Nitro seco analogues of the duocarmycins containing sulfonate leaving groups as hypoxia-activated prodrugs for cancer therapy

Article abstract of DOI:10.1021/jm201717y

The synthesis of 19 (5-nitro-2,3-dihydro-1H-benzo[e]indol-1-yl)methyl sulfonate prodrugs containing sulfonate leaving groups and 7-substituted electron-withdrawing groups is reported. These were designed to undergo hypoxia-selective metabolism to form potent DNA minor groove-alkylating agents. Analogues 17 and 24, containing the benzyl sulfonate leaving group and a neutral DNA minor groove-binding side chain, displayed hypoxic cytotoxicity ratios (HCRs) of >1000 in HT29 human cancer cells in vitro in an antiproliferative assay. Four analogues maintained large HCRs across a panel of eight human cancer cell lines. In a clonogenic assay, 19 showed an HCR of 4090 in HT29 cells. Ten soluble phosphate preprodrugs were also prepared and evaluated in vivo, alone and in combination with radiation in SiHa human tumor xenografts at a nontoxic dose. Compounds 34 and 39 displayed hypoxic log₁₀ cell kills (LCKs) of 1.78 and 2.71, respectively, equivalent or superior activity to previously reported chloride or bromide analogues, thus showing outstanding promise as hypoxia-activated prodrugs.

Full text of DOI:10.1021/jm201717y

Article
pubs.acs.org/jmc
Nitro seco Analogues of the Duocarmycins Containing Sulfonate
Leaving Groups as Hypoxia-Activated Prodrugs for Cancer Therapy
Ralph J. Stevenson,* William A. Denny, Moana Tercel, Frederik B. Pruijn, and Amir Ashoorzadeh
Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019,
Auckland 1142, New Zealand
S
* Supporting Information
ABSTRACT: The synthesis of 19 (5-nitro-2,3-dihydro-1H-
benzo[e]indol-1-yl)methyl sulfonate prodrugs containing
sulfonate leaving groups and 7-substituted electron-with-
drawing groups is reported. These were designed to undergo
hypoxia-selective metabolism to form potent DNA minor
groove-alkylating agents. Analogues 17 and 24, containing the
benzyl sulfonate leaving group and a neutral DNA minor
groove-binding side chain, displayed hypoxic cytotoxicity ratios (HCRs) of >1000 in HT29 human cancer cells in vitro in an
antiproliferative assay. Four analogues maintained large HCRs across a panel of eight human cancer cell lines. In a clonogenic
assay, 19 showed an HCR of 4090 in HT29 cells. Ten soluble phosphate preprodrugs were also prepared and evaluated in vivo,
alone and in combination with radiation in SiHa human tumor xenografts at a nontoxic dose. Compounds 34 and 39 displayed
hypoxic log10 cell kills (LCKs) of 1.78 and 2.71, respectively, equivalent or superior activity to previously reported chloride or
bromide analogues, thus showing outstanding promise as hypoxia-activated prodrugs.
Figure 1) under hypoxia. The latter amines are potent
cytotoxins11 with a similar mechanism to the phenolic seco-
duocarmycins, alkylating at N3 of adenine in AT-rich regions of
DNA,9,12 presumably via an imino form (Figure S1 in the
Supporting Information) of the spirocyclohexadienone. In
contrast, the hypoxia-activated prodrug (HAP) nitro com-
pounds are relatively nontoxic, consistent with their inability to
undergo spirocyclization. The hypoxic cytotoxicity ratios [HCR
= IC50(oxic)/IC50(hypoxic)] of compounds such as 7 were
variable among cell lines, suggested due to their relatively low
one-electron reduction potentials [E(1) = −512 mV for 8,
Figure 1].13 Analogues bearing electron-withdrawing groups
(EWGs) on ring A (as defined in Figure 1) at the 7-position
had the greatest effect [E(1) values from −350 to −420 mV] in
raising the reduction potential. The 7-sulfonamide 9 (Figure 1)
had HCRs of 19−330 across an 11-cell line panel13 and also
presented the opportunity of further modification to provide
increased aqueous solubility. The sulfonamide alcohol 10
(Figure 1) showed HCRs from 10 to 250 across a 14-cell line
panel, and the corresponding phosphate preprodrug 11 (Figure
1, which rapidly hydrolyzed to 10 in plasma) showed significant
hypoxic log10 cell kills [additional cell kill, on a log scale, for the
combination of radiation and nitroCBI as compared to
radiation alone; log10 cell kills (LCKs) of 0.41−1.82] in mice
in combination with radiation (to sterilize the oxygenated cells)
in excision assays with five different human tumor xenograft
models.14 In SiHa cervical carcinoma xenografts, the combina-
tion of 11 and radiation gave complete tumor sterilization in 3/
INTRODUCTION
■
The DNA minor groove (adenine N3) alkylating agents
exemplified by the natural antitumor antibiotic duocarmycin
SA1 (1) and by a range of simpler synthetic precursor seco-form
a n a l o g u e s [ e . g . , h y d r o x y C B I ( s e c o - 1 , 2 , 9 , 9 a -
tetrahydrocyclopropa[c]benz[e]indol-4-one) (2, Figure 1)]2
are extremely potent (sub-nM IC50) cytotoxins. Their high
potency is considered to be due to their ability to become much
more reactive alkylators following initial noncovalent DNA
binding3 and to then form DNA monoadducts following a
single alkylation event, with these lesions generating little DNA
distortion and thus evading some routes of DNA repair. Some
analogues of this class [e.g., adozelesin (3) and carzelesin (4),
Figure 1]4,5 did undergo clinical trials for cancer treatment but
were too myelotoxic.
This class has become a major focus for the development of
selective prodrug forms, including those designed to release
these potent “effectors” by exploiting the specific physiological
property of hypoxia in solid tumors. A recent review outlines
the challenges, opportunities, and strategies in targeting tumor
hypoxia in this regard.6 A CBI analogue (5, Figure 1) with a
quinone “trigger” showed no oxic/hypoxic differential, which
was suggested to be due to prodrug instability.7 Other
analogues (e.g., 6, Figure 1) were tested against cells
overexpressing or lacking the two-electron reductase DT-
diaphorase, but selective hypoxic cytotoxicity was not
evaluated.8
We have previously shown9,10 that nitro analogues (e.g., 7,
Figure 1) of duocarmycins are hypoxia-selective, undergoing
oxygen-reversible one-electron reduction that ultimately results
in generation of the corresponding amino compounds (e.g., 7a,
Received: December 20, 2011
Published: February 16, 2012
© 2012 American Chemical Society
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Figure 1. (+)-Duocarmycin SA, adozelesin, carzelesin, and some previously reported analogues of the duocarmycins.
5 mice, whereas the corresponding aminoCBI 10 was
completely inactive. Similar trends were observed for 7-
carboxamide analogues.14 We recently showed that related
nitroCBIs bearing a bromide leaving group also provide high
HCRs (10−286) in HT29 colon carcinoma and SiHa cell lines,
with their phosphate preprodrugs also showing high hypoxic
LCKs (0.87−2.80) in SiHa human tumor xenografts.15
Finally,16 we have also compared chloride versus sulfonate
leaving groups (e.g., 12 and 13, Figure 1), since the latter are
better leaving groups than chloride, with significantly greater
nucleofugalities.17 In nitroCBIs lacking an A ring substituent,
sulfonates with neutral DNA minor groove-binding side
chains18 had consistently higher HCRs (IC50 ratios) than the
corresponding chloride compounds; a comparison of 7, 12, and
13 in SKOV3 ovarian carcinoma cells showed HCRs of 2.8, 39,
and 246, respectively.16 The general conclusion was that
sulfonate leaving groups offer a way of increasing the hypoxic
selectivity of nitroCBIs without introducing A ring substituents.
In this manuscript, we now systematically explore combinations
of sulfonate leaving groups, A ring E(1)-raising 7-EWGs, and
various DNA minor groove-binding side chains and evaluate
the products as HAPs in vitro and phosphate preprodrugs in
vivo.
shown in Schemes 11−14. The syntheses of precursors and
important intermediates are shown in Schemes 1−4.
We previously demonstrated that nitroCBIs containing a
chloride leaving group and A ring EWGs raise E(1) relative to
unsubstituted parent compounds, thus providing HAPs with
enhanced reductive metabolism and improved hypoxia-selective
cytotoxicity.9,13 While all A ring substituents (i.e., 6-, 7-, 8-, and
9-) raised E(1), the effect was strongest when the substituent
was in the 7-position [ΔE(1) = 83−159 mV]. Four examples of
7-substituents (NO2, SO2Me, CN, and SO2NH2) raised E(1)
values to above −400 mV. Although higher E(1) alone was not
sufficient to observe hypoxic selectivity and significant HCRs
(>10) were found for several nitroCBIs (e.g., 7-CONH2) with
E(1) < −400 mV,13 the combination of 7-substituents with H-
bond donor capacity and higher E(1) generally provided the
most highly selective compounds. In the current work, we
chose to limit our targets to 7-substituted nitroCBIs. This
provides a direct comparison of the new nitroCBIs as HAPs
with the 7-substituted prodrugs13,14 and 7-substituted phos-
phate preprodrugs14 previously reported. It was demonstrated
in the chloride series that while the final products are
structurally more complex than the corresponding unsubsti-
tuted analogues, their syntheses were generally easier.13 This
was a direct consequence of the A ring EWGs, which direct
nitration of the benzindoline intermediates to the desired 5-
regioisomer. Although this key step was not completely
selective, the major isomer was readily purified by recrystalliza-
tion or chromatography, and we postulated that this may also
be the case in the sulfonate series. In the current study, we
RESULTS AND DISCUSSION
■
Chemistry. The new nitroCBI prodrugs 14−32 described
in this manuscript are listed in Table 1, and their syntheses are
shown in Schemes 5−10. New nitroCBI phosphate prepro-
drugs 33−42 are listed in Table 3, and their syntheses are
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a
Table 1. Cytotoxicity Data for NitroCBIs 14−32 (Sulfonates) and Known14 Chlorides 10 and 144−146
b
IC50 (μM)
HT29
SiHa
c
c
compd
Z
R
Y
oxic
hypoxic
HCR
oxic
hypoxic
HCR
14
15
16
17
18
19
20
21
22
23
CN
A
B
C
A
D
E
OSO2CH2Ph
OSO2CH2Ph
OSO2CH2Ph
OSO2CH2Ph
OSO2CH2Ph
OSO2CH2Ph
OSO2CH2Ph
OSO2CH2Ph
OSO2CH2Ph
OSO2CH2Ph
Cl
2.6 0.6
2.5 0.3
3.4 0.3
9.2 2.4
2.2 0.1
7.0 0.5
0.043 0.01
0.49 0.06
0.62 0.03
0.016 0.006
0.038 0.01
0.17 0.02
0.22 0.05
0.43 0.22
0.61 0.22
0.57 0.19
3.2 0.2
94 24
0.65 0.14
0.71 0.12
0.57 0.07
0.99 0.29
0.53 0.08
2.1 0.5
0.026 0.007
0.035 0.008
0.019 0.002
0.18 0.02
0.12 0.03
0.046 0.005
0.24 0.04
0.096 0.015
0.16 0.05
0.21 0.06
ND
31 11
CN
5.2 1.2
5.4 0.3
1370 502
23
31
7
2
CN
CONH2
CONH2
CONH2
CONH2
CONH2
5.8 1.9
4.9 1.4
58
4
4
40
45
64
8
3
d
B
C
A
D
A
D
A
D
A
D
E
22
4.6 1.7
15
7
105
18.5 0.3
2.1 1.3
21 14
22 14
CONH(CH2)2OH
CONH(CH2)2OH
CONH(CH2)2OH
CONH(CH2)2OH
SO2NH2
3
13
12
2
6
0.90 0.11
2.3 0.23
ND
10
12
ND
20
70
10
3
3
4.3 0.2
5.7 1.8
e
144
1.7 0.5
e
145
Cl
12
35
2
4
0.11 0.07
0.042 0.007
0.090 0.029
485 196
1010 188
4.2 1.1
0.24 0.14
0.18 0.01
0.045 0.012
2.1 0.3
4
8
3
24
25
26
27
28
29
30
31
32
OSO2CH2Ph
OSO2CH2Ph
OSO2Me
13
2
SO2NH2
1.6 0.3
18
3
0.37 0.06
6.3 0.7
4.1 0.8
SO2NH(CH2)2OH
SO2NH(CH2)2OH
SO2NH(CH2)2OH
SO2NH(CH2)2OH
SO2NH(CH2)2OH
SO2NH(CH2)2OH
SO2NH(CH2)2OH
SO2NH(CH2)2OH
14
13
26
1
1
3
14
7
8.6 7.9
7.4 1.7
31 13
3.2 0.3
OSO2Me
2.2 0.7
0.38 0.17
0.32 0.03
0.11 0.008
0.055 0.012
0.37 0.03
0.039 0.003
3.5 2.5
17
27
8
4
OSO2Me
4.1 2.3
11
1
A
D
E
OSO2CH2Ph
OSO2CH2Ph
OSO2CH2Ph
OSO2CH2Ph
Cl
8.4 0.7
0.36 0.03
3.9 0.4
23
2
0.94 0.11
4.5 1.6
7.3 1.5
8.4 1.0
66 17
f
38
47
50
28
8
9.7 3.0
d
f
0.95 0.39
50
19
600 51
16
46 10
3
d
f
F
2
2
>20
<2.5
23
13
2
1
e
146
A
D
11
2
2.1 0.1
4
e
10
SO2NH(CH2)2OH
Cl
9.3
1
0.09 0.02
160 40
2.0 0.2
0.07 0.01
a
b
ND, not determined. Drug concentration to reduce cell density to 50% of that of controls, 5 days after 4 h of exposure (mean SEM for 3−9
c
d
experiments). HCR = IC50(oxic)/IC50(hypoxic). Values are means of intraexperiment ratios ( SEM for 3−6 experiments). Single determination.
e
f
Synthesis and cytotoxicity data of nitroCBIs with chloride leaving groups previously reported.14 Values are interexperimental.
b
a
Table 2. HCRs of Compounds 17, 19, 20, and 24 in a Panel of Human Tumor Cell Lines
c
d
d
d
d
d
d
d
d
compd
SKOV3
A549
C33A
76 62
184 149
H1299
86 45
H460
32 22
HCT116
5
HCT8
PC3
ND
17
19
20
24
85 17
ND
6.7 0.5
13
55 40
34
38
20
1
3
4
644 153
104 74
403 280
127 37
193 70
283 201
ND
ND
e
e
e
ND
93
6
255
>114
374
e
e
119 17
177
226 28
0.67
ND
278 72
a
b
c
ND, not determined. HCR = IC50(oxic)/IC50(hypoxic). Values are means of intraexperiment ratios ( SEM for 2−5 experiments). See Table 1.
d
SKOV3, ovarian carcinoma; A549, nonsmall-cell lung carcinoma; C33A, cervical carcinoma; H1299, lung carcinoma; H460, large-cell lung
e
carcinoma; HCT116, colorectal carcinoma; HCT8, colon adenocarcinoma; and PC3, prostate carcinoma. Single determination.
chose EWGs [Z in Table 1, i.e., CN,13 CONH2,13 SO2NH2,13
CONH(CH2)2OH,14 and SO2NH(CH2)2OH14] based on our
established syntheses for chloride analogues and/or improved
defined in Figure 1). Thus, once sulfonate leaving groups were
incorporated into the nitroCBI scaffolds, extra care was
required (e.g., use of dilute base and cold temperatures) to
avoid elimination of RSO2OH. Generally, we chose to
introduce sulfonate leaving groups late in the synthesis.
The intermediate 7-cyano acetate 47 was obtained by several
routes (Scheme 1), the best of which was halogen exchange of
43 to give iodide 44 followed by protecting group exchange to
give 45 and quantitative conversion to 47. This sequence
provided the highest overall yield with no separation issues.
Nitration of 47 was conducted using concentrated H2SO4 with
hypoxia-selective cytotoxicity for chloride analogues.9,13,14
A
series of DNA minor groove-binding side chains (R in Table 1)
were selected based on our previous work with chloride,13,14
bromide,15 and sulfonate16 analogues. We have recently noted
that alternative leaving groups (i.e., bromide15 or sulfonates16)
are especially susceptible to halide ion scrambling and
elimination in a basic environment. These potential side
reactions are enhanced by having EWGs in rings A and B (as
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a
Scheme 1. Synthesis of Intermediates 58, 63, 70, and 72
a
Reagents and conditions: (a) NaI, 2-butanone, reflux. (b) (i) TFA, CH2Cl2; (ii) TFAA, pyridine, 0 °C. (c) (From 45) AgOAc, HOAc, reflux. (d)
AcOCH2CHCHOAc, DMF, NaH, 0 °C. (e) (From 49) Bu3SnH, AIBN, benzene, reflux. (f) NIS, TsOH, CH3CN, 35 °C. (g) NaH, CH2
CHCH2Br, DMF, 0 °C. (h) (From 53) TEMPO, Bu3SnH, benzene, reflux. (i) Zn, HOAc, THF, H2O, reflux. (j) (From 55) Ac2O, Et3N, DMAP,
THF. (k) KNO3, 98% H2SO4, −10 °C. (l) Cs2CO3, KOH, THF, reflux. (m) H2O2, K2CO3, DMSO. (n) TFA, EDCI·HCl, TsOH, DMA. (o) (From
69) NaNO2, TFA, 0 °C. (p) THPO(CH2)2NH2, PyBOP, DIPEA, THF, 0 °C. (q) Cs2CO3, MeOH, 0 °C.
a slight excess of KNO3, giving the desired 5-NO2 isomer 57
(structure confirmed by 2D NMR experiments) (65%) and 9-
NO2 isomer 56 (5%). Short reaction times and low
temperatures in this and subsequent nitrations were required
to preserve the acetate protecting group. Base-promoted
cleavage of the trifluoroacetamide and acetate gave indoline
58. The neutral minor groove-binding side chain 5,6,7-
trimethoxyindole (TMI) was introduced using 5,6,7-trime-
thoxy-1H-indole-2-carboxylic acid and N-ethyl-N′-(3-
dimethylaminopropyl)carbodiimide hydrochloride (ED-
CI·HCl) as a coupling reagent (Scheme 5). As previously
reported, the EDCI-mediated reactions were most successful
under acidic conditions (catalytic anhydrous TsOH).13 The
alcohol 59 thus obtained was converted to the benzyl sulfonate
14 using benzylsulfonyl chloride in pyridine. Two polar neutral
side chains were introduced by first forming benzyl sulfonate 61
from Boc-protected indoline 60, followed by Boc-deprotection
to give 62, and then coupling with the carboxylic acids in the
final step giving benzyl sulfonates 15 and 16 (Scheme 5).
Five primary 7-carboxyamido benzyl sulfonates (Scheme 6)
were synthesized using the 5-nitro-7-cyano acetate 57 (Scheme
1) as a starting material. Attempts to hydrolyze nitrile 57 under
acidic conditions (90% H2SO4) led to loss of the acetate and
further reaction of the primary alcohol. Instead, hydrolysis was
achieved using H2O2 in DMSO under basic conditions19 that
also resulted in cleavage of the trifluoroacetamide and acetate to
provide 63 in good overall yield (Scheme 1). Coupling of 63
with 5,6,7-trimethoxy-1H-indole-2-carboxylic acid, 5-(2-
(dimethylamino)ethoxy)-1H-indole-2-carboxylic acid hydro-
chloride, introducing the 5-[(dimethylamino)ethoxy]indole
(DEI) basic side chain, and (E)-3-(4-methoxyphenyl)acrylic
acid, introducing the 4-methoxystyrene (MS) side chain, gave
7-carboxyamido alcohols 64−66 (Scheme 6). Sulfonylation of
64−66 gave the benzyl sulfonates 17−19. Benzyl sulfonates
19−21 were synthesized by an alternative route via Boc-
protected indoline alcohol 67, which was first converted to
benzyl sulfonate 68 before undergoing Boc deprotection and
couplings.
Two secondary 7-carboxamido benzyl sulfonates (22 and 23,
Scheme 7) were synthesized using 63 (Scheme 1) as a starting
material. Coupling with trifluoroacetic acid (TFA) allowed
selective protection of the indoline to give 69, which was
converted into carboxylic acid 70 by diazotization (NaNO2/
TFA). The secondary amide was formed by benzotriazol-1-yl-
oxytripyrrolidinophosphonium hexafluorophosphate (pyBOP)-
mediated coupling to give the tetrahydropyranyl (THP)-
protected alcohol 71. Coupling of the deprotected amine 72
with carboxylic acids introduced the TMI and DEI side chains
giving 73 and 74 (Scheme 7). Sulfonylation of the alcohols 73
and 74 gave the benzyl sulfonates 75 and 76. The final step
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a
Scheme 2. Synthesis of Intermediate 91
a
Reagents and conditions: (a) (i) KNO3, SO2Cl2, CH3CN, 0 °C; (ii) Bn2NH, Et3N, THF. (b) KOH, MeOH, H2O, reflux. (c) DPPA, tert-BuOH,
Et3N, reflux. (d) NIS, TsOH, CH2Cl2, CH3CN. (e) NaH, allyl bromide, DMF, 0 °C to rt. (f) (From 82) TEMPO, Bu3SnH, C6H6. (g) H2SO4, 0 °C.
(h) Boc2O, THF, reflux. (i) Zn, HOAc, THF, H2O, reflux. (j) Ac2O, Et3N, DMAP, THF. (k) (i) HCl, dioxane; (ii) TFAA, Et3N, THF, 0 °C. (l)
(From 88) KNO3, H2SO4, 0 °C. (m) Cs2CO3, MeOH, THF.
a
Scheme 3. Synthesis of Intermediates 99 and 131
a
Reagents and conditions: (a) NaI, 2-butanone, reflux. (b) AgOMs, CH3CN. (c) (i) ClSO3H, CH2Cl2, −78 to 15 °C; (ii) oxalyl chloride, DMF, 0
°C. (d) (From 97) KNO3, 98% H2SO4, −5 °C. (e) (i) (t-BuO)2(O)PO(CH2)2NH2, DIPEA, CH2Cl2, 0 °C; (ii) Cs2CO3, MeOH, H2O, 0 °C.
required the acid-mediated deprotection of the THP-protected
alcohols 75 and 76 giving 22 and 23, respectively.
86, followed by acetylation to 87 (using only a slight excess of
Ac2O to avoid acetylation of the sulfonamide) and a protecting
group exchange to give the trifluoroacetamide 88. Nitration
gave the desired 5-NO2 product 90 (28%) and a significant
amount of the 9-NO2 isomer 89 (17%). Base treatment of 90
removed both the trifluoroacetamide and the acetate groups to
give alcohol 91. Coupling of 91 with carboxylic acids
introduced the TMI (92) and DEI (93) side chains (Scheme
8). The final step required benzylsulfonylation of 92 to give 24
and of 93 to give 25.
