Weight loss effects of quaternary salts of 5-amino-1-(chloromethyl)-1,2- dihydro-3H-benz[e]indoles; Structure-activity relationships

Article abstract of DOI:10.1016/j.bmc.2011.12.007

Quaternary salt analogues based on the DNA minor groove binder and adenine N3 alkylating agent 5-amino-1-(chloromethyl)-1,2-dihydro-3H-benz[e]indole (aminoCBI) show remarkable effects on the body weight of mice (a long-term failure to gain weight relative

Full text of DOI:10.1016/j.bmc.2011.12.007

Bioorganic & Medicinal Chemistry 20 (2012) 734–749
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Weight loss effects of quaternary salts of 5-amino-1-(chloromethyl)-1,
2-dihydro-3H-benz[e]indoles; structure–activity relationships
Moana Tercel a, Ralph J. Stevenson a, Guo-Liang Lu a, Stephen M. Stribbling a, William R. Wilson a,
Michele A. Tatnell b, Rebecca N. Marnane b, Kathleen G. Mountjoy b,c, William A. Denny a,
⇑
a Auckland Cancer Society Research Centre, School of Medical Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
b Department of Physiology, School of Medical Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
c Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
Quaternary salt analogues based on the DNA minor groove binder and adenine N3 alkylating agent
5-amino-1-(chloromethyl)-1,2-dihydro-3H-benz[e]indole (aminoCBI) show remarkable effects on the
body weight of mice (a long-term failure to gain weight relative to matched controls with no loss of appe-
tite or perceptible deterioration in health) following administration of a single (non-toxic) dose between
Received 1 October 2011
Revised 27 November 2011
Accepted 3 December 2011
Available online 10 December 2011
about 0.5–5 lmol/kg. The nature of the quaternizing group was not important, but a related hydroxyCBI
analogue was much less effective. Compounds where the chloro group was replaced by a hydrogen or
hydroxy group (thus abrogating DNA alkylating capability) showed no weight control activity. It is spec-
ulated, based on other studies, that the marked long-term weight control effect is due to inhibition of bile
flow into the intestine and reduced absorption of triglycerides, together with accelerated cell death in
spleen and white adipose tissues due to drug accumulation there. This class of compound may serve
as interesting tools for further study of these phenomena.
Keywords:
Benz[e]indole
AminoCBI
Quaternary salt
Weight loss
SAR study
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
have significant side-effects, which have limited their use.1
Sibutramine has sympathetic nervous system effects that can
Obesity is a major and rising health problem world-wide, as it is
associated with many other disease states, including type II diabe-
tes, coronary artery diseases, osteoarthritis, and some types of can-
cer.1 Much is now known about the biochemical mechanisms that
regulate energy metabolism and body weight,2 and drugs are avail-
able that lower food intake by modulating the hypothalamic sys-
tem,1 block nutrient absorption3 or increase energy output
(thermogenesis).4 Of these only orlistat (1) and sibutramine (2)
(Fig. 1) have been approved by the FDA for long-term treatment
(and orlistat was withdrawn in 2010).
Orlistat is an inhibitor of pancreatic lipase that reduces energy
intake by preventing fat absorption,3 and sibutramine is a selective
inhibitor of serotonin and noradrenalin re-uptake, although much
of the activity appears to reside in its desmethyl metabolites.5
However, these compounds must be taken continuously, and both
result in elevated blood pressure and heart rate,6 while the side-
effects of orlisat are primarily gastrointestinal (flatulence and
frequent loose stools).7
The 5-amino-1-(chloromethyl)-1,2-dihydro-3H-benz[e]indole
(aminoCBI) class of DNA minor groove alkylators are very potent
cytotoxins,8 and have been utilised as effector units in a number
of classes of prodrugs designed for cancer therapy. These have
included antibody conjugates,9 prodrugs for gene therapy based
on nitrobenzyl carbamates,10 and the corresponding nitroCBIs as
hypoxia-activated prodrugs.11 In studies of the antiproliferative
effects of the N,N-dimethyl-N-(pyrrolylmethyl) quaternary salt 3
(see Table 1) (designed as a putative hypoxia- and radiation-
activated cytotoxin based on previous studies with related mustard
NHCHO
Abbreviations: AIBN, 2,20-azobis[isobutyronitrile]; CBI, seco-1,2,9,9a-tetrahydro-
cyclopropa[c]benz[e]indol-4-one; CSA, camphorsulfonic acid; DIPEA, diisopropyl-
ethylamine; DMA, dimethylacetamide; DMAP, 4-dimethylaminopyridine;
EDCIꢀHCl, N-ethyl-N0-(3-dimethylaminopropyl)carbodiimide hydrochloride; ip,
intra-peritoneal; NMP, N-methylpyrrolidone; pyBOP, benzotriazol-1-yl-oxytripyrr-
olidinophosphonium hexafluorophosphate.
O
O
O
NMe2
(CH2)4Me
Cl
Me(CH2)10
2
1
⇑
Corresponding author. Tel.: +64 9 923 6144; fax: +64 9 373 7502.
E-mail address: b.denny@auckland.ac.nz (W.A. Denny).
Figure 1. Structures of orlistat (1) and sibutramine (2).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.12.007

M. Tercel et al. / Bioorg. Med. Chem. 20 (2012) 734–749
735
Table 1
Structural and biological data for benzindole quaternary salts
X
R
N
O
Y
No.
X
Y
R
Dosea
(l
mol/kg)
Weight gainb (%)
Significance
Treated
Control
0.47
1.4
4.2
38.3 4.9
19.0 1.8
ꢁ4.1 2.8
27.7 4.6
31.5 8.2
30.1 5.9
P <0.02c
Me
N
-
+
NS
Br
P <0.0037c
3
Cl
NH2
N
NO2
Me Me
0.14
0.42
1.4
44.1 5.3
41.6 8.7
23.6 3.7
26.1 7.9
NS
Me
N
-
ꢁ7.6 6.2
P <0.0035c
Br
P <0.0007c
+
4
5
Cl
Cl
NH2
NH2
ꢁ22.8 7.0
N
SO2Me
NO2
Me Me
Me
N
4.2
21.0 0.4d
26.1 7.8
27.7 4.6
NS
N
Me
0.47
4.2
17.0 2.6
Early deaths
P <0.03c
Me
Me
-
6
7
Cl
Cl
NH2
OH
+
N
Cl
Me
Me
N
-
+
Br
N
4.2
18.8 6.8
27.7 4.6
NS
NO2
Me Me
12.6
23.7 3.7
18.8 2.5
18.8 1.8
31.5 8.2
NS
NS
Me
N
-
+
37.8e
Br
N
8
OH
OH
H
NH2
NH2
NH2
NO2
SO2Me
NO2
Me Me
37.8
50.2
117
36.8 4.9
42.6 17.0
Early deaths
23.9 4.6
27.4 9.4
P <0.0166f
P <0.0287c
Me
-
+
Br
N
N
9
Me Me
4.2
37.8
26.0 11.9
43.2 14.6
27.7 4.6
35.6 3.1
NS
NS
Me
N
-
+
Br
N
10
Me Me
Me
N
-
Br
+
N
SO2Me
11
12
H
NH2
37.8
30.4 5.0
35.6 3.1
33.3 5.0
NS
Me Me
Cl
4.2
12.6
16.8 5.2
Early deaths
P <0.0178c
-
TFA
Me
N
(CH2)5
N
+
N
Me
SO2Me
Me
O
NH2
(continued on next page)

736
M. Tercel et al. / Bioorg. Med. Chem. 20 (2012) 734–749
Table 1 (continued)
No.
X
Y
R
Dosea
(l
mol/kg)
Weight gainb (%)
Significance
Treated
Control
4.2
12.6
11.6 5.9
2.9 7.9
33.3 5.0
41.6 8.7
P <0.0124c
P <0.0035c
Cl
-
Me
N
TFA
(CH2)6
N
+
13
N
Me
SO2Me
Me
O
NH2
OH
377
23.7 4.2
ꢁ22.1 10.1
24.5 3.5
12.8 1.5
NS
1130g
P <0.0003h
O
HO
HO
OH
14 (STZ)
NHCONCH3
NO
a
Dose in 42% aqueous DMSO, given ip as a single treatment on day 1 to groups of 4 animals.
Body weight was measured at day 60, averaged for each treatment group and for the control (vehicle-treated) group, and is reported as the mean % loss (or gain) in weight
b
relative to the pre-treatment value.
c
Paired Student’s ‘t’ test.
2/4 died by day 15.
d
somewhat unstable bromide 16. Similar treatment of the known16
methylsulfone alcohol 17 gave the bromide 18.
To prepare the aminoCBI agents 3–6 of Table 1 (Scheme 2), the
5-amino substituent of the known8 trifluoroacetamide 26 was pro-
tected as the bisBoc derivative 27 (Boc2O/DMAP) to avoid alkyl-
ation during the quaternization step. This was then treated with
aqueous Cs2CO3 to cleave the trifluoroacetamide and provide the
free amine, which was reacted directly with the acid chloride of
4-(dimethylaminomethyl)cinnamic acid 20 (prepared as shown
in Scheme 1 from ester 19) to generate 29. Quaternization of 29
was performed with 16, 18, or with methyl iodide, in THF or EtOAc,
to give the intermediates 30a–c. Cleavage of the Boc protecting
groups from these with HBr or HCl in dioxane then gave the de-
sired quaternary salts 3, 4 and 6. For 6 the chloride salt was ob-
tained by ion exchange chromatography. The compounds were
shown by HPLC analysis to be contaminated with 0.3–1.0% of the
unquaternized amine 31, an authentic sample of which was pre-
pared by Boc-deprotection of 29. The tertiary amine 5 was pre-
pared by triflate deprotection of 27 and coupling with the acid
chloride of 25 (prepared as shown in Scheme 1) to give 28, which
was deprotected with HCl to generate 5.
Figure 2. Effect of a single ip dose of 3 (4.2 lM/kg) on the body weight of female
C3H/HeN mice over time. Values are mean and SEM for 6 mice/group.
The phenol CBI 7 was prepared from the previously described17
phenol 32, which was Boc-deprotected and allowed to react with
4-(dimethylaminomethyl)cinnamic acid (20) in the presence of
EDCI (Scheme 3). The crude amine could then be directly quatern-
ized using 16 as above. The quaternary salt product still contained
traces of 33 (0.15% by HPLC analysis).
The 1-(hydroxymethyl) derivatives 8 and 9 (Scheme 4) were
prepared from the known18 1-(hydroxymethyl)-5-nitroCBI deriva-
tive 34. The Boc protecting group was first changed to trifluoroac-
etamide, then 35 was hydrogenated over PtO2 to give the
somewhat unstable 5-amino compound that was protected as
the trisBoc derivative 36. Deprotection of the trifluoroacetamide
and coupling with E-4-(dimethylaminomethyl)cinnamoyl chloride
gave the key benzylamine intermediate 37. Quaternization of this
with bromides 16 or 18 gave 38a and 38b, respectively, which
were deprotected with HBr in dioxane to give the hydroxy com-
pounds 8 and 9.
quaternary salts12,13) we observed a remarkable body weight effect
in female C3H mice. Following a single ip dose the animals showed
a long-term failure to gain weight relative to matched controls
with no perceptible deterioration in health and no loss of appetite
or difficulty in eating or drinking14 (see below, and Fig. 2). We
undertook an initial exploration of this by seeking to define struc-
ture–activity relationships (SAR) for the phenomenon, and report
the results in the present paper.
2. Results and discussion
2.1. Chemistry
The (bromomethyl)heterocycles were prepared as in Scheme 1.
The known15 alcohol 2-(hydroxymethyl)-1-methyl-5-nitropyrrole
(15) was treated with bromine/triphenylphosphine to give the
The 1-methyl compounds 10 and 11 were synthesized from the
known19 aminoCBI precursor 39, which was cyclized with Bu3SnH/
AIBN to 40 (Scheme 5). This was deprotected with TFA, then

M. Tercel et al. / Bioorg. Med. Chem. 20 (2012) 734–749
737
i
OH
Br
R
R
N
N
Me
Me
15: R=NO2
16: R=NO2
17: R=SO2Me
18: R=SO2Me
ii
NMe2.HCl
NMe2
HO2C
RO2C
MeO2C
20
19
Me
N
NO2
R
N
Me
v
tBuO2C
21: R=H
22: R=Br
iii
iv
24: R=tBu
25: R=H
vi
23: R=NHMe
Scheme 1. Reagents and conditions: (i) Br2, PPh3, MeCN, 15–35 min, 20 °C; (ii) NaOH, MeOH/H2O, 2 h, reflux, then HCl; (iii) AIBN, NBS, CCl4, reflux, 15 h; (iv) aqueous MeNH2,
EtOH, DCM, 2 h, 20 °C; (v) DIPEA, THF, 16, 15 h, 20 °C (N2); (vi) TFA, 1 h, 20 °C.
Me
N
NO2
N
Me
Cl
R
N
O
28: R=N(Boc)2
5: R=NH2
v
Cl
Cl
ii
NMe2
N
NCOCF3
ii
O
N(Boc)2
R
29
26: R=NH2
i
27: R=N(Boc)2
v
iii
Cl
Cl
R
NMe2
N
O
N
O
31
N(Boc)2
30a-c
NH2
iv
3, 4 and 6 of Table 1
compounds
a
R =
Me2
N
Br-
Me2
b
R =
c
R =
+
N
NMe3
NO2
SO2Me
+
N
Me
+
N
Me
Cl-
Br-
Scheme 2. Reagents and conditions: (i) (Boc)2O, DMAP, dioxane, 18 h, reflux (N2); (ii) Cs2CO3, THF/H2O, 8 h, 20 °C, then E-4-(dimethylaminomethyl)cinnamoyl chloride or the
acid chloride of 25, MeCN, DMF, 2–5.5 h, 20 °C; (iii) 16, 18, or MeI, THF or EtOAc, 16 h–3 days, 20 °C (dark); (iv) HBr or HCl, dioxane, 12–16 h, 20 °C (dark); (v) HCl(g), dioxane,
1 h, 20 °C.

738
M. Tercel et al. / Bioorg. Med. Chem. 20 (2012) 734–749
47a and 47b and re-protected to give the 5-N(Boc)2 derivatives
Cl
Cl
48a and 48b. These compounds were then quaternized with 18
to give the intermediates 49a and 49b, which were deprotected
with HCl to give the desired compounds 12 and 13.
R
NBoc
N
Following an analysis of the SAR studies, which suggested that 4
was a highly active compound, a radio-labelled sample of this was
prepared, to enable drug disposition studies22 (Scheme 7). The
most accessible site for introducing a radiolabel is the methyl
group of the quaternary nitrogen, allowing use of commercially
available radiolabelled methylating agents. To this end, the mono-
methyl version (55) of compound 30b (Scheme 2) was prepared
starting from 18 and 23 and progressing through allylamine 53
(Scheme 7). This was the deallylated and re-protected as the bisBoc
compound 55, which was alkylated with tritium-enriched methyl
iodide to give 56. Deblocking then gave tritium-labelled 4 (3H-4)
(chemical purity 85%, radiochemical purity 71%, specific activity
107 GBq/mmol). Intensive attempts to improve the radiochemical
purity by fractional crystallisation or HPLC purification were not
successful.
i
O
32 OH
OH
33
7
: R=CH2NMe2
ii
Me2
: R =
NO2
N
N
Me
+
Br-
Scheme 3. Reagents and conditions: (i) HCl(g), dioxane, 1 h, 20 °C, then 20,
EDCIꢀHCl, DMA, 16 h, 20 °C; (ii) 16, THF, 18 days, 20 °C (dark).
coupled with (E)-4-(dimethylaminomethyl)cinnamoyl chloride to
give 41 selectively. The allyl group was removed using the method
of Honda et al.20 with Pd(Ph3P)4/PhSO2Na/camphorsulfonic acid
(CSA) to give 42 in excellent yield. Re-protection of 42 as the bisBoc
derivative 43 was followed by quaternization with bromides 16
and 18 to give 44a and 44b, respectively. Boc-deprotection of these
with HCl, followed by ion exchange chromatography, gave the re-
quired compounds 10 and 11.
Finally, the longer-chain quaternary compounds 12 and 13
were prepared from the known19 5-allylaminoCBI derivative 45
(Scheme 6). The Boc groups were removed with TFA, and the
resulting diamine was selectively coupled with 6-(dimethyl-
amino)hexanoyl chloride21 or 7-(dimethylamino)heptanoyl chlo-
ride21 to give 46a and 46b. These were then de-allylated to give
2.2. Biology
The structures of the compounds studied are shown in Table 1.
Compounds 3 (SN28127) and 4 (SN29220) were studied in greater
detail elsewhere22 to explore the scope and mechanism of their ef-
fects. In the present paper, we study a wider range of analogues in
a single assay, seeking to define structure–activity relationships.
Male C3H/HeN mice (ꢂ50 days old at time of drug treatment;
groups of 4) were monitored for 7–10 days to establish normal eat-
ing and weight gain behaviour, then were given drug as a single ip
injection in 42% aqueous DMSO. Body weight and food and water
intake were measured for a further 60 days. Individual animals in
OH
OBoc
NR
ii
NCOCF3
iii
NO2
N(Boc)2
36
34: R=Boc
i
35: R=COCF3
OBoc
OBoc
R
NMe2
N
N
iv
O
O
38a, b
37
N(Boc)2
v
N(Boc)2
compounds 8 and 9 of Table 1
b
a
R =
Me2
Me2
SO2Me
R =
NO2
N
N
+
N
N
Me
+
Br-
Br-
Me
Scheme 4. Reagents and conditions: (i) HCl(g), dioxane, 15 h, 20 °C, then (CF3CO)2O, pyridine, 30 min, 0 °C; (ii) PtO2ꢀxH2O, THF, MeOH, H2, 40 psi, 1 h, then (Boc)2O, DMAP,
dioxane, 20 h, reflux (N2); (iii) Cs2CO3, DCM, MeOH 1 h, 20 °C, then E-4-(dimethylaminomethyl)cinnamoyl chloride, Et3N, THF, 15 h, 20 °C; (iv) 16 or 18, THF, 5–6 days, 20 °C
(dark); (v) HBr(g), dioxane, 15 h, 20 °C (dark).

