hz, 1H), 7.14 (d, J = 15.2 Hz, 1H),
7.11 (d, J = 8.4 Hz, 2H), 4.73–4.63 (m, 2H), 4.60–4.52 (m, 1H),
4.46–4.36 (m, 2H), 4.07–3.94 (m, 2H), 3.79 (q, J = 5.9 Hz, 4H),
3.02 (t, J = 5.6 Hz, 2H), 11 protons not observed (large water peak).
After D2O addition the following peaks became visible: d 3.54 (t,
J = 5.6 Hz, 2H), 3.50–3.10 (br m, 6H). Anal. Calcd for
References and notes
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