Three secondary 7-sulfonamido methane sulfonates (26−28,
Scheme 9) were synthesized using known chloride 9413
(Scheme 3) as a starting material. Halide exchange of 94
gave iodide 95, and reaction of 95 with AgOMs gave methane
sulfonate 96. During the formation of 7-sulfonyl chloride 97
Two primary 7-sulfonamido benzyl sulfonates (24 and 25,
Scheme 8) were synthesized via a multistep route starting with
the synthesis20 of the protected dibenzylsulfonamide 78 from
known13 naphthoate 77 (Scheme 2). Ester hydrolysis, modified
Curtius reaction, iodination, and allylation followed by radical
ring cyclization21,22 produced (2,2,6,6-tetramethylpiperidin-1-
yl)oxyl (TEMPO) adduct23 83. Acid-mediated deprotection
removed both the Boc and the benzyl groups of 83 giving 7-
sulfonamide 84. Reductive cleavage of the TEMPO group at
this stage gave an unstable product, while the corresponding
trifluoroacetamide was also unstable to the cleavage conditions,
so the Boc protecting group was reinstalled to give 85. Zn/
HOAc-mediated reduction of the N−O bond then gave alcohol
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a
Scheme 4. Synthesis of Intermediates 114 and 135
a
Reagents and conditions: (a) NaH, AcOCHCH(CH2)OAc, DMF, 0 °C to rt. (b) Bu3SnH, AIBN, benzene, reflux. (c) (i) HCl(g), dioxane; (ii)
TFAA, py, 0 °C. (d) AgOAc, HOAc, 80 °C. (e) KOAc, 18-crown-6, HOAc, 100 °C. (f) AgOAc, HOAc. (g) (From 109) (i) ClSO3H, CH2Cl2, −80
to 5 °C; (ii) oxalyl chloride, DMF, −10 to 5 °C. (h) KNO3, 98% H2SO4, −12 °C. (i) (i) TBDMSO(CH2)2NH2, DIPEA, CH2Cl2, 0 °C; (ii) Cs2CO3,
MeOH, H2O, 0 °C to rt. (j) (i) (t-BuO)2(O)PO(CH2)2NH2, DIPEA, CH2Cl2, 0 °C; (ii) Cs2CO3, MeOH, H2O, 0 °C.
a
a
Scheme 5. Synthesis of Compounds 14−16 of Table 1
Scheme 6. Synthesis of Compounds 17−21 of Table 1
a
Reagents and conditions: (a) RCO2H, EDCI·HCl, TsOH, DMA. (b)
PhCH2SO2Cl, pyridine or Et3N, 0 °C. (c) Boc2O, dioxane, reflux. (d)
(i) TFA, CH2Cl2; (ii) RCO2H, EDCI·HCl, TsOH, DMA.
a
Reagents and conditions: (a) RCO2H, EDCI·HCl, TsOH, DMA. (b)
PhCH2SO2Cl, pyridine, CH2Cl2, 0 °C. (c) Boc2O, THF, reflux. (d)
TFA, CH2Cl2.
alcohol-protected (THP or TBDMS) ethanolamine, followed
by cleavage of the trifluoroacetamide, to give compounds 100−
102 (Scheme 9). Coupling of 7-sulfonamides 100−102 with
carboxylic acids introducing TMI, DEI, and MS side chains,
followed by TFA-promoted deprotection where required, gave
methane sulfonates 26−28. EDCI·HCl-mediated coupling of
100 with 4-methoxycinnamic acid resulted in a long reaction
time and significant exchange of methane sulfonate to chloride,
from methane sulfonate 96, a considerable amount of the
corresponding sulfonic acid formed as a precipitate. However,
this could be reconverted to 97 by treating a solution of the
acid in CH2Cl2/DMF with oxalyl chloride, ultimately giving a
94% yield of 97 from 96. Nitration of 97 gave the desired 5-
NO2 isomer 99 (57%) and some 9-NO2 isomer 98 (15%). The
sulfonamide was formed by reacting 99 with ethanolamine or
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a
a
Scheme 7. Synthesis of Compounds 22 and 23 of Table 1
Scheme 10. Synthesis of Compounds 29−32 of Table 1
a
a
Reagents and conditions: (a) RCO2H, EDCI·HCl, TsOH, DMA. (b)
Reagents and conditions: (a) RCO2H, EDCI·HCl, TsOH, DMA. (b)
PhCH2SO2Cl, pyridine, 0 °C. (c) MeSO3H, MeOH or TFA, CH2Cl2.
PhCH2SO2Cl, pyridine, 0 °C. (c) TFA, CH2Cl2, MeOH.
a
and the final TFA-mediated cleavage of the TBDMS group
gave 29−32.
The phosphate preprodrug 33 (Scheme 11) was prepared by
coupling of 63 (Scheme 1) with a phosphate ester-containing
Scheme 8. Synthesis of Compounds 24 and 25 of Table 1
a
Scheme 11. Synthesis of Compound 33 of Table 3
a
Reagents and conditions: (a) RCO2H, EDCI·HCl, TsOH, DMA. (b)
PhCH2SO2Cl, pyridine, 0 °C.
prompting the use of EDCI·TsOH with the THP-protected
101. In the event, the two-step route gave an improved overall
yield of 63% (up from 43%) for 28.
Four secondary 7-sulfonamido benzyl sulfonates (29−32,
Scheme 10) were synthesized by several routes (all Scheme 4).
The reaction of methane sulfonate 96 with AgOAc in acetic
acid proved to be the most convenient method of forming
acetate 109 (97%) (Scheme 4). Chlorosulfonylation followed
by nitration gave the desired 5-NO2 isomer 113 (62%) and 9-
NO2 isomer 112 (20%). The sulfonamide was introduced by
reacting 113 with alcohol-protected (TBDMS) ethanolamine
to give 114. The presence of base [diisopropylethylamine
(DIPEA) and subsequent addition of aqueous Cs2CO3] in this
step also removed both trifluoroacetamide and acetate groups.
Coupling of 114 with carboxylic acids introduced the TMI
(115), DEI (116), MS (117), and 5-[(morpholino)ethoxy]-
indole24 (MEI) (118) side chains (Scheme 10). Benzylsulfo-
nylation of alcohols 115−118 gave benzyl sulfonates 119−122,
a
Reagents and conditions: (a) 5-(2-((Di-tert-butoxyphosphoryl)oxy)-
ethoxy)-1H-indole-2-carboxylic acid, EDCI·HCl, TsOH, DMA. (b)
PhCH2SO2Cl, pyridine, CH2Cl2, 0 °C. (c) TFA, CH2Cl2.
carboxylic acid14 to give 123, followed by formation of benzyl
sulfonate 124 and TFA-mediated deprotection of the
phosphate ester.
Phosphates 34−42 (Table 3) are more water-soluble
preprodrugs of alcohols 22, 23, and 26−32 of Table 1, and
their syntheses (Schemes 12−14) are based on methodology
previously reported.14
In Vitro Cytotoxicity. In accordance with our previous
studies,14 hypoxia-selective cytotoxicity in vitro was assessed
with the cell-permeable alcohols and other 7-substituted
compounds rather than the highly polar phosphates. Thus
a
Scheme 9. Synthesis of Compounds 26−28 of Table 1
a
Reagents and conditions: (a) (From 99 to 100) (i) ethanolamine, CH2Cl2, 0 °C; (ii) Cs2CO3, MeOH, H2O, 0 °C. (b) (From 99 to 101) (i)
THPO(CH2)2NH2, CH2Cl2, 0 °C; (ii) Cs2CO3, MeOH, H2O, 0 °C. (c) (From 99 to 102) (i) TBDMSO(CH2)2NH2, Et3N, CH2Cl2, 0 °C; (ii)
Cs2CO3, MeOH, H2O, 0 °C. (d) RCO2H, EDCI·HCl or EDCI·TsOH, TsOH, DMA or CH2Cl2. (e) TFA, CH2Cl2, MeOH.
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a
Scheme 12. Synthesis of Compounds 34 and 35 of Table 3
a
Reagents and conditions: (a) (t-BuO)2(O)PO(CH2)2NH2, PyBOP, DIPEA, 0 °C. (b) Cs2CO3, MeOH, 0 °C. (c) RCO2H, EDCI·HCl, TsOH,
DMA. (d) PhCH2SO2Cl, pyridine, 0 °C. (e) TFA, CH2Cl2.
a
Scheme 13. Synthesis of Compounds 36−38 of Table 3
displaced from the line indicating HCR = 1 (equivalent toxicity
under both conditions). In HT29, the majority of the benzyl
sulfonates are more toxic under both oxic and hypoxic
conditions and also more hypoxia-selective than the methane
sulfonates. The most selective benzyl sulfonates 17 and 24,
containing a neutral TMI side chain, have very large HCRs of
1370 and 1010, respectively. As previously suggested,13 it is
possible that H-bond donor capacity of sulfonamides or
carboxamides enhances their reductase-substrate ability for
hypoxic activation. This feature combined with higher E(1) (7-
EWG in A ring) both contribute to the high selectivity. In
contrast, their previously reported chloride analogues (TMI
side chain) showed low HCRs of 2.8 and 3.6, respectively, while
the chlorides with a basic DEI side chain exhibited large HCRs
of 60 and 330, respectively.13 Other benzyl sulfonates exhibiting
significant hypoxic selectivity are 14, 18−21, 22, 25, 29, and 31
(HCR = 13−105), the majority of which have neutral side
chains. The two highly selective previously reported14 chlorides
10 (HCR = 160) and 145 (HCR = 485) containing a basic DEI
side chain, compare well on the basis of hypoxia-selectivity with
most of the benzyl sulfonates in HT29 cells.
The SAR in regards to leaving group and side chain in SiHa
cells is more difficult to interpret than in HT29 cells (Figure 2).
For instance, methane sulfonates 27 and 28 do show significant
HCRs (17 and 27), faring better than several benzyl sulfonates.
However, in general, the benzyl sulfonates are more selective.
Notably, two of the most selective, 30 (HCR = 66) and 32
(HCR = 600), contain basic side chains. Other benzyl
sulfonates exhibiting significant HCRs (19−70) are 14−16,
19−21, 24, and 31, none of which contain a basic side chain. In
SiHa cells, the selective chlorides14 10 (HCR = 46) and 145
(HCR = 20) were comparable to the benzyl sulfonates.
However, in both HT29 (17 and 24) and SiHa (32) cell lines,
we here provide examples of nitroCBIs containing a benzyl
sulfonate leaving group that appear markedly more hypoxia-
a
Reagents and conditions: (a) RCO2H, EDCI·HCl, TsOH, DMA. (b)
TFA, CH2Cl2.
compounds 14−32 were evaluated by measuring IC50 values
under oxic (20% O2) and hypoxic (<20 ppm O2) conditions
and deriving a HCR [HCR = IC50(oxic)/IC50(hypoxic)]
(Table 1 and Figures S2 and S3 in the Supporting
Information). Data for previously reported14 alcohols contain-
ing a chloride leaving group (10, 144−146) are presented for
comparison. These chlorides are suitable for comparison
because of their closely matching structures, and 10 and 145
are of interest as they provided the highest HCRs of the eight
sulfonamides or carboxamides in our previous report.14 In our
recent study on nitroCBIs containing sulfonate leaving groups
but lacking A ring substituents, we reported that sulfonates with
neutral side chains (e.g., TMI) showed consistently higher
HCRs than the corresponding chloride analogues using IC50-
based assays.16 We have also shown that nitroCBIs containing a
bromide leaving group follow a similar trend to chlorides in
regards to the side chain structure−activity relationship (SAR):
those with basic side chains provide larger HCRs.15
Figure 2 illustrates data from Table 1 where IC50 values
under oxic versus hypoxic conditions are plotted separately for
HT29 and SiHa. In both cell lines, almost all compounds are
a
Scheme 14. Synthesis of Compounds 39−42 of Table 3
a
Reagents and conditions: (a) RCO2H, EDCI·HCl, TsOH, DMA. (b) PhCH2SO2Cl, pyridine, 0 °C. (c) TFA, CH2Cl2.
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shared between SiHa and HT29. It appears that in general SiHa
is more sensitive than HT29. A combined summed ranking of
HCRs for the 19 sulfonates evaluated in HT29 and SiHa
indicates that compounds 24 and 20 receive the highest ranks
(Figure S4 in the Supporting Information).
Benzyl sulfonates 17, 19, 20, and 24 with high hypoxic
selectivity in either HT29 or SiHa cells were examined in a
panel of eight human tumor cell lines of various tissue origins
using an antiproliferative assay (Tables 2 and S1 in the
Supporting Information). All four compounds have neutral side
chains, and compound 20 contains an alcohol that could be
converted into a solubilizing phosphate group. Sizable HCRs in
all cell lines (with the exception of 24 in H460 cells) were
observed, indicating that the unidentified enzymes responsible
for nitro reduction are widely expressed and that the bulky
benzyl sulfonate does not hinder reduction to the point of
compromising hypoxic selectivity. The TMI side chain presents
no special advantages as compounds 19 and 20 exhibit HCRs
of 34−644 and 38−374, respectively, that is, at least as large as
for 17. While 17, 19, and 20 contain a 7-CONH2 substituent,
24 contains a 7-SO2NH2 group and, with the exception of
H460, similarly showed substantial HCRs of 20−278.
The question arises as to whether there is an HCR advantage
to combining both a bulky sulfonate leaving group and an A
ring EWG. For a given A ring substituent, a switch from
chloride to benzyl sulfonate almost always results in an
improvement in HCR when the side chain is neutral and a
reduction when the side chain is strongly basic, with the effect
mostly being a product of different hypoxic IC50 values. If a
bulky sulfonate leaving group is present, then a compound with
an A ring EWG will generally show an improvement in HCR
over a compound lacking such a substituent, but the effect
depends on both the side chain and the cell line. The two
effects (A ring substitution vs nature of the leaving group) have
such different SARs that making a generalization is difficult.
We have previously reported cytotoxicities in both an
antiproliferative assay (based on IC50 values) and a clonogenic
assay (based on C10 values).13,14 In the current study, the
cytotoxicity of 19 was further evaluated in HT29 cells and 16,
17, 20, 21, and 23 in SiHa cells using a clonogenic assay.27 All
compounds exhibit much higher toxicity under hypoxic (<20
ppm O2) than oxic (20% O2) conditions [HCR = C10(oxic)/
C10(hypoxic)], where cell killing (C10) was quantified as the
drug concentration required to lower the surviving fraction to
10% of that of controls. For instance, compound 19 provided a
remarkably high HCR of 4090 in HT29 cells (cf. HCR = 40 in
antiproliferative assay). The compounds evaluated in SiHa cells
showed HCRs of 41−422 (cf. HCR = 5.8−64 in antiprolifer-
ative assay), and although the clonogenic assay ratios are higher
(1.3−100-fold) than those of the antiproliferative assay, both
assays demonstrate similar trends.
In Vivo Activity. We have previously shown that nitroCBIs
incorporating a solubilizing phosphate group have excellent
aqueous solubility (in the mM range), that the phosphate
moiety of nitroCBI 11 is rapidly hydrolyzed in plasma to the
corresponding alcohol, and that 11 at a nontoxic dose of 42
μmol/kg provides significant hypoxic tumor cell kill in a range
of human tumor xenografts.14 Higher doses (>100 μmol/kg)
caused acute toxicity in some animals, and a maximum tolerated
dose was difficult to define.14 Thus, in the present study, the
phosphates were administered at an intravenous (iv) dose of 42
μmol/kg to SiHa tumor-bearing immunocompromised mice
either alone or 5 min after a single 15 Gy dose of ionizing
Figure 2. Cytotoxicity of sulfonates 14−32 (Table 1) and known14
chlorides 10 and 144−146 under oxic and hypoxic conditions in
HT29 and SiHa human tumor cell lines assessed using an
antiproliferative assay. Z, R, and Y: see Table 1. Dashed lines: HCR
= 1, 10, or 100.
selective than previously reported nitroCBIs containing a
chloride leaving group. It is noteworthy that 17 is much less
selective in SiHa than HT29 cells, and 32 is much less selective
in HT29 than SiHa cells. Figure S4 (Supporting Information)
illustrates cytotoxicity under oxic and hypoxic conditions in
SiHa versus HT29 cells for sulfonates 14−32 and known14
chlorides 10 and 144−146 of Table 1. For the oxic data, the
intercept is significantly different (from zero) and indicates that
HT29 IC50 values are, on average, 4.2-fold higher than in SiHa.
None of the compounds are more toxic in HT29 than in SiHa
under oxic conditions. There is no significant correlation under
hypoxia, and this suggests different reductases at work in SiHa
versus HT29. Furthermore, the spread of hypoxic IC50 data is
much greater in HT29 (∼1000-fold) than in SiHa (∼20-fold).
We recently reported25 that for chloride 10 in SiHa, the oxic
toxicity is a consequence of a low level of oxic reduction. If the
same mechanism is true for the new sulfonates, then Figure S4
in the Supporting Information suggests that oxic reductases are
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radiation (sufficient to sterilize aerobic tumor cells). At this
dose, no acute toxicity was observed for any of the analogues.
Eighteen hours later, the tumors were excised, and surviving
clonogens were assessed after 14 days to determine the number
of colony-forming cells per gram of tumor tissue (Figure S5 in
the Supporting Information).
The 7-carboxamide 33, a phosphate analogue of alcohol 20,
contains a benzyl sulfonate leaving group and a phosphate
moiety incorporated in the DNA-binding side chain. It
exhibited weak activity as a single agent (nitroCBI alone),
and the hypoxic LCK of radiation plus drug (vs radiation alone)
was relatively low (0.37), with neither measure being
statistically significant (Table 3 and Figure 3 where values for
leaving group. These compounds exhibited hypoxic LCKs of
0.18−2.71. An irradiation dose of 15 Gy provided 2.14 logs of
cell kill as compared to control (Table 3 for 39). When 39 was
combined with radiation, a greater cell kill was achieved (4.85
log), indicating elimination of radioresistant hypoxic cells
within the tumors by the nitroCBI. Compound 39 (with a
neutral TMI side chain) was easily the most active of all
compounds tested with no clonogens detected in four of four
treated tumors (the number quoted is calculated assuming one
colony detected for each tumor at the highest number of cells
plated; i.e., the true hypoxic LCK will be greater than the
reported value of 2.71). This compares favorably with the
previously reported14 two chloride comparators 148 (hypoxic
LCK = 0.54) and 11 (hypoxic LCK = 1.83). In the sulfonate
series, compounds 39 and 41 with neutral side chains provide
greater hypoxic LCKs than 40 (having a DEI side chain) and
42 (having a MEI side chain) with basic side chains, whereas in
the chloride series the activity is reversed with 13 (with a basic
DEI side chain) exhibiting the greater hypoxic LCK. The
moderate in vivo data for 40 and 42 is in contrast to their
alcohol analogues 30 and 32, which display high selectivity in
SiHa cells in vitro (Table 1). In a recent report,15 we have
shown that nitroCBIs containing a bromide leaving group
follow a similar trend to chlorides in regards to side chain with
the bromide analogue of 42 (with a basic side chain) providing
a hypoxic LCK of 2.80 in SiHa tumor-bearing mice and the
bromide analogue of 39 (with a neutral side chain) providing a
hypoxic LCK of 1.84.
Six phosphates were also assayed in H460 (human large-cell
lung tumor) xenografts at 75 μmol/kg, which again proved to
be a nontoxic dose (Table S2 in the Supporting Information).
Although large HCRs were observed in vitro (Tables 2 and S1
in the Supporting Information) for H460, the relatively high
IC50 values (Table S1 in the Supporting Information) indicate
that it seems to be a relatively resistant cell line. In the event,
hypoxic LCKs were mostly disappointing ranging from 0 to
0.60 with little evidence of any single agent activity. The most
active compound was the methane sulfonate 36 (having a TMI
side chain) with a hypoxic LCK of 0.60.
Figure 3. Antitumor activity of sulfonates 19 and 33−42 (Table 3)
and known14 chlorides 11, 147, and 148 in SiHa human tumor
xenografts assayed by tumor excision 18 h after iv dosing with
nitroCBI (42 or 13 μmol/kg for 19) only or γ-irradiation (15 Gy) plus
nitroCBI, followed by culturing for 14 days. Values are means SEMs
for groups of three mice (control or nitroCBI alone) or five mice
(radiation alone or radiation + nitroCBI). Values for control and
radiation alone are averages
SEMs from seven independent
experiments. Downward arrow: the number of tumors for which no
surviving clonogens could be detected within sample size and
detection limits of the assay. *, p < 0.05; **, p < 0.01; calculated
using intraexperiment controls.
CONCLUSIONS
control alone and radiation alone are averages of all
independent experiments). Interestingly, 7-carboxamide 19
with the neutral MS side chain and the absence of a phosphate
moiety provided a statistically significant hypoxic LCK of 1.16
at the lower dose (limited due to poor solubility) of 13 μmol/
kg.
Secondary 7-carboxamides 34 and 35, which incorporate a
phosphate in the carboxamide side chain and contain a benzyl
sulfonate leaving group, both provided statistically significant
hypoxic LCKs of 1.78 and 0.98, respectively. They also
exhibited weak but statistically significant single agent activity.
Although 34 (having a neutral TMI side chain) is superior to
35 (having a basic DEI side chain), it was not as active as the
previously reported14 chloride analogue 147 (having a basic
DEI side chain) with hypoxic LCK of 2.21.
■
The current study was performed to investigate SARs
influencing cytotoxicity and hypoxic selectivity for a series of
nitroCBI prodrugs and their more water-soluble phosphate
preprodrugs. The new compounds contain a sulfonate leaving
group (benzyl sulfonate or methane sulfonate) and a 7-EWG,
two structural modifications that were shown to independently
increase the hypoxic selectivity of related nitroCBIs.13−16
Although the target nitroCBIs are structurally more complex
than the unsubstituted parents,16 their syntheses proved
generally easier, as previously reported for chlorides.13,14
Sulfonate leaving groups16 are more prone to elimination
under basic conditions than the chloride leaving group;
however, if the sulfonate group was introduced late in the
synthesis and appropriate care was taken, this side reaction was
greatly minimized. Moreover, we found during syntheses of
nitroCBIs that the sulfonate leaving group was less susceptible
to elimination than a bromide.15 In general, the very good
hypoxic selectivity and antitumor activity observed for a
number of the sulfonates justified the synthetic effort.
Secondary 7-sulfonamides 36−38 contain a methane
sulfonate leaving group and provided a hypoxic LCK range of
0.33−0.71 (Table 1); 36 (with a neutral TMI side chain) and
38 (with a neutral MS side chain) are superior to 37 (with a
basic DEI side chain).