M. Tercel et al. / Bioorg. Med. Chem. 20 (2012) 734–749
739
Me
Me
NMe2
I
NBoc
NBoc
N
i
ii
O
BocN
41
: R=NHCH2CH=CH2
BocN
R
iii
iv
42: R=NH2
40
39
43: R=N(Boc)2
Me
R
a
R =
Me2
N
NO2
N
Me
N
v
+
Br-
O
b
R =
Me2
44a, b
SO2Me
N
Me
N(Boc)2
vi
N
+
Br-
compounds 10 and 11 of Table 1
Scheme 5. Reagents and conditions: (i) AIBN, Bu3SnH, benzene, 15 min, reflux; (ii) TFA, 2 h, 20 °C, then E-4-(dimethylaminomethyl)cinnamoyl chloride (from acid 20), THF,
12 h, 20 °C; (iii) Pd(Ph3P)4, PhSO2Na, CSA, DCM , 3 h, 20 °C (N2); (iv) (Boc)2O, DMAP, dioxane, 48 h, reflux; (v) 16 or 18, THF, 48–85 h, 20 °C (N2, dark); (vi) HCl(g), dioxane, 15 h,
20 °C (dark).
treatment and control groups (given vehicle only) were weighed
regularly and the mean body weight change at the end of the
experiment for the treatment and control (vehicle-only) groups
are recorded in Table 1. Food and water intake for each group were
also monitored over the course of the experiment. As explored in
more detail elsewhere,22 with most compounds there was a sub-
stantial dose-dependent but transient increase in both food and
water intake from about days 15–25 after treatment (up to a max-
imal 50% increase in average food intake, and up to a maximal
threefold increase in water consumption). In all cases, food and
water intakes had returned to control levels before 60 days.22
The original nitroimidazole analogue 3 demonstrated a consis-
resulted in substantial and prolonged weight control with respect
to untreated animals at 60 days post-dosing (Table 1 and Fig. 2).
To explore the possible influence of metabolic reduction of the
nitro group (which would be expected to result in fragmentation
and release of the very cytotoxic DNA minor groove alkylator
32),12 the nitro group was replaced in compound 4 with a methyl-
sulfonyl group, which has similar electronic properties, but is not a
ready substrate for bioreduction. This compound proved more po-
tent as a weight controller, showing substantial prolonged effects
at doses of both 0.42 and 1.4
lmol/kg, but caused some deaths at
12.6 mol/kg. To explore whether the quaternary salt function
l
was necessary, a limited study was done with compound 5 (the
tent dose–response effect for body weight; a dose of 0.47
kg was ineffective (both with regard to body weight and food
and water intake), but the higher doses of 1.4 and 4.2 mol/kg
lmol/
des-methyl, non-quaternized analogue of 3), but this was not effec-
tive at a dose (4.2 lmol/kg) where 3 showed substantial effects,
and also caused some deaths at that dose. Finally, to evaluate the
importance of the nature of the quaternary centre, we prepared
the simple trimethylammonium compound 6. This showed modest
l
Cl
Cl
weight control (P <0.03) at a non-toxic dose (0.47
lmol/kg), and
was toxic at 4.2 mol/kg (Fig. 3). An experiment with repeat dosing
l
n
was conducted with 6, which was given at both day 0 and day 40.
The results (Fig. 3) show that with this compound a second dose
generated a similar but attenuated response.
N
NBoc
NMe2
i
iv
O
We then turned to exploration of the benzindole chromophore.
Compound 7 is a hydroxyCBI (the phenol analogue of 3). Non-qua-
ternary hydroxy- and aminoCBIs have broadly similar mechanisms
of cytotoxicity, through alkylation of DNA at the N3 of adenines in
poly-AT sequences.23 However, in the weight loss assay 7 showed
R
BocN
46a, b: R=NHCH2CH=CH2
47a, b: R=NH2
45
ii
iii
a
: n=5
b: n=6
48a, b
: R=N(Boc)2
no significant effect on mouse body weight at a dose (4.2 lmol/kg)
Cl
where 3 was significantly active (P <0.0037).
Me
N
Of more potential interest were analogues where the chloro
leaving group on the chromophore was replaced, to abrogate the
DNA alkylating capability of the compounds. This was investigated
firstly with the corresponding alcohols 8 and 9 (the direct ana-
logues of chloro compounds 3 and 4, respectively) since these were
synthetically accessible from available intermediates. Even at
n Br-
N
+
SO2Me
N
Me2
v
compounds 12
O
and 13 of Table 1
49a, b
N(Boc)2
much higher doses (38 for compound 8 and 50
pound 9), the former showed no and the latter only modest weight
control activity. Compound 9 was also tested at 117 mol/kg, but
was acutely toxic at that dose. Because it was not possible to
conclusively prove that 8 and 9 did not contain small amounts of
potentially active bromide from the HBr deblocking step (different
lmol/kg for com-
Scheme 6. Reagents and conditions: (i) TFA, 3 h, 20 °C, then 6-(dimethyl-
amino)hexanoyl chloride hydrochloride or 7-(dimethylamino)heptanoyl chloride
hydrochloride, DCM, 30 min, 20 °C; or HCl, dioxane, 3 h, 20 °C, then 7-(dimethyl-
amino)heptanoyl chloride hydrochloride, DMF, DIPEA, 16 h, 20 °C; (ii) Pd(Ph3P)4,
PhSO2Na, CSA, DCM, 30 min, 20 °C (N2); (iii) (Boc)2O, DMAP, THF, dioxane, 12–15 h,
reflux; (iv) 18, K2CO3, THF, 2 h, 20 °C; (v) TFA, DCM, 6 h, 20 °C.
l

740
M. Tercel et al. / Bioorg. Med. Chem. 20 (2012) 734–749
Cl
Me
N
SO2Me
23
+
i
N
Me
NBoc
18
+
R
50: R=CO2tBu
51: R=CO2H
52 R=COCl
ii
iii
NBoc
45
Cl
Me
N
SO2Me
N
N
Me
iv
O
v
53: R=NHCH2CH=CH2
54: R=NH2
R
N
vi
55 R=N(Boc)2
vii
Cl
R
Me
X-
N
N
SO2Me
Me
*
Me
O
56: R=N(Boc)2; X=I
3H-4: R=NH2; X=Cl
viii
Scheme 7. Reagents and conditions: (i) DIPEA, THF, 15 h, 20 °C; (ii) TFA, 1 h, 20 °C; (iii) (COCl)2, DMF, DCM, 3 h, 20 °C; (iv) TFA, 3 h, 0 °C; then 52, DIPEA, DCM, 3 h, 20 °C; (v)
CSA, PhSO2Na, Pd(PPh3)4, DCM, 3 h, 20 °C; (vi) (Boc)2O, DMAP, THF, dioxane, reflux overnight; (vii) 3H-MeI, NMP, sealed vial, 8 days, 40 °C; (viii) 4 N HCl, dioxane, 15 h, 20 °C.
batches of 8 gave considerably different biological results), we also
prepared and evaluated the corresponding methyl compounds 10
and 11 as unequivocal non-alkylating analogues. These are the
analogues of compounds 3 and 4, respectively, but with the chloro-
methyl group replaced by methyl, and were completely ineffective
body weight without any apparent toxicity, compared to control-
treated animals in a mouse model. The reduction is sustained for
60 days post injection (and for up to 160 days in other experi-
ments21), while the mice continue to eat as much food as control
mice. Drug-treated mice showed an increase in water intake be-
tween about 15–25 days post injection, but this then returned to
normal. In all cases where dose–response studies were done,
reduction in body weight and transient increase in thirst were both
dose-dependent.22 The body weight loss appeared to maximize at
about 25 days post drug injection and then remained fairly stable.
More detailed studies22 indicated that the weight loss was largely
due to substantial (50–75%) reductions in body fat. The SAR shows
that a DNA alkylating capability is a necessary but not sufficient
(compound 7) condition for these compounds to show this sus-
tained body weight control. Mechanism of action studies22 with
compounds 3 and 4 tentatively suggest that these cause inhibition
of bile flow into the intestine and reduced absorption of triglycer-
ides, together with accelerated cell death in spleen and white adi-
pose tissues due to drug accumulation there. Tissue distribution
studies22 with 3H-4 showed that within 1–2 days most of the
radioactivity accumulated in the pancreas, spleen, liver, bile duct,
stomach, kidneys, visceral fat, and retroperitoneal fat, and was re-
tained in the spleen, visceral fat and retroperitoneal fat for up to
30 days after treatment. While this class of compound may not
be suitable as drugs due to their DNA-alkylating capability, the
observation that they appear to reset the ‘set-point’ body weight
in mice is intriguing.
in terms of weight control at doses of 38 lmol/kg, suggesting that
DNA alkylating ability is required for the weight loss effects.
Having thus established the requirement for the 5-amino-1-
(chloromethyl)-1,2-dihydro-3H-benz[e]indole (potentially DNA
alkylating) chromophore and the quaternary centre for weight loss
in this series, we finally looked at the effect of varying the link be-
tween these units, to see whether their relative positioning was
important. Replacing the cinnamide unit with more flexible alkyl
linkers gave compounds 12 and 13, which showed effective long-
term weight control at a dose of 4.2
lmol/kg (P <0.0178). Com-
pound 13 was also effective (P <0.0035) at a dose of 12.6
lmol/
kg, whereas 12, different only by one methylene unit) was toxic
at that dose.
While no true positive controls are available for this unique
long-term weight control effect, the compounds were compared
with the glucosylnitrosourea streptozotocin (STZ; 14), since both
compounds show selective toxicity to the pancreas.22,24 However,
14 showed no weight loss effect at 377
lmol/kg, and was toxic
at 1130 mol/kg, with excessive and continuing weight loss result-
l
ing in termination of the experiment at day 18. The mechanism of
action and spectrum of action may not be the same; 14 shows pro-
miscuous methylation of DNA, including at adenine N-3 sites,25
whereas the 5-aminoCBIs are exclusive adenine-N3 alkylators.23
More importantly, 14 readily induces type 1 diabetes, whereas a
detailed study22 of compounds 3 and 4 shows that these do not.
4. Experimental
4.1. Chemistry
3. Conclusions
All reagents used were of analytical grade. Analyses were car-
ried out in the Microchemical Laboratory, University of Otago,
Dunedin, NZ. Melting points were determined on an Electrother-
mal 2300 Melting Point Apparatus. Final products were analysed
Single non-toxic doses of N,N-dimethyl-N-(pyrrolylmethyl) and
other quaternary salts of 5-amino-1-(chloromethyl)-1,2-dihydro-
3H-benz[e]indole can cause substantial and permanent control of

M. Tercel et al. / Bioorg. Med. Chem. 20 (2012) 734–749
741
evaporator. Thin-layer chromatography was carried out on alumi-
num-backed silica gel plates (Merck 60 F254), with visualization of
components by UV light (254 nm) or exposure to I2. Column chro-
matography was carried out on silica gel, (Merck 230–400 mesh).
DCM refers to dichloromethane; DMSO refers to dimethylsulfox-
ide; EtOAc refers to ethyl acetate; MeOH refers to methanol; MeCN
refers to acetonitrile; petroleum ether refers to petroleum ether,
boiling range 40–60 °C. All solvents were freshly distilled.
4.1.1. 2-(Bromomethyl)-1-methyl-5-nitropyrrole (16) (Scheme 1)
A solution of Br2 (0.45 mL, 8.7 mmol) in dry MeCN (5 mL) was
added dropwise to a solution of PPh3 (2.28 g, 8.7 mmol) in MeCN
(30 mL) at 0 °C, and the pale yellow solution was allowed to warm
to 20 °C. A solution of 2-(hydroxymethyl)-1-methyl-5-nitropyr-
role15 (15) (1.04 g, 6.7 mmol) in MeCN (15 mL) was added and
the purple solution was stirred at 20 °C for 35 min. The mixture
was evaporated and the residue purified by column chromatogra-
phy (EtOAc/petroleum ether, 1:9) to give 16 as a pale yellow crys-
talline solid (1.35 g, 92%): mp 92–92 °C; 1H NMR (CDCl3) d 7.16 (d,
J = 4.4 Hz, 1H), 6.27 (d, J = 4.4 Hz, 1H), 4.48 (s, 2H), 3.99 (s, 3H).
Anal. Calcd for C6H7BrN2O2: C, 32.90; H, 3.22; N, 12.79. Found: C,
33.31; H, 2.58; N, 12.71. Compound 16 was stable for several
months when stored in the freezer, but underwent solvent dis-
placement of the bromide on attempted recrystallization from
MeOH.
4.1.2. 2-(Bromomethyl)-1-methyl-5-(methylsulfonyl)pyrrole (18)
A solution of Br2 (373 mg, 2.33 mmol) in dry MeCN (5 mL) was
added dropwise to a cooled solution of Ph3P (527 mg, 2.33 mmol)
in dry MeCN (9 mL) at 0 °C. After the mixture became colorless, a
solution of 2-(hydroxymethyl)-1-methyl-5-(methylsulfonyl)pyr-
role16 (17) (400 mg, 2.12 mmol) in dry MeCN (3 mL) was added
at 20 °C. After 15 min MeCN was removed and column chromatog-
raphy (petroleum ether/EtOAc, 1:1) gave 18 (437 mg, 82%) as a
white crystalline solid: mp 131–134 °C; 1H NMR (CDCl3) d 6.84
(d, J = 4.1 Hz, 1H), 6.27 (d, J = 4.1 Hz, 1H), 4.49 (s, 2H), 3.90 (s,
3H), 3.10 (s, 3H). 13C NMR d 134.8, 130.7, 117.0, 110.4, 45.4, 31.9,
22.8. Anal. Calcd for C7H10BrNO2S: C, 33.35; H, 4.00; N, 5.56.
Found: C, 33.88; H, 4.08; N, 5.35. Compound 18 was stable for sev-
eral months when stored in the freezer.
Figure 3. Variation in (from top) average weight change and food and water intake
following treatment of male C3H/HeN mice with compound 6 at 0.42 mol/kg at
l
4.1.3. E-4-(Dimethylaminomethyl)cinnamic acid hydrochloride
(20)
day 0 and again at day 40. Body weight changes are shown as a % of mouse body
weight on day 0. Food and water intake are averaged per mouse. Data are shown as
means (n = 4 mice).
A solution of NaOH (974 mg, 24.4 mmol) in water (8 mL) was
added to a solution of methyl E-4-(dimethylaminomethyl)cinna-
mate26 (19) (2.67 g, 12.2 mmol) in MeOH (40 mL), and the mixture
was stirred at reflux for 2 h, then cooled to 20 °C. Aqueous HCl
(12.2 mL of 2.0 N solution, 24.4 mmol) was added and the solution
was evaporated to dryness. The residue was dried in a vacuum des-
iccator and then extracted overnight with MeCN in a Soxhlet appa-
ratus. The MeCN was reduced to a small volume (ca. 30 mL),
allowed to cool to 20 °C, and the white solid was filtered off and
dried to give acid 20 (1.10 g, 44%): mp 181–183 °C; 1H NMR
(DMSO-d6) d 7.63 (d, J = 8.1 Hz, 2H), 7.56 (d, J = 16 Hz, 1H), 7.33
(d, J = 8.1 Hz, 2H), 6.49 (d, J = 16 Hz, 1H), 3.42 (s, 2H), 2.15 (s,
6H); 13C NMR d 167.6, 143.4, 141.0, 133.0, 129.1, 128.0, 119.0,
62.8, 44.8. Anal. Calcd for C12H15NO2ꢀ1/4H2O: C, 68.71; H, 7.45;
N, 6.68. Found: C, 68.82; H, 7.70; N, 6.87.
by reverse-phase HPLC (Alltima C18 5
l
m column, 150 ꢃ 3.2 mm;
Alltech Associated, Inc., Deerfield, IL) using an Agilent HP1100
equipped with a diode-array detector. Mobile phases were gradi-
ents of 80% MeCN/20% H2O (v/v) in 45 mM NH4O2CH at pH 3.5
and 0.5 mL/min. Purity was determined by monitoring at
330 50 nm and was P95%, unless otherwise stated. NMR spectra
were obtained on a Bruker Avance 400 spectrometer at 400 MHz
for 1H spectra. Spectra were obtained in DMSO-d6 or CDCl3 unless
otherwise specified, and are referenced to Me4Si. Chemical shifts
and coupling constants were recorded in units of ppm and Hz,
respectively. Assignments were determined using COSY, HSQC,
and HMBC two-dimensional experiments where appropriate. Mass
spectra were determined on a VG-70SE mass spectrometer using
an ionizing potential of 70 eV at a nominal resolution of 1000.
High-resolution spectra were obtained at nominal resolutions of
3000, 5000, or 10000 as appropriate. FAB+ spectra used m-nitro-
benzyl alcohol as the matrix and a xenon atom gun. Accurate mass
calculations were referenced to polyethyleneglycol (PEG). Solu-
tions in organic solvents were dried with anhydrous Na2SO4. Sol-
4.1.4. E-[4-({Methyl[(1-methyl-5-nitro-2-pyrrolyl)methyl]-
amino}methyl)cinnamic acid trifluoroacetate (25)
AIBN (80 mg, 0.459 mmol) and NBS (830 mg, 4.59 mmol) were
added to a solution of tert-butyl E-4-methylcinnamate27 (21)
(1.00 g, 4.59 mmol) in CCl4 (20 mL) at reflux. After 15 h the mixture
was cooled and DCM was added. The solution was washed with
water and brine and then dried (Na2SO4) and evaporated. The
vents were evaporated under reduced pressure on
a rotary