The more bulky benzyl sulfonates proved more hypoxia-
selective in vitro than methane sulfonates (Table 1 and Figure
2), as observed previously with A ring unsubstituted
Secondary 7-sulfonamides 39−42 incorporate a phosphate in
the sulfonamide side chain and contain a benzyl sulfonate
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sulfonates.16 A subset of the 7-substituted nitroCBIs with
neutral side chains (e.g., 17 and 24) was significantly more
hypoxia-selective than those with basic side chains, as also
shown previously for unsubstituted parents.16 Although a
subset of three primary 7-carboxamides and one 7-sulfonamide
with neutral side chains showed significant hypoxic selectivity
across a cell line panel, there were some exceptions to this
correlation. For instance, compound 32 (having a weakly basic
MEI side chain) showed the largest HCR (IC50 ratio) in SiHa
cells of all of the compounds tested. Unlike their primary
parents (e.g., 17 and 24), neutral side chains combined with
secondary 7-carboxamides (e.g., 22) or 7-sulfonamides (e.g., 26
and 29) did not appear to provide a marked increase in hypoxic
selectivity in vitro. This leaves open the possibility that the very
high HCRs seen for some sulfonates bearing a TMI side chain
are not a consequence of the fact that the side chain is neutral
but of some other as yet unexplained property. It would be
useful to understand toxicity differentials between sulfonate
prodrug and effector, made difficult by the intrinsic instability
of the latter due to the powerful leaving group effect of the
sulfonate, as better leaving groups might be expected to
increase cytotoxic potency. However, several sulfonates
displayed nanomolar hypoxic cytotoxicities (Table 1).
We have described two ways of increasing the hypoxic
selectivity of nitroCBIs: adding an appropriate EWG to the A
ring13,14 or incorporating a bulky leaving group.16 Although
both are capable of providing compounds with high HCRs, the
former is possibly more powerful since the A ring substituent
alone can give consistently high HCRs across a cell line
panel,13,14 whereas the effect of the sulfonate leaving group is
more cell line-dependent,16 as shown for 17 and 32. The
current study demonstrates that combining both effects can
give highly hypoxia-selective compounds.
The antitumor activity for the phosphate analogues also
demonstrated the previously observed correlation in regard to
side chain, with 34 and 39 (both having a benzyl sulfonate
leaving group and neutral TMI side chain) providing the
greatest hypoxic LCKs of the new compounds tested. Overall,
phosphates with neutral side chains provided greater hypoxic
LCKs than those with basic side chains, which was opposite to
that shown previously for chloride14 or bromide15 analogues.
Although we do not have an explanation for this observation, it
is potentially useful as basic side chains may compromise
distribution (by sequestration in acidic organelles) or
metabolism. The association between in vitro and in vivo
data is not complete; for instance, the alcohol analogue (29) of
phosphate 39 exhibited relatively moderate HCRs (IC50 ratios)
in vitro in the two cell lines in which it was examined.
Nevertheless, 39 was extremely active against hypoxic cells in
xenografts of one of these two cell lines. This outstanding in
vivo activity of 39 advocates it as a viable hypoxia-activated
preprodrug for cancer therapy, effective against the most
resistant tumor cell population.
(Dunedin, New Zealand). Melting points were determined on an
Electrothermal 2300 Melting Point Apparatus. NMR spectra were
1
obtained on a Bruker Avance 400 spectrometer at 400 MHz for H
and 100 MHz for 13C spectra. “Methods” indicate alternative routes to
the same compound.
Synthesis of Compounds in Table 1 and their Intermediates.
tert-Butyl 7-Cyano-1-(iodomethyl)-1H- benzo[e]indole-3(2H)-car-
boxylate (44) (Scheme 1). A solution of tert-butyl 1-
(chloromethyl)-7-cyano-1H-benzo[e]indole-3(2H)-carboxy-
late13 (43) (10.00 g, 29.2 mmol) in anhydrous 2-butanone
(100 mL) was treated with NaI (26.30 g, 175.5 mmol) and
heated under reflux for 5 days. The mixture was cooled to room
temperature and then evaporated to dryness, and the residue
was dissolved in CHCl3, washed with 5% aqueous sodium
disulfite and brine, dried, and concentrated under reduced
pressure. The residue was purified by column chromatography
on silica gel eluting with petroleum ether/EtOAc (5:1) and
further recrystallized from CH2Cl2/petroleum ether to give 44
1
as a pale yellow solid (12.40 g, 98%): mp 141−143 °C. H
NMR [(CD3)2SO]: δ 8.52 (d, J = 1.2 Hz, 1H), 8.11 (br, s, 1H),
8.04 (dd, J = 14.3, 8.7 Hz, 1H), 8.01 (dd, J = 8.8, 3.7 Hz, 1H),
7.73 (dd, J = 8.7, 1.5 Hz, 1H), 4.22−4.10 (m, 2H), 3.89 (dd, J =
11.1, 2.4 Hz, 1H), 3.68 (dd, J = 10.2, 3.0 Hz, 1H), 3.64−3.60
(m, 1H), 1.55 (s, 9H). Anal. calcd for C19H19IN2O2: C, 52.55;
H, 4.41; N, 6.45. Found: C, 52.83; H, 4.41; N, 6.50.
1-(Iodomethyl)-3-(2,2,2-trifluoroacetyl)-2,3-dihydro-1H-benzo[e]-
indole-7-carbonitrile (45) (Scheme 1). Method A. A solution of 44
(12.30 g, 28.3 mmol) in CH2Cl2 (350 mL) was treated with TFA (65
mL, 849.0 mmol) at 0 °C. The mixture was stirred overnight at room
temperature and concentrated under reduced pressure to give 1-
(iodomethyl)-7-cyano-1,2-dihydro-3H-benzo[e]indole as a red foam.
The residue was then dissolved in pyridine (200 mL), cooled to 0 °C,
and treated with trifluoroacetic anhydride (TFAA) (10 mL, 71.8
mmol). The mixture was stirred at 0 °C for 2 h and then poured into a
beaker of ice water, and the resulting precipitate was extracted into
CH2Cl2 (3×). The combined organic layers were washed with HCl (1
M), water, and brine, dried, and concentrated under reduced pressure.
The residue was purified by column chromatography on silica gel
eluting with petroleum ether/EtOAc (7:3) and further recrystallized
from CH2Cl2/petroleum ether to give 45 as a pale yellow solid (10.93
1
g, 90%): mp 187−189 °C. H NMR [(CD3)2SO]: δ 8.66 (d, J = 1.5
Hz, 1H), 8.44 (d, J = 9.0 Hz, 1H), 8.20 (d, J = 8.7 Hz, 1H), 8.12 (d, J
= 9.0, 1H), 7.86 (dd, J = 8.7, 1.5 Hz, 1H), 4.57 (dd, J = 10.8, 9.3 Hz,
1H), 4.37−4.33 (m, 1H), 4.28 (dt, J = 11.3, 1.8 Hz, 1H), 3.79 (dd, J =
10.4, 3.1 Hz, 1H), 3.72 (dd, J = 10.3, 5.9 Hz, 1H). HRMS (FAB) calcd
for C16H11F3IN2O (MH+) m/z, 430.9868; found, 430.9867.
(7-Cyano-3-(2,2,2-trifluoroacetyl)-2,3-dihydro-1H-benzo[e]indol-
1-yl)methyl Acetate (47) (Scheme 1). Method A. A solution of 45
(15.40 g, 35.8 mmol) in glacial acetic acid (300 mL) was treated with
AgOAc (18.80 g, 112.6 mmol) and heated under reflux for 48 h. The
mixture was cooled to room temperature and then evaporated to
dryness, and the dark brown residue was dissolved in EtOAc, passed
through a short pad of Celite, washed with water and brine, dried, and
concentrated under reduced pressure. The residue was purified by
chromatography eluting with petroleum ether/EtOAc (5:1, 4:1 and
3:1) to give 47 as a pale yellow solid (12.97 g, 100%): mp 158−160
°C. 1H NMR [(CD3)2SO]: δ 8.64 (d, J = 1.3 Hz, 1H), 8.43 (d, J = 9.0
Hz, 1H), 8.21 (d, J = 8.7 Hz, 1H), 8.12 (d, J = 9.0 Hz, 1H), 7.87 (dd, J
= 8.7, 1.6 Hz, 1H), 4.53 (dd, J = 10.8, 8.8 Hz, 1H), 4.46−4.36 (m,
2H), 4.35−4.29 (m, 1H), 4.28−4.22 (m, 1H), 1.89 (s, 3 H). 13C NMR
[(CD3)2SO]: δ 170.0, 153.6 (q, JCF = 37 Hz), 142.1, 135.0, 130.6,
130.3, 130.1, 127.5, 127.0, 125.1, 118.9, 118.0, 116.4 (q, JCF = 288 Hz),
107.7, 64.8, 52.2, 38.7, 20.3. Anal. calcd for C18H13F3N2O3: C, 59.67;
H, 3.62; N, 7.73. Found: C, 59.72; H, 3.72; N, 7.81.
EXPERIMENTAL SECTION
■
General Chemistry Methods. Final products were analyzed by
reverse-phase HPLC (Alltima C18 5 μm column, 150 mm × 3.2 mm;
Alltech Associated, Inc., Deerfield, IL) using an Agilent HP1100
equipped with a diode array detector. The mobile phase was 80%
CH3CN/20% H2O (v/v) in 45 mM HCO2NH4 at pH 3.5 and 0.5
mL/min. The purity was determined by monitoring at 330 50 nm
and was ≥95% for final products unless otherwise stated. The final
product purity was also assessed by combustion analysis carried out in
the Campbell Microanalytical Laboratory, University of Otago
(7-Cyano-5-nitro-3-(2,2,2-trifluoroacetyl)-2,3-dihydro-1H-benzo-
[e]indol-1-yl)methyl Acetate (57) and (7-Cyano-9-nitro-3-(2,2,2-
trifluoroacetyl)-2,3-dihydro-1H-benzo[e]indol-1-yl)methyl Acetate
(56) (Scheme 1). Compound 47 (2.36 g, 6.51 mmol) was ground
to a powder and dissolved in 98% H2SO4 (20 mL) at −10 °C and
stirred for 10 min. Anhydrous KNO3 (857 mg, 8.49 mmol) was added
portion wise, and the mixture was stirred for a further 7 min, then
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(14) (Scheme 5). α-Toluenesulfonyl chloride (39 mg, 0.20 mmol)
was added to a solution of 59 (85 mg, 0.17 mmol) in pyridine (2 mL)
at 0 °C, and the mixture was stirred at this temperature for 10 min.
More α-toluenesulfonyl chloride (19 mg, 0.10 mmol) was added, and
after a further 20 min, ice-cold water (5 mL) was added. The mixture
was stirred at 0 °C for 10 min, and then, the precipitated solid was
filtered off, washed with water, and dried in a vacuum desiccator. The
crude product was triturated with EtOAc at room temperature to give
14 as a yellow solid (85 mg, 77%): mp 219−222 °C. 1H NMR
[(CD3)2SO]: δ 11.62 (d, J = 1.6 Hz, 1H), 9.24 (s, 1H), 8.87 (d, J = 1.1
Hz, 1H), 8.31 (d, J = 8.8 Hz, 1H), 8.03 (dd, J = 8.8, 1.5 Hz, 1H),
7.23−7.12 (m, 6H), 6.98 (s, 1H), 4.92−4.86 (m, 1H), 4.62−4.49 (m,
6H), 3.94 (s, 3H), 3.84 (s, 3H), 3.82 (s, 3H). Anal. calcd for
C33H28N4O9S·H2O: C, 58.75; H, 4.48; N, 8.30. Found: C, 58.78; H,
4.28; N, 8.23.
poured into a beaker of ice water, and stirred for 10 min. The resulting
precipitate was extracted with EtOAc (3×), and the combined organic
layers were washed with water and brine, dried, and concentrated
under reduced pressure. The residue was purified by column
chromatography on silica gel eluting with petroleum ether/EtOAc
(3:2) and further recrystallized from CH2Cl2/MeOH to give 57 as a
yellow solid (1.72 g, 65%): mp 188−190 °C. 1H NMR [(CD3)2SO]: δ
9.10 (s, 1H, H-4), 8.90 (d, J = 1.0 Hz, 1H, H-6), 8.44 (d, J = 8.7 Hz,
1H, H-9), 8.09 (dd, J = 8.8, 1.5 Hz, 1H, H-8), 4.59 (dd, J = 11.0, 9.1
Hz, 1H, H-2), 4.52−4.41 (m, 3H, H-1, H-2, CH2OAc), 4.34 (dd, J =
11.0, 5.4 Hz, 1H, CH2OAc), 1.88 (s, 3H, COCH3). 13C NMR
[(CD3)2SO]: δ 170.0 (COCH3), 154.1 (q, JCF = 37 Hz, COCF3),
146.4 (C-5), 140.7 (C-3a), 134.1 (C-9b), 130.7 (C-9a), 130.1 (C-6),
128.9 (C-8), 126.2 (C-9), 121.6 (C-5a), 119.7 (CN), 118.3 (C-4),
115.5 (q, JCF = 288 Hz, CF3), 111.0 (C-7), 64.7 (CH2OAc), 52.5 (C-
2), 39.2 (C-1), 20.2 (COCH3) (full assignments and 5-nitro structure
determined by 2D NMR experiments: COSY, NOESY, HSQC,
HMBC). Anal. calcd for C18H12F3N3O5: C, 53.08; H, 2.97; N, 10.32.
Found: C, 53.03; H, 2.93; N, 10.28; and 56 as a yellow solid (140 mg,
5%): mp 176−178 °C (MeOH). 1H NMR [(CD3)2SO]: δ 9.00 (d, J =
1.6 Hz, 1H), 8.67 (d, J = 9.1 Hz, 1H), 8.62 (d, J = 1.6 Hz, 1H), 8.36
(d, J = 9.1 Hz, 1H), 4.55 (dd, J = 11.1, 8.5 Hz, 1H), 4.32−4.26 (m,
1H), 4.05 (dd, J = 11.1, 5.3 Hz, 1H), 3.96 (dd, J = 11.1, 5.8 Hz, 1H),
3.89−3.82 (m, 1H), 1.76 (s, 3H). 13C NMR [(CD3)2SO]: δ 169.6,
154.0 (q, JCF = 37 Hz), 146.6, 145.5, 139.9, 132.3, 131.7, 125.3, 123.9,
122.0, 119.4, 117.0, 115.5 (q, JCF = 288 Hz), 106.4, 65.3, 51.8, 39.6,
20.0. Anal. calcd for C18H12F3N3O5: C, 53.08; H, 2.97; N, 10.32.
Found: C, 53.15; H, 3.01; N, 10.27.
tert-Butyl 7-Cyano-1-(hydroxymethyl)-5-nitro-1H-benzo[e]-
indole-3(2H)-carboxylate (60) (Scheme 5). A solution of 58 (187
mg, 0.69 mmol) in anhydrous THF (30 mL) and under N2 was treated
with di-tert-butyl dicarbonate (606 mg, 2.78 mmol). The mixture was
heated under reflux for 24 h, cooled to room temperature, and then
evaporated to dryness, and the residue was purified by column
chromatography eluting with petroleum ether/EtOAc (4:1, 3:2 and
1:1) to give 60 as a yellow solid (171 mg, 67%): mp (petroleum ether/
1
EtOAc) 107−111 °C. H NMR [(CD3)2SO]: δ 8.87 (br, s, 1H), 8.82
(d, J = 1.1 Hz, 1H), 8.23 (d, J = 8.8 Hz, 1H), 7.93 (dd, J = 8.8, 1.5 Hz,
1H), 5.02 (m, 1H), 4.21−4.12 (m, 2H), 4.07−4.00 (m, 1H), 3.72−
3.69 (m, 1H), 3.63−3.59 (m, 1H), 1.56 (s, 9H). Anal. calcd for
C19H19N3O5·0.2EtOAc: C, 61.45; H, 5.37; N, 10.86. Found: C, 61.92;
H, 5.77; N, 10.51.
1-(Hydroxymethyl)-5-nitro-2,3-dihydro-1H-benzo[e]indole-7-car-
bonitrile (58) (Scheme 1). A solution of 57 (320 mg, 0.79 mmol) in
THF (20 mL) and water (6 mL) was treated with Cs2CO3 (768 mg,
2.36 mmol). The mixture was heated under reflux for 2 h, cooled to
room temperature, treated with KOH (142 mg, 2.53 mmol) in water
(1 mL), and heated under reflux for a further 3 h. The solution was
concentrated under reduced pressure, and the residue was dissolved in
EtOAc, washed with water and brine, dried, and concentrated under
reduced pressure. The crude product was purified by column
chromatography on silica gel using petroleum ether/EtOAc (1:1
then neat EtOAc) to give 58 as a red solid (206 mg, 97%): mp
(EtOAc/petroleum ether) 206−209 °C. 1H NMR [(CD3)2SO]: δ 8.55
(d, J = 1.4 Hz, 1H, H-6), 7.93 (d, J = 8.8 Hz, 1H, H-9), 7.75 (s, 1H, H-
4), 7.70 (dd, J = 8.8, 1.6 Hz, 1H, H-8), 6.71 (s, 1H, NH), 4.99 (t, J =
5.5 Hz, 1H, OH), 3.88−3.76 (m, 2H, H-1, H-2), 3.71 (dd, J = 9.6, 2.5
Hz, 1H, H-2), 3.65−3.58 (m, 1H, CH2OH), 3.51−3.42 (m, 1H,
CH2OH). 13C NMR [(CD3)2SO]: δ 151.8 (C-3a), 146.7 (C-5), 131.8
(C-9a), 129.8 (C-6), 127.3 (C-8), 127.0 (C-9b), 124.4 (C-9), 119.3
(CN), 116.7 (C-5a), 110.5 (C-4), 105.4 (C-7), 62.3 (CH2OH), 50.2
(C-2), 43.5 (C-1) (full assignments and structure determined by 2D
NMR experiments: COSY, NOESY, HSQC, HMBC). HRMS (EI)
calcd for C14H11N3O3 (M+) m/z, 269.08004; found, 269.07969.
1-(Hydroxymethyl)-5-nitro-3-(5,6,7-trimethoxy-1H-indole-2-car-
bonyl)-2,3-dihydro-1H-benzo[e]indole-7-carbonitrile (59) (Scheme
5). A mixture of 58 (101 mg, 0.38 mmol), 5,6,7-trimethoxy-1H-indole-
2-carboxylic acid (123 mg, 0.49 mmol), anhydrous TsOH (71 mg,
0.42 mmol), and EDCI·HCl (0.29 g, 1.52 mmol) in dry
dimethylacetamide (DMA) (2 mL) was stirred at room temperature
for 17 h. Dilute aqueous NaHCO3 was added, and the mixture was
stirred for several minutes. The precipitated solid was filtered off,
washed with water, and dried in a vacuum desiccator. The solid was
triturated with hot EtOAc to give 59 as a yellow solid (155 mg, 82%):
mp 254−256 °C. 1H NMR [(CD3)2SO]: δ 11.57 (d, J = 1.4 Hz, 1H),
9.26 (s, 1H), 8.87 (d, J = 1.1 Hz, 1H), 8.33 (d, J = 8.6 Hz, 1H), 7.98
(dd, J = 8.8, 1.5 Hz, 1H), 7.21 (d, J = 2.2 Hz, 1H), 6.98 (s, 1H), 5.06
(t, J = 5.6 Hz, 1H), 4.86−4.79 (m, 1H), 4.65 (dd, J = 10.5, 2.3 Hz,
1H), 4.22−4.16 (m, 1H), 3.94 (s, 3H), 3.83 (s, 3H), 3.81 (s, 3H),
3.78−3.71 (m, 1H), 3.70−3.63 (m, 1H). Anal. calcd for
C26H22N4O7·H2O: C, 60.00; H, 4.65; N, 10.76. Found: C, 59.94; H,
4.39; N, 10.75.
tert-Butyl 1-(((Benzylsulfonyl)oxy)methyl)-7-cyano-5-nitro-1H-
benzo[e]indole-3(2H)-carboxylate (61) (Scheme 5). Compound 60
(170 mg, 0.46 mmol) was dissolved in CH2Cl2 (15 mL) and pyridine
(149 μL, 1.84 mmol), cooled to 0 °C, and then treated with α-
toluenesulfonyl chloride (263 mg, 1.38 mmol). The mixture was
stirred at room temperature for a further 6 h, diluted with CH2Cl2,
washed with water and brine, dried, and concentrated under reduced
pressure. The residue was purified by column chromatography eluting
with petroleum ether/EtOAc (4:1) to give 61 as a yellow solid (201
mg, 83%): mp (petroleum ether/EtOAc) 139−141 °C. 1H NMR
[(CD3)2SO]: δ 8.82 (d, J = 1.2 Hz, 1H), 8.82 (br, s, 1H), 8.19 (d, J =
8.8 Hz, 1H), 7.97 (dd, J = 8.8, 1.5 Hz, 1H), 7.34−7.22 (m, 5H), 4.65
(br, s, 2H), 4.47 (d, J = 2.0 Hz, 1H), 4.45 (br, s, 1H), 4.41−4.35 (m,
1H), 4.27−4.22 (m, 1H), 4.08 (dd, J = 11.5, 2.7 Hz, 1H), 1.56 (s, 9H).
Anal. calcd for C26H25N3O7S: C, 59.65; H, 4.81; N, 8.03. Found: C,
59.68; H, 4.92; N, 8.15. Preparation of this intermediate using Et3N as
a base was also successful, but a lower yield (64%) was obtained.
(7-Cyano-5-nitro-2,3-dihydro-1H-benzo[e]indol-1-yl)methyl Phe-
nylmethanesulfonate (62) (Scheme 5). A solution of 61 (200 mg,
0.38 mmol) in CH2Cl2 (10 mL) was treated with TFA (883 μL, 11.5
mmol) and stirred overnight at room temperature. The mixture was
then poured into a beaker of ice water and extracted with CH2Cl2
(3×), and the combined organic layers were washed with water and
brine, dried, and concentrated under reduced pressure to give a red
solid. The residue was purified by column chromatography eluting
with petroleum ether/EtOAc (1:1) and further recrystallized from
CH2Cl2/i-Pr2O to give 62 as a red solid (120 mg, 75%): mp 154−156
°C. 1H NMR [(CD3)2SO]: δ 8.55 (d, J = 1.1 Hz, 1H), 7.86 (d, J = 8.7
Hz, 1H), 7.77 (s, 1H), 7.75 (dd, J = 8.8, 1.6 Hz, 1H), 7.34−7.30 (m,
5H), 6.87 (br s, 1H), 4.69 (s, 2H), 4.30−4.29 (m, 2H), 4.22−4.15 (m,
1H), 3.86−3.80 (m, 1H), 3.63 (dd, J = 10.5, 2.5 Hz, 1H). Anal. calcd
for C21H17N3O5S·0.2EtOAc: C, 59.37; H, 4.25; N, 9.53. Found: C,
59.07; H, 4.18; N, 9.59.