742
M. Tercel et al. / Bioorg. Med. Chem. 20 (2012) 734–749
residue was purified by column chromatography (petroleum ether/
EtOAc, 49:1) to give tert-butyl E-4-(bromomethyl)cinnamate (22)
(686 mg, 50%) as a colorless oil: 1H NMR (CDCl3) d 7.57 (d,
J = 16.1 Hz, 1H), 7.46 (d, J = 8.1 Hz, 2H), 7.38 (d, J = 8.2 Hz, 2H),
6.35 (d, J = 16.0 Hz, 1H), 4.80 (s, 2H), 1.53 (s, 9H); HRMS (79Br) m/
z 296.0401 (M+), C14H17BrO2 requires 296.0412.
A solution of 22 (380 mg, 1.28 mmol) in EtOH (10 mL) and DCM
(3 mL) was added to a solution of methylamine (1.49 g, 19.2 mmol,
40% w/w) in water and the mixture was stirred for 2 h. DCM
(30 mL) was added and the organic layer was separated. The organ-
ic layer was washed with 2 M NaOH, water and brine and then
dried (Na2SO4) and evaporated. The residue was purified by col-
umn chromatography (DCM/MeOH, 99:1 then 49:1) to give tert-
butyl E-4-(methylaminomethyl)cinnamate (23) (195 mg, 62%) as
a yellow oil: 1H NMR (CDCl3) d 7.57 (d, J = 16.0 Hz, 1H), 7.47 (d,
J = 8.1 Hz, 2H), 7.32 (d, J = 8.1 Hz, 2H), 6.34 (d, J = 16.0 Hz, 1H),
3.77 (s, 2H), 2.44 (s, 3H), 2.04 (s, 1H), 1.52 (s, 9H); HRMS m/z
247.1572 (M+), C15H21NO2 requires 247.1572.
A solution of 23 (69 mg, 0.279 mmol), bromide 16 (51 mg,
0.233 mmol) and DIPEA (39 mg, 0.303 mmol) in THF (3 mL) was
stirred under N2 for 15 h. The solvent was removed under reduced
pressure and the residue was purified by column chromatography
(petroleum ether/EtOAc, 9:1) to give tert-butyl E-[4-({methyl[(1-
methyl-5-nitro-2-pyrrolyl)methyl]amino}methyl)cinnamate (24)
(102 mg, 94%) as a yellow oil: 1H NMR (CDCl3) d 7.57 (d,
J = 16.0 Hz, 1H), 7.46 (d, J = 8.1 Hz, 2H), 7.29 (d, J = 8.1 Hz, 2H),
7.14 (d, J = 4.3 Hz, 1H), 6.35 (d, J = 16.0 Hz, 1H), 6.10 (d, J = 4.3 Hz,
1H), 3.96 (s, 3H), 3.52 (s, 2H), 3.48 (s, 2H), 2.17 (s, 3H), 1.53 (s,
9H); HRMS m/z 385.2002 (M+), C21H27N3O4 requires 385.2002.
TFA (1 mL) was added to cooled (0 °C) 24 (40 mg, 0.104 mmol)
and the resulting solution was stirred for 1 h. The TFA was re-
moved under reduced pressure and the residue was triturated with
Et2O to give 25 as a hygroscopic colorless solid that turned to an oil
in air: 1H NMR (DMSO-d6) d 7.74 (d, J = 8.0 Hz, 2H), 7.59 (d,
J = 16.0 Hz, 1H), 7.48 (d, J = 7.9 Hz, 2H), 7.25 (d, J = 4.4 Hz, 1H),
6.57 (d, J = 16.0 Hz, 1H), 6.47 (d, J = 4.4 Hz, 1H), 4.23 (s, 2H), 4.14
(s, 2H), 3.88 (s, 3H), 4 protons not observed; HRMS (FAB) m/z
330.1454 (M+1ꢁCF3CO2H) C17H20N3O4 requires 330.1454.
washed with aqueous NaCl, dried (Na2SO4), and evaporated to pro-
vide crude amine as a yellow oil, which was immediately dissolved
in THF (40 mL) and treated with E-4-(dimethylaminomethyl)cin-
namoyl chloride [prepared from E-4-(dimethylaminomethyl)cin-
namic acid (20) (256 mg, 1.24 mmol) and oxalyl chloride in dry
MeCN 20 mL and DMF (one drop) stirred at 20 °C for 5.5 h, fol-
lowed by evaporation], followed by Et3N (0.52 mL, 3.73 mmol).
The resulting light brown suspension was stirred at 20 °C for
45 min, then a solution of KOH (0.58 g, 10 mmol) in H2O (6 mL)
was added and the mixture was stirred at 20 °C for a further 12 h
(to remove co-eluting by-product methyl E-4-(dimethylamino-
methyl)cinnamate). Solvent was evaporated and the aqueous resi-
due was extracted with EtOAc (ꢃ3) and purified by column
chromatography (EtOAc then EtOAc/MeOH, 4:1) to give 5-[bis
(tert-butoxycarbonyl)]amino-1-(chloromethyl)-3-[(E)-4-(dimethyl-
aminomethyl)cinnamoyl]-1,2-dihydro-3H-benz[e]indole (29) as a
yellow foam (480 mg, 77%): 1H NMR (CDCl3) d 8.58 (br s, 1H),
7.87 (d, J = 15.3 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.76 (d, J = 8.2 Hz,
1H), 7.58 (d, J = 8.0 Hz, 2H) 7.53 (t, J = 7.5 Hz, 1H), 7.45 (t,
J = 7.6 Hz, 1H), 7.37 (d, J = 8.0 Hz, 2H), 6.90 (d, J = 15.3 Hz, 1H),
4.59–4.53 (m, 1H), 4.46 (t, J = 9.5 Hz, 1H), 4.24–4.14 (m, 1H), 3.99
(dd, J = 11.3, 2.9 Hz, 1H), 3.53 (t, J = 10.8 Hz, 1H), 3.50 (s, 2H),
2.29 (s, 6H), 1.36 (s, 18H); 13C NMR d 164.4, 151.8, 151.7, 144.0,
141.1, 140.8, 137.3, 133.9, 129.7, 129.6, 128.4, 128.2, 127.3,
125.3, 123.9, 123.6, 122.7, 118.3, 118.0, 82.9, 63.8, 53.1, 46.1,
45.2, 42.7, 27.8; HRMS (FAB, 35Cl) m/z 620.29015 (M+H),
C35H43ClN3O5 requires 620.28912.
A solution of 29 (181 mg, 0.29 mmol) and bromide 16 (76.7 mg,
0.35 mmol) in THF (6 mL) was stirred in the dark at 20 °C for
3 days, giving a fine yellow precipitate. The precipitate was al-
lowed to settle and the supernatant was decanted off. The remain-
ing solid was triturated several times with MeCN, then filtered off
and dried to give N-4-((E)-3-{5-[bis(tert-butoxycarbonyl)]amino-
1-(chloromethyl)-1,2-dihydro-3H-benz[e]indol-3-yl}-3-oxo-1-pro-
penyl)benzyl-N,N-dimethyl-N-[(1-methyl-5-nitro-2-pyrrolyl)
methyl]ammonium bromide (30a) as a pale yellow solid (166 mg,
68%): mp 221 °C (dec.); 1H NMR (DMSO-d6) d 8.40 (s, 1H), 8.03 (d,
J = 8.3 Hz, 1H), 7.98 (d, J = 8.0 Hz, 2H), 7.76 (d, J = 15.4 Hz, 1H), 7.68
(d, J = 8.3 Hz, 1H), 7.64 (d, J = 8.3 Hz, 2H), 7.60 (t, J = 7.6 Hz, 1H),
7.53 (t, J = 7.5 Hz, 1H), 7.38 (d, J = 4.4 Hz, 1H), 7.35 (d, J = 16 Hz,
1H), 6.70 (d, J = 4.5 Hz, 1H), 4.83 (s, 2H), 4.66 (s, 2H), 4.65–4.47
(m, 3H), 4.09–3.99 (m, 2H), 4.02 (s, 3H), 2.95 (s, 6H), 1.37 (s, 9H),
1.36 (s, 9H). Anal. Calcd for C41H49BrClN5O7: C, 58.68; H, 5.88; N,
8.35; Br, 9.52. Found: C, 58.91; H, 5.77; N, 8.23; Br, 9.74.
Dioxane (3 mL) saturated with HBr gas was added to a suspen-
sion of 30a (144 mg, 0.17 mmol) in dioxane (6 mL); the mixture
was stirred in the dark at 20 °C for 12 h. Excess HBr was removed
under reduced pressure, then the solid was filtered off and dried to
give 3 as a yellow solid (120 mg, 97%): mp 170–180 °C (dec.); 1H
NMR (DMSO-d6) d 8.20 (br s, 1H), 8.04 (d, J = 8.5 Hz, 1H), 7.97 (d,
J = 8.2 Hz, 2H), 7.86 (d, J = 8.4 Hz, 1H), 7.73 (d, J = 15.3 Hz, 1H),
7.64 (d, J = 8.2 Hz, 2H), 7.54 (t, J = 7.5 Hz, 1H), 7.41 (t, J = 7.6 Hz,
1H), 7.37 (d, J = 4.5 Hz, 1H), 7.35 (d, J = 16 Hz, 1H), 6.70 (d,
J = 4.5 Hz, 1H), 4.84 (s, 2H), 4.67 (s, 2H), 4.55–4.46 (m, 2H), 4.31–
4.26 (m, 1H), 4.03 (s, 3H), 3.99 (dd, J = 11.1, 2.7 Hz, 1H), 3.85 (dd,
J = 10.4, 8.1 Hz, 1H), 2.96 (s, 6H); HRMS (FAB, 35Cl) m/z
558.22205, C31H33ClN5O3 requires 558.22719.
4.1.5. (E)-N-(4-(3-(5-Amino-1-(chloromethyl)-1H-benzo[e]
indol-3(2H)-yl)-3-oxoprop-1-enyl)benzyl)-N,N-dimethyl-1-
(1-methyl-5-nitro-1H-pyrrol-2-yl)methanaminium bromide
hydrobromide (3) (Scheme 2)
A solution of 5-amino-1-(chloromethyl)-3-(trifluoroacetyl)-1,2-
dihydro-3H-benz[e]indole8 (26) (0.64 g, 1.95 mmol) in dioxane
(50 mL) was treated with di-tert-butyl dicarbonate (1.5 g,
6.9 mmol) and DMAP (5 mg) and the solution was stirred at reflux
under N2 for 16 h. More di-tert-butyl dicarbonate (1.2 g, 5.5 mmol)
was added and the solution stirred at reflux for a further 2 h. The
solvent was evaporated and the residue purified by column chro-
matography (EtOAc/petroleum ether, 4:1) to give 5-[bis(tert-
butoxycarbonyl)]amino-1-(chloromethyl)-3-(trifluoroacetyl)-1,2-
dihydro-3H-benz[e]indole (27) as a cream solid (0.94 g, 91%). A
sample was recrystallized from benzene/petroleum ether as white
prisms: mp 181–182 °C; 1H NMR (CDCl3) d 8.37 (s, 1H), 7.87 (d,
J = 8.3 Hz, 1H), 7.80 (d, J = 8.1 Hz, 1H), 7.59 (dt, J = 7.6, 1.2 Hz,
1H), 7.53 (dt, J = 7.6, 1.3 Hz, 1H), 4.64 (d, J = 11.4 Hz, 1H), 4.45
(dt, J = 11.4, 8.5 Hz, 1H), 4.26–4.18 (m, 1H), 3.97 (dd, J = 11.5,
3.2 Hz, 1H), 3.57 (dd, J = 11.5, 9.4 Hz, 1H), 1.38 (s, 9H), 1.35 (s,
9H). Anal. Calcd for C25H28ClF3N2O5: C, 56.77; H, 5.34; N, 5.30.
Found: C, 56.63; H, 5.49; N, 5.23.
HPLC analysis indicated the presence of 0.3% of 31, an authentic
sample of which was prepared from 30 as described below.
4.1.6. 5-Amino-1-(chloromethyl)-3-[(E)-4-(dimethylami
nomethyl)cinnamoyl]-1,2-dihydro-(3H)benz[e]indole (31)
A solution of 29 (188 mg, 0.30 mmol) in dioxane (15 mL) was
saturated with HCl gas and stirred at 20 °C for 1 h, during which
time the HCl salt of the product precipitated. Aqueous NaHCO3
was added and the mixture was extracted with EtOAc (ꢃ3). The
A solution of Cs2CO3 (1.4 g, 4.3 mmol) in water (30 mL) was
added to a solution of 27 (440 mg, 0.83 mmol) in THF (40 mL),
and the two-phase mixture was stirred vigorously at 20 °C for
8 h. The THF was evaporated and the residue was diluted with
aqueous NaCl and extracted with EtOAc (ꢃ3). The extracts were