(7-Cyano-3-(5-(2-hydroxyethoxy)-1H-indole-2-carbonyl)-5-nitro-
2,3-dihydro-1H-benzo[e]indol-1-yl)methyl Phenylmethanesulfo-
nate (15) (Scheme 5). A solution of 62 (39 mg, 0.09 mmol) in dry
DMA (1 mL) was treated with anhydrous TsOH (17 mg, 0.10 mmol),
5-(2-hydroxyethoxy)-1H-indole-2-carboxylic acid (26 mg, 0.12 mmol),
and EDCI·HCl (71 mg, 0.37 mmol). The mixture was stirred at room
temperature for 2 h, then cooled to 0 °C, treated with 5% aqueous
NaHCO3, and stirred for further 10 min. The precipitate was collected
(7-Cyano-5-nitro-3-(5,6,7-trimethoxy-1H-indole-2-carbonyl)-2,3-
dihydro-1H-benzo[e]indole-1-yl)methyl phenylmethanesulfonate
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by filtration, washed with cold water and small portions of ice-cold
EtOAc, and then dried in a vacuum desiccator. The resulting solid was
recrystallized from CH2Cl2/diethyl ether in a cold room (+5 °C) to
filtered off, washed with water, and dried. The crude product was
triturated with EtOAc to give 17 as a yellow-orange solid (53 mg,
49%): mp 164−168 °C. 1H NMR [(CD3)2SO]: δ 11.67 (s, 1H), 9.14
(s, 1H), 8.86 (d, J = 1.1 Hz, 1H), 8.33 (br, s, 1H), 8.21 (d, J = 8.8 Hz,
1H), 8.15 (dd, J = 8.8, 1.5 Hz, 1H), 7.66 (br, s, 1H), 7.22−7.11 (m,
6H), 6.98 (s, 1H), 4.92−4.85 (m, 1H), 4.66−4.49 (m, 6H), 3.94 (s,
3H), 3.83 (s, 3H), 3.81 (s, 3H). Anal. calcd for
C33H30N4O10S·0.5H2O: C, 57.97; H, 4.57; N, 8.19. Found: C,
57.81; H, 4.51; N, 8.08.
1
give 15 as a yellow solid (30 mg, 53%): mp 239−242 °C. H NMR
[(CD3)2SO]: δ 11.74 (d, J = 1.6 Hz, 1H), 9.29 (s, 1H), 8.86 (d, J = 1.2
Hz, 1H), 8.32 (d, J = 8.8 Hz, 1H), 8.03 (dd, J = 8.8, 1.5 Hz, 1H), 7.44
(d, J = 8.9 Hz, 1H), 7.32−7.11 (m, 7H), 6.98 (dd, J = 8.9, 2.4 Hz, 1H),
4.95−4.90 (m, 1H), 4.84 (t, J = 5.5 Hz, 1H), 4.67−4.51 (m, 6H), 4.02
(t, J = 5.1 Hz, 2H), 3.76 (q, J = 5.3 Hz, 2H). Anal. calcd for
C32H26N4O8S·0.5CH2Cl2: C, 58.34; H, 4.07; N, 8.37. Found: C, 58.10;
H, 4.21; N, 8.53.
tert-Butyl 7-Carbamoyl-1-(hydroxymethyl)-5-nitro-1H-benzo[e]-
indole-3(2H)-carboxylate (67) (Scheme 6). A solution of 63 (110
mg, 0.38 mmol) in dioxane (30 mL) and under N2 was treated with di-
tert-butyl dicarbonate (498 mg, 2.28 mmol). The mixture was heated
under reflux for 24 h and concentrated to dryness under reduced
pressure. The residue was purified by column chromatography eluting
with CH2Cl2/MeOH (19:1) followed by trituration with i-Pr2O to
1-(Hydroxymethyl)-5-nitro-2,3-dihydro-1H-benzo[e]indole-7-car-
boxamide (63) (Scheme 1). A solution of 57 (1.71 g, 4.20 mmol) in
DMSO (35 mL) was treated with H2O2 (10 mL, 35% aqueous
solution, 103.4 mmol) and K2CO3 (2.10 g, 15.2 mmol). The mixture
was stirred overnight at room temperature and then poured into a
beaker of ice water and stirred for further 2 h. The solution was
extracted with EtOAc (3×), and the combined organic layers were
washed with water and brine and concentrated under reduced
pressure. The residue was triturated with diethyl ether and dried in
a vacuum oven (35 °C) for 48 h. Purification by column
chromatography eluting with EtOAc/MeOH (20:1) followed by
recrystallization from CH2Cl2/MeOH afforded 63 as dark red needles
(910 mg, 75%): mp 219−221 °C. 1H NMR [(CD3)2SO]: δ 8.63 (d, J
= 1.3 Hz, 1H), 8.06 (br, s, 1H), 7.93 (dd, J = 8.9, 1.7 Hz, 1H), 7.83 (d,
J = 8.8 Hz, 1H), 7.64 (s, 1H), 7.37 (br s, 1H), 6.38 (d, J = 1.4 Hz, 1H),
4.96 (t, J = 5.5 Hz, 1H), 3.87−3.80 (m, 1H), 3.78−3.62 (m, 3H),
3.47−3.39 (m, 1H). 13C NMR [(CD3)2SO]: δ 167.6, 150.4, 147.5,
131.8, 129.2, 126.7, 125.5, 123.4, 122.8, 117.3, 109.4, 62.1, 50.1, 43.9.
Anal. calcd for C14H13N3O4: C, 58.53; H, 4.56; N, 14.63. Found: C,
58.26; H, 4.69; N, 14.40.
1
give 67 as a yellow solid (97 mg, 66%): mp 199−201 °C. H NMR
[(CD3)2SO]: δ 8.82 (s, 1H), 8.72 (br, s, 1H), 8.21 (br, s, 1H), 8.11 (d,
J = 8.8 Hz, 1H), 8.07 (dd, J = 8.8, 1.5 Hz, 1H), 7.54 (br, s, 1H), 5.01
(t, J = 5.5 Hz, 1H), 4.15 (br, s, 1H), 4.17 (br, s, 1H), 4.04−3.98 (m,
1H), 3.71−3.76 (m, 1H), 3.57−3.62 (m, 1H), 1.56 (s, 9H). Anal. calcd
for C19H21N3O6·0.2MeOH·0.1i-Pr2O: C, 58.89; H, 5.74; N, 10.41.
Found: C, 58.86; H, 5.64; N, 10.03.
tert-Butyl 1-(((Benzylsulfonyl)oxy)methyl)-7-carbamoyl-5-nitro-
1H-benzo[e]indole-3(2H)-carboxylate (68) (Scheme 6). Compound
67 (95 mg, 0.25 mmol) was dissolved in CH2Cl2 (10 mL) and Et3N
(101 μL, 0.65 mmol), cooled to 0 °C, treated with α-toluenesulfonyl
chloride (124 mg, 0.65), and stirred for 20 min. The mixture was
diluted with CH2Cl2, washed with water and brine, dried, and
concentrated under reduced pressure. The residue was purified by
column chromatography eluting with CH2Cl2/MeOH (16:1) to give
1
68 as a yellow solid (128 mg, 95%): mp 173−175 °C. H NMR
TLC and product analysis showed that the order of reaction was as
follows: hydrolysis of the trifluoroacetamide, then nitrile, then acetate,
to give sequentially more polar products. If the reaction was worked up
at shorter times, the product with the acetate still intact could be
isolated by column chromatography eluting with EtOAc to give (7-
carbamoyl-5-nitro-2,3-dihydro-1H-benzo[e]indol-1-yl)methyl acetate
[(CD3)2SO]: δ 8.82 (s, 1H), 8.73 (br, s, 1H), 8.25 (br, s, 1H), 8.13−
8.07 (m, 2H), 7.57 (br, s, 1H), 7.34−7.22 (m, 5H), 4.65 (br, s, 2H),
4.48 (br, s, 1H), 4.47 (br, s, 1H), 4.38−4.33 (m, 1H), 4.26−4.21 (m,
1H), 3.14 (dd, J = 11.4, 2.3 Hz, 1H), 1.56 (s, 9H). Anal. calcd for
C26H27N3O8S: C, 57.66; H, 5.03; N, 7.76. Found: C, 57.79; H, 5.22;
N, 7.46.
1
as a red-orange solid: mp 211−214 °C. H NMR [(CD3)2SO]: δ
(E)-(7-Carbamoyl-3-(3(4-methoxyphenyl)acryloyl)-5-nitro-2,3-di-
hydro-1H-benzo[e]indol-1-yl)methyl phenylsulfonate (19) (Scheme
6). Method A. Compound 66 (140 mg, 0.31 mmol) was dissolved in
CH2Cl2 (6 mL) and pyridine (6 mL) at 38 °C in a warm water bath.
The solution was cooled to 0 °C, treated with α-toluenesulfonyl
chloride (298 mg, 1.56 mmol), and stirred for 10 min, then poured
into a beaker of ice water. The precipitate was collected by filtration,
washed with water, and dried in a vacuum desiccator. The residue was
purified by column chromatography eluting with CH2Cl2/MeOH
(99:1 and 49:1) and further triturated with small portions of CH2Cl2
and then ice-cold MeOH to provide 19 as a yellow solid (133 mg,
8.63 (s, 1H), 8.08 (br, s, 1H), 7.97 (d, J = 8.8 Hz, 1H), 7.86 (d, J = 8.8
Hz, 1H), 7.67 (s, 1H), 7.40 (br, s, 1H), 6.49 (br, s, 1H), 4.28 (dd, J =
10.0, 4.4 Hz, 1H), 4.13−4.00 (m, 2H), 3.82−3.74 (m, 1H), 3.65−3.57
(m, 1H), 2.00 (s, 3H). 13C NMR [(CD3)2SO]: δ 170.3, 167.5, 150.5,
148.0, 131.8, 129.3, 125.9, 124.7, 123.4, 122.4, 117.3, 109.5, 63.9, 50.4,
39.9, 20.6. Anal. calcd for C16H15N3O5: C, 58.36; H, 4.59; N, 12.76.
Found: C, 58.33; H, 4.66; N, 12.31.
1-(Hydroxymethyl)-5-nitro-3-(5,6,7-trimethoxy-1H-indole-2-car-
bonyl)-2,3-dihydro-1H-benzo[e]indole-7-carboxamide (64)
(Scheme 6). A mixture of 63 (62 mg, 0.22 mmol), 5,6,7-trimethoxy-
1H-indole-2-carboxylic acid (70 mg, 0.29 mmol), anhydrous TsOH
(41 mg, 0.24 mmol), and EDCI·HCl (165 mg, 0.88 mmol) in dry
DMA (1.5 mL) was stirred at room temperature for 16 h. Dilute
aqueous NaHCO3 was added, and the brown suspension was stirred at
room temperature for 20 min. The solid was filtered off, washed with
aqueous NaHCO3 and then water, dried, and triturated with hot
EtOAc to give 64 as a brown solid (87 mg, 77%): mp 242−246 °C
(dec). 1H NMR [(CD3)2SO]: δ 11.54 (s, 1H), 9.14 (s, 1H), 8.86 (d, J
= 1.3 Hz, 1H), 8.26 (br, s, 1H), 8.22 (d, J = 8.8 Hz, 1H), 8.13 (dd, J =
8.8, 1.6 Hz, 1H), 7.58 (br s, 1H), 7.19 (s, 1H), 6.98 (s, 1H), 5.07 (t, J
= 5.2 Hz, 1H), 4.84−4.76 (m, 1H), 4.65 (dd, J = 10.5, 2.2 Hz, 1H),
4.19−4.12 (m, 1H), 3.95 (s, 3H), 3.83 (s, 3H), 3.81 (s, 3H), 3.80−
3.75 (m, 1H), 3.68−3.62 (m, 1H). Anal. calcd for C26H24N4O8·H2O:
C, 57.99; H, 4.87; N, 10.40. Found: C, 58.31; H, 4.75; N, 10.36.
(7-Carbamoyl-5-nitro-3-(5,6,7-trimethoxy-1H-indole-2-carbon-
yl)-2,3-dihydro-1H-benzo[e]indol-1-yl)methyl Phenylmethanesulfo-
nate (17) (Scheme 6). α-Toluenesulfonyl chloride (62 mg, 0.32
mmol) was added to a solution of 64 (85 mg, 0.16 mmol) in pyridine
(5 mL) at 0 °C, and the mixture was stirred at this temperature for 20
min. More α-toluenesulfonyl chloride (14 mg, 0.08 mmol) was added,
and after a further 15 min, ice water (10 mL) was added. The mixture
was stirred at 0 °C for several minutes, and the precipitated solid was
1
71%): mp 207−209 °C. H NMR [(CD3)2SO]: δ 9.22 (s, 1H), 8.83
(s, 1H), 8.27 (br, s, 1H), 8.17−8.12 (m, 2H), 7.81−7.78 (m, 2H), 7.74
(d, J = 15.3 Hz, 1H), 7.59 (br, s, 1H), 7.26−7.21 (m, 5H), 7.07−7.02
(m, 3H), 4.66−4.59 (m, 3H), 4.56−4.51 (m, 4H), 3.83 (s, 3H).
HRMS (FAB) calcd for C31H28N3O8S (MH+) m/z, 602.1597; found,
602.1595. HPLC purity, 94.1%.
1-(Hydroxymethyl)-5-nitro-3-(2,2,2-trifluoroacetyl)-2,3-dihydro-
1H-benzo[e]indole-7-carboxamide (69) (Scheme 1). A solution of 63
(586 mg, 2.04 mmol) in DMA (10 mL) was treated with anhydrous
TsOH (386 mg, 2.24 mmol), TFA (616 μL, 8.16 mmol), and
EDCI·HCl (1.56 g, 8.14 mmol). The mixture was stirred at room
temperature overnight, then cooled to 0 °C, treated with 5% aqueous
NaHCO3, and stirred for further 10 min. The resulting solution was
poured into a beaker of ice water and extracted with EtOAc, and the
combined organic layers were washed with brine, dried with Na2SO4,
and concentrated under reduced pressure to give an orange solid. The
solid was triturated with i-Pr2O and small portions of cold diethyl
ether to provide 69 as an orange solid (615 mg, 79%): mp 249−251
1
°C. H NMR [(CD3)2SO]: δ 9.01 (s, 1H), 8.87 (d, J = 1.3 Hz, 1H),
8.29 (br, s, 1H), 8.28 (d, J = 8.9 Hz, 1H), 8.17 (dd, J = 8.8, 1.6 Hz,
1H), 7.62 (br, s, 1H), 5.05 (t, J = 5.4 Hz, 1H), 4.61−4.49 (m, 2H),
2793
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4.24−4.20 (m, 1H), 3.83−3.78 (m, 1H), 3.75−3.69 (m, 1H). Anal.
calcd for C16H12F3N3O5: C, 50.14; H, 3.16; N, 10.96. Found: C, 50.39;
H, 3.45; N, 10.76.
1.80−1.71 (m, 1H), 1.67−1.60 (m, 1H), 1.50−1.44 (m, 4H). HRMS
(FAB) calcd for C33H36N4O10 (M+) m/z, 648.2431; found, 648.2423.
(5-Nitro-7-((2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)carbamoyl)-
3-(5,6,7-trimethoxy-1H-indole-2-carbonyl)-2,3-dihydro-1H-benzo-
[e]indol-1-yl)methyl Phenylmethanesulfonate (75) (Scheme 7). A
solution of 73 (137 mg, 0.21 mmol) in dry pyridine (10 mL) and was
treated with α-toluenesulfonyl chloride (121 mg, 0.63 mmol) at 0 °C.
The mixture was stirred for 10 min and then poured into a beaker of
ice water and stirred for further 10 min. The solution was extracted
with CH2Cl2 (3×), and the combined organic layers were washed with
water and brine, dried, and concentrated under reduced pressure. The
residue was purified by column chromatography eluting with CH2Cl2/
MeOH (19:1 and 9:1) and further recrystallized from CH2Cl2/diethyl
ether in cold room (+5 °C) to give 75 as a yellow solid (113 mg,
67%): mp (CH2Cl2/diethyl ether) 126−129 °C. 1H NMR
[(CD3)2SO]: δ 11.58 (d, J = 1.6 Hz, 1H), 9.13 (br, s, 1H), 8.85 (t,
J = 5.4 Hz, 1H), 8.81 (d, J = 1.1 Hz, 1H), 8.21 (d, J = 8.8 Hz, 1H),
8.12 (dd, J = 8.8, 1.4 Hz, 1H), 7.21−7.12 (m, 6H), 6.98 (br, s, 1H),
4.90−4.85 (m, 1H), 4.64−4.53 (m, 7H), 3.94 (s, 3H), 3.84 (s, 3H),
3.82 (s, 3H), 3.80−3.75 (m, 2H), 3.60−3.42 (m, 4H), 1.80−1.71 (m,
1H), 1.67−1.61 (m, 1H), 1.50−1.45 (m, 4H). HRMS (FAB) calcd for
C40H42N4O12S (M+) m/z, 802.2520; found, 802.2526.
(7-((2-Hydroxyethyl)carbamoyl)-5-nitro-3-(5,6,7-trimethoxy-1H-
indole-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-1-yl)methyl Phe-
nylmethanesulfonate (22) (Scheme 7). A solution of 75 (28 mg,
0.035 mmol) in MeOH (2 mL) was treated with methane sulfonic acid
(4 drops), stirred at room temperature for 1 h, and concentrated under
reduced pressure. The residue was dissolved in CH2Cl2, washed with
water and brine, dried, and concentrated under reduced pressure. The
crude product (88% HPLC purity) was further purified by preparative
HPLC (Synergi-Max RP column; flow rate, 14 mL/min; pump 1:
H2O/TFA, pH 2.5, gradient 60%−1%−1%−60%; pump 2: CH3CN/
H2O, 9:1, gradient 40%−99%−99%−40%) to provide 22 as a yellow
solid (13 mg, 52%): mp 126−130 °C. 1H NMR [(CD3)2SO]: δ 11.59
(d, J = 1.7 Hz, 1H), 9.12 (br, s, 1H), 8.82 (d, J = 1.2 Hz, 1H), 8.77 (t, J
= 5.5 Hz, 1H), 8.21 (d, J = 8.8 Hz, 1H), 8.14 (dd, J = 8.8, 1.5 Hz, 1H),
7.23−7.12 (m, 6H), 6.98 (s, 1H), 4.90−4.85 (m, 1H), 4.75 (t, J = 5.6
Hz, 1H), 4.53 (m, 5H), 3.94 (s, 3H), 3.84 (s, 3H), 3.82 (s, 3H), 3.60−
3.55 (m, 2H), 3.43−3.39 (m, 3H). HRMS (FAB) calcd for
C35H35N4O11S (MH+) m/z, 719.2023; found, 719.2017. HPLC purity,
96.9%. Attempted deprotection of the THP group by TFA/CH2Cl2
(1:1) did not go to completion, and a byproduct was formed.
1-(Hydroxymethyl)-5-nitro-3-(2,2,2-trifluoroacetyl)-2,3-dihydro-
1H-benzo[e]indole-7-carboxylic acid (70) (Scheme 1). Compound
69 (288 mg, 0.75 mmol) was dissolved in TFA (10 mL) at room
temperature, cooled to 0 °C in an ice water bath, and stirred for 5 min.
The mixture was treated with NaNO2 (569 mg, 8.25 mmol), further
stirred for 10 min, and then poured into a beaker of ice water with
stirring. The precipitate was collected by filtration, dissolved in EtOAc,
washed with water and brine, dried, and concentrated under reduced
pressure. The residue was triturated with small portions of cold
CH2Cl2 to provide 70 as a yellow solid (241 mg, 84%): mp 246−250
°C. 1H NMR [(CD3)2SO]: δ 13.44 (br s, 1H), 9.06 (s, 1H), 9.02 (d, J
= 1.1 Hz, 1H), 8.28 (d, J = 8.8 Hz, 1H), 8.19 (dd, J = 8.8, 1.5 Hz, 1H),
5.06 (br, s, 1H), 4.63−4.49 (m, 2H), 4.24−4.21 (m, 1H), 3.83−3.80
( m , 1 H ) , 3 . 7 4 − 3 . 7 0 ( m , 1 H ) . A n a l . c a l c d f o r
C16H11F3N2O6.0.1CH2Cl2: C, 49.24; H, 2.87; N, 7.13. Found: C,
49.32; H, 3.06; N, 7.39.
1-(Hydroxymethyl)-5-nitro-N-(2-((tetrahydro-2H-pyran-2-yl)oxy)-
ethyl)-3-(2,2,2-trifluoroacetyl)-2,3-dihydro-1H-benzo[e]indole-7-
carboxamide (71) (Scheme 1). A solution of 70 (110 mg, 0.29 mmol)
in anhydrous THF (15 mL) was treated with DIPEA (150 μL, 0.86
mmol) at room temperature. The solution was cooled to 0 °C and
treated with 2-(tetrahydro-2H-2-pyranyloxy)ethanamine26 (46 mg,
0.32 mmol) and PyBOP (194 mg, 0.37 mmol), stirred at room
temperature for 1 h, and then evaporated to dryness. The residue was
dissolved in EtOAc, washed with ice water and brine, dried, and
concentrated under reduced pressure, and the crude product was
purified by column chromatography eluting with diethyl ether/MeOH
(24:1) to provide 71 as a yellow solid (110 mg, 74%): mp 122−124
1
°C. H NMR [(CD3)2SO]: δ 9.01 (s, 1H), 8.87 (t, J = 5.4 Hz, 1H),
8.83 (d, J = 1.3 Hz, 1H), 8.27 (d, J = 8.9 Hz, 1H), 8.13 (dd, J = 8.8, 1.6
Hz, 1H), 5.05 (t, J = 5.4 Hz, 1H), 4.63 (t, J = 3.6 Hz, 1H), 4.58−4.48
(m, 2H), 4.23−4.20 (m, 1H), 3.83−3.69 (m, 4H), 3.59−3.49 (m, 3H),
3.46−3.40 (m, 1H), 1.78−1.72 (m, 1H), 1.66−1.61 (m, 1H), 1.50−
1.43 (m, 4H). HRMS (ESI) calcd for C23H24F3N3NaO7 (MNa+) m/z,
534.1459; found, 534.1454.