M. Tercel et al. / Bioorg. Med. Chem. 20 (2012) 734–749
743
extracts were dried (Na2SO4) and evaporated, and the residue was
purified by column chromatography (EtOAc then EtOAc/MeOH, 9:1
then 4:1), and recrystallized from benzene, giving 31 (as a yellow
solid (91 mg, 71%): mp 230–240 °C (dec.): 1H NMR (CDCl3) d 8.04
(br s, 1H), 7.86 (d, J = 15.3 Hz, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.68
(d, J = 8.3 Hz, 1H), 7.57 (d, J = 8.1 Hz, 2H), 7.50 (t, J = 7.3 Hz, 1H),
7.39–7.34 (m, 3H), 6.91 (d, J = 15.2 Ha, 1H), 4.51 (d, J = 10.6 Hz,
1H), 4.37 (t, J = 9.5 Hz, 1H), 4.32 (s, 2H), 4.09–4.02 (m, 1H), 3.95
(dd, J = 11.2, 2.8 Hz, 1H), 3.47 (s, 2H), 3.43 (t, J = 11.1 Hz, 1H),
2.27 (s, 6H); 13C NMR d 164.6, 144.2, 143.7, 142.1, 141.2, 133.9,
130.1, 129.6, 128.1, 127.2, 123.3, 122.9, 122.1, 121,1, 118,3,
114.6, 101.4, 64.0, 53.3, 46.3, 45.4, 42.6. Anal. Calcd for
(t, J = 7.6 Hz, 1H), 7.34 (d, J = 15.4 Hz, 1H), 4.67–4.47 (m, 5H),
4.08–3.98 (m, 2H), 3.05 (s, 9H), 1.37 (s, 9H), 1.36 (s, 9H). Anal.
Calcd for C36H45ClIN3O5ꢀH2O: C, 55.43; H, 6.07; N, 5.39. Found:
C, 55.66; H, 5.99; N, 5.33.
A solution of 30c (175 mg, 0.23 mmol) in dioxane (4 mL) was
treated with dioxane (2 mL) saturated with HCl gas. A precipitate
began to form within a few minutes. The mixture was stirred at
20 °C for 16 h, and the excess HCl was removed under reduced
pressure. The solid was filtered off, washed with dioxane and dried
to give a yellow-orange solid (122 mg): mp >310 °C; 1H NMR
(DMSO-d6) d 8.15 (br s, 1H), 8.07 (d, J = 8.5 Hz, 1H), 7.96 (d,
J = 8.2 Hz, 2H), 7.85 (d, J = 8.3 Hz, 1H), 7.73 (d, J = 15.4 Hz, 1H),
7.61 (d, J = 8.2 Hz, 2H), 7.53 (d, J = 7.6 Hz, 1H), 7.39 (t, J = 7.6 Hz,
1H), 7.33 (d, J = 15.4 Hz, 1H), 4.57 (s, 2H), 4.54–4.45 (m, 2H),
4.30–4.23 (m, 1H), 3.99 (dd, J = 11.0, 3.0 Hz, 1H), 3.84 (dd,
J = 11.0, 7.8 Hz, 1H), 3.07 (s, 9H). Microanalysis indicated a mixture
of chloride and iodide counterions. The mixed salt (110 mg) was
dissolved in MeOH (2 mL) and purified by ion exchange chroma-
tography (Biorad AG-1X4 resin in Clꢁ form), eluting with MeOH.
The yellow product-containing fractions were concentrated to a
small volume and diluted with EtOAc to give 6 as a yellow solid
C25H26ClN3O: C, 71.50; H, 6.24; N, 10.01. Found: C, 71.66; H,
6.18; N, 9.93.
4.1.7. (E)-N-(4-(3-(5-Amino-1-(chloromethyl)-1H-benzo[e]
indol-3(2H)-yl)-3-oxoprop-1-enyl)benzyl)-N,N-dimethyl-1-
(1-methyl-5-(methylsulfonyl)-1H-pyrrol-2-yl)methanaminium
bromide hydrobromide (4)
A solution of 29 (159 mg, 0.256 mmol) and bromide 18 (97 mg,
0.385 mmol) in THF (4 mL) was stirred in the dark at 20 °C for 3 days.
The precipitate was filtered off and triturated with Et2O to give N-4-
((E)-3-{5-[bis(tert-butoxycarbonyl)]amino-1-(chloromethyl)-1,2-
dihydro-3H-benz[e]indol-3-yl}-3-oxo-1-propenyl)benzyl-N,N-di-
methyl-N-[(1-methyl-5-methylsulfonyl-2-pyrrolyl)methyl]ammo-
nium bromide (30b) (206 mg, 92%) as a pale yellow solid: mp 182–
185 °C (dec.); 1H NMR (DMSO-d6) d 8.40 (s, 1H), 8.03 (d, J = 8.3 Hz,
1H), 7.98 (d, J = 8.0 Hz, 2H), 7.77 (d, J = 15.4 Hz, 1H), 7.72–7.64 (m,
3H), 7.61 (td, J = 7.5, 1.1 Hz, 1H), 7.54 (td, J = 7.6, 1.0 Hz, 1H), 7.35
(d, J = 11.5 Hz, 1H), 6.92 (d, J = 4.1 Hz, 1H), 6.64 (d, J = 4.1 Hz, 1H),
4.77 (s, 2H), 4.65 (s, 2H), 4.62–4.54 (m, 2H), 4.53–4.46 (m, 1H),
4.07 (dd, J = 11.3, 3.3 Hz, 1H), 4.01 (dd, J = 11.2, 6.4 Hz, 1H), 3.97 (s,
3H), 3.32 (s, 3H), 2.92 (s, 6H), 1.37 (s, 9H), 1.35 (s, 9H). Anal. Calcd
for C42H52BrClN4O7S: C, 57.83; H, 6.01; N, 6.42. Found: C, 57.55; H,
6.01; N, 6.34.
A solution of 30b (40 mg, 0.046 mmol) in dioxane (1 mL) was
treated with dioxane (2 mL) saturated with HBr gas and the mix-
ture was stirred in the dark at 20 °C for 15 h. Excess HBr was re-
moved under reduced pressure, then the solid was filtered off
and triturated with Et2O to give 4 as a yellow solid (28 mg, 80%):
mp 178–182 °C (dec.); 1H NMR (DMSO-d6) d 8.07 (d, J = 8.5 Hz,
1H), 8.01 (s, 1H), 7.96 (d, J = 8.2 Hz, 2H), 7.80 (d, J = 8.4 Hz, 1H),
7.71 (d, J = 15.4 Hz, 1H), 7.65 (d, J = 8.2 Hz, 2H), 7.50 (t, J = 7.5 Hz,
1H), 7.34 (t, J = 7.5 Hz, 1H), 7.33 (d, J = 15.4 Hz, 1H), 6.92 (d,
J = 4.1 Hz, 1H), 6.65 (d, J = 4.1 Hz, 1H), 4.78 (s, 2H), 4.64 (s, 2H),
4.53–4.42 (m, 2H), 4.25–4.16 (m, 1H), 4.00–3.93 (m, 1H), 3.95 (s,
3H), 3.79 (dd, J = 11.1, 8.1 Hz, 1H), 3.28 (s, 3H), 2.91 (s, 6H); HRMS
(FAB, 35Cl) m/z 591.2213 (M+1), C32H36ClN4O3S requires 591.2197.
HPLC purity 98.4%.
(83 mg, 79%). Anal. Calcd for
59.11; H, 6.38; N, 7.39; Cl, 18.69. Found: C, 58.90; H, 6.41; N,
7.77; Cl, 18.24.
C
26H30Cl3N3OꢀH2Oꢀ1/2EtOAc: C,
4.1.9. (E)-1-(5-Amino-1-(chloromethyl)-1H-benzo[e]indol-3(2H)
-yl)-3-(4-((methyl((1-methyl-5-nitro-1H-pyrrol-2-yl)methyl)
amino)methyl)phenyl)prop-2-en-1-one dihydrochloride (5)
A solution of 27 (60 mg, 0.114 mmol) and Cs2CO3 (153 mg,
0.456 mmol) in DCM (5 mL) and MeOH (5 mL) was stirred for
15 min. DCM (20 mL) and water were added and the organic layer
was separated. The organic layer was washed with water, brine,
and dried (Na2SO4), and then evaporated to provide crude amine
(49 mg), which was immediately dissolved in THF (3 mL) with
Et3N (58 mg, 0.57 mmol). The solution was then treated with a
solution of E-4-(dimethylaminomethyl)cinnamoyl chloride [pre-
pared from acid 20 (76 mg, 0.171 mmol) and oxalyl chloride
(187 mg, 1.54 mmol) in dry MeCN (3 mL) and DMF (one drop) stir-
red for 2 h and then evaporated under reduced pressure] in THF
(2 mL)] and the mixture was stirred for 2 h at 20 °C. After 2 h the
solvents were removed under reduced pressure and EtOAc and
H2O were added to the residue. The EtOAc extract was washed
with brine and dried (Na2SO4). Column chromatography (petro-
leum ether/EtOAc, 7:3 then 3:2) followed by trituration with
petroleum ether gave 5-[bis(tert-butoxycarbonyl)]amino-1-(chlo-
romethyl)-3-{(E)-3-[4-({methyl[(1-methyl-5-nitro-2-pyrrolyl)
methyl]amino}methyl)phenyl]-2-propenoyl}-1,2-dihydro-3H-ben-
z[e]indole (28) (53 mg, 63%) as a yellow powder: mp 110–115 °C
(dec.); 1H NMR (CDCl3) d 8.57 (s, 1H), 7.88 (d, J = 15.3 Hz, 1H),
7.82 (d, J = 8.4 Hz, 1H), 7.75 (d, J = 8.3 Hz, 1H), 7.58 (d, J = 8.0 Hz,
2H), 7.53 (td, J = 7.1, 0.9 Hz, 1H), 7.44 (td, J = 7.2, 1.0 Hz, 1H), 7.34
(d, J = 7.9 Hz, 2H), 7.15 (d, J = 4.3 Hz, 1H), 6.91 (d, J = 15.2 Hz, 1H),
6.12 (d, J = 4.3 Hz, 1H), 4.58 (d, J = 10.8 Hz, 1H), 4.47 (t, J = 9.1 Hz,
1H), 4.27–4.16 (m, 1H), 4.03–3.96 (m, 1H), 9.98 (s, 3H), 3.58–
3.49 (m, 1H), 3.57 (s, 2H), 3.52 (s, 2H), 2.20 (s, 3H), 1.36 (s, 18H).
Anal. Calcd for C40H46ClN5O7: C, 64.55; H, 6.23; N, 9.41. Found: C,
64.20; H, 6.07; N, 9.05.
Compound 4 was also obtained as a mixture of bromide and tri-
fluoroacetate salts by Boc-deprotection of 30b with TFA/DCM (1:1).
4.1.8. (E)-1-(4-(3-(5-Amino-1-(chloromethyl)-1H-benzo[e]
indol-3(2H)-yl)-3-oxoprop-1-enyl)phenyl)-N,N,N-trimethylme
thanaminium chloride hydrochloride (6)
A solution of 29 (180 mg, 0.29 mmol) and methyl iodide
(22 lL, 0.35 mmol) in EtOAc (2 mL) was allowed to stand in
the dark at 20 °C for 16 h. Et2O was added dropwise, causing
an oil to separate, and the supernatant was decanted off. The
remaining oil was triturated with EtOAc, and the resulting solid
was filtered off to give N-4-((E)-3-{5-[bis(tert-butoxycar-
bonyl)]amino-1-(chloromethyl)-1,2-dihydro-3H-benz[e]indol-3-
yl}-3-oxo-1-propenyl)benzyl-N,N,N-trimethylammonium iodide
(30c) (201 mg, 91%): mp >320 °C; 1H NMR (DMSO-d6) d 8.40
(s, 1H), 8.03 (d, J = 8.3 Hz, 1H), 7.97 (d, J = 8.0 Hz, 2H), 7.75 (d,
J = 15.4 Hz, 1H), 7.68 (d, J = 8.5 Hz, 1H), 7.63–7.58 (m, 3H), 7.53
A solution of 28 (55 mg, 0.074 mmol) in dioxane (1 mL) was
treated with dioxane (2 mL) saturated with HCl gas and the mix-
ture was stirred in the dark at 20 °C for 15 h. Excess HCl was re-
moved under reduced pressure, then the precipitate was filtered
off and triturated with Et2O to give 5 as a yellow solid (45 mg,
100%): mp 192–197 °C (dec.); 1H NMR (DMSO-d6) d 7.95 (d,
J = 8.4 Hz, 1H), 7.83 (s, 1H), 7.82 (d, J = 8.1 Hz, 2H), 7.64 (d,
J = 15.4 Hz, 1H), 7.59–7.47 (m, 2H), 7.55 (d, J = 8.2 Hz, 2H), 7.39
(t, J = 7.8 Hz, 1H), 7.25 (d, J = 4.5 Hz, 1H), 7.19 (d, J = 15.4 Hz, 1H),