1-(Hydroxymethyl)-5-nitro-N-(2-((tetrahydro-2H-pyran-2-yl)oxy)-
ethyl)-2,3-dihydro-1H-benzo[e]indole-7-carboxamide (72) (Scheme
1). A solution of 71 (160 mg, 0.31 mmol) in MeOH (16 mL) was
cooled to 0 °C in an ice water bath and stirred for 10 min. The
solution was then treated with Cs2CO3 (102 mg, 0.31 mmol), stirred
for further 20 min, and then evaporated to dryness. The residue was
purified by column chromatography eluting with CH2Cl2/MeOH
(19:1) and followed by recrystallization from CH2Cl2 to provide 72 as
Methyl 6-(N,N-dibenzylsulfamoyl)-2-naphthoate (78) (Scheme
2). Solid KNO3 (9.32 g, 92 mmol) and then SO2Cl2 (7.40 mL, 92
mmol) were added to a suspension of methyl 6-(dimethylcarbamoylth-
io)-2-naphthoate13 (77) (11.11 g, 38.4 mmol) in CH3CN (250 mL) at
0 °C, and the mixture was stirred at this temperature for 3 h. More
SO2Cl2 (7.40 mL, 92 mmol) was added, and after a further 30 min at 0
°C, the mixture was cautiously diluted with ice-cold aqueous NaHCO3.
The mixture was extracted with EtOAc, and the extract was washed
with aqueous NaHCO3 and brine and then dried (MgSO4) and
evaporated. The crude sulfonyl chloride (9.9 g, ca. 35 mmol) was
dissolved in THF (100 mL), and Bn2NH (8.0 mL, 41.8 mmol) and
Et3N (5.8 mL, 41.8 mmol) were added. The mixture was stirred at
room temperature for 4 days, then diluted with aqueous HCl (2 N),
and extracted with EtOAc. The EtOAc extract was washed with brine
and then dried (MgSO4) and evaporated. The resulting solid was
triturated with hot MeOH (350 mL) to give 78 as a cream solid (5.65
1
a red solid (109 mg, 85%): mp (CH2Cl2) 119−121 °C. H NMR
[(CD3)2SO]: δ 8.61 (m, 1H), 8.59 (d, J = 1.1 Hz, 1H), 7.90 (dd, J =
8.9, 1.6 Hz, 1H), 7.85 (d, J = 8.9 Hz, 1H), 7.64 (s, 1H), 6.37 (d, J = 1.1
Hz, 1H), 4.95 (t, J = 5.5 Hz, 1H), 4.61 (t, J = 3.6 Hz, 1H), 3.86−3.62
(m, 6H), 3.57−3.52 (m, 1H), 3.49−3.40 (m, 4H), 1.80−1.71 (m, 1H),
1.66−1.60 (m, 1H), 1.50−1.42 (m, 4H). Anal. calcd for C21H25N3O6:
C, 60.71; H, 6.07; N, 10.11. Found: C, 60.65; H, 6.13; N, 9.80.
1-(Hydroxymethyl)-5-nitro-N-(2-((tetrahydro-2H-pyran-2-yl)oxy)-
ethyl)-3-(5,6,7-trimethoxy-1H-indole-2-carbonyl)-2,3-dihydro-1H-
benzo[e]indole-7-carboxamide (73) (Scheme 7). A solution of 72
(147 mg, 0.35 mmol) in DMA (2 mL) was treated with anhydrous
TsOH (67 mg, 0.39 mmol), 5,6,7-trimethoxy-1H-indole-2-carboxylic
acid (116 mg, 0.46 mmol), and EDCI·HCl (270 mg, 1.41 mmol). The
mixture was stirred at room temperature for 2 h, then cooled to 0 °C,
treated with 5% aqueous NaHCO3, and stirred for a further 10 min.
The precipitate was collected by filtration, washed with cold water,
diethyl ether, and a small portion of cold MeOH, and then dried in a
vacuum desiccator. The residue was triturated with diethyl ether to
1
g, 33%). H NMR [(CD3)2SO]: δ 8.77 (s, 1H), 8.61 (d, J = 1.6 Hz,
1H), 8.37 (d, J = 8.8 Hz, 1H), 8.28 (d, J = 8.8 Hz, 1H), 8.13 (dd, J =
8.6, 1.7 Hz, 1H), 7.97 (dd, J = 8.7, 1.9 Hz, 1H), 7.22−7.15 (m, 6H),
7.13−7.06 (m, 4H), 4.39 (s, 4H), 3.96 (s, 3H). 1H NMR analysis also
showed the presence of some impurities, one of which was later
identified as the corresponding 5-chloro analogue [methyl 5-chloro-6-
(N,N-dibenzylsulfamoyl)-2-naphthoate]. An analytically pure sample
of 78 was obtained by recrystallization from MeOH, mp 150−152 °C.
Anal. calcd for C26H23NO4S: C, 70.09; H, 5.20; N, 3.14. Found: C,
70.14; H, 5.10; N, 3.09. A second crop of 78 was obtained from the
MeOH triturate (1.29 g, 8%).
1
give 73 as a yellow solid (154 mg, 68%): mp 243−247 °C. H NMR
[(CD3)2SO]: δ 11.53 (d, J = 1.6 Hz, 1H), 9.14 (br, s, 1H), 8.83−8.81
(m, 2H), 8.22 (d, J = 8.8 Hz, 1H), 8.09 (dd, J = 8.8, 1.5 Hz, 1H), 7.18
(d, J = 2.1 Hz, 1H), 6.98 (br, s, 1H), 5.06 (t, J = 5.6 Hz, 1H), 4.83−
4.77 (m, 1H), 4.67−4.62 (m, 2H), 4.18−4.12 (m, 1H), 3.94 (s, 3H),
3.83 (s, 3H), 3.81 (s, 3H), 3.80−3.76 (m, 3H), 3.467−3.41 (m, 5H),
2794
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6-(N,N-Dibenzylsulfamoyl)-2-naphthoic Acid (79) (Scheme 2). A
solution of KOH (4.52 g, 81 mmol) in water (100 mL) was added to a
suspension of ester 78 (6.92 g, 15.5 mmol) in MeOH (200 mL), and
the mixture was stirred at reflux for 30 min, by which time all of the
suspended solid had dissolved. The mixture was cooled and acidified
with aqueous HCl (2 N, 60 mL) and then diluted with water. After the
mixture was stirred overnight, the precipitated solid was filtered off,
washed with water, and dried to give 79 as a white solid (6.21 g, 93%),
(m, 5H), 4.06−3.88 (m, 1H), 1.51 and 1.29 (2 s, 9H). HRMS (FAB)
calcd for C32H34IN2O4S (MH+) m/z 669.12841; found, 669.12875.
tert-Butyl 7-(N,N-Dibenzylsulfamoyl)-1-(((2,2,6,6-tetramethylpi-
peridin-1-yl)oxy)methyl)-1H-benzo[e]indole-3(2H)-carboxylate (83)
(Scheme 2). TEMPO (1.91 g, 12.2 mmol) was added to a solution of
iodide 82 (6.86 g, 10.2 mmol) in dry benzene (350 mL) under
nitrogen at room temperature. The mixture was stirred at reflux, and
Bu3SnH (97%, 3.29 mL, 12.2 mmol) was added dropwise over 5 min.
After 10 min, the mixture was cooled to room temperature. The
addition of TEMPO and Bu3SnH was repeated under the same
conditions a further two times until TLC analysis indicated complete
consumption of the starting material. The mixture was evaporated, and
the residue was purified by column chromatography (eluting with
EtOAc/petroleum ether, 1:20 then 1:15 then 1:10). The product-
containing fractions were evaporated to dryness and triturated with
MeOH. A small quantity of white solid was filtered off, and the filtrate
1
mp 260−261 °C. H NMR [(CD3)2SO]: δ 13.31 (br s, 1H), 8.74 (s,
1H), 8.60 (d, J = 1.5 Hz, 1H), 8.35 (d, J = 8.9 Hz, 1H), 8.25 (d, J = 8.8
Hz, 1H), 8.12 (dd, J = 8.6, 1.6 Hz, 1H), 7.95 (dd, J = 8.7, 1.9 Hz, 1H),
7.22−7.16 (m, 6H), 7.13−7.08 (m, 4H), 4.39 (s, 4H). Anal. calcd for
C25H21NO4S: C, 69.59; H, 4.91; N, 3.25. Found: C, 69.66; H, 4.98; N,
3.26.
tert-Butyl (6-(N,N-Dibenzylsulfamoyl)naphthalen-2-yl)-
carbamate (80) (Scheme 2). A mixture of acid 79 (6.21 g, 14.4
mmol), diphenylphosphoryl azide (DPPA; 3.41 mL, 15.8 mmol), and
Et3N (2.4 mL, 17.3 mmol) in dry tert-BuOH (100 mL) was stirred at
reflux under a CaCl2 drying tube for 17 h. The yellow solution was
allowed to cool, and the tert-BuOH was evaporated. The residue was
dissolved in CH2Cl2, and the solution was washed with dilute aqueous
HCl (2×) and then brine. The CH2Cl2 layer was dried (MgSO4),
diluted with MeOH, and concentrated under reduced pressure until a
solid began to precipitate. The mixture was allowed to stand at room
temperature until precipitation was complete. The solid was filtered off
and dried to give 80 as a cream solid (6.62 g, 92%). A sample was
recrystallized from EtOAc/petroleum ether as a white solid, mp 191−
1
was evaporated to give 83 as an orange oil-foam (7.00 g, 98%). H
NMR (CDCl3): δ 8.31 (d, J = 1.7 Hz, 1H), 7.88 (d, J = 8.9 Hz, 1H),
7.80 (d, J = 8.9 Hz, 1H), 7.76 (dd, J = 8.9, 1.9 Hz, 1H), 7.23−7.17 (m,
6H), 7.11−7.06 (m, 4H), 4.36 (s, 4H), 4.31−4.24 (m, 1H), 4.17−4.11
(m, 1H), 4.05−3.98 (m, 1H), 3.93−3.86 (m, 2H), 1.61 (s, 9H), 1.45−
1.26 (m, 6H), 1.15−0.94 (m, 12H), 1 proton not observed. HRMS
(FAB) calcd. for C41H52N3O5S (MH+) m/z, 698.36277; found,
698.36256.
1
Analysis of the white solid by H NMR and MS showed that it
consisted largely of the 6-chloro analogue [tert-butyl 6-chloro-7-(N,N-
dibenzylsulfamoyl)-1-((2,2,6,6-tetramethylpiperidin-1-yloxy)methyl)-
1H-benzo[e]indole-3(2H)-carboxylate] formed as an impurity in the
chlorosulfonylation step. Compound 83 isolated as above was free of
this impurity.
1
193 °C. H NMR [(CD3)2SO]: δ 9.80 (s, 1H), 8.39 (d, J = 1.6 Hz,
1H), 8.25 (d, J = 1.4 Hz, 1H), 8.04 (d, J = 9.0 Hz, 1H), 7.98 (d, J = 8.9
Hz, 1H), 7.78 (dd, J = 8.7, 1.9 Hz, 1H), 7.64 (dd, J = 8.9, 2.1 Hz, 1H),
7.22−7.16 (m, 6H), 7.12−7.07 (m, 4H), 4.34 (s, 4H), 1.53 (s, 9H).
Anal. calcd for C29H30N2O4S: C, 69.30; H, 6.02; N, 5.57. Found: C,
69.05; H, 6.22; N, 5.61.
1-(((2,2,6,6-Tetramethylpiperidin-1-yl)oxy)methyl)-2,3-dihydro-
1H-benzo[e]indole-7-sulfonamide (84) (Scheme 2). Ice-cold con-
centrated H2SO4 (20 mL) was added to dibenzylsulfonamide 83 (4.58
g, 6.56 mmol) cooled in an ice bath, and the mixture was stirred at 0
°C until TLC analysis indicated complete debenzylation of the
sulfonamide (a total of 14 h, interrupted overnight when the reaction
mixture was allowed to stand in the freezer). The mixture was poured
into ice-cold water, EtOAc was added, and then concentrated NH3 was
cautiously added with stirring until the aqueous phase was alkaline.
The mixture was extracted with EtOAc (3×), and the combined
extracts were washed with brine (2×), then dried (MgSO4), and
evaporated. The residue was triturated with EtOAc to give 84 as a
white solid (0.68 g, 22%), mp 199−201 °C (MeOH). 1H NMR
[(CD3)2SO]: δ 8.18 (d, J = 1.7 Hz, 1H), 7.77 (d, J = 8.6 Hz, 1H), 7.72
(d, J = 8.9 Hz, 1H), 7.68 (dd, J = 8.9, 1.8 Hz, 1H), 7.22 (s, 2H), 7.04
(d, J = 8.6 Hz, 1H), 6.13 (d, J = 2.4 Hz, 1H), 3.90−3.82 (m, 2H),
3.77−3.62 (m, 2H), 3.62−3.57 (m, 1H), 1.57−1.22 (m, 6H), 1.09 (s,
6H), 1.06 (s, 3H), 0.99 (s, 3H). Anal. calcd for C22H31N3O3S: C,
63.28; H, 7.48; N, 10.06. Found: C, 63.50; H, 7.31; N, 10.07. The
mother liquor was evaporated, and the residue was purified by column
chromatography (eluting with EtOAc/petroleum ether, 1:2 then 1:1)
to give more 84 (1.29 g, 42%).
tert-Butyl (6-(N,N-Dibenzylsulfamoyl)-1-iodonaphthalen-2-yl)-
carbamate (81) (Scheme 2). Carbamate 80 (6.37 g, 12.7 mmol)
was dissolved in CH2Cl2 (150 mL), and the solution was diluted with
CH3CN (120 mL). N-Iodosuccinimide (3.14 g, 14.0 mmol) and
TsOH·H2O (60 mg, 0.32 mmol) were added, and the resulting
orange-brown solution was stirred at room temperature in the dark for
6 days. The solution was washed with aqueous Na2S2O5 (10%), then
dried (MgSO4), and evaporated. The residue was dissolved in the
minimum amount of CH2Cl2, and the solution was diluted with
EtOAc and allowed to stand at room temperature. The precipitated
solid was filtered off and dried to give 81 as a cream solid (3.16 g,
40%). A sample was recrystallized from EtOAc as a white solid, mp
1
194−195 °C. H NMR [(CD3)2SO]: δ 8.94 (s, 1H), 8.52 (d, J = 1.9
Hz, 1H), 8.28 (d, J = 9.0 Hz, 1H), 8.14 (d, J = 8.7 Hz, 1H), 7.98 (dd, J
= 9.0, 2.0 Hz, 1H), 7.76 (d, J = 8.8 Hz, 1H), 7.22−7.16 (m, 6H),
7.13−7.08 (m, 4H), 4.36 (s, 4H), 1.51 (s, 9H). Anal. calcd for
C29H29IN2O4S: C, 55.42; H, 4.65; N, 4.46. Found: C, 55.42; H, 4.82;
N, 4.55. The mother liquor was evaporated, and the residue was
purified by column chromatography (eluting with EtOAc/petroleum
ether/CH2Cl2, 10:75:15) and recrystallization (CH2Cl2/EtOAc) to
give more 77 as a cream solid (3.09 g, 39%).
tert-Butyl 7-Sulfamoyl-1-(((2,2,6,6-tetramethylpiperidin-1-yl)-
oxy)methyl)-1H-benzo[e]indole-3(2H)-carboxylate (85) (Scheme
2). A mixture of indoline 84 (1.97 g, 4.72 mmol) and di-tert-
butyldicarbonate (4.12 g, 18.9 mmol) in THF (70 mL) was stirred at
reflux for 16 h and then cooled to room temperature. The THF was
evaporated, and the residue was purified by column chromatography
(eluting with EtOAc/petroleum ether, 1:2) to give 85 as a cream solid
tert-Butyl Allyl(6-(N,N-dibenzylsulfamoyl)-1-iodonaphthalen-2-
yl)carbamate (82) (Scheme 2). Sodium hydride (60% dispersion in
oil, 0.53 g, 13.3 mmol) was added to a solution of iodide 81 (6.43 g,
10.2 mmol) and allyl bromide (1.77 mL, 20.4 mmol) in dry DMF (100
mL) at 0 °C. The mixture was stirred at this temperature for 15 min
and then allowed to warm to room temperature. After 2.5 h, the
mixture was cooled again in an ice bath, ice-cold water (3 mL) was
added, and the mixture was allowed to warm to room temperature
once more. The solvent was evaporated under reduced pressure, and
the residue was partitioned between EtOAc and brine. The aqueous
layer was extracted again with EtOAc (2×), and the combined organic
layer was washed with brine (3×) and then dried (MgSO4) and
1
(1.93 g, 79%), mp 200−202 °C (MeOH).; H NMR [(CD3)2SO]: δ
8.35 (d, J = 1.8 Hz, 1H), 8.13 (v br s, 1H), 8.03 (d, J = 8.7 Hz, 1H),
8.01 (d, J = 8.8 Hz, 1H), 7.83 (dd, J = 8.9, 1.9 Hz, 1H), 7.34 (s, 2H),
4.23 (dd, J = 11.1, 1.4 Hz, 1H), 4.13−4.06 (m, 1H), 4.00−3.86 (m,
3H), 1.54 (s, 9H), 1.46−1.18 (m, 6H), 1.12 (s, 3H), 0.98 (s, 3H), 0.76
(s, 3H), 0.64 (s, 3H). Anal. calcd for C27H39N3O5S: C, 62.64; H, 7.59;
N, 8.12. Found: C, 62.67; H, 7.35; N, 8.14.
1
evaporated to give 82 as a yellow foam (6.86 g, 100%). H NMR
tert-Butyl 1-(Hydroxymethyl)-7-sulfamoyl-1H-benzo[e]indole-
3(2H)-carboxylate (86) (Scheme 2). A mixture of TEMPO adduct
85 (1.93 g, 3.73 mmol) and Zn powder (2.44 g, 37 mmol) in THF (40
mL), HOAc (30 mL), and H2O (10 mL) was stirred at reflux. More
[(CD3)2SO]: δ 8.58−8.53 (m, 1H), 8.33 (d, J = 9.0 Hz, 1H), 8.22−
8.15 (m, 1H), 8.00 (d, J = 8.6 Hz, 1H), 7.53 (d, J = 8.5 Hz, 1H), 7.23−
7.04 (m, 10H), 6.05−5.90 (m, 1H), 5.19−5.02 (m, 2H), 4.58−4.31
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Zn powder (3 × 2.4 g) was added after 7, 25, and 29 h, and more
HOAc was added after 29 h. After a total of 33 h, TLC analysis showed
complete consumption of the starting material. The mixture was
cooled to room temperature, diluted with EtOAc, and filtered through
Celite washing with EtOAc. The solvents were evaporated, the residue
was dissolved in EtOAc, and the solution was washed with aqueous
NaHCO3 and brine and then dried (MgSO4) and evaporated. The
residue was purified by column chromatography (eluting with EtOAc/
petroleum ether, 1:2 then 1:1 then 2:1) to give 87 (152 mg, 10%,
identical to the product from the following step) and 86 as a pale
yellow solid (997 mg, 71%). A sample was triturated with CH2Cl2/
incomplete reaction, so further portions of TFAA (a total of 204 μL,
1.45 mmol) and Et3N (0.27 mL, 1.9 mmol) were added over 3 h until
the starting material was consumed. Ice-cold water and a slight excess
of aqueous HCl (2 N) were added, and the THF was evaporated. The
aqueous residue was warmed with EtOAc (100 mL) until all
suspended solid dissolved, the EtOAc layer was separated, and the
aqueous phase was extracted again with EtOAc (2×). The combined
organic layers were washed with brine, dried (MgSO4), and
concentrated to a small volume. On standing at room temperature a
solid separated which was filtered off and dried to give 88 as a light tan
solid (543 mg, 68%), mp 200−202 °C. 1H NMR [(CD3)2SO]: δ 8.47
(d, J = 1.7 Hz, 1H), 8.41 (d, J = 9.0 Hz, 1H), 8.24 (d, J = 8.9 Hz, 1H),
8.19 (d, J = 9.0 Hz, 1H), 7.96 (dd, J = 8.8, 1.9 Hz, 1H), 7.45 (s, 2H),
4.57−4.51 (m, 1H), 4.47−4.37 (m, 2H), 4.36−4.25 (m, 2H), 1.89 (s,
3H). Anal. calcd for C17H15F3N2O5S: C, 49.04; H, 3.63; N, 6.73.
Found: C, 49.31; H, 3.71; N, 6.63. The mother liquor was evaporated,
and the residue was purified by column chromatography (eluting with
EtOAc/petroleum ether, 2:3) to give more 88 (200 mg, 21%).
(5-Nitro-7-sulfamoyl-3-(2,2,2-trifluoroacetyl)-2,3-dihydro-1H-
benzo[e]indol-1-yl)methyl Acetate (90) and (9-Nitro-7-sulfamoyl-3-
(2,2,2-trifluoroacetyl)-2,3-dihydro-1H-benzo[e]indol-1-yl)methyl Ac-
etate (89) (Scheme 2). Ice-cold concentrated H2SO4 (8 mL) was
added to trifluoroacetamide 88 (730 mg, 1.75 mmol) cooled in an ice
bath, and the mixture was stirred at this temperature for 20 min until
all of the suspended solid had dissolved. Solid KNO3 (213 mg, 2.1
mmol) was added, giving a color change from brown to green to
yellow-brown over several minutes. After 12 min at 0 °C, ice-cold
water was added, and the mixture was extracted with EtOAc (2×). The
combined extracts were washed with brine (2×) and then dried
(MgSO4) and evaporated. The residue was dissolved in THF, and the
solution was evaporated onto silica gel. Purification by column
chromatography (eluting with EtOAc/petroleum ether, 1:2 then 1:1
then 3:2) gave 89 as a yellow solid (138 mg, 17%), mp 254−256 °C
(MeOH). 1H NMR [(CD3)2SO]: δ 8.81 (d, J = 1.8 Hz, 1H), 8.66 (d, J
= 9.0 Hz, 1H), 8.51 (d, J = 9.1 Hz, 1H), 8.50 (d, J = 1.8 Hz, 1H), 7.69
(s, 2H), 4.56 (dd, J = 11.1, 8.5 Hz, 1H), 4.34−4.26 (m, 1H), 4.09−
3.95 (m, 2H), 3.93−3.86 (m, 1H), 1.75 (s, 3H). Anal. calcd for
C17H14F3N3O7S: C, 44.26; H, 3.06; N, 9.11. Found: C, 44.55; H, 3.18;
N, 8.89; and 90 as a yellow-orange solid (224 mg, 28%), mp 240−241
1
petroleum ether to give a white solid, mp 198−200 °C. H NMR
[(CD3)2SO]: δ 8.35 (d, J = 1.8 Hz, 1H), 8.12 (v br s, 1H), 8.02 (d, J =
8.7 Hz, 2H), 7.84 (dd, J = 8.9, 1.9 Hz, 1H), 7.35 (s, 2H), 4.98 (t, J =
5.4 Hz, 1H), 4.17−4.04 (m, 2H), 3.91−3.83 (m, 1H), 3.78−3.70 (m,
1H), 3.51−3.40 (m, 1H), 1.55 (s, 9H). Anal. calcd for
C18H22N2O5S·0.25H2O: C, 56.46; H, 5.92; N, 7.32. Found: C,
56.49; H, 5.66; N, 7.19.
tert-Butyl 1-(Acetoxymethyl)-7-sulfamoyl-1H-benzo[e]indole-
3(2H)-carboxylate (87) (Scheme 2). Acetic anhydride (261 μL, 2.76
mmol) was added dropwise to a solution of alcohol 86 (997 mg, 2.63
mmol), Et3N (0.73 mL, 5.3 mmol), and DMAP (3 mg, 0.03 mmol) in
THF (60 mL), and the mixture was stirred at room temperature for 2
h. TLC analysis showed the presence of starting material, so more
acetic anhydride (52 μL, 0.53 mmol) was added. The mixture was
stirred for a further 2 h, and then, the THF was evaporated. The
residue was dissolved in EtOAc, and the solution was washed with
aqueous HCl (1 N), aqueous NaHCO3, and brine, then dried
(MgSO4), and evaporated. The residue was purified by column
chromatography (eluting with EtOAc/petroleum ether, 2:3 then 1:1)
to give acetate 87 as a pale yellow solid (953 mg, 86%). A sample was
recrystallized from CH2Cl2/petroleum ether as a white solid, mp 161−
162 °C. 1H NMR [(CD3)2SO]: δ 8.37 (d, J = 1.7 Hz, 1H), 8.17−8.01
(m, 3H), 7.87 (dd, J = 8.9, 1.9 Hz, 1H), 7.37 (s, 2H), 4.42−4.35 (m,
1H), 4.18−4.04 (m, 4H), 1.96 (s, 3H), 1.55 (s, 9H). Anal. calcd for
C20H24N2O6S: C, 57.13; H, 5.75; N, 6.66. Found: C, 57.03; H, 5.79;
N, 6.70.