744
M. Tercel et al. / Bioorg. Med. Chem. 20 (2012) 734–749
6.54 (d, J = 4.5 Hz, 1H), 4.49–4.33 (m, 2H), 4.45 (s, 2H), 4.37 (s, 2H),
4.30–4.22 (m, 1H), 3.77 (s, 3H), 2.66 (s, 3H), 2 protons not ob-
served. Anal. Calcd for C30H32Cl3N5O3ꢀH2Oꢀ1/2dioxane: C, 56.60;
H, 5.64; N, 10.31. Found: C, 56.91; H, 5.48; N, 10.47.
chromatography (petroleum ether/EtOAc, 4:1–1:1) gave 1-
(hydroxymethyl)-5-nitro-3-(trifluoroacetyl)-1,2-dihydro-3H-benz
[e]indole (35) (177 mg, 89%) as an orange powder: mp 150–151 °C;
1H NMR (CDCl3) d 9.11 (s, 1H), 8.49–8.40 (m, 1H), 7.99–7.92 (m,
1H), 7.74–7.67 (m, 2H), 4.67 (dt, J = 11.2, 1.5 Hz, 1H), 4.46 (dd,
J = 11.1, 8.7 Hz, 1H), 4.15–4.05 (m, 2H), 3.90–3.80 (m, 1H), 1.68
(t, J = 4.2 Hz, 1H). Anal. Calcd for C15H11F3N2O4: C, 53.00; H, 3.26;
N, 8.23. Found: C, 53.48; H, 3.34; N, 8.26.
A solution of 35 (200 mg, 0.588 mmol) in THF (15 mL) and
MeOH (3 mL) was hydrogenated over PtO2 ꢃ H2O (Acros, Pt con-
tent 79–84%, 30 mg) at 40 psi for 1 h. The mixture was filtered
through Celite and the filtrate was evaporated to give crude 5-ami-
no-1-(hydroxymethyl)-3-(trifluoroacetyl)-1,2-dihydro-3H-benz[e]-
indole as a cream solid (180 mg, 99%), which was used directly in
the next step. The entire sample (180 mg, 0.581 mmol) was
dissolved in dioxane (10 mL) with di-tert-butyl dicarbonate
(1.01 g, 4.65 mmol) and DMAP (5 mg) and the solution was stirred
at reflux under N2 for 15 h. More di-tert-butyl dicarbonate
(506 mg, 2.32 mmol) was added and the solution was stirred at re-
flux for a further 5 h. The solvent was evaporated and the residue
purified by column chromatography (petroleum ether/EtOAc,
9:1). Trituration with petroleum ether gave 5-[bis(tert-butoxycar-
bonyl)]amino-1-{[(tert-butoxycarbonyl)oxy]methyl}-3-(trifluoro-
4.1.10. (E)-N-(4-(3-(1-(Chloromethyl)-5-hydroxy-1H-benzo[e]
indol-3(2H)-yl)-3-oxoprop-1-enyl)benzyl)-N,N-dimethyl-1-
(1-methyl-5-nitro-1H-pyrrol-2-yl)methanaminium bromide
hydrobromide (7) (Scheme 3)
A solution of 3-(tert-butoxycarbonyl)-1-(chloromethyl)-5-hy-
droxy-1,2-dihydro-3H-benz[e]indole17 (32) (181 mg, 0.54 mmol)
in dioxane (10 mL) was saturated with HCl gas and allowed to
stand at 20 °C until TLC indicated complete deprotection of the
Boc group (ca. 1 h). The dioxane was evaporated, and 20
(111 mg, 0.54 mmol), EDCIꢀHCl (207 mg, 1.08 mmol), and dry
DMA (3 mL) were added to the residue. The resulting mixture
was stirred at 20 °C for 16 h, then diluted with aqueous NaHCO3
and extracted with EtOAc (ꢃ3). The extracts were dried (Na2SO4)
and evaporated, and the residue was triturated with DCM to give
1-(chloromethyl)-3-[(E)-4-(dimethylaminomethyl)cinnamoyl]-5-
hydroxy-1,2-dihydro-3H-benz[e]indole (33) as
a yellow solid
(72 mg, 32%): mp 225–233 °C (dec.). 1H NMR (DMSO-d6) d 10.42
(s, 1H), 8.12–8.07 (m, 2H), 7.81 (d, J = 8.3 Hz, 1H), 7.76 (d,
J = 8.1 Hz, 2H), 7.67 (d, J = 15.4 Hz, 1H), 7.51 (dt, J = 7.6, 0.8 Hz,
1H), 7.38–7.31 (m, 3H), 7.21 (d, J = 15.4 Hz, 1H), 4.57–4.45 (m,
2H), 4.27–4.18 (m, 1H), 3.99 (dd, J = 11.0, 2.9 Hz, 1H), 3.84 (dd,
J = 11.0, 7.8 Hz, 1H), 3.42 (s, 2H), 2.16 (s, 6H); 13C NMR d 163.5,
154.2, 142.0, 140.8, 133.6, 129.8, 129.1, 128.4, 128.2, 127.2,
123.1, 122.8, 122.5, 121.8, 119.6, 114.6, 100.0, 62.9, 52.9, 47.7,
44.8, 40.6. Anal. Calcd for C25H25ClN2O2: C, 71.33; H, 5.99; N,
6.65. Found: C, 71.24; H, 6.07; N, 6.81.
acetyl)-1,2-dihydro-3H-benz[e]indole (36) (263 mg, 74%) as
a
cream solid: mp 147–150 °C; 1H NMR (CDCl3) d 8.37 (s, 1H), 7.94
(d, J = 8.3 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.59 (td, J = 7.6, 1.1 Hz,
1H), 7.52 (td, J = 7.7, 1.1 Hz, 1H), 4.61 (dd, J = 11.3, 3.4 Hz, 1H),
4.55 (d, J = 11.4 Hz, 1H), 4.38 (dd, J = 11.3, 8.5 Hz, 1H), 4.25–4.17
(m, 1H), 3.90 (dd, J = 11.1, 10.0 Hz, 1H), 1.52 (s, 9H), 1.37 (s, 9H),
1.36 (s, 9H). Anal. Calcd for C30H37F3N2O8: C, 59.01; H, 6.11; N,
4.59. Found: C, 59.03; H, 6.14; N, 4.54.
The triturate was evaporated and the residue was purified by
column chromatography (EtOAc/MeOH, 9:1 then 4:1). The frac-
tions containing product were evaporated, and the residue was dis-
solved in THF and filtered. The filtrate was evaporated and the
residue triturated with EtOAc to give further 33 (69 mg, 30%).
A solution of 33 (28 mg, 0.066 mmol) in THF (6 mL) was treated
with bromide 16 (17.5 mg, 0.11 mmol) and the mixture was stirred
in the dark at 20 °C for 18 days. The precipitate was filtered off to
give 7 as a yellow powder (33 mg, 78%): 1H NMR (DMSO-d6) d
10.46 (s, 1H), 8.12 (d, J = 8.5 Hz, 1H), 8.08 (s, 1H), 7.97 (d,
J = 8.1 Hz, 2H), 7.82 (d, J = 8.3 Hz, 1H), 7.73 (d, J = 15.4 Hz, 1H),
7.65 (d, J = 8.1 Hz, 2H), 7.52 (t, J = 7.5 Hz, 1H), 7.39–7.32 (m, 3H),
6.70 (d, J = 4.5 Hz, 1H), 4.89 (s, 2H), 4.72 (s, 2H), 4.57–4.45 (m,
2H), 4.27–4.20 (m, 1H), 4.04 (s, 3H), 4.01 (dd, J = 11.0, 2.8 Hz,
1H), 3.84 (dd, J = 11.0, 8.1 Hz, 1H), 2.96 (s, 6H); 13C NMR d 163.2,
154.2, 141.9, 141.0, 139.7, 136.6, 133.5, 129.8, 129.2, 128.6,
127.7, 127.2, 123.1, 122.9, 122.6, 121.9, 121.8, 115.7, 114.7,
113.1, 99.9, 66.0, 58.1, 53.0, 48.2, 47.6, 40.6, 34.9. HPLC analysis
indicated the presence of 0.15% of 33.
A solution of 36 (150 mg, 0.246 mmol) and Cs2CO3 (400 mg,
1.23 mmol) in DCM (8 mL) and MeOH (8 mL) was stirred for 1 h.
The solvents were removed under reduced pressure and the resi-
due partitioned between EtOAc and water. The organic layer was
washed with brine, then dried (Na2SO4) and evaporated. The crude
product (126 mg) was dissolved in THF (4 mL) with Et3N (124 mg,
1.23 mmol) and treated with a solution of E-4-(dimethylamino-
methyl)cinnamoyl chloride (0.369 mmol) in THF (4 mL). The mix-
ture was stirred for 15 h, then solvents were removed and the
residue was partitioned between DCM and water. The DCM extract
was washed with brine, and then dried (Na2SO4) and evaporated.
Column chromatography (petroleum ether/EtOAc, 1:1 then DCM/
MeOH, 95:5) followed by trituration with petroleum ether gave
5-[bis(tert-butoxycarbonyl)]amino-1-[(tert-butoxycarbonyl)oxy]-
methyl-3-[(E)-4-(dimethylaminomethyl)cinnamoyl]-1,2-dihydro-
3H-benz[e]indole (37) (160 mg, 93%) as a yellow powder: mp 127–
131 °C; 1H NMR (CDCl3) d 8.57 (s, 1H), 7.91 (d, J = 8.3 Hz, 1H), 7.87
(d, J = 15.3 Hz, 1H), 7.80 (d, J = 8.3 Hz, 1H), 7.58 (d, J = 8.1 Hz, 2H),
7.53 (t, J = 7.2 Hz, 1H), 7.43 (t, J = 7.1 Hz, 1H), 7.37 (d, J = 7.9 Hz,
2H), 4.68 (d, J = 9.6 Hz, 1H), 4.53 (d, J = 10.2 Hz, 1H), 4.42 (t,
J = 8.7 Hz, 1H), 4.26–4.13 (m, 1H), 3.85 (t, J = 10.9 Hz, 1H), 3.48 (s,
2H), 2.28 (s, 6H), 1.52 (s, 9H), 1.35 (s, 18H). Anal. Calcd for
A sample was prepared as the chloride salt: Anal. Calcd for
C
31H32Cl2N4O2ꢀ2H2O: C, 58.96; H, 5.75; N, 8.87. Found: C, 59.12;
H, 5.50; N, 8.83.
C
40H51N3O8ꢀMeOH: C, 67.10; H, 7.55; N, 5.73. Found: C, 67.05; H,
4.1.11. (E)-N-(4-(3-(5-Amino-1-(hydroxymethyl)-1H-benzo[e]
indol-3(2H)-yl)-3-oxoprop-1-enyl)benzyl)-N,N-dimethyl-1-
(1-methyl-5-nitro-1H-pyrrol-2-yl)methanaminium bromide
hydrobromide (8) (Scheme 4)
7.20; N, 5.80.
A solution of 37 (46 mg, 0.066 mmol) and bromide 16 (17 mg,
0.079 mmol) in THF (2 mL) was stirred under N2 in the dark for
6 days. The THF was removed and the residue was dissolved in
EtOAc. Et2O was added and the resulting precipitate was filtered
off and triturated with Et2O to give N-4-((E)-3-{5-[bis(tert-butoxy
carbonyl)]amino-1-[(tert-butoxycarbonyl)oxy]methyl-1,2-dihydro-
3H-benz[e]indol-3-yl}-3-oxo-1-propenyl)benzyl-N,N-dimethyl-
N-[(1-methyl-5-nitro-2-pyrrolyl)methyl]ammoniumbromide(38a)
(50 mg, 83%) as a yellow powder: mp 166–170 °C (dec.); 1H NMR
(DMSO-d6) d 8.41 (s, 1H), 8.06 (d, J = 8.4 Hz, 1H), 7.98 (d, J = 8.1 Hz,
2H), 7.76 (d, J = 15.4 Hz, 1H), 7.69 (d, 8.4 Hz, 1H), 7.65–7.58 (m,
A
solution of 1-(hydroxymethyl)-5-nitro-3-(tert-butoxycar-
bonyl)-1,2-dihydro-3H-benz[e]indole18 (34) (200 mg, 0.581 mmol)
in dioxane (20 mL) saturated with HCl gas was stirred for 15 h. The
dioxane was removed under reduced pressure and pyridine (5 mL)
and (CF3CO)2O (439 mg, 2.09 mmol) were added to the residue at
0 °C. After 30 min the mixture was poured into ice water and
extracted with DCM. The DCM extract was washed with 1 M HCl
and brine and then dried (Na2SO4) and evaporated. Column

M. Tercel et al. / Bioorg. Med. Chem. 20 (2012) 734–749
745
1H), 7.63 (d, J = 8.4 Hz, 2H), 7.53 (td, J = 7.7, 1.0 Hz, 1H), 7.36 (d,
J = 4.5 Hz, 1H), 7.35 (d, J = 15.4 Hz, 1H), 6.70 (d, J = 4.5 Hz, 1H), 4.82
(s, 2H), 4.65 (s, 2H), 4.60–4.53 (m, 2H), 4.47 (dd, J = 10.8, 3.7 Hz,
1H), 4.36–4.28 (m, 1H), 4.10 (dd, J = 10.7, 8.1 Hz, 1H), 4.02 (s, 3H),
2.94 (s, 6H), 1.37 (s, 27H); HRMS m/z 840.4187 (M+), C46H58N5O10 re-
quires 840.4184.
Dioxane (2 mL) saturated with HBr gas was added to a solution
of 38a (50 mg, 0.054 mmol) in dioxane (1 mL) and the mixture was
stirred in the dark at 20 °C for 15 h. Excess HBr was removed under
reduced pressure and the precipitate was filtered off and triturated
with Et2O to give 8 as a yellow solid (38 mg, 100%): mp 180–185 °C
(dec.); 1H NMR (DMSO-d6) d 8.21 (s, 1H), 8.02 (d, J = 8.5 Hz, 1H),
7.95 (d, J = 8.3 Hz, 2H), 7.83 (d, J = 8.4 Hz, 1H), 7.72 (d, J = 15.4 Hz,
1H), 7.63 (d, J = 8.2 Hz, 2H), 7.52 (t, J = 7.3 Hz, 1H), 7.39 (t,
J = 7.2 Hz, 1H), 7.35 (d, J = 4.5 Hz, 1H), 7.33 (d, J = 15.5 Hz, 1H),
6.69 (d, J = 4.5 Hz, 1H), 4.83 (s, 2H), 4.66 (s, 2H), 4.52 (d,
J = 10.8 Hz, 1H), 4.45–4.36 (m, 1H), 4.02 (s, 3H), 3.92–3.82 (m,
1H), 3.78 (dd, J = 10.8, 3.9 Hz, 1H), 2.95 (s, 6H), 2 protons not ob-
served; HRMS m/z 540.2614 (M+), C31H34N5O4 requires 540.2611.
HPLC purity 95.2%.
1-iodo-2,4-naphthalenediamine19 (39) (2.40 g, 4.25 mmol) in dry
benzene at reflux. After 15 min the mixture was cooled and the
solvent was removed under reduced pressure. The residue was
partitioned between EtOAc and water, then the EtOAc layer was
washed with brine, dried (Na2SO4) and evaporated. The residue
was purified by column chromatography (petroleum ether then
petroleum ether/EtOAc, 99:1–93:7) gave 5-[allyl(tert-butoxycar-
bonyl)amino]-3-(tert-butoxycarbonyl)-1-methyl-1,2-dihydro-3H-
benzo[e]indole (40) (1.44 g, 77%) as a brown oil: 1H NMR (CDCl3) d
8.10 (br s, 1H), 7.80–7.70 (m, 2H), 7.45 (t, J = 8.3 Hz, 1H), 7.35 (t,
J = 8.1 Hz, 1H), 6.08–5.88 (m, 1H), 5.21–4.98 (m, 2H), 4.65–4.30
(m, 1H), 4.25–4.09 (m, 2H), 3.88–3.70 (m, 2H), 1.58 (s, 9H), 1.41
(d, J = 6.9 Hz, 3H), 1.23 (s, 9H); HRMS m/z 438.2516 (M+),
C26H34N2O4 requires 438.2519.
TFA (15 mL) was added to 40 (1.40 g, 3.20 mmol) at 0 °C and the
solution was stirred at 20 °C for 2 h. The TFA was removed under
reduced pressure and the residue was partitioned between DCM
and aqueous NaHCO3. The DCM layer was washed with brine, dried
(Na2SO4), and evaporated to give the crude amine (760 mg, 100%).
This was dissolved in THF (10 mL) and treated with a solution of
(E)-3-(4-((dimethylamino)methyl)cinnamoyl chloride [4.79 mmol,
prepared from (E)-3-(4-((dimethylamino)methyl)cinnamic acid
(20) with oxalyl chloride/catalytic DMF] in THF (10 mL). The mix-
ture was stirred for 12 h, then diluted with H2O and extracted with
EtOAc. The EtOAc extract was washed with brine, dried (Na2SO4),
and evaporated. The residue was purified by column chromatogra
phy (DCM then DCM/MeOH, 99:1–9:1) to give 5-allylamino-3-[(E)-
4-(dimethylaminomethyl)cinnamoyl]-1-methyl-1,2-dihydro-3H-
benz[e]indole (41) (836 mg, 62%) as a brown oil: 1H NMR (CDCl3) d
7.98 (s, 1H), 7.86 (d, J = 15.7 Hz, 1H), 7.82 (d, J = 8.5 Hz, 1H), 7.72 (d,
J = 8.3 Hz, 1H), 7.56 (d, J = 8.1 Hz, 2H), 7.47 (t, J = 7.2 Hz, 1H), 7.37–
7.31 (m, 1H), 7.36 (d, J = 8.1 Hz, 2H), 6.89 (d, J = 15.3 Hz, 1H), 6.17–
6.05 (m, 1H), 5.41 (d, J = 17.1 Hz, 1H), 5.25 (d, J = 10.0 Hz, 1H),
4.55–4.40 (m, 1H), 4.46 (br s, 1H), 4.06–3.97 (m, 2H), 3.87–3.79
(m , 1H), 3.51–3.43 (m, 1H), 3.47 (s, 2H), 2.28 (s, 6H), 1.42 (d,
J = 6.8 Hz, 3H); HRMS m/z 425.2466 (M+), C28H31N3O requires
425.2467.
Allyl amine 41 (70 mg, 0.165 mmol) was dissolved in dry DCM
(5 mL) and the solution was purged with N2. Pd(Ph3P)4 (13 mg,
0.012 mmol), PhSO2Na (68 mg, 0.412 mmol) and CSA (115 mg,
0.494 mmol) were added20 and the solution was stirred under N2
for 3 h, then diluted with aqueous NaHCO3 and extracted with
DCM. The DCM extract was washed with brine, dried (Na2SO4),
and evaporated. The residue was purified by column chromatogra-
phy (DCM then DCM/MeOH, 99:1–23:2) to give 5-amino-3-[(E)-4-
(dimethylaminomethyl)cinnamoyl]-1-methyl-1,2-dihydro-3H-benz
[e]indole (42) (80 mg, 91%) as a yellow foam: 1H NMR (CDCl3) d
8.06 (s, 1H), 7.87–7.82 (m, 1H), 8.81 (d, J = 8.2 Hz, 1H), 7.74 (d,
J = 8.6 Hz, 1H), 7.56 (d, J = 8.1 Hz, 2H), 7.46 (t, J = 7.4 Hz, 1H),
7.38–7.31 (m, 1H), 7.37 (d, J = 8.1 Hz, 2H), 6.87 (d, J = 15.1 Hz,
1H), 4.45 (t, J = 8.8 Hz, 1H), 4.22 (s, 2H), 4.02 (d, J = 9.5 Hz, 1H),
3.91–3.78 (m, 1H), 3.47 (s, 2H), 2.27 (s, 6H), 1.42 (d, J = 6.8 Hz,
3H); HRMS m/z 386.2235 (M+), C25H27N3O requires 386.2232.
A sample was prepared as the hydrochloride salt: Anal. Calcd for
C
31H32Cl2N4O2ꢀ2H2O: C, 58.96; H, 5.75; N, 8.87. Found: C, 59.12; H,
5.50; N, 8.83.
4.1.12. (E)-N-(4-(3-(5-Amino-1-(hydroxymethyl)-1H-benzo[e]
indol-3(2H)-yl)-3-oxoprop-1-enyl)benzyl)-N,N-dimethyl-1-
(1-methyl-5-(methylsulfonyl)-1H-pyrrol-2-yl)methanaminium
bromide hydrobromide (9)
A solution of 36 (40 mg, 0.057 mmol) and bromide 18 (17 mg,
0.068 mmol) in THF (1.5 mL) was stirred under N2 in the dark for
5 days. The THF was removed, the residue was dissolved in EtOAc,
and this solution was diluted with Et2O. The resulting precipitate
was filtered off and triturated with Et2O to give N-4-((E)-3-{5-[bis(-
tert-butoxycarbonyl)]amino-1-[(tert-butoxycarbonyl)oxy]methyl-
1,2-dihydro-3H-benz[e]indol-3-yl}-3-oxo-1-propenyl)benzyl-N,N-
dimethyl-N-[(1-methyl-5-methylsulfonyl-2-pyrrolyl)methyl]
ammonium bromide (38b) (38 mg, 78%) as a yellow powder: mp
170 °C (dec.); 1H NMR (DMSO-d6)
d
8.41 (s, 1H), 8.06 (d,
J = 8.4 Hz, 1H), 7.98 (d, J = 8.0 Hz, 2H), 7.75 (d, J = 15.3 Hz, 1H),
7.70–7.59 (m, 2H), 7.64 (d, J = 8.4 Hz, 2H), 7.52 (t, J = 8.0 Hz, 1H),
7.34 (d, J = 15.5 Hz, 1H), 6.92 (d, J = 4.1 Hz, 1H), 6.65 (d, J = 4.1 Hz,
1H), 4.77 (s, 2H), 4.65 (s, 2H), 4.59–4.52 (m, 2H), 4.47 (dd,
J = 10.7, 3.9 Hz, 1H), 4.36–4.28 (m, 1H), 4.11 (dd, J = 10.6, 8.0 Hz,
1H), 3.95 (s, 3H), 3.31 (s, 3H), 2.92 (s, 6H), 1.37 (s, 27H); HRMS
m/z 873.4105 (M+), C47H61N4O10S requires 873.4108.
Dioxane (3 mL) saturated with HBr gas was added to a solution
of 38b (98 mg, 0.103 mmol) in dioxane (2 mL) and the mixture was
stirred in the dark at 20 °C for 15 h. Excess HBr was removed under
reduced pressure, then the precipitate was filtered off and tritu-
rated with Et2O to give 9 as a yellow solid (68 mg, 90%): mp
176–180 °C (dec.); 1H NMR (DMSO-d6) d 8.10 (s, 1H), 8.02 (d,
J = 8.5 Hz, 1H), 7.96 (d, J = 8.2 Hz, 2H), 7.80 (d, J = 8.6 Hz, 1H),
7.72 (d, J = 15.4 Hz, 1H), 7.66 (d, J = 8.2 Hz, 2H), 7.50 (t, J = 7.3 Hz,
1H), 7.36 (t, J = 7.3 Hz, 1H), 7.32 (d, J = 15.4 Hz, 1H), 6.92 (d,
J = 4.1 Hz, 1H), 6.65 (d, J = 4.1 Hz, 1H), 4.78 (s, 2H), 4.64 (s, 2H),
4.51 (d, J = 11.6 Hz, 1H), 4.43–4.34 (m, 1H), 3.96 (s, 3H), 3.88–
3.79 (m, 1H), 3.77 (dd, J = 11.0, 4.0 Hz, 1H), 2.91 (s, 6H), 5 protons
not observed; HRMS m/z 573.2529 (M+), C32H37N4O4S requires
573.2536. HPLC purity 94.2%.
A
solution of 42 (80 mg, 0.208 mmol), (Boc)2O (905 mg,
4.15 mmol), and DMAP (8 mg) in dioxane (2 mL) was heated at re-
flux for 48 h. The mixture was diluted with H2O and extracted with
EtOAc. The EtOAc layer was washed with H2O, brine, and dried
(Na2SO4) and evaporated. Column chromatography (DCM then
DCM/MeOH, 99:1–97:3) gave 5-[bis(tert-butoxycarbonyl)]amino-
3-[(E)-4-(dimethylaminomethyl)cinnamoyl]-1-methyl-1,2-dihy-
dro-3H-benz[e]indole (43) (45 mg, 37%) as a yellow gum: 1H NMR
(CDCl3) d 8.57 (s, 1H), 7.87 (d, J = 15.4 Hz, 1H), 7.80 (d, J = 8.2 Hz,
1H), 7.79 (d, J = 8.2 Hz, 1H), 7.55 (d, J = 8.1 Hz, 2H), 7.48 (t,
J = 6.9 Hz, 1H), 7.41 (t, J = 7.7 Hz, 1H), 7.34 (d, J = 8.0 Hz, 2H), 6.86
(d, J = 14.9 Hz, 1H), 4.50 (t, J = 9.4 Hz, 1H), 4.09 (d, J = 9.1 Hz, 1H),
3.99–3.89 (m, 1H), 3.46 (s, 2H), 2.26 (s, 6H), 1.47 (d, J = 6.9 Hz,
4.1.13. (E)-N-(4-(3-(5-Amino-1-methyl-1H-benzo[e]indol-3(2H)
-yl)-3-oxoprop-1-enyl)benzyl)-N,N-dimethyl-1-(1-methyl-5-
nitro-1H-pyrrol-2-yl)methanaminium bromide hydrobromide
(10) (Scheme 5)
AIBN (140 mg, 0.851 mmol) and Bu3SnH (1.49 g, 5.11 mmol)
were added to a solution of N,N0-bis[allyl(tert-butoxycarbonyl)]-

746
M. Tercel et al. / Bioorg. Med. Chem. 20 (2012) 734–749
3H), 1.37 (s, 9H), 1.35 (s, 9H); HRMS m/z 586.3286 (M+),
1H), 3.40 (s, 3H), 2.93 (s, 6H), 1.34 (d, J = 6.6 Hz, 3H); HRMS m/z
C35H43N3O5 requires 586.3281.
557.2576 (M+), C32H37N4O3S requires 557.2586. HPLC purity 95.8%.
A solution of 43 (45 mg, 0.077 mmol) and bromide 16 (20 mg,
0.092 mmol) in THF (2 mL) was stirred under N2 in the dark for
48 h. The resulting precipitate was filtered off and washed with
THF to give N-4-((E)-3-{5-[bis(tert-butoxycarbonyl)]-amino-1-
methyl-1,2-dihydro-3H-benz[e]indol-3-yl}-3-oxo-1-propenyl)
benzyl-N,N-dimethyl-N-[(1-methyl-5-nitro-2-pyrrolyl)methyl]
ammonium bromide (44a) (51 mg, 82%) as a yellow powder: mp
168–172 °C (dec.); 1H NMR (DMSO-d6) d 8.38 (s, 1H), 7.99–7.92
(m, 3H), 7.74 (d, J = 15.4 Hz, 1H), 7.69–7.62 (m, 3H), 7.58 (t,
J = 7.5 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.37 (d, J = 4.5 Hz, 1H), 7.32
(d, J = 15.4 Hz, 1H), 6.70 (d, J = 4.5 Hz, 1H), 4.84 (s, 2H), 4.67 (s,
2H), 4.57 (t, J = 9.9 Hz, 1H), 4.31 (d, J = 9.4 Hz, 1H), 4.09–4.01 (m,
1H), 4.02 (s, 3H), 2.94 (s, 6H), 1.42–1.37 (m, 3H), 1.39 (s, 9H),
1.36 (s, 9H). Anal. Calcd for C41H50BrN5O7ꢀ11/2H2O: C, 59.20; H,
6.42; N, 8.42. Found: C, 59.13; H, 6.03; N, 8.33.
Dioxane (10 mL) saturated with HCl gas was added to a solution
of 44a (80 mg, 0.100 mmol) in dioxane (10 mL). The mixture was
stirred in the dark at 20 °C for 15 h and the solvent was removed
under reduced pressure. The residue was dissolved in MeOH
(1 mL) and purified by ion exchange chromatography (Biorad AG-
1X4 resin in Clꢁ form), eluting with MeOH to give 10 as a yellow
solid (38 mg, 64%): mp >300 °C (dec.); 1H NMR (DMSO-d6) d 8.15
(s, 1H), 8.05 (d, J = 8.5 Hz, 1H), 7.95 (d, J = 8.2 Hz, 2H), 7.80 (d,
J = 8.0 Hz, 1H), 7.72 (d, J = 15.4 Hz, 1H), 7.64 (d, J = 8.2 Hz, 2H),
7.51 (t, J = 7.9 Hz, 1H), 7.41–7.32 (m, 1H), 7.38 (d, J = 4.5 Hz, 1H),
7.32 (d, J = 15.4 Hz, 1H), 6.69 (d, J = 4.5 Hz, 1H), 4.85 (s, 2H), 4.67
(s, 2H), 4.54–4.43 (m, 1H), 4.18 (d, J = 9.8 Hz, 1H), 4.03 (s, 3H),
2.94 (s, 6H), 1.34 (d, J = 6.8 Hz, 3H); HRMS m/z 524.2670 (M+),
4.1.15. (E)-5-(4-(3-(5-Amino-1-(chloromethyl)-1H-benzo[e]
indol-3(2H)-yl)-3-oxoprop-1-enyl)phenyl)-N,N-dimethyl-N-((1-
methyl-5-(methylsulfonyl)-1H-pyrrol-2-yl)methyl)pentan-1-
aminium trifluoroacetate hydrotrifluoroacetate (12) (Scheme 6)
TFA (5 mL) was added to 5-[allyl(tert-butoxycarbonyl)amino]-
3-(tert-butoxycarbonyl)-1-(chloromethyl)-1,2-dihydro-3H-benz
[e]indole19 (45) (300 mg, 0.636 mmol) at 0 °C and the solution was
stirred at 20 °C for 3 h. The TFA was removed under reduced pres-
sure and the residue was partitioned between DCM and aqueous
NaHCO3. The DCM layer was washed with brine, dried (Na2SO4),
and evaporated to give the crude amine (173 mg, 100%) which
was used directly. The entire sample was dissolved in DCM
(5 mL) and treated slowly with
a solution of 6-(dimethyl-
amino)hexanoyl chloride hydrochloride21 (203 mg, 0.953 mmol)
in THF (5 mL). The mixture was stirred for 30 min, then diluted
with H2O and extracted with DCM. The DCM extract was washed
with brine, dried (Na2SO4), and evaporated. The residue was puri-
fied by column chromatography (DCM/MeOH/NH3, 9:1:12 drops/
100 mL of solution) to give 5-allylamino-1-(chloromethyl)-3-[6-
(dimethylamino)hexanoyl]-1,2-dihydro-3H-benz[e]indole
(46a)
(130 mg, 50%) as a brown powder: mp 185–190 °C (dec.); 1H
NMR (DMSO-d6) d 8.13 (d, J = 8.5 Hz, 1H), 7.71 (d, J = 8.2 Hz, 1H),
7.62 (s, 1H), 7.45 (t, J = 7.3 Hz, 1H), 7.28 (t, J = 7.4 Hz, 1H), 6.54 (s,
1H), 6.20–5.89 (m, 1H), 5.25 (dd, J = 17.2, 1.3 Hz, 1H), 5.13 (d,
J = 9.4 Hz, 1H), 4.27 (t, J = 9.6 Hz, 1H), 4.14–4.02 (m, 2H), 3.94
(dd, J = 10.9, 2.9 Hz, 1H), 3.90–3.82 (m, 2H), 3.71 (dd, J = 10.5,
8.7 Hz, 1H), 2.93–2.83 (m, 2H), 2.63 (s, 6H), 2.57–2.48 (m, 2H),
1.70–1.57 (m, 4H), 1.43–1.34 (m, 2H). This was unstable, and
was used directly in the next step.
C31H34N5O3 requires 524.2662. HPLC purity 95.4%.
Compound 46a (200 mg, 0.484 mmol) was dissolved in dry
DCM (10 mL) and the solution was purged with N2. Pd(Ph3P)4
(39 mg, 0.034 mmol), PhSO2Na (200 mg, 1.21 mmol), and CSA
(337 mg, 1.45 mmol) were added and the solution was stirred un-
der N2 for 30 min. The mixture was diluted with aqueous NaHCO3
and extracted with DCM. The DCM extract was washed with
brine, dried (Na2SO4), and evaporated to give crude 5-amino-1-
(chloromethyl)-3-[6-(dimethylamino)hexanoyl]-1,2-dihydro-3H-
benz[e]indole (47a) (87 mg, 48%), which was unstable and was
used directly. A solution of 47a (45 mg, 0.121 mmol), (Boc)2O
(237 mg, 1.09 mmol), and DMAP (1 mg) in THF (6 mL) and diox-
ane (2 mL) was heated at reflux for 12 h. The mixture was diluted
with H2O and extracted with EtOAc. The EtOAc extract was
washed with H2O, aqueous Na2CO3, and brine, and then dried
(Na2SO4) and evaporated. The residue was purified by column
chromatography [DCM/MeOH, 9:1 containing c.NH3 (12 drops
per 100 mL of eluant)] to give 5-[bis(tert-butoxy-carbonyl)] ami-
no-1-(chloromethyl)-3-[6-(dimethylamino)hexanoyl]-1,2-dihy-
dro-3H-benz[e]indole (48a) (47 mg, 68%) as a yellow foam. 1H
NMR (DMSO-d6) d 8.28 (s, 1H), 7.98 (d, J = 8.3 Hz, 1H), 7.65 (d,
J = 8.1 Hz, 1H), 7.56 (td, J = 7.5, 1.2 Hz, 1H), 7.48 (td, J = 7.6,
1.0 Hz, 1H), 4.49–4.34 (m, 2H), 4.23 (d, J = 8.7 Hz, 1H), 4.05 (dd,
J = 11.3, 2.8 Hz, 1H), 3.50 (dd, J = 11.1, 6.2 Hz, 1H), 2.56–2.25 (m,
2H), 2.28–2.22 (m, 2H), 2.15 (s, 6H), 1.67–1.59 (m, 2H), 1.51–
1.42 (m, 2H), 1.40–1.30 (m, 2H), 1.35 (s, 18H); HRMS (FAB, 35Cl)
m/z 574.3048 (M+1), C32H45ClN3O5 requires 574.3048.
4.1.14. (E)-N-(4-(3-(5-Amino-1-methyl-1H-benzo[e]indol-3(2H)-
yl)-3-oxoprop-1-enyl)benzyl)-N,N-dimethyl-1-(1-methyl-5-
(methylsulfonyl)-1H-pyrrol-2-yl)methanaminium bromide
hydrobromide (11)
A solution of 43 (100 mg, 0.171 mmol) and bromide 18 (52 mg,
0.205 mmol) in THF (10 mL) was stirred under N2 in the dark for
85 h, then concentrated under reduced pressure to a small vol-
ume. EtOAc was added and the resulting precipitate was filtered
off and washed with EtOAc to give N-4-((E)-3-{5-[bis(tert-butoxy
carbonyl)]-amino-1-methyl-1,2-dihydro-3H-benz[e]indol-3-yl}-3-
oxo-1-propenyl)benzyl]-N,N-dimethyl-N-[(1-methyl-5-methylsul-
fonyl-2-pyrrolyl)methyl]ammonium bromide (44b) (132 mg, 92%)
as a yellow powder: mp 171–174 °C (dec.); 1H NMR (DMSO-d6) d
8.38 (s, 1H), 8.01–7.92 (m, 3H), 7.74 (d, J = 15.4 Hz, 1H), 7.68–7.62
(m, 3H), 7.59 (t, J = 7.5 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.32 (d,
J = 15.5 Hz, 1H), 6.93 (d, J = 4.1 Hz, 1H), 6.64 (d, J = 4.1 Hz, 1H),
4.77 (s, 2H), 4.63 (s, 2H), 4.57 (t, J = 9.9 Hz, 1H), 4.31 (d,
J = 9.4 Hz, 1H), 4.10–4.02 (m, 1H), 3.95 (s, 3H), 3.31 (s, 3H), 2.92
(s, 6H), 1.43–1.38 (m, 3H), 1.39 (s, 9H), 1.36 (s, 9H). Anal. Calcd
for C42H53BrN4O7SꢀH2O: C, 58.94; H, 6.48; N, 6.55. Found: C,
59.04; H, 6.20; N, 6.43.
Dioxane (5 mL) saturated with HCl gas was added to a solution
of 44b (50 mg, 0.060 mmol) in dioxane (5 mL). The mixture was
stirred in the dark at 20 °C for 15 h and the solvent was removed
under reduced pressure. The residue was dissolved in MeOH
(1 mL) and purified by ion exchange chromatography (Biorad AG-
1X4 resin in Clꢁ form), eluting with MeOH to give 11 as a yellow
solid (33 mg, 89%): mp >300 °C (dec.); 1H NMR (DMSO-d6) d 8.21
(s, 1H), 8.04 (d, J = 8.5 Hz, 1H), 7.94 (d, J = 8.0 Hz, 2H), 7.86–7.78
(m, 1H), 7.72 (d, J = 15.3 Hz, 1H), 7.66 (d, J = 7.9 Hz, 2H), 7.57–
7.48 (m, 1H), 7.44–7.35 (m, 1H), 7.31 (d, J = 15.4 Hz, 1H), 6.92 (d,
J = 4.1 Hz, 1H), 6.65 (d, J = 4.1 Hz, 1H), 4.81 (s, 2H), 4.67 (s, 2H),
4.54–4.43 (m, 1H), 4.25–4.17 (m, 1H), 3.97 (s, 3H), 3.94–3.84 (m,
A solution of 48a (43 mg, 0.075 mmol), bromide 18 (21 mg,
0.083 mmol) and dry K2CO3 (25 mg) in THF (2 mL) was stirred under
N2 in the dark at room temperature for 2 h, and the inorganic solids
were then filtered off. The filtrate was evaporated and the resulting
residue was triturated with EtOAc/Et2O and then with Et2O to
give N-6-{5-[bis(tert-butoxycarbonyl)amino]-1-(chloromethyl)-
1,2-dihydro-3H-benz[e]indol-3-yl}-6-oxo-1-hexyl-N,N-dimethyl-
N -{[1-methyl-5-(methylsulfonyl)-pyrrol-2-yl]methyl}ammonium