Further elution (EtOAc/petroleum ether, 3:2) gave a sample that
was shown by 1H NMR and MS analysis to be a close-running mixture
of recovered starting material (6%) and the corresponding diacetate
[tert-butyl 1-(acetoxymethyl)-7-(N-acetylsulfamoyl)-1H-benzo[e]-
indole-3(2H)-carboxylate, 5%]. Reaction with a larger excess of acetic
1
°C (MeOH). H NMR [(CD3)2SO]: δ 9.10 (s, 1H), 8.89 (d, J = 1.5
Hz, 1H), 8.47 (d, J = 8.9 Hz, 1H), 8.12 (dd, J = 8.9, 1.7 Hz, 1H), 7.65
(s, 2H), 4.65−4.56 (m, 1H), 4.51−4.43 (m, 3H), 4.41−4.34 (m, 1H),
1.87 (s, 3H). Anal. calcd for C17H14F3N3O7S: C, 44.26; H, 3.06; N,
9.11. Found: C, 44.31; H, 3.11; N, 8.99.
1
anhydride gave a clean sample of this diacetate as a white foam. H
NMR [(CD3)2SO]: δ 12.08 (br s, 1H), 8.52 (d, J = 1.7 Hz, 1H), 8.17−
8.13 (m, 2H), 8.08 (d, J = 8.9 Hz, 1H), 7.86 (dd, J = 8.9, 1.9 Hz, 1H),
4.44−4.36 (m, 1H), 4.18−4.03 (m, 4H), 1.96 (s, 3H), 1.91 (s, 3H),
1.55 (s, 9H). LRMS (APCI, -ve) calcd for C22H26N2O7S (M − H) m/
z, 461; found, 461.5 (100%).
1-(Hydroxymethyl)-5-nitro-2,3-dihydro-1H-benzo[e]indole-7-sul-
fonamide (91) (Scheme 2). Cs2CO3 (0.47 g, 1.5 mmol) was added to
a solution of acetate 90 (224 mg, 0.49 mmol) in THF (10 mL) and
MeOH (6 mL), and the mixture was stirred at room temperature for 1
h. During this time, the color changed from yellow to red-brown to
brown, and a brown solid separated. The mixture was diluted with
more MeOH, and the solid was filtered off. The solid was redissolved
in a mixture of THF and EtOAc, and the solution was diluted with
water. The mixture was concentrated to a small volume and allowed to
stand at room temperature. The precipitated solid was filtered off and
dried to give 91 as a red-brown solid (99 mg, 63%), mp 229−231 °C.
1H NMR [(CD3)2SO]: δ 8.62 (d, J = 1.6 Hz, 1H), 7.98 (d, J = 8.9 Hz,
1H), 7.83 (dd, J = 9.0, 1.8 Hz, 1H), 7.74 (s, 1H), 7.39 (s, 2H), 6.53 (d,
J = 1.2 Hz, 1H), 4.98 (t, J = 5.5 Hz, 1H), 3.90−3.82 (m, 1H), 3.80−
3.68 (m, 2H), 3.67−3.60 (m, 1H), 3.50−3.42 (m, 1H). Anal. calcd for
C13H13N3O5S·0.5H2O: C, 46.98; H, 4.25; N, 12.64. Found: C, 46.77;
H, 4.44; N, 12.23.
1-(Hydroxymethyl)-5-nitro-3-(5,6,7-trimethoxy-1H-indole-2-car-
bonyl)-2,3-dihydro-1H-benzo[e]indole-7-sulfonamide (92) (Scheme
8). A mixture of amine 91 (52 mg, 0.16 mmol), 5,6,7-trimethoxy-1H-
indole-2-carboxylic acid (53 mg, 0.21 mmol), anhydrous TsOH (30
mg, 0.18 mmol), and EDCI·HCl (123 mg, 0.64 mmol) in dry DMA (2
mL) was stirred at room temperature for 19 h. Dilute aqueous
NaHCO3 was added, and the mixture was stirred at room temperature
for 1 h. The precipitated solid was filtered off, washed with H2O, dried,
and triturated with EtOAc to give 92 as a yellow solid (72 mg, 80%),
(7-Sulfamoyl-3-(2,2,2-trifluoroacetyl)-2,3-dihydro-1H-benzo[e]-
indol-1-yl)methyl Acetate (88) (Scheme 2). A solution of carbamate
87 (953 mg, 2.27 mmol) in HCl-dioxane (4 M, 20 mL) was stirred at
room temperature for 2 h and then evaporated. The residue was
partitioned between EtOAc and aqueous NaHCO3, and the aqueous
layer was again extracted with EtOAc (6×). The combined EtOAc
layers were dried (MgSO4) and evaporated to give crude (7-sulfamoyl-
2,3-dihydro-1H-benzo[e]indol-1-yl)methyl acetate as a pale yellow
solid (0.76 g). TLC analysis showed the presence of minor impurities
that were not completely separated by attempted trituration (EtOAc)
or column chromatography (eluting with EtOAc/petroleum ether, 2:3
then 3:2). The remaining apparently unstable material (614 mg, 85%)
was used directly in the next step. 1H NMR [(CD3)2SO]: δ 8.19 (d, J
= 1.7 Hz, 1H), 7.82−7.77 (m, 2H), 7.73 (dd, J = 8.8, 1.9 Hz, 1H), 7.20
(s, 2H), 7.06 (d, J = 8.6 Hz, 1H), 6.18 (d, J = 2.4 Hz, 1H), 4.29−4.21
(m, 1H), 4.00−3.92 (m, 2H), 3.74−3.67 (m, 1H), 3.56−3.51 (m, 1H),
2.02 (s, 3H). LRMS (APCI, -ve) calcd for C15H16N2O4S (M − H) m/
z, 319; found, 319.3 (80%), 212.3 (100%).
The crude indoline (614 mg, 1.92 mmol) and Et3N (0.53 mL, 3.8
mmol) were dissolved in THF (30 mL), and the solution was cooled
to 0 °C. TFAA (284 μL, 2.02 mmol) was added dropwise, and the
mixture was stirred at 0 °C. TLC analysis after 20 min indicated
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1
mp 280−285 °C (dec). H NMR [(CD3)2SO]: δ 11.56 (s, 1H), 9.26
washed with cold water and cold brine and dried (Na2SO4). After
filtration through a plug of Celite/silica gel and removal of EtOAc, the
crude product was precipitated (CH2Cl2/i-Pr2O) to give 97 (1.64 g,
(s, 1H), 8.88 (d, J = 1.6 Hz, 1H), 8.37 (d, J = 8.9 Hz, 1H), 8.05 (dd, J
= 8.9, 1.7 Hz, 1H), 7.60 (s, 2H), 7.20 (s, 1H), 6.98 (s, 1H), 5.07 (t, J =
5.6 Hz, 1H), 4.87−4.79 (m, 1H), 4.66 (dd, J = 10.6, 2.2 Hz, 1H),
4.21−4.14 (m, 1H), 3.95 (s, 3H), 3.83 (s, 3H), 3.81 (s, 3H), 3.80−
3.75 (m, 1H), 3.71−3.64 (m, 1H). Anal. calcd for C25H24N4O9S: C,
53.95; H, 4.35; N, 10.07. Found: C, 53.65; H, 4.56; N, 9.87.
1
94%) as a white powder: mp 169−172 °C. H NMR [(CD3)2SO) δ
8.32 (d, J = 8.9 Hz, 1H), 8.21 (d, J = 1.4 Hz, 1H), 8.06 (d, J = 9.0 Hz,
1H), 8.00 (d, J = 8.7 Hz, 1H), 7.81 (dd, J = 8.7, 1.6 Hz, 1H), 4.58 (dd,
J = 10.0, 3.2 Hz, 1H), 4.58−4.54 (m, 1H), 4.49−4.36 (m, 3H), 3.05 (s,
3H). HRMS calcd for C16H1335ClF3NO6S2 m/z, 470.9825; found,
470.9824; calcd for C16H1337ClF3NO6S2 m/z, 472.9795; found,
472.9801.
(5-Nitro-7-sulfamoyl-3-(5,6,7-trimethoxy-1H-indole-2-carbonyl)-
2,3-dihydro-1H-benzo[e]indol-1-yl)methyl Phenylmethanesulfo-
nate (24) (Scheme 8). α-Toluenesulfonyl chloride (28 mg, 0.14
mmol) was added to a solution of alcohol 92 (62.5 mg, 0.11 mmol) in
pyridine (10 mL) at 0 °C, and the mixture was stirred at this
temperature for 20 min. More α-toluenesulfonyl chloride (28 mg, 0.14
mmol) was added, and after a further 20 min, ice-cold water was
added. The mixture was stirred at 0 °C for several minutes, and the
precipitated solid was filtered off and dried. 1H NMR analysis
indicated that the reaction had only proceeded to ca. 20% completion
but that there was no competing elimination or reaction on the
sulfonamide. The procedure was repeated using a large excess of α-
toluenesulfonyl chloride until TLC analysis showed complete
conversion of the starting material. The crude product obtained was
dissolved in THF, and the solution was evaporated onto silica gel.
Purification by column chromatography (eluting with EtOAc/
petroleum ether, 1:1 then 3:1) followed by trituration with EtOAc
gave 24 as a yellow-orange solid (44 mg, 55%), mp 243−247 °C (dec).
1H NMR [(CD3)2SO]: δ 11.62 (d, J = 1.6 Hz, 1H), 9.24 (s, 1H), 8.86
(7-(Chlorosulfonyl)-5-nitro-3-(2,2,2-trifluoroacetyl)-2,3-dihydro-
1H-benzo[e]indol-1-yl)methyl Methanesulfonate (99) and (7-
(Chlorosulfonyl)-9-nitro-3-(2,2,2-trifluoroacetyl)-2,3-dihydro-1H-
benzo[e]indol-1-yl)methyl Methanesulfonate (98) (Scheme 3). A
cooled (0 °C) solution of KNO3 (450 mg, 4.44 mmol) in 98% H2SO4
(2 mL) was added dropwise to a cooled (−5 °C) solution of 97 (1.90
g, 4.03 mmol) in 98% H2SO4 (30 mL). After 20 min at −5 °C, the
mixture was poured into ice water and extracted with cold EtOAc. The
organic layer was washed with cold water and cold brine and then
dried (Na2SO4) and evaporated. The residue was purified by flash
chromatography (petroleum ether/EtOAc; gradient, 100:0 to 2:3)
followed by recrystallization (CH2Cl2/i-Pr2O) to give 99 (1.19 g,
57%) as a white powder: mp 180−182 °C. 1H NMR (CDCl3): δ 9.32
(s, 1H), 9.27 (d, J = 1.7 Hz, 1H), 8.27 (dd, J = 9.0, 1.8 Hz, 1H), 8.20
(d, J = 9.0 Hz, 1H), 4.71 (d, J = 11.6 Hz, 1H), 4.65−4.53 (m, 2H),
4.47−4.40 (m, 1H), 4.24 (dd, J = 11.0, 8.2 Hz, 1H), 3.05 (s, 3H). Anal.
calcd for C16H12ClF3N2O8S2: C, 37.18; H, 2.34; N, 5.42. Found: C,
37.05; H, 2.43; N, 5.31; and 98 (313 mg, 15%) as a white powder: mp
206−210 °C. 1H NMR (CDCl3): δ 8.93 (d, J = 9.1 Hz, 1H), 8.82 (d, J
= 2.0 Hz, 1H), 8.40 (d, J = 2.0 Hz, 1H), 8.29 (d, J = 9.0 Hz, 1H),
4.55−4.50 (m, 2H), 4.19−4.04 (m, 3H), 2.95 (s, 3H). Anal. calcd for
C16H12ClF3N2O8S2: C, 37.18; H, 2.34; N, 5.42. Found: C, 37.22; H,
2.35; N, 5.47.
(d, J = 1.7 Hz, 1H), 8.34 (d, J = 8.9 Hz, 1H), 8.07 (dd, J = 8.9, 1.7 Hz,
1H), 7.64 (s, 2H), 7.23−7.12 (m, 6H), 6.98 (s, 1H), 4.94−4.86 (m,
1H), 4.66−4.51 (m, 6H), 3.95 (s, 3H), 3.84 (s, 3H), 3.82 (s, 3H).
Anal. calcd for C32H30N4O11S2: C, 54.08; H, 4.26; N, 7.88. Found: C,
54.28; H, 4.39; N, 7.68.
2,2,2-Trifluoro-1-(1-(iodomethyl)-1H-benzo[e]indol-3(2H)-yl)-
ethanone (95) (Scheme 3). A solution of NaI (7.19 g, 47.9 mmol) in
2-butanone (30 mL) was heated (85 °C) for 1 h. Compound 9413 (5.0
g, 16.0 mmol) was added, and the mixture was heated (85 °C) for 17 h
after which EtOAc and water were added. The organic layer was
washed with aqueous sodium disulfite (10%), water, and brine, dried
(Na2SO4), and evaporated. The crude product was precipitated
(EtOAc/petroleum ether) to give 95 (4.91 g, 76%) as a cream powder:
mp 132−135 °C. 1H NMR (CDCl3): δ 8.42 (d, J = 9.0 Hz, 1H), 7.92
(d, J = 7.9 Hz, 1H), 7.89 (d, J = 8.9 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H),
7.58 (td, J = 8.1, 1.1 Hz, 1H), 7.48 (td, J = 8.1, 1.1 Hz, 1H), 4.49 (d, J
= 11.5 Hz, 1H), 4.43 (dd, J = 11.5, 8.2 Hz, 1H), 4.21 (tt, J = 9.8, 2.3
Hz, 1H), 3.65 (dd, J = 10.5, 2.3 Hz, 1H), 3.23 (t, J = 10.2 Hz, 1H).
Anal. calcd for C15H11F3INO: C, 44.47; H, 2.74; N, 3.46. Found: C,
44.74; H, 3.04; N, 3.41.
(3-(2,2,2-Trifluoroacetyl)-2,3-dihydro-1H-benzo[e]indol-1-yl)-
methyl Methanesulfonate (96) (Scheme 3). A solution of iodide 95
(3.6 g, 8.89 mmol) and AgOMs (10.8 g, 53.3 mmol) in CH3CN (50
mL) was stirred in the dark for 24 h. CH3CN was removed, EtOAc
was added, and the mixture was filtered through Celite. After removal
of EtOAc, the crude product was recrystallized (EtOAc/petroleum
ether) to give 96 (3.15 g, 95%) as colorless crystals: mp 122−124 °C.
1H NMR (CDCl3) δ 8.43 (d, J = 9.0 Hz, 1H), 7.91 (d, J = 7.8 Hz, 1H),
7.88 (d, J = 8.8 Hz, 1H), 7.83 (dd, J = 8.3, 0.5 Hz, 1H), 7.61 (td, J =
8.2, 1.2 Hz, 1H), 7.51 (td, J = 8.1, 1.1 Hz, 1H), 4.65 (dd, J = 3.8, 0.6
Hz, 1H), 4.59 (dt, J = 11.5, 1.4 Hz, 1H), 4.42 (dd, J = 11.5, 8.2 Hz,
1H), 4.29 (td, J = 8.2, 3.4 Hz, 1H), 4.14 (dd, J = 10.6, 9.1 Hz, 1H),
2.95 (s, 3H). Anal. calcd for C16H14F3NO4S: C, 51.47; H, 3.78; N,
3.75. Found: C, 51.77; H, 3.82; N, 3.86.
(7-(Chlorosulfonyl)-3-(2,2,2-trifluoroacetyl)-2,3-dihydro-1H-
benzo[e]indol-1-yl)methyl Methanesulfonate (97) (Scheme 3). A
solution of mesylate 96 (1.40 g, 3.75 mmol) in CH2Cl2 (5 mL) was
added dropwise to a cooled (−78 °C) solution of ClSO3H (1.74 g,
15.0 mmol) in CH2Cl2 (20 mL). The stirred mixture was allowed to
warm to 15 °C over 12 h, producing a gray precipitate. The mixture
was then cooled (0 °C). A minimum amount of DMF was slowly
added to dissolve the precipitate, and oxalyl chloride (1.5 mL) was
added dropwise. After 1 h, the mixture was poured into ice water. The
system was extracted with cold EtOAc, and the organic portion was
(7-(N-(2-Hydroxyethyl)sulfamoyl)-5-nitro-2,3-dihydro-1H-benzo-
[e]indol-1-yl)methyl Methanesulfonate (100) (Scheme 9). A solution
of ethanolamine (46 mg, 0.76 mmol) in CH2Cl2 (0.5 mL) was added
dropwise to a cooled (0 °C) solution of 99 (130 mg, 0.25 mmol) in
CH2Cl2 (10 mL). After 15 min at 0 °C, Cs2CO3 (164 mg, 0.50 mmol),
MeOH (5 mL), and water (1 mL) were added. After a further 15 min,
ice was added, and the mixture was poured into cold water/cold
EtOAc. The organic layer was separated and then washed with cold
water and cold brine, then dried (Na2SO4), and evaporated to give 100
(100 mg, 89%). A portion was recrystallized (EtOAc/CH2Cl2) to give
1
red crystals: mp 142−146 °C. H NMR [(CD3)2SO]: δ 8.59 (d, J =
1.6 Hz, 1H), 8.0 (d, J = 9.0 Hz, 1H), 7.82 (dd, J = 9.0, 1.7 Hz, 1H),
7.79 (s, 1H), 7.69 (br s, 1H), 6.74 (d, J = 1.4 Hz, 1H), 4.63 (t, J = 5.6
Hz, 1H), 4.40−4.34 (m, 1H), 4.31−4.19 (m, 2H), 3.86 (td, J = 9.6, 2.0
Hz, 1H), 3.70 (dd, J = 9.9, 2.3 Hz, 1H), 3.36 (q, J = 5.8 Hz, 2H), 3.14
(s, 3H), 2.81 (t,
J = 6.3 Hz, 2H). Anal. calcd for
C16H19N2O8S2·0.1EtOAc: C, 43.36; H, 4.39; N, 9.25. Found: C,
43.09; H, 4.39; N, 8.91.
(7-(N-(2-Hydroxyethyl)sulfamoyl)-5-nitro-3-(5,6,7-trimethoxy-1H-
indole-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-1-yl)methyl
Methanesulfonate (26) (Scheme 9). A solution of EDCI·HCl (58 mg,
0.31 mmol), TsOH (3 mg, 0.02 mmol), TMI-2-carboxylic acid (25
mg, 0.10 mmol), and 100 (34 mg, 0.08 mmol) in DMA (1 mL) was
stirred for 15 h. Cold 5% aqueous NaHCO3 was added, and the
precipitate was filtered and washed with cold water to give 26 (52 mg,
1
100%): mp 169−173 °C (dec.). H NMR [(CD3)2SO]: δ 11.59 (s,
1H), 9.24 (s, 1H), 8.86 (s, 1H), 8.41 (d, J = 8.9 Hz, 1H), 8.03 (dd, J =
8.9, 1.5 Hz, 1H), 7.92 (t, J = 5.7 Hz, 1H), 4.96−4.85 (m, 1H), 4.71−
4.61 (m, 2H), 4.61−4.49 (m, 3H), 3.94 (s, 3H), 3.83 (s, 3H), 3.81 (s,
3H), 3.05 (s, 3H), 2.88 (q, J = 6.0 Hz, 2H), 2 protons not observed.
Anal. calcd for C28H30N4O12S2·0.25H2O: C, 49.23; H, 4.50; N, 8.20.
Found: C, 49.16; H, 4.43; N, 8.03.
(3-(2,2,2-Trifluoroacetyl)-2,3-dihydro-1H-benzo[e]indol-1-yl)-
methyl Acetate (109) (Scheme 4). Method D. A solution of 96 (2.0 g,
4.94 mmol) and AgOAc (2.47 g, 14.8 mmol) in HOAc (50 mL) was
stirred in the dark for 48 h. EtOAc was added, and the mixture was
filtered through Celite. The organic layer was washed with water and
brine, dried (Na2SO4), and evaporated. The residue was purified by
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flash chromatography (petroleum ether/EtOAc; gradient, 100:0 to
9:1) to give 109 (1.61 g, 97%) as a white powder.
mg, 0.08 mmol), TsOH (2 mg, 0.01 mmol), and TMI-2-carboxylic
acid (5 mg, 0.01 mmol) were added, and after 2 h, cold 5% aqueous
NaHCO3 was added, and the precipitate was filtered and washed with
cold water. The residue was purified by trituration (Et2O) followed by
precipitation (CH2Cl2/MeOH) to give 115 (55 mg, 92%) as a yellow
(7-(Chlorosulfonyl)-3-(2,2,2-trifluoroacetyl)-2,3-dihydro-1H-
benzo[e]indol-1-yl)methyl Acetate (111) (Scheme 4). A solution of
109 (5.50 g, 16.3 mmol) in CH2Cl2 (40 mL) was added dropwise to a
cooled (−80 °C) solution of ClSO3H (7.57 g, 65.3 mmol) in CH2Cl2
(80 mL). The stirred mixture was allowed to warm to 5 °C over 5 h
producing a gray precipitate. The mixture was kept at 5 °C overnight
and then cooled (−10 °C). A minimum amount of DMF was slowly
added to dissolve the precipitate, oxalyl chloride (3 mL) was added
dropwise, and the mixture was allowed to warm to 5 °C over 3 h. The
mixture was poured into ice water and extracted with cold EtOAc, and
the organic layer was separated and washed with cold water and cold
brine, dried (Na2SO4), and evaporated. After filtration through a plug
of silica gel, the crude product was precipitated (CH2Cl2/i-Pr2O) to
give 111 (5.77 g, 81%). A portion was recrystallized (petroleum ether/
EtOAc) to give colorless crystals: mp 153−157 °C. 1H NMR (CDCl3)
δ 8.64 (d, J = 8.6 Hz, 1H), 8.62 (d, J = 1.9 Hz, 1H), 8.16 (d, J = 9.0
Hz, 1H), 8.08 (2dd, J = 9.0, 2.1 Hz, 2H), 4.58 (dd, J = 11.4, 4.0 Hz,
1H), 4.53 (d, J = 11.4 Hz, 1H), 4.44 (dd, J = 11.3, 8.4 Hz, 1H), 4.24−
4.16 (m, 1H), 4.02 (dd, J = 11.4, 8.3 Hz, 1H), 2.08 (s, 3H). Anal. calcd
for C17H13ClF3NO5S·0.3EtOAc: C, 47.29; H, 3.36; N, 3.03. Found: C,
47.50; H, 3.09; N, 3.20.