M. Tercel et al. / Bioorg. Med. Chem. 20 (2012) 734–749
747
bromide (49a) (57 mg, 92%) as a cream powder: mp 150–153 °C
(dec.).1H NMR (DMSO-d6) d 8.29 (s, 1H), 7.99 (d, J = 8.3 Hz, 1H),
7.65 (d, J = 8.4 Hz, 1H), 7.57 (td, J = 7.5, 1.0 Hz, 1H), 7.49 (td, J = 7.6,
1.0 Hz, 1H), 6.90 (d, J = 4.1 Hz, 1H), 6.58 (d, J = 4.1 Hz, 1H), 4.66 (s,
2H), 4.48–4.40 (m, 2H), 4.23 (d, J = 8.1 Hz, 1H), 4.09–4.04 (m, 1H),
3.97 (dd, J = 11.1, 6.2 Hz, 1H), 3.91 (s, 3H), 3.41–3,28 (m, 2H), 3.28
(s, 3H), 2.98 (s, 6H), 2.71–2.54 (m, 2H), 1.87–1.78 (m, 2H), 1.74–
1.67 (m, 2H), 1.46–1.38 (m, 2H), 1.35 (s, 9H), 1.34 (s, 9H). Anal. Calcd
for C38H54BrClN4O7Sꢀ11/2H2O: C, 53.49; H, 6.73; N, 6.57. Found: C,
53.49; H, 6.37; N, 6.43.
TFA (1 mL) was added to a cooled (0 °C) solution of 49a (54 mg,
0.065 mmol) in DCM (2 mL) and stirred for 6 h. Solvents were re-
moved and the residue was triturated with EtOAc/Et2O and then
with Et2O to give 12 (39 mg) as a pale yellow powder: mp 158–
162 °C (dec.). 1H NMR (DMSO-d6) d 8.02 (d, J = 8.4 Hz, 1H), 7.76
(s, 1H), 7.70 (d, J = 8.3 Hz, 1H), 7.43 (t, J = 7.3 Hz, 1H), 7.23
(t, J = 7.4 Hz, 1H), 6.90 (d, J = 4.1 Hz, 1H), 6.57 (d, J = 4.1 Hz, 1H),
5.80 (br s, 1H), 4.66 (s, 2H), 4.28 (t, J = 9.8 Hz, 1H), 4.14–4.02 (m,
2H), 3.94 (dd, J = 11.0, 2.8 Hz, 1H), 3.91 (s, 3H), 3.69 (dd, J = 10.3,
8.8 Hz, 1H), 3.48–3.34 (m, 2H), 3.28 (s, 3H), 2.97 (s, 6H), 2.60–
2.40 (m, 2H), 1.88–1.76 (m, 2H), 1.75–1.63 (m, 2H), 1.48–1.34
(m, 2H); HRMS (FAB, 35Cl) m/z 545.22353 (M+), C28H38ClN4O3S
requires 545.2353. HPLC purity 92.3%.
(td, J = 8.4, 1.0 Hz, 1H), 7.34 (td, J = 8.4, 1.0 Hz, 1H), 4.26 (br, 3H
including NH2), 4.19 (t, J = 9.5 Hz, 1H), 4.00 (br t, J = 9.2 Hz, 1H),
3.92 (dd, J = 11.3, 3.1 Hz, 1H), 3.39 (t, J = 10.8 Hz, 1H), 2.40–2.60
(m, 2H), 2.26 (t, J = 7.4 Hz, 2H), 2.22 (s, 6H), 1.74–1.82 (m, 2H),
1.35–1.54 (m, 6H); HRMS (FAB, 35Cl) m/z 388.21532 (M+H),
C
22H3135ClN3O requires 388.21557.
A
solution of 47b (644 mg, 1.71 mmol), (Boc)2O (3.73 g,
17.1 mmol), and DMAP (15 mg) in THF (60 mL) and dioxane
(20 mL) was heated at reflux for 15 h. The solvents were evapo-
rated under reduced pressure and the residue was dissolved in
DCM. The DCM solution was washed with H2O, aqueous NaHCO3,
and brine, and then dried (Na2SO4) and evaporated. The residue
was purified by column chromatography [EtOAc then Et3N/EtOAc,
1:40 then 1:20] to give 5-[bis(tert-butoxycarbonyl)]amino-1-(chlo-
romethyl)-3-[7-(dimethylamino)heptanoyl-1,2-dihydro-3H-benz
[e]indole (48b) (742 mg, 74%) as a white gum: 1H NMR (CDCl3): d
8.48 (s, 1H), 7.80 (d, J = 8.4 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.51
(td, J = 7.6, 1.0 Hz, 1H), 7.42 (td, J = 7.6, 1.0 Hz, 1H), 4.33 (d,
J = 10.1, 1H), 4.27 (t, J = 9.6 Hz, 1H), 4.13 (br t, J = 9.0 Hz, 1H),
3.97 (dd, J = 11.3, 3.1 Hz, 1H), 3.48 (t, J = 10.8 Hz, 1H), 2.42–2.61
(m, 2H), 2.26 (t, J = 7.5 Hz, 2H), 2.22 (s, 6H), 1.75–1.82 (m, 2H),
1.39–1.54 (m, 6H), 1.37 (s, 9H), 1.35 (s, 9H); HRMS (FAB, 35Cl)
m/z 588.3192 (M+1), C32H47ClN3O5 requires 588.3204.
A solution of 48b (31 mg, 0.053 mmol), bromide 18 (15 mg,
0.058 mmol) and dry K2CO3 (25 mg) in THF (2 mL) was stirred un-
der N2 in the dark for 2 h. Inorganic solids were then filtered off
and the filtrate was evaporated and the resulting residue was trit-
urated with EtOAc/Et2O and then with Et2O gave 6-[5-[bis(tert-
butoxycarbonyl)amino]-1-(chloromethyl)-1,2-dihydro-3H-benzo
[e]indol-3-yl]-N,N-dimethyl-N-{[1-methyl-5-(methylsulfonyl)-1H-
pyrrol-2-yl]methyl}-6-oxo-1-heptane ammonium bromide (49b)
(40 mg, 91%) as a cream powder: mp 148–151 °C (dec.); 1H NMR
4.1.16. (E)-6-(4-(3-(5-Amino-1-(chloromethyl)-1H-benzo[e]
indol-3(2H)-yl)-3-oxoprop-1-enyl)phenyl)-N,N-dimethyl-N-((1-
methyl-5-(methylsulfonyl)-1H-pyrrol-2-yl)methyl)hexan-1-
aminium trifluoroacetate hydrotrifluoroacetate (13)
A solution of 45 (1.25 g , 2.64 mmol) in DCM (5 mL) was treated
with HCl in dioxane (4 M, 30 mL) and the mixture was stirred at
room temperature for 3 h, during which time a white precipitate
formed. The solvent was evaporated and the off-white solid ob-
tained (the amine hydrochloride) was dissolved in dry DMF
(30 mL). To this solution was added 7-(dimethylamino)heptanoyl
chloride hydrochloride21 (611 mg, 2.92 mmol), DIPEA (2.13 mL,
13.25 mmol), and (benzotriazol-1-yloxy)tripyrrolidinophosphoni-
um hexafluorophosphate, (1.793 g, 3.44 mmol), and the mixture
was stirred at room temperature for 16 h. The solvent was evapo-
rated under reduced pressure and the residue was dissolved in
DCM. The solution was washed with dilute aqueous NaHCO3, dried
(Na2SO4), and evaporated. The crude product was purified by col-
umn chromatography [Et3N/MeOH/DCM, 1:10:400 then 1:10:200
then 1:10:100 followed by a second column EtOAc then Et3N/
EtOAc, 1:40 then 1:20 then 1:10] to give N-allyl-1-(chloro-
methyl)-3-[7-(dimethylamino)heptanoyl]-2,3-dihydro-1H-benzo[e]
indol-5-amine (46b) as white solid (867 mg, 77%): mp 168–170 °C
(dec.); 1H NMR (DMSO-d6) d 8.13 (d, J = 8.5 Hz, 1H), 7.71 (d,
J = 8.1 Hz, 1H), 7.61 (s, 1H), 7.44 (t, J = 7.3 Hz, 1H), 7.27 (t,
J = 7.7 Hz, 1H), 6.59 (s, 1H), 6.02–5.90 (m, 1H), 5.26 (dd, J = 17.3,
1.4 Hz, 1H), 5.12 (d, J = 10.3 Hz, 1H), 4.28 (t, J = 9.6 Hz, 1H), 4.15–
4.00 (m, 2H), 3.93 (dd, J =10.9, 2.8 Hz, 1H), 3.86 (s, 2H), 3.70 (dd,
J = 10.4, 8.6 Hz, 1H), 2.90–2.83 (m, 2H), 2.62 (s, 6H), 2.56–2.45
(m, 2H), 1.68–1.56 (m, 4H), 1.43–1.30 (m, 4H). This was unstable,
and was used directly for the next step.
(DMSO-d6)
d 8.29 (s, 1H), 7.99 (d, J = 8.3 Hz, 1H), 7.65 (d,
J = 8.2 Hz, 1H), 7.57 (t, J = 7.9 Hz, 1H), 7.49 (t, J = 8.1 Hz, 1H), 6.90
(d, J = 4.1 Hz, 1H), 6.58 (d, J = 4.1 Hz, 1H), 4.66 (s, 2H), 4.47–4.37
(m, 2H), 4.23 (d, J = 8.3 Hz, 1H), 4.07 (dd, J = 9.9, 2.7 Hz, 1H), 3.97
(dd, J = 11.3, 6.2 Hz, 1H), 3.91 (s, 3H), 3.33–3,28 (m, 2H), 3.27 (s,
3H), 2.97 (s, 6H), 2.61–2.51 (m, 2H), 1.83–1.74 (m, 2H), 1.71–
1.62 (m, 2H), 1.50–1.40 (m, 2H), 1.40–1.32 (m, 2H), 1.35 (s, 9H),
1.34 (s, 9H). Anal. Calcd for C39H56BrClN4O7SꢀH2O: C, 54.58; H,
6.81; N, 6.53. Found: C, 54.39; H, 6.72; N, 6.32.
TFA (1 mL) was added to a cooled (0 °C) solution of 49b (37 mg,
0.044 mmol) in DCM (2 mL) and stirred for 6 h. Solvents were re-
moved and the residue was triturated with EtOAc/Et2O and then
with Et2O to give 13 (21 mg, 100%) as a pale yellow powder: mp
160–163 °C (dec.). 1H NMR (DMSO-d6) d 8.02 (d, J = 8.4 Hz, 1H),
7.76 (s, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.43 (t, J = 7.7 Hz, 1H), 7.23
(t, J = 7.7 Hz, 1H), 6.90 (d, J = 4.1 Hz, 1H), 6.57 (d, J = 4.1 Hz, 1H),
5.80 (br s, 1H), 4.65 (s, 2H), 4.28 (t, J = 9.1 Hz, 1H), 4.15–4.02 (m,
2H), 3.95 (dd, J = 10.9, 2.8 Hz, 1H), 3.91 (s, 3H), 3.69 (dd, J = 10.3,
8.9 Hz, 1H), 3.46–3.31 (m, 2H), 3.28 (s, 3H), 2.95 (s, 6H), 2.60–
2.40 (m, 2H), 1.85–1.72 (m, 2H), 1.71–1.61 (m, 2H), 1.50–1.31
(m, 4H); HRMS (FAB, 35Cl) m/z 559.2506 (M+), C29H40ClN4O5S re-
quires 559.2510. HPLC purity 92.4%.
Compound 46b (867 mg, 2.03 mmol), PhSO2Na (836 mg,
5.08 mmol), and CSA (1.415 g, 6.09 mmol) were dissolved in dry
DCM (100 mL) and the solution was purged with N2. Pd(Ph3P)4
(164 mg, 0.14 mmol) was added and the solution was stirred under
N2 for 30 min. The mixture was diluted with aqueous NaHCO3 and
extracted with DCM. The DCM extract was washed with brine,
dried (Na2SO4), and evaporated. The crude product was purified
by column chromatography [EtOAc then Et3N/EtOAc, 1:40 then
1:20 then 1:10] to give to give 5-amino-1-(chloromethyl)-3-[7-
(dimethylamino)heptanoyl]-1,2-dihydro-3H-benz[e]indole (47b)
(650 mg, 83%) as a white solid: mp 195 °C (dec.); 1H NMR (CDCl3)
d 7.95 (s, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.48
4.1.17. Tritium-labeled (E)-N-(4-(3-(5-amino-1-(chloromethyl)-
1H-benzo[e]indol-3(2H)-yl)-3-oxoprop-1-enyl)benzyl)-N,N-
dimethyl-1-(1-methyl-5-(methylsulfonyl)-1H-pyrrol-2-yl)
methanaminium bromide hydrobromide (3H-4) (Scheme 7)
A
solution of 18 (960 mg, 3.81 mmol) and 23 (1.035 g,
4.19 mmol) and DIPEA (1.062 mL, 6.10 mmol) in THF (50 mL) was
stirred under N2 for 15 h. The solvent was removed under reduced
pressure and the residue was purified by column chromatography
(petroleum ether/EtOAc, 1:1) to give (E)-tert-butyl 3-(4-
((methyl((1-methyl-5-(methylsulfonyl)-1H-pyrrol-2-yl)methyl)
amino)methyl)phenyl)acrylate (50) (1.53 g, 96%) as a white solid:

748
M. Tercel et al. / Bioorg. Med. Chem. 20 (2012) 734–749
1H NMR (CDCl3) d 7.56 (d, J = 16.0 Hz, 1H), 7.45 (d, J = 8.1 Hz, 2H),
7.29 (d, J = 8.1 Hz, 2H), 6.82 (d, J = 4.0 Hz, 1H), 6.35 (d, J = 16.0 Hz,
1H), 6.10 (d, J = 4.0 Hz, 1H), 3.85 (s, 3H), 3.50 (s, 2H), 3.47 (s, 2H),
3.06 (s, 3H), 2.16 (s, 3H), 1.53 (s, 9H); LRMS (ACPI+): m/z 419.5
(M+H), C22H31N2O4S requires 419.2005.
(20 mL) was allowed to reflux overnight. All solvent was removed
and the residue was dissolved in DCM. After a basic (aqueous NaH-
CO3) workup, the crude product was purified through silica gel col-
umn chromatography. A mixture of EtOAc/DCM/petroleum ether
(v/v/v = 2:1:1) was used to elute out (E)-di(tert-butyl) 1-(chloro
methyl)-3-((2E)-3-(4-((methyl((1-methyl-5-(methylsulfonyl)-1H-
pyrrol-2-yl)methyl)amino)methyl)phenyl)-2-propenoyl)-2,3-dihy-
dro-1H-benzo[e]indol-5-ylimidodicarbonate (55) as bright yellow
solid (797 mg, 86%). 1H NMR (MeOH-d4): d 8.45 (br s, 1H), 7.96
(d, J = 8.3 Hz, 1H), 7.78 (d, J = 5.9 Hz, 1H), 7.75 (s, 1H), 7.67 (d,
J = 8.1 Hz, 2H), 7.61–7.57 (td, J = 7.6, 1.0 Hz, 1H), 7.53–7.49 (td,
J = 7.6, 1.0 Hz, 1H), 7.39 (d, J = 8.1 Hz, 2H), 7.13 (d, J = 15.4 Hz,
1H), 6.77 (d, J = 4.0 Hz, 1H), 6.16 (d, J = 4.0 Hz, 1H), 4.62–4.58 (m,
2H), 4.12 (br s, 1H), 4.06–4.03 (dd, J = 11.3, 3.1 Hz, 1H), 3.90–3.86
(dd, J = 11.3, 7.0 Hz, 1H), 3.87 (s, 3H), 3.57 (s, 2H), 3.56 (s, 2H),
3.12 (s, 3H), 2.19 (s, 3H), 1.33 (s, 9H), 1.30 (s, 9H). HRMS (ESI):
m/z 777.3071 (M+H), C41H5035ClN4O7S requires 777.3089.
TFA (1 mL) was added to cooled (0 °C) 50 (1.23 g, 2.95 mmol)
and the resulting solution was stirred at 20 °C for 1 h. The TFA
was removed under reduced pressure and the residue was tritu-
rated with DCM and Et2O to give (E)-3-(4-((methyl((1-methyl-5-
(methylsulfonyl)-1H-pyrrol-2-yl)methyl)amino)methyl)phenyl)
acrylic acid (51) as a hygroscopic colorless solid (trifluorosulfonate
salt) that turned to a gum in air: 1H NMR (DMSO-d6) d 12.49 (br,
1H), 9.93 (br, 1H), 7.79 (d, J = 7.5 Hz, 2H), 7.62 (d, J = 16.0 Hz,
1H), 7.54 (d, J = 7.0 Hz, 2H), 6.82 (d, J = 3.6 Hz, 1H), 6.60 (d,
J = 16.0 Hz, 1H), 6.51(br, 1H), 4.51 (br, 2H), 4.37 (br, 2H), 3.80 (s,
3H), 3.25 (s, 3H), 2.62 (br, 3H); HRMS (ESI): m/z 363.1381
(MꢁOTFA), C18H23N2O4S requires 363.1373.
Oxalyl chloride (620
lL, 7.10 mmol) and then DMF (two drops)
A solution of 55 (40 mg, 0.051 mmol) in N-methyl-2-pyrrolidi-
none (2 mL) in a 5 mL vial with a screw cap was treated with
were added to a suspension of 51 (TFA salt) (678 mg, 1.42 mmol) in
DCM (10 mL). The mixture was stirred for 3 h and then the solvents
were removed under high vacuum to give the crude HCl salt of the
acyl chloride 52.
methyl iodide (0.5 lL, 0.008 mmol). The vial was sealed and the
mixture was stirred at 40 °C for 4 h, then cooled with ice and 3H-
methyl iodide (0.003 mmol, 250 mCi, in 0.25 mL of toluene, Amer-
ican Radiolabeled Chemicals, Inc.) was added. The mixture was
then stirred at 40 °C for 4 days, cooled with ice, further MeI
A solution of 45 (645 mg, 1.37 mmol) in TFA was held at 0 °C for
3 h, then solvent was pumped off and the dark brown gum ob-
tained (amine HCl salt) was suspended in DCM (10 mL) and the
solution was cooled in an ice bath. DIPEA (2.50 mL, 14.00 mmol)
was added before a solution of 52 (0.54 g, 1.432 mmol) in DCM
(10 mL) was added slowly to give a yellow solution. This solution
was stirred in an ice bath for 1 h and at room temperature for
3 h, and then washed with aqueous NaHCO3. The crude product
purified by silica gel column chromatography twice both using
EtOAc/petroleum ether (gradient: v/v = 1;2, 1:1, 2;1) as eluents to
(2.6 lL, 0.042 mmol) was added and the mixture was stirred at
40 °C for 4 further days. The solution was transferred into a flask
and the vial was washed by a little DCM. Et2O (150 mL) was added
and the resulting suspension was left at ꢁ20 °C overnight. The pre-
cipitate was collected by decantation and then recrystallized from
DCM and Et2O (ꢃ2) to give 56 as a white solid (40 mg, 85%); 1H
NMR (MeOH-d4): d 8.45 (br s, 1H), 7.98 (d, J = 8.3 Hz, 1H), 7.91
(d, J = 8.1 Hz, 1H), 7.83 (d, J = 15.4 Hz, 1H), 7.77 (d, J = 8.3 Hz, 1H),
7.68 (d, J = 8.2 Hz, 2H), 7.64–7.60 (td, J = 8.2, 1.0 Hz, 1H), 7.55–
7.51 (m, 1H), 7.30 (d, J = 15.4 Hz, 2H), 7.02 (d, J = 4.2 Hz, 1H),
6.68 (d, J = 4.2 Hz, 1H), 4.78 (s, 2H), 4.67 (s, 2H), 4.64 (d,
J = 5.3 Hz, 2H), 4.44 (br s, 1H), 4.08–4.05 (dd, J = 11.3, 3.1 Hz, 1H),
4.00 (s, 3H), 3.91–3.87 (dd, J = 11.3, 7.1 Hz, 1H), 3.24 (s, 3H), 3.02
(s, 6H), 1.33 (s, 9H), 1.31 (s, 9H). This was used directly in the next
step.
A solution of 56 (40 mg, 0.043 mmol) in 4 M HCl in dioxane
(10 mL) was kept at room temperature for 15 h, and the resulting
precipitate was collected by filtration and recrystallized from
MeOH/dioxane, then triturated with Et2O ether to give 3H-4
(27 mg, 79%) as a white solid. HPLC chemical purity 85%, radio-
chemical purity 71%, specific activity 107 GBq/mmol. 1H NMR
give
(E)-1-(1-(chloromethyl)-5-(vinylamino)-1H-benzo[e]indol-
3(2H)-yl)-3-(4-((methyl((1-methyl-5-(methylsulfonyl)-1H-pyrrol-
2-yl)methyl)amino)methyl)phenyl)prop-2-en-1-one (53) as a yel-
low solid (475 mg, 56%). 1H NMR (CDCl3): 7.93 (br, 1H), 7.87 (d,
J = 15.1 Hz, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.69 (d, J = 8.3 Hz, 1H),
7.57 (d, J = 8.1 Hz, 2H), 7.50 (d, J = 7.1 Hz, 1H), 7.38–7.33 (m, 3H),
6.93 (br d, J = 15.2 Hz, 1H), 6.84 (d, J = 4.0 Hz, 1H), 6.11 (d,
J = 4.0 Hz, 1H), 6.11–6.05 (br, 1H), 5.40 (d, J = 17.1 Hz, 1H), 5.25
(d, J = 10.4 Hz, 1H), 4.62 (br, 1H), 4.52 (d, J = 10.7 Hz, 1H), 4.40 (t,
J = 9.1 Hz, 1H), 4.06–4.02 (br s, 2H), 3.98–3.94 (dd, J = 11.3,
2.9 Hz, 1H), 3.88 (s, 3H), 3.54 (s, 2H), 3.50 (s, 2H), 3.43 (t,
J = 11.0 Hz, 1H), 3.08 (s, 3H), 2.19 (s, 3H). LRMS (ACPI+): m/z
617.2 (M+H), C34H3835ClN4O3S requires 617.2353.
A
solution of 53 (866 mg, 1.40 mmol), PhSO2Na (577 mg,
(MeOH-d4) d 8.83 (s, 1H), 8.09 (d, J = 8.3 Hz, 1H), 7.91 (d,
3.51 mmol) and CSA (978 mg, 4.21 mmol) in dry DCM (60 mL)
was purged with N2. Pd(PPh3)4 (114 mg, 0.10 mmol) was then
added and the mixture was stirred for 3 h. After NaHCO3 work-
up, the residue obtained was subjected to silica gel column chro-
matography twice using MeOH/DCM (v/v 5%) as eluent firstly
and EtOAc/petroleum ether/DCM/TEA (v/v 3:1:1:0.02) secondly
to give 688 mg (85%) of (E)-1-(5-amino-1-(chloromethyl)-1H-ben-
zo[e]indol-3(2H)-yl)-3-(4-((methyl((1-methyl-5-(methylsulfonyl)-
1H-pyrrol-2-yl)methyl)amino)methyl)phenyl)prop-2-en-1-one (54)
as a bright yellow solid. 1H NMR (CDCl3): d 8.03 (br s, 1H), 7.87–
7.80 (m, 2H), 7.69 (d, J = 8.2 Hz, 1H), 7.57 (d, J = 8.1 Hz, 2H), 7.52
(t, J = 7.6 Hz, 1H), 7.39–7.33 (m, 3H), 6.92 (br d, J = 15.6 Hz, 1H),
6.83 (d, J = 4.0 Hz, 1H), 6.11 (d, J = 4.0 Hz, 1H), 4.51 (d, J = 9.6 Hz,
1H), 4.39 (t, J = 9.4 Hz, 1H), 4.31 (s, 2H), 4.09–4.05 (m, 1H), 3.98–
3.94 (dd, J = 11.2, 2.8 Hz, 1H), 3.88 (s, 3H), 3.54 (s, 2H), 3.50 (s,
2H), 3.44 (t, J = 10.9 Hz, 1H), 3.08 (s, 3H), 2.19 (s, 3H). HRMS
(FAB, 35Cl) m/z 577.20413 (M+H), calcd for C31H3435ClN4O3S:
577.20402.
J = 8.2 Hz, 1H), 7.93 (d, J = 8.2 Hz, 2H), 7.87 (d, J = 15.5 Hz, 1H),
7.77–7.66 (m, 4H), 7.32 (d, J = 15.5 Hz, 2H), 7.02 (d, J = 4.2 Hz,
1H), 6.69 (d, J = 4.2 Hz, 1H), 4.80 (s, 2H), 4.70 (s, 2H), 4.67 (d,
J = 5.1 Hz, 2H), 4.48 (br s, 1H), 4.09–4.06 (dd, J = 11.3, 3.2 Hz, 1H),
4.02 (s, 3H), 3.96–3.92 (dd, J = 11.4, 6.7 Hz, 1H), 3.24 (s, 3H), 3.02
(s, H).
4.2. Biology
4.2.1. In vivo weight loss experiments
C3H/HeN mice (ꢂ50 days old at time of treatment) were ob-
tained from the Vernon Jansen Animal Unit, University of Auck-
land, and housed in groups of four per cage with unrestricted
access to Teklad Global 18% protein rodent diet was purchased
from Harlan Teklad Global Diet (Oxford, UK) and tap water. Base-
line body weight and food and water intake were measured for
7–10 days prior to drug injection to determine that the animals
were eating and gaining weight normally (data not shown). A sin-
gle ip injection of test compound in 42% aqueous DMSO, or 42%
aqueous DMSO alone (vehicle control), was administered on day
A
mixture of 54 (685 mg, 1.19 mmol), Boc2O (2.60 g,
11.87 mmol) and DMAP (10 mg) in THF (60 mL) and dioxane

M. Tercel et al. / Bioorg. Med. Chem. 20 (2012) 734–749
5. Nisoli, E.; Carruba, M. O. Obes. Rev. 2000, 1, 127.
749
1 to groups of 4 animals, and body weight and food and water in-
take were monitored 2–3 times per week for 60 days. The results
are shown in Table 1. Tissue distribution studies with 3H-4 were
conducted as described.22
The University of Auckland Animal Ethics Committee approved
the animal protocols, which permitted continuing observation of
animals with severe body weight loss provided that daily monitor-
ing of clinical signs indicated the animals to be in good general
health.
6. Florentin, M.; Liberopoulos, E. N.; Elisaf, M. S. Obes. Rev. 2008, 9, 378.
7. Harp, J. B. J. Nutr. Biochem. 1998, 9, 516.
8. Atwell, G. J.; Milbank, J. J. B.; Wilson, W. R.; Hogg, A.; Denny, W. A. J. Med. Chem.
1999, 42, 3400.
9. Jeffrey, S. C.; Torgov, M. Y.; Andreyka, J. B.; Boddington, L.; Cerveny, C. G.;
Denny, W. A.; Gordon, K. A.; Gustin, D.; Haugen, J.; Kline, T.; Nguyen, M. T.;
Senter, P. D. J. Med. Chem. 2005, 48, 1344.
10. Hay, M. P.; Atwell, G. J.; Wilson, W. R.; Pullen, S. M.; Denny, W. A. J. Med. Chem.
2003, 46, 2456.
11. Tercel, M.; Atwell, G. J.; Yang, S.; Ashoorzadeh, A.; Stevenson, R. J.; Botting, K. J.;
Gu, Y.; Mehta, S. Y.; Wilson, W. R.; Pruijn, F. B. Angew. Chem. 2011, 123, 2654.
Angew. Chem., Int. Ed. 2011, 50, 2606.
12. Wilson, W. R.; Tercel, M.; Anderson, R. F.; Denny, W. A. Anti-Cancer Drug Des.
1998, 13, 663.
Acknowledgements
13. Tercel, M.; Lee, A. E.; Hogg, A.; Anderson, R. F.; Lee, H. H.; Siim, B. G.; Denny, W.
A.; Wilson, W. R. J. Med. Chem. 2001, 44, 3511.
14. Stribbling, S. M.; Mountjoy, K. G.; Tercel, M.; Wilson, W. R.; Denny, W. A.;
Stevenson, R. J.; Lu, G.-L. WO 08039087, published 3rd April 2008.
15. Borch, R. F.; Liu, J.; Schmidt, J. P.; Marakovits, J. T.; Joswig, C.; Gipp, J. J.;
Mulcahy, R. T. J. Med. Chem. 2000, 43, 2258.
The authors are grateful to Dr. Maruta Boyd, Sisira Kumara,
Yongchuan Gu and Olga Shevstova for technical assistance. This
work was funded by the Marsden Fund and the UniServices Pre-
Seed Accelerator Fund.
16. Chou, S.-S. P.; Yeh, Y.-H. Tetrahedron Lett. 2001, 42, 1309.
17. Boger, D. L.; McKie, J. A. J. Org. Chem. 1995, 60, 1271.
18. Atwell, G. J.; Tercel, M.; Boyd, M.; Wilson, W. R.; Denny, W. A. J. Org. Chem.
1998, 63, 9414.
Supplementary data
19. Yang, S.; Denny, W. A. J. Org. Chem. 2002, 67, 8958.
20. Honda, M.; Morita, H.; Nagakura, I. J. Org. Chem. 1997, 62, 8932.
21. Olomucki, M. Bull. Soc. Chim. Fr. 1963, 10, 2067.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.12.007.
22. Tercel, M.; Marnane, R.; Tatnell, M. A.; Stevenson, R. J.; Halim, A.; Lu, G.-L.;
Duchesnes, C.; Truong, M.; Denny, W. A.; Wilson, W. R.; Mountjoy, K. G. Int. J.
Obes., submitted for publication.
References and notes
23. Gieseg, M. A.; Matejovic, J.; Denny, W. A. Anti-Cancer Drug Des. 1999, 14, 77.
24. Adeghate, E.; Hameed, R. S.; Ponery, A. S.; Tariq, S.; Sheen, R. S.; Shaffiullah, M.;
Donath, T. Exp. Clin. Endocrinol. Diabetes 2010, 118, 699.
25. Bennett, R. A.; Pegg, A. E. Cancer Res. 1981, 41, 2786.
26. Happer, D. A. R.; Steenson, B. E. J. Chem. Soc., Perkin Trans. 2 1983, 843.
27. Palomo, C.; Aizpurua, J. M.; Garcia, J. M.; Ganboa, I.; Cossio, F. P.; Lecea, B.;
Lopez, C. J. Org. Chem. 1990, 55, 2498.
1. Hofbauer, K. G.; Nicholson, J. R.; Boss, O. Annu. Rev. Pharmacol. Toxicol. 2007, 47,
565.
2. Isidro, M. L.; Cordido, F. Mini-Rev. Med. Chem. 2009, 9, 664.
3. Hadváry, P.; Sidler, W.; Meister, W.; Vetter, W.; Wolfer, H. J. Biol. Chem. 1991,
266, 2021.
4. Akbas, F.; Gasteyger, C.; Sjodin, A.; Astrup, A.; Larsen, T. M. Obes. Rev. 2009, 10,
58.