1
powder: mp 233−237 °C. H NMR [(CD3)2SO]: δ 11.56 (s, 1H),
9.27 (s, 1H), 8.87 (d, J = 1.6 Hz, 1H), 8.36 (d, J = 8.9 Hz, 1H), 8.01
(dd, J = 8.9, 1.6 Hz, 1H), 7.97 (br s, 1H), 7.20 (d, J = 2.0 Hz, 1H),
6.98 (s, 1H), 5.07 (t, J = 5.5 Hz, 1H), 4.82 (t, J = 10.2 Hz, 1H), 4.67
(dd, J = 10.5, 2.0 Hz, 1H), 4.21−4.13 (m, 1H), 3.94 (s, 3H), 3.83 (s,
3H), 3.81 (s, 3H), 3.80−3.74 (m, 1H), 3.69−3.62 (m, 1H), 3.56 (t, J =
6.1 Hz, 2H), 2.96−2.85 (m, 2H), 0.80 (s, 9H), −0.03 (s, 6H). Anal.
calcd for C34H42N4O10SSi·0.5H2O: C, 55.50; H, 5.89; N, 7.61. Found:
C, 55.44; H, 6.00; N, 7.95.
(7-(N-(2-((tert-Butyldimethylsilyl)oxy)ethyl)sulfamoyl)-5-nitro-3-
(5,6,7-trimethoxy-1H-indole-2-carbonyl)-2,3-dihydro-1H-benzo[e]-
indol-1-yl)methyl Phenylmethanesulfonate (119) (Scheme 10). α-
Toluenesulfonyl chloride (42 mg, 0.22 mmol) was added to a cooled
(0 °C) solution of 115 (40 mg, 0.06 mmol) in N2-purged dry pyridine
(4 mL). After 1 h, a further portion of α-toluenesulfonyl chloride (84
mg, 0.44 mmol) was added, and after 1 h at 0 °C, ice water was added,
and the precipitate was filtered and washed with cold water and Et2O
1
to give 119 (37 mg, 76%) as a yellow powder: mp 114−117 °C. H
NMR [(CD3)2SO]: δ 11.62 (s, 1H), 9.25 (s, 1H), 8.85 (d, J = 1.6 Hz,
1H), 8.34 (d, J = 8.9 Hz, 1H), 8.02 (dd, J = 8.9, 1.7 Hz, 1H), 7.99 (t, J
= 5.8 Hz, 1H), 7.25−7.09 (m, 6H), 6.98 (s, 1H), 4.89 (t, J = 9.3 Hz,
1H), 4.66−4.56 (m, 3H), 4.56−4.48 (m, 3H), 3.94 (s, 3H), 3.84 (s,
3H), 3.82 (s, 3H), 3.57 (t, J = 6.2 Hz, 2H), 2.91 (q, J = 6.0 Hz, 2H),
0.80 (s, 9H), −0.03 (s, 6H). Anal. calcd for C41H48N4O12S2Si·0.5H2O:
C, 55.33; H, 5.55; N, 6.30. Found: C, 55.32; H, 5.56; N, 6.24.
(7-(N-(2-Hydroxyethyl)sulfamoyl)-5-nitro-3-(5,6,7-trimethoxy-1H-
indole-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-1-yl)methyl Phe-
nylmethanesulfonate (29) (Scheme 10). TFA (0.5 mL) was added
dropwise to a solution of 119 (34 mg, 0.04 mmol) in CH2Cl2 (2 mL)
and MeOH (0.5 mL). After 30 h, solvents were removed, and the
residue was triturated (petroleum ether/Et2O) to give 29 (29 mg,
100%). 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, gradient 20%−95%−
(7-(Chlorosulfonyl)-5-nitro-3-(2,2,2-trifluoroacetyl)-2,3-dihydro-
1H-benzo[e]indol-1-yl)methyl Acetate (113) and (7-(Chlorosulfon-
yl)-9-nitro-3-(2,2,2-trifluoroacetyl)-2,3-dihydro-1H-benzo[e]indol-1-
yl)methyl Acetate (112) (Scheme 4). A cooled (0 °C) solution of
KNO3 (56 mg, 0.55 mmol) in 98% H2SO4 (0.5 mL) was added
dropwise to a cooled (−12 °C) solution of 111 (200 mg, 0.46 mmol)
in 98% H2SO4 (20 mL). After 15 min at −12 °C, the mixture was
poured into ice water and extracted with cold EtOAc. The organic
layer was separated and washed with cold water and cold brine, dried
(Na2SO4), and evaporated. The residue was purified by flash
chromatography (petroleum ether/EtOAc; gradient, 100:0 to 3:2)
followed by recrystallization (CH2Cl2/i-Pr2O) to give 113 (137 mg,
62%) as colorless crystals: mp 162−164 °C. 1H NMR (CDCl3): δ 9.33
(s, 1H), 9.28 (d, J = 1.6 Hz, 1H), 8.30 (d, J = 9.0 Hz, 1H), 8.22 (dd, J
= 9.0, 1.9 Hz, 1H), 4.64−4.57 (m, 2H), 4.52 (dd, J = 11.3, 8.5 Hz,
1H), 4.34−4.27 (m, 1H), 4.07 (dd, J = 11.4, 7.8 Hz, 1H), 2.08 (s, 3H).
Anal. calcd for C17H12ClF3N2O7S: C, 42.47; H, 2.52; N, 5.83. Found:
C, 42.32; H, 2.49; N, 5.57; and 112 (45 mg, 20%) as a white powder:
1
20%) gave a yellow powder (99.9% purity): mp 191−193 °C. H
NMR [(CD3)2SO]: δ 11.61 (s, 1H), 9.25 (s, 1H), 8.85 (s, 1H), 8.34
(d, J = 8.9 Hz, 1H), 8.03 (d, J = 8.5 Hz, 1H), 7.92 (br s, 1H), 7.24−
7.10 (m, 6H), 6.98 (s, 1H), 4.94−4.84 (m, 1H), 4.68 (t, J = 5.5 Hz,
1H), 4.64−4.50 (m, 6H), 3.95 (s, 3H), 3.85 (s, 3H), 3.82 (s, 3H), 3.40
(q, J = 5.8 Hz, 2H), 2.87 (q, J = 5.7 Hz, 2H). HRMS (FAB) calcd for
C34H35N4O12S2 m/z, 755.1693; found, 755.1699.
1
mp 152−154 °C. H NMR (CDCl3) δ 8.89 (d, J = 9.0 Hz, 1H), 8.79
(d, J = 2.0 Hz, 1H), 8.41 (d, J = 2.0 Hz, 1H), 8.24 (d, J = 9.1 Hz, 1H),
4.48 (dd, J = 11.1, 7.9 Hz, 1H), 4.39 (dt, J = 11.2, 1.4 Hz, 1H), 4.12−
3.96 (m, 3H), 1.90 (s, 3H). Anal. calcd for C17H12ClF3N2O7S: C,
42.47; H, 2.52; N, 5.83. Found: C, 42.60; H, 2.46; N, 5.65.
N-(2-((tert-Butyldimethylsilyl)oxy)ethyl)-1-(hydroxymethyl)-5-
nitro-2,3-dihydro-1H-benzo[e]indole-7-sulfonamide (114) (Scheme
4). A solution of TBDMSO(CH2)2NH2 (142 mg, 0.81 mmol)27 and
DIPEA (242 mg, 1.88 mmol) in CH2Cl2 (2 mL) was added dropwise
to a cooled (0 °C) solution of 113 (300 mg, 0.63 mmol) in CH2Cl2
(15 mL). After 20 min at 0 °C, Cs2CO3 (400 mg, 1.23 mmol), MeOH
(5 mL), and water (1 mL) were added. After a further 30 min, a
further portion of Cs2CO3 (400 mg, 1.23 mmol) was added, and the
mixture was allowed to warm to room temperature over 2 h. The
mixture was poured into water/EtOAc, and the organic layer was
separated and washed with cold water and cold brine and dried
(Na2SO4). Filtration through a silica gel plug followed by removal of
EtOAc and trituration (petroleum ether) gave 114 (280 mg, 93%) as a
red oil. 1H NMR [(CD3)2SO]: δ 8.60 (d, J = 1.6 Hz, 1H), 7.97 (d, J =
9.0 Hz, 1H), 7.77 (dd, J = 9.0, 1.7 Hz, 1H), 7.77−7.71 (m, 2H), 6.57
(s, 1H), 4.98 (t, J = 5.4 Hz, 1H), 3.88−3.82 (m, 1H), 3.79−3.68 (m,
2H), 3.66−3.61 (m, 1H), 3.52 (t, J = 6.3, 2H), 2.85 (q, J = 6.1 Hz,
2H), 0.78 (s, 9H), −0.05 (s, 6H). HRMS (FAB) calcd for
C21H32N3O6SSi m/z, 482.1781; found, 482.1786.
Synthesis of Compounds in Table 3 and Their Intermediates.
Di-tert-butyl (2-((2-(7-Carbamoyl-1-(hydroxymethyl)-5-nitro-2,3-di-
hydro-1H-benzo[e]indole-3-carbonyl)-1H-indol-5-yl)oxy)ethyl)
Phosphate (123) (Scheme 11). A solution of 63 (110 mg, 0.38
mmol) in DMA (3 mL) was treated with anhydrous TsOH (72
mg, 0.42 mmol), 5-(2-((di-tert-butoxyphosphoryl)oxy)ethoxy)-
1H-indole-2-carboxylic acid14 (207 mg, 0.50 mmol), and
EDCI·HCl (300 mg, 1.57 mmol). The mixture was stirred at
room temperature for 4 h, then cooled to 0 °C, treated with 5%
aqueous NaHCO3 (10 mL), and stirred for further 10 min. The
precipitate was collected by filtration, washed with cold water
and small portions of ice-cold EtOAc, and then dried in a
vacuum desiccator. The residue was triturated with diethyl
ether to give 123 as a yellow solid (140 mg, 54%): mp 206−
1
210 °C. H NMR [(CD3)2SO]: δ 11.71 (d, J = 1.7 Hz, 1H),
9.19 (s, 1H), 8.85 (d, J = 1.3 Hz, 1H), 8.25 (br, s, 1H), 8.22 (d,
J = 8.9 Hz, 1H), 8.13 (dd, J = 8.8, 1.5 Hz, 1H), 7.57 (br, s, 1H),
7.43 (d, J = 8.9 Hz, 1H), 7.20 (dd, J = 8.1, 1.7 Hz, 2H), 6.96
(dd, J = 8.9, 2.4 Hz, 1H), 5.06 (t, J = 5.6 Hz, 1H), 4.84 (t, J =
10.3 Hz, 1H), 4.72 (dd, J = 10.4, 2.1 Hz, 1H), 4.22−4.18 (m,
5H), 3.78 (m, 1H), 3.70−3.64 (m, 1H), 1.43 (s, 18H). HRMS
(FAB) calcd for C33H39N4O10P (M+) m/z, 682.2404; found,
682.2405.
N-(2-((tert-Butyldimethylsilyl)oxy)ethyl)-1-(hydroxymethyl)-5-
nitro-3-(5,6,7-trimethoxy-1H-indole-2-carbonyl)-2,3-dihydro-1H-
benzo[e]indole-7-sulfonamide (115) (Scheme 10). A solution of
EDCI·HCl (64 mg, 0.33 mmol), TsOH (3 mg, 0.02 mmol), TMI-2-
carboxylic acid (25 mg, 0.10 mmol), and 114 (40 mg, 0.08 mmol) in
DMA (2 mL) was stirred for 1.5 h. Further portions of EDCI·HCl (15
(7-Carbamoyl-3-(5-(2-((di-tert-butoxyphosphoryl)oxy)ethoxy)-
1H-indole-2-carbonyl)-5-nitro-2,3-dihydro-1H-benzo[e]indol-1-yl)-
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methyl Phenylmethanesulfonate (124) (Scheme 11). A solution of
123 (100 mg, 0.15 mmol) in dry pyridine (3 mL) was treated with α-
toluenesulfonyl chloride (140 mg, 0.73 mmol) at 0 °C. The mixture
was stirred for 1 h and then poured into a beaker of ice water and
stirred for further 10 min. The solution was extracted with CH2Cl2
(3×), and the combined organic layers were washed with water and
brine, dried, and concentrated under reduced pressure. The residue
was triturated with diethyl ether to give 124 as a yellow solid (43 mg,
34%): mp 166−170 °C. 1H NMR [(CD3)2SO]: δ 11.75 (d, J = 1.7 Hz,
1H), 9.18 (s, 1H), 8.85 (d, J = 1.2 Hz, 1H), 8.29 (br, s, 1H), 8.22 (d, J
= 8.8 Hz, 1H), 8.16 (dd, J = 8.8, 1.4 Hz, 1H), 7.61 (br, s, 1H), 7.45 (d,
J = 8.9 Hz, 1H), 7.20−7.11 (m, 7H), 6.97 (dd, J = 8.9, 2.4 Hz, 1H),
4.93−4.91 (m, 1H), 4.66−4.55 (m, 6H), 4.22−4.20 (m, 4H), 1.43 (s,
18H). HRMS (FAB) calcd for C40H45N4O12 PS (M+) m/z, 836.2492;
found, 836.2497.
(7-Carbamoyl-5-nitro-3-(5-(2-(phosphonooxy)ethoxy)-1H-in-
dole-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-1-yl)methyl phenyl-
methanesulfonate (33) (Scheme 11). A solution of 124 (40 mg, 0.05
mmol) in CH2Cl2 (2 mL) was treated with TFA (41 μL, 0.53 mmol),
stirred at room temperature for 24 h, and then concentrated under
reduced pressure. The residue was repeatedly dissolved in CH2Cl2 and
then concentrated to remove the excess TFA. The crude product was
triturated with diethyl ether to give 33 as a yellow solid (26 mg, 72%):
mp 191−194 °C. 1H NMR [(CD3)2SO]: δ 11.74 (s, 1H), 11.20 (br, s,
1H), 9.18 (s, 1H), 8.85 (d, J = 1.0 Hz, 1H), 8.28 (br, s, 1H), 8.21 (d, J
= 8.8 Hz, 1H), 8.16 (dd, J = 8.8, 1.3 Hz, 2H), 7.61 (br, s, 1H), 7.45 (d,
J = 8.9 Hz, 1H), 7.21−7.11 (m, 6H), 6.99 (dd, J = 8.9, 2.3 Hz, 1H),
4.94−4.90 (m, 1H), 4.66−4.55 (m, 6H), 4.19−4.13 (m, 4H), 2
protons not observed. HRMS (ESI) calcd for C32H28N4O12PS (MH−)
m/z, 723.1168; found, 723.1157. HPLC purity, 99.7%.
cold water and i-Pr2O, and then dried in a vacuum desiccator to give
127 as a yellow solid (140 mg, 69%): mp 150−154 °C.; H NMR
1
[(CD3)2SO]: δ 11.53 (d, J = 1.8 Hz, 1H), 9.14 (br, s, 1H), 8.94 (t, J =
5.4 Hz, 1H), 8.83 (d, J = 1.3 Hz, 1H), 8.23 (d, J = 8.8 Hz, 1H), 8.11
(dd, J = 8.8, 1.5 Hz, 1H), 7.19 (d, J = 2.3 Hz, 1H), 6.98 (br, s, 1H),
5.06 (t, J = 5.5 Hz, 1H), 4.80 (t, J = 10.4 Hz, 1H), 4.66 (dd, J = 10.6,
2.1 Hz, 1H), 4.18−4.12 (m, 1H), 4.03 (m, 2H), 3.94 (s, 3H), 3.83 (s,
3H), 3.81 (s, 3H), 3.80−3.76 (m, 1H), 3.68−3.61 (m, 1H), 3.59−3.52
(m, 2H), 1.40 (s, 18H). HRMS (ESI) calcd for C36H45N4NaO12P
(MNa+) m/z, 779.2664; found, 779.2684.
(7-((2-((Di-tert-butoxyphosphoryl)oxy)ethyl)carbamoyl)-5-nitro-
3-(5,6,7-trimethoxy-1H-indole-2-carbonyl)- 2,3-dihydro-1H-benzo-
[e]indol-1-yl)methyl Phenylmethanesulfonate (129) (Scheme 12).
A solution of 127 (140 mg, 0.19 mmol) in dry pyridine (5 mL) was
treated with α-toluenesulfonyl chloride (210 mg, 1.11 mmol) at 0 °C.
The mixture was stirred for 1 h, then poured into a beaker of ice water,
stirred for further 10 min, and then extracted with EtOAc (3×). The
combined organic layers were washed with water and brine, dried, and
concentrated under reduced pressure. The residue was purified by
column chromatography eluting with CH2Cl2/MeOH (19:1) to give
129 as a yellow solid (106 mg, 61%): mp 109 °C. 1H NMR
[(CD3)2SO]: δ 11.59 (d, J = 1.7 Hz, 1H), 9.12 (br, s, 1H), 8.97 (t, J =
5.5 Hz, 1H), 8.82 (d, J = 1.2 Hz, 1H), 8.22 (d, J = 8.8 Hz, 1H), 8.13
(dd, J = 8.8, 1.5 Hz, 1H), 7.22−7.12 (m, 6H), 6.98 (br, s, 1H), 4.90−
4.85 (m, 1H), 4.60−4.53 (m, 6H), 4.04 (q, J = 5.7 Hz, 2H), 3.94 (s,
3H), 3.84 (s, 3H), 3.82 (s, 3H), 3.57 (q, J = 5.4 Hz, 2H), 1.39 (s,
18H). Anal. calcd for C43H51N4O14PS: C, 56.70; H, 5.64; N, 6.15.
Found: C, 56.57; H, 5.86; N, 6.07.
(5-Nitro-7-((2-(phosphonooxy)ethyl)carbamoyl)-3-(5,6,7-trime-
thoxy-1H-indole-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-1-yl)-
methyl Phenylmethanesulfonate (34) (Scheme 12). A solution of
129 (100 mg, 0.11 mmol) in CH2Cl2 (5 mL) was treated with TFA
(93 μL, 1.21 mmol), stirred at room temperature for 24 h, and then
evaporated to dryness. The residue was repeatedly dissolved in CH2Cl2
and then concentrated under reduced pressure to remove the excess
TFA. The crude product was triturated with diethyl ether to give 34 as
a yellow solid (88 mg, 100%): mp 163−167 °C. 1H NMR
[(CD3)2SO]: δ 11.59 (d, J = 1.7 Hz, 1H), 9.13 (br, s, 1H), 8.98 (t,
J = 5.3 Hz, 1H), 8.84 (d, J = 1.1 Hz, 1H), 8.22 (d, J = 8.9 Hz, 1H),
8.15 (dd, J = 8.8, 1.4 Hz, 1H), 7.22−7.13 (m, 6H), 6.98 (br, s, 1H),
4.88−4.83 (m, 1H), 4.61−4.53 (m, 6H), 4.04−3.98 (m, 2H), 3.94 (s,
3H), 3.84 (s, 3H), 3.82 (s, 3H), 3.57 (q, J = 5.7 Hz, 2H), 2 protons
not observed. HRMS (FAB) calcd for C35H36N4O14PS (MH+) m/z,
799.1686; found, 799.1678. HPLC purity, 96.5%.
Di-tert-butyl (2-(1-(Hydroxymethyl)-5-nitro-3-(2,2,2-trifluoroace-
tyl)-2,3-dihydro-1H-benzo[e]indole-7-carbonylamino)ethyl) Phos-
phate (125) (Scheme 12). A solution of 70 (120 mg, 0.31 mmol)
in anhydrous THF (15 mL) was treated with DIPEA (160 μL, 0.86
mmol) at room temperature. The mixture was cooled to 0 °C, treated
with 2-aminoethyl di(tert-butyl) phosphate14 (153 mg, 0.64 mmol)
and PyBOP (211 mg, 0.41 mmol), and stirred at room temperature for
1 h. The mixture was evaporated to dryness, and the residue was
dissolved in EtOAc, washed with ice-cold water and brine, dried, and
concentrated under reduced pressure. The crude product was purified
by column chromatography eluting with diethyl ether/MeOH (24:1)
1
to provide 125 as a yellow solid (190 mg, 99%): mp 76−79 °C. H
NMR [(CD3)2SO]: δ 9.00 (s, 1H), 8.98 (t, J = 5.5 Hz, 1H), 8.84 (d, J
= 1.3 Hz, 1H), 8.28 (d, J = 8.8 Hz, 1H), 8.15 (dd, J = 8.8, 1.6 Hz, 1H),
5.05 (t, J = 5.4 Hz, 1H), 4.58−4.49 (m, 2H), 4.23−4.21 (m, 1H), 4.03
(q, J = 5.9 Hz, 2H), 3.83−3.78 (m, 1H), 3.75−3.69 (m, 1H), 3.56 (q, J
= 5.7 Hz, 2H), 1.39 (s, 18H). HRMS (ESI) calcd for
C26H33F3N3NaO9P (MNa+) m/z, 642.1799; found, 642.1792.
Di-tert-butyl (2-(1-(Hydroxymethyl)-5-nitro-2,3-dihydro-1H-
benzo[e]indole-7-carbonylamino)ethyl) Phosphate (126) (Scheme
12). A solution of 125 (100 mg, 0.16 mmol) in MeOH (10 mL) was
cooled to 0 °C in an ice water bath and stirred for 10 min. The mixture
was then treated with Cs2CO3 (53 mg, 0.16 mmol), stirred for a
further 30 min, and then evaporated to dryness. The residue was
purified by column chromatography eluting with CH2Cl2/MeOH
(49:1 and 48:1) to provide 126 as a red gum (83 mg, 99%). 1H NMR
[(CD3)2SO]: δ 8.73 (t, J = 5.5 Hz, 1H), 8.60 (d, J = 1.3 Hz, 1H), 7.91
(dd, J = 8.9, 1.6 Hz, 1H), 7.85 (d, J = 8.8 Hz, 1H), 7.63 (s, 1H), 7.39
(s, 1H), 4.95 (t, J = 5.4 Hz, 1H), 4.00 (q, J = 6.0 Hz, 2H), 3.86−3.82
(m, 1H), 3.76−3.63 (m, 3H), 3.52 (q, J = 5.6 Hz, 2H), 3.46−3.41 (m,
1H), 1.39 (s, 18H). HRMS (FAB) calcd for C24H34N3O8P (M+) m/z,
523.2084; found, 523.2083.
(7-(N-(2-((Di-tert-butoxyphosphoryl)oxy)ethyl)sulfamoyl)-5-nitro-
2,3-dihydro-1H-benzo[e]indol-1-yl)methyl Methanesulfonate (131)
(Scheme 3). A solution of DIPEA (90 mg, 0.70 mmol) in CH2Cl2 (1
mL) followed by a solution of 2-aminoethyl di(tert-butyl) phosphate
(90 mg, 0.35 mmol) in CH2Cl2 (4 mL) were added dropwise to a
cooled (0 °C) solution of 99 (120 mg, 0.23 mmol) in CH2Cl2 (18
mL). After 15 min, Cs2CO3 (160 mg, 0.46 mmol), MeOH (7 mL),
and water (1 mL) were added. After a further 15 min, ice was added,
and the mixture was poured into cold water/cold EtOAc. The organic
layer was separated and then washed with cold water and cold brine,
then dried (Na2SO4), and evaporated to give 131 (110 mg, 74%). A
portion was recrystallized (petroleum ether/EtOAc) to give red
1
crystals: mp 146−151 °C. H NMR [(CD3)2SO]: δ 8.60 (d, J = 1.6
Hz, 1H), 8.01 (d, J = 8.9 Hz, 1H), 7.95 (br s, 1 H), 7.81 (dd, J = 8.9,
1.7 Hz, 1H), 7.80 (s, 1 H), 4.41−4.33 (m, 1H), 4.29−4.19 (m, 2H),
3.90−3.77 (m, 3H), 3.71 (dd, J = 9.8, 2.0 Hz, 1H), 3.15 (s, 3H), 2.90
(t, J = 5.8 Hz, 2H), 1.35 (s, 18H). Anal. calcd for C24H36N3O11PS2: C,
45.21; H, 5.69; N, 6.59. Found: C, 45.50; H, 5.82; N, 6.45.
Di-tert-butyl (2-(1-(Hydroxymethyl)-5-nitro-3-(5,6,7-trimethoxy-
1H-indole-2-carbonyl)-2,3-dihydro-1H-benzo[e]indole-7-
carbonylamino)ethyl) Phosphate (127) (Scheme 12). A solution of
126 (142 mg, 0.27 mmol) in DMA (3 mL) was treated with
anhydrous TsOH (51 mg, 0.30 mmol), 5,6,7-trimethoxy-1H-indole-2-
carboxylic acid (88 mg, 0.35 mmol), and EDCI·HCl (206 mg, 1.08
mmol). The mixture was stirred at room temperature for 2 h, then
cooled to 0 °C, treated with 5% aqueous NaHCO3, and stirred for
further 10 min. The precipitate was collected by filtration, washed with
(7-(N-(2-((Di-tert-butoxyphosphoryl)oxy)ethyl)sulfamoyl)-5-nitro-
3-(5,6,7-trimethoxy-1H-indole-2-carbonyl)-2,3-dihydro-1H-benzo-
[e]indol-1-yl)methyl Methanesulfonate (132) (Scheme 13). A
solution of EDCI·HCl (280 mg, 1.44 mmol), TsOH (12 mg, 0.07
mmol), TMI-2-carboxylic acid (118 mg, 0.47 mmol), and 131 (230
mg, 0.36 mmol) in DMA (2 mL) was stirred for 5 h. Cold 5% aqueous
NaHCO3 was added, and the precipitate was filtered and washed with
1
cold water to give 132 (295 mg, 94%) as a yellow powder. H NMR
[(CD3)2SO]: δ 11.60 (s, 1H), 9.25 (s, 1H), 9.88 (d, J = 1.6 Hz, 1H),
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8.42 (d, J = 8.9 Hz, 1H), 8.15 (br s, 1H), 8.03 (dd, J = 8.9, 1.7 Hz,
1H), 7.20 (d, J = 2.0 Hz, 1H), 6.98 (s, 1H), 4.90 (t, J = 10.5 Hz, 1H),
4.66 (d, J = 11.1 Hz, 1H), 4.62−4.48 (m, 3H), 3.95 (s, 3H), 3.86−3.79
(m, 2H), 3.83 (s, 3H), 3.81 (s, 3H), 3.08−3.02 (m, 2H), 3.07 (s, 3H),
1.37 (s, 18H).
Hz, 1H), 8.02 (dd, J = 8.9, 1.7 Hz, 1H), 7.23−7.11 (m, 6H), 6.98 (s,
1H), 4.88 (t, J = 9.3 Hz, 1H), 4.67−4.48 (m, 4H), 4.62 (d, J = 3.0 Hz,
2H), 3.95 (s, 3H), 3.88−3.80 (m, 2H), 3.84 (s, 3H), 3.82 (s, 3H), 3.06
(q,
J = 5.6 Hz, 2H), 1.36 (s, 18H). Anal. calcd for
C42H51N4O15PS2·H2O: C, 52.28; H, 5.54; N, 5.81. Found: C, 52.36;
H, 5.41; N, 5.76.
(5-Nitro-7-(N-(2-(phosphonooxy)ethyl)sulfamoyl)-3-(5,6,7-trime-
thoxy-1H-indole-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-1-yl)-
methyl Methanesulfonate (36) (Scheme 13). A solution of TFA (380
mg, 3.33 mmol) in CH2Cl2 (1 mL) was added dropwise to a solution
of 132 (290 mg, 0.33 mmol) in CH2Cl2 (15 mL). After 15 h, solvents
were removed, and the residue was triturated with Et2O to give 36
(5-Nitro-7-(N-(2-(phosphonooxy)ethyl)sulfamoyl)-3-(5,6,7-trime-
thoxy-1H-indole-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-1-yl)-
methyl Phenylmethanesulfonate (39) (Scheme 14). TFA (140 mg,
1.23 mmol) was added dropwise to a solution of 140 (116 mg, 0.12
mmol) in CH2Cl2 (2 mL). After 15 h, solvents were removed to give
1
1
(260 mg, 100%): mp 137−141 °C (dec.). H NMR [(CD3)2SO]: δ
39 (102 mg, 100%) as a yellow powder: mp 212−215 °C. H NMR
11.59 (s, 1H), 9.24 (s, 1H), 8.87 (s, 1H), 8.42 (d, J = 8.9 Hz, 1H), 8.26
(br s, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.18 (d, J = 1.8 Hz, 1H), 6.98 (s,
1H), 4.91 (t, J = 9.5 Hz, 1H), 4.66 (d, J = 10.7 Hz, 1H), 4.62−4.49 (m,
3H), 3.95 (s, 3H), 3.84 (s, 3H), 3.84−3.75 (m, 2H), 3.82 (s, 3H), 3.06
(s, 3H), 3.06−3.00 (m, 2H), 3 protons not observed. Anal. calcd for
C28H31N4O15PS2·H2O: C, 43.30; H, 4.28; N, 7.21. Found: C, 43.42; H,
4.53; N, 7.04.
[(CD3)2SO]: δ 11.70 (s, 1H), 9.27 (s, 1H), 8.87 (d, J = 1.5 Hz, 1H),
8.36 (d, J = 8.9 Hz, 1H), 8.19 (t, J = 5.7 Hz, 1H), 8.02 (dd, J = 8.9, 1.6
Hz, 1H), 7.2−7.10 (m, 6H), 6.98 (s, 1H), 4.95−4.85 (m, 1H), 4.65−
4.50 (m, 6H), 3.94 (s, 3H), 3.83 (s, 3H), 3.83−3.80 (m, 2H), 3.81 (s,
3H), 3.02 (q, J = 5.8 Hz, 2H), 2 protons not observed. Anal. calcd for
C34H35N4O15PS2·0.5H2O: C, 48.40; H, 4.30; N, 6.64. Found: C, 48.51;
H, 4.19; N, 6.61.
In Vitro Cytotoxicity. Inhibition of proliferation of log-phase
monolayers was assessed in 96-well plates as previously described.13,28
The drug exposure 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
concentration required to inhibit cell density to 50% of that of the
controls on the same plate.
Di-tert-butyl (2-(1-(Hydroxymethyl)-5-nitro-2,3-dihydro-1H-
benzo[e]indole-7-sulfonamido)ethyl) Phosphate (135) (Scheme 4).
A solution of DIPEA (350 mg, 2.69 mmol) and 2-aminoethyl di(tert-
butyl) phosphate (272 mg, 1.08 mmol) in CH2Cl2 (2 mL) was added
dropwise to a cooled (0 °C) solution of 113 (430 mg, 0.90 mmol) in
CH2Cl2 (10 mL). After 30 min, Cs2CO3 (580 mg, 1.78 mmol), MeOH
(5 mL), and water (1 mL) were added, and the mixture was allowed to
warm to room temperature over 2 h. A further portion of Cs2CO3
(580 mg, 1.78 mmol) was added, and after 1 h, water/EtOAc were
added. The organic layer was separated and washed with cold water
and cold brine, dried (Na2SO4), and evaporated. The residue was
purified by flash chromatography (CH2Cl2/MeOH; gradient, 100:0 to
95:5) followed by trituration (Et2O) to give 135 (350 mg, 70%) as a
Clonogenic Assay. Clonogenic assays were performed as previously
described.25 Drug exposures were performed in 96-well plates (Nunc)
using either a 37 °C humidified incubator (95% air, 5% CO2) or in the
incubator compartment (37 °C) of an anaerobic chamber where
palladium catalyst-scrubbed gas (90% N2, 5% H2, 5% CO2) ensured
severe anoxia (<0.001% O2). Cell cultures were grown in αMEM
(Gibco BRL, Grand Island, NY) containing 5% heat-inactivated fetal
calf serum (FCS; Gibco BRL, Auckland, New Zealand) and
maintained in exponential growth phase. For each experiment, 3 ×
105 cells in 150 μL were seeded into wells in αMEM + 10% FCS + 10
mmol L−1 added D-glucose + 200 μmol L−1 2′-deoxycytidine (2′-dCyd)
containing penicillin (100 units mL−1) and streptomycin (100 μg
mL−1) (P/S) and allowed to attach for at least 2 h. High glucose (final
concentration, 17 mmol L−1) and the presence of 2′-dCyd minimize
hypoxia-induced cell-cycle arrest. Drug solutions were added (150 μL),
and cells were exposed in duplicate for 4 h. At the end of the
incubation, the extracellular medium was removed, and the cells were
washed with 100 μL of PBS and trypsinized. Subsequently, cells were
plated for clonogenic survival in plastic Petri dishes in αMEM + 10%
FCS containing P/S. The dishes were stained with methylene blue 14
days later, and colonies containing more than 50 cells were counted.
In Vivo Activity. Antitumor activity by excision assay was performed
as previously described.13,14 SiHa and H460 tumors were grown in
male mice by subcutaneous inoculation of 107 cells from tissue culture,
and mice were randomized to treatment groups when tumors reached
a mean diameter of 8−10 mm. In each experiment, mice received
vehicle alone (phosphate-buffered saline; n = 3), compound alone
(dissolved in phosphate-buffered saline with 4 equiv of sodium
bicarbonate; n = 3), radiation alone (15 Gy, whole body cobalt-60 γ
irradiation, n = 5), or radiation followed 5 min later by compound (n =
5) administered via the tail vein. Significance of treatment effects was
tested using ANOVA with Holm−Sidak posthoc test on log-
transformed data with SigmaPlot v11.2 (Sysat Software, Inc.).
Compound 19 was formulated in 10% DMSO, 40% PEG-400, and
50% water. All animal studies were approved by the University of
Auckland Animal Ethics Committee. Experiments were performed
using CD1-Foxn1nu/nu (nude) mice.
1
red powder: mp 90 °C (dec.). H NMR [(CD3)2SO]: δ 8.61 (d, J =
1.6 Hz, 1H), 7.99 (d, J = 9.0 Hz, 1H), 7.91 (br s, 1H), 7.77 (dd, J =
9.0, 1.7 Hz, 1H), 7.75 (s, 1H), 6.58 (s, 1H), 4.98 (t, J = 5.4 Hz, 1H),
3.88−3.75 (m, 4H), 3.71 (dd, J = 9.7, 2.8 Hz, 1H), 3.67−3.60 (m,
1H), 3.50−3.42 (m, 1H), 2.98 (t, J = 5.6 Hz, 2H), 1.34 (s, 18H). Anal.
calcd for C23H34N3O9PS·0.5H2O: C, 48.59; H, 6.21; N, 7.39. Found:
C, 48.81; H, 6.24; N, 7.39. HRMS (FAB) calcd for C23H35N3O9PS m/
z, 560.1832; found, 560.1843.
Di-tert-butyl (2-(1-(Hydroxymethyl)-5-nitro-3-(5,6,7-trimethoxy-
1H-indole-2-carbonyl)-2,3-dihydro-1H-benzo[e]indole-7-
sulfonamido)ethyl) Phosphate (136) (Scheme 14). A solution of
EDCI·HCl (154 mg, 0.81 mmol), TsOH (6 mg, 0.03 mmol), TMI-2-
carboxylic acid (49 mg, 0.19 mmol), and 135 (90 mg, 0.16 mmol) in
DMA (5 mL) was stirred for 2 h. Further portions of EDCI·HCl (20
mg, 0.11 mmol), TsOH (2 mg, 0.01 mmol), and TMI-2-carboxylic
acid (12 mg, 0.05 mmol) were added, and after 1 h, cold 5% aqueous
NaHCO3 was added, and the precipitate was filtered and washed with
cold water. The residue was purified by precipitation (CH2Cl2/
MeOH) to give 136 (112 mg, 88%) as a yellow powder: mp 216−220
°C (dec.). 1H NMR [(CD3)2SO]: δ 11.56 (d, J = 1.4 Hz, 1H), 2.28 (s,
1H), 8.88 (d, J = 1.5 Hz, 1H), 8.38 (d, J = 8.9 Hz, 1H), 8.13 (t, J = 5.8
Hz, 1H), 8.00 (dd, J = 8.9, 1.6 Hz, 1H), 7.20 (d, J = 2.1 Hz, 1H), 6.98
(s, 1H), 5.07 (t, J = 5.6 Hz, 1H), 4.82 (t, J = 9.6 Hz, 1H), 4.66 (dd, J =
10.5, 2.0 Hz, 1H), 4.21−4.13 (m, 1H), 3.94 (s, 3H), 3.87−3.82 (m,
2H), 3.83 (s, 3H), 3.81 (s, 3H), 3.81−3.74 (m, 1H), 3.71−3.62 (m,
1H), 3.06 (q, J = 5.8 Hz, 2H), 1.36 (s, 18H). Anal. calcd for
C35H45N4O13PS: C, 53.03; H, 5.72; N, 7.07. Found: C, 52.77; H, 5.81;
N, 7.00.
(7-(N-(2-((Di-tert-butoxyphosphoryl)oxy)ethyl)sulfamoyl)-5-nitro-
3-(5,6,7-trimethoxy-1H-indole-2-carbonyl)-2,3-dihydro-1H-benzo-
[e]indol-1-yl)methyl Phenylmethanesulfonate (140) (Scheme 14).
α-Toluenesulfonyl chloride (120 mg, 0.63 mmol) was added to a
cooled (0 °C) solution of 136 (100 mg, 0.13 mmol) in N2-purged dry
pyridine (5 mL). After 15 min, a further portion of α-toluenesulfonyl
chloride (120 mg, 0.63 mmol) was added, and after 1 h at 0 °C, ice
water was added, and the precipitate was filtered and washed with cold
water to give 140 (118 mg, 100%) as a yellow powder: mp 156−160
°C (dec.). 1H NMR [(CD3)2SO]: δ 11.62 (d, J = 1.6 Hz, 1H), 9.26 (s,
1H), 8.86 (d, J = 1.6 Hz, 1H), 8.35 (d, J = 8.9 Hz, 1H), 8.17 (t, J = 5.6
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details for the syntheses of intermediates 46, 45,
49, 50, 52−55, 50 (method B), 47, 65, 66, 74, 76, 93, 101,
103, 102, 105, 104, 107, 108 (method A), 108 (method B),
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Journal of Medicinal Chemistry
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EORTC early clinical studies group (ECSG). Cancer Chemother.
Pharmacol. 2000, 46, 167−171.
109 (method A), 109 (method B), 116, 120, 117, 121, 118,
122, 128, 130, 133, 134, 137, 141, 138, 142, 139, and 143.
Syntheses of compounds in Table 1: 16, 18, 19 (method B),
20, 21, 23, 25, 27 (method A), 27 (method B), 27 (method
C), 28 (method A), 28 (method B), and 30−32. Syntheses of
compounds in Table 3: 35, 37, 38, and 40−42. Figures and
tables. This material is available free of charge via the Internet
at http://pubs.acs.org.
(6) Wilson, W. R.; Hay, M. P. Targeting hypoxia in cancer therapy.
Nat. Rev. Cancer 2011, 11, 393−410.
(7) Townes, H.; Summerville, K.; Purnell, B.; Hooker, M.; Madsen,
E.; Hudson, S.; Lee, M. Investigation of a novel reductively-activatable
anticancer prodrug of seco-CBI-TMI, an analog of duocarmycin SA.
Med. Chem. Res. 2002, 11, 248−253.
(8) Wei, J.; Trzupek, J. D.; Rayl, T. J.; Broward, M. A.; Vielhauer, G.
A.; Weir, S. J.; Hwang, I.; Boger, D. L. A unique class of duocarmycin
and CC-1065 analogues subject to reductive activation. J. Am. Chem.
Soc. 2007, 129, 15391−15397.
(9) 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. Nitro-chloromethylbenzindolines: Hypoxia-activated prodrugs of
potent adenine N3 DNA minor groove alkylators. Mol. Cancer Ther.
2009, 8, 2903−2913.
AUTHOR INFORMATION
Corresponding Author
*Tel: +64 9 923 86788. Fax: +64 9 3737502. E-mail: r.
stevenson@auckland.ac.nz.
■
Notes
The authors declare no competing financial interest.
(10) Tercel, M.; Denny, W. A.; Wilson, W. R. A novel nitro-
substituted seco-CI: Application as a reductively activated ADEPT
prodrug. Bioorg. Med. Chem. Lett. 1996, 6, 2741−2744.
(11) Atwell, G. J.; Tercel, M.; Boyd, M.; Wilson, W. R.; Denny, W. A.
Synthesis and cytotoxicity of 5-amino-1-(chloromethyl)-3-[(5,6,7-
trimethoxyindol-2-yl)carbonyl]-1,2-dihydro-3H-benz[e]indole
(amino-seco-CBI-TMI) and related 5-alkylamino analogues: New
DNA minor groove alkylating agents. J. Org. Chem. 1998, 63, 9414−
9420.
(12) Gieseg, M. A.; Matejovic, J.; Denny, W. A. Comparison of the
patterns of DNA alkylation by phenol and amino seco-CBI-TMI
compounds: Use of a PCR method for the facile preparation of single
end-labelled double stranded DNA. Anti-Cancer Drug Des. 1999, 14,
77−84.
(13) 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. Hypoxia-activated prodrugs: substituent effects on the
properties of nitro seco-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-
4-one (nitroCBI) prodrugs of DNA minor groove alkylating agents. J.
Med. Chem. 2009, 52, 7258−7272.
(14) 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. Selective treatment of hypoxic tumor cells in vivo:
Phosphate pre-prodrugs of nitro analogues of the duocarmycins.
Angew. Chem., Int. Ed. Engl. 2011, 50, 2606−2609.
(15) Stevenson, R. J.; Denny, W. A.; Ashoorzadeh, A.; Pruijn, F. B.;
Tercel, M. 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. Bioorg. Med. Chem.
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(16) Ashoorzadeh, A.; Atwell, G. J.; Pruijn, F. B.; Wilson, W. R.;
Tercel, M.; Denny, W. A.; Stevenson, R. J. The effect of sulfonate
leaving groups on the hypoxia-selective toxicity of nitro analogs of the
duocarmycins. Bioorg. Med. Chem. 2011, 19, 4851−4860.
(17) Jaramillo, P.; Domingo, L. R.; Perez, P. Towards an intrinsic
nucleofugality scale: The leaving group (LG) ability in CH3LG model
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ACKNOWLEDGMENTS
■
We are grateful for the assistance of Sisira Kumara and Karin
Tan for HPLC determinations, Dr. Maruta Boyd and Dr.
Shannon Black for NMR spectrometry, and Wouter van
Leeuwen, Caroline McCulloch, Sally Bai, and Sunali Mehta for
cytotoxicity studies and in vivo antitumor assays. This work was
funded by the Foundation for Research, Science and
Technology (NERF grant) and the Auckland Division of the
Cancer Society of New Zealand.
ABBREVIATIONS USED
■
AIBN, azobisisobutyronitrile; CBI, seco-1,2,9,9a-
tetrahydrocyclopropa[c]benz[e]indol-4-one; DIPEA, diisopro-
pylethylamine; DEI, 5-[(dimethylamino)ethoxy]indole; DMA,
dimethylacetamide; DPPA, diphenylphosphoryl azide; E(1),
one-electron reduction potential; EDCI·HCl, N-ethyl-N′-(3-
dimethylaminopropyl)carbodiimide hydrochloride; EWG, elec-
tron-withdrawing group; HAP, hypoxia-activated prodrug;
HCR, hypoxic cytotoxicity ratio; iv, intravenous; LCK, log10
cell kill; MEI, 5-[(morpholino)ethoxy]indole; MS, 4-methox-
ystyrene; pyBOP, benzotriazol-1-yl-oxytripyrrolidinophospho-
nium hexafluorophosphate; SAR, structure−activity relation-
ship; TEMPO, (2,2,6,6-tetramethylpiperidin-1-yl)oxyl; TFA,
trifluoroacetic acid; TFAA, trifluoroacetic anhydride; THP,
tetrahydropyranyl; TMI, 5,6,7-trimethoxyindole
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