Optimised synthesis of a nitroCBI hypoxia-activated prodrug with substantial anticancer activity

Article abstract of DOI:10.1016/j.tet.2019.04.027

An optimised synthesis of a hypoxia-activated anticancer prodrug related to the duocarmycin family of natural products is described. The improved 10-step synthesis increases the overall yield from 4.4% to over 40% while requiring just 2 chromatography-bas

Full text of DOI:10.1016/j.tet.2019.04.027

Accepted Manuscript
Optimised synthesis of a nitroCBI hypoxia-activated prodrug with substantial
anticancer activity
Ho H. Lee, Benjamin D. Dickson, Ralph J. Stevenson, Shangjin Yang, Moana Tercel
PII:
S0040-4020(19)30429-6
https://doi.org/10.1016/j.tet.2019.04.027
TET 30272
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 7 March 2019
Revised Date: 9 April 2019
Accepted Date: 11 April 2019
Please cite this article as: Lee HH, Dickson BD, Stevenson RJ, Yang S, Tercel M, Optimised synthesis
of a nitroCBI hypoxia-activated prodrug with substantial anticancer activity, Tetrahedron (2019), doi:
https://doi.org/10.1016/j.tet.2019.04.027.
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Optimised synthesis of a nitroCBI hypoxia-
activated prodrug with substantial
anticancer activity
Leave this area blank for abstract info.
Ho H. Lee, Benjamin D. Dickson, Ralph J. Stevenson, Shangjin Yang, Moana Tercel
Auckland Cancer Society Research Centre, School of Medical Sciences, Faculty of Medical and Health
Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand

1
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Tetrahedron
journal homepage: www.elsevier.com
Optimised synthesis of a nitroCBI hypoxia-activated prodrug with substantial
anticancer activity
a
a
a
b
a
Ho H. Lee , Benjamin D. Dickson Ralph J. Stevenson Shangjin Yang and Moana Tercel ∗
,
,
,
a
Auckland Cancer Society Research Centre, School of Medical Sciences, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag
2019, Auckland 1142, New Zealand
Jiyuan Research Institute, Zhengzhou University, China
9
b
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
An optimised synthesis of a hypoxia-activated anticancer prodrug related to the duocarmycin
family of natural products is described. The improved 10-step synthesis increases the overall
yield from 4.4% to over 40% while requiring just 2 chromatography-based purifications. The
most significant improvements optimise the isomer distribution in a key chlorosulfonylation
reaction, and facilitate the removal of tin residues from a tributyltin hydride-mediated radical
reaction. Improved preparations of two side chains are also reported. The new method provides
practical access to sufficient material to support advanced efficacy and toxicology assessments.
Available online
Keywords:
NitroCBI
2
009 Elsevier Ltd. All rights reserved.
Hypoxia-activated prodrug
Optimised synthesis
OMe
1
. Introduction
N
Inspired by the structure and mechanism of action of the
N
OMe
H
1
MeO C
2
O
duocarmycin natural products (such as duocarmycin SA, 1,
Figure 1) we have developed a class of anticancer prodrugs that
N
OMe
H
O
2–6
are selectively toxic to cells under low oxygen conditions. The
prodrugs are named nitroCBIs (2) after the synthetic 1,2,9,9a-
tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) variant of the
alkylating subunit, modified such that the active phenol of the
minor groove-binding
subunit
alkylating subunit
1
(+)-duocarmycin SA
Cl
Cl
2,3
ring-opened form of CBI is replaced with a nitro group. In the
general structure 2, R is a side chain that can bind in the minor
groove of DNA, and Rʹ is a substituent that modifies the
NCOR
NCOR
1e
5e
R'
R'
3
reduction potential of the nitro group. With appropriate Rʹ
substituents 2 undergoes efficient bioreduction, via an initial 1-
electron oxygen-sensitive step, to yield the corresponding
aminoCBI 3. Under physiological conditions 3 spontaneously
NH2
aminoCBI
NO2
O .- O2
2
3
2
nitroCBI
NH2
5
-HCl
cyclises to an unstable intermediate 4 that, like the natural
products, selectively alkylates the N3 position of adenine in the
minor groove of DNA, creating a highly toxic lesion 5 (where dR
represents the deoxyribose connection to DNA). Hypoxic cells
capable of activating nitroCBIs are not commonly found in
normal tissues, but are a prevalent feature of tumours, where they
are widely associated with multiple types of treatment
N
N
N
3
N
+
NCOR
dR
DNA
R'
NCOR
NH2+
R'
7
resistance. Thus hypoxia-activated prodrugs such as nitroCBIs
4
have great potential to be combined with standard agents to
5
NH2
8,9
deliver more effective cancer therapy.
Figure 1: Structure of duocarmycin SA, and mechanism of
action of nitroCBI hypoxia-activated prodrugs.
—
——
∗
Corresponding author. Tel.: +64-9-373-7599; fax: +64-9-373-7502; e-mail: m.tercel@auckland.ac.nz

2
Tetrahedron
ACCEPTED MAba
N
se
U
, t
S
he
C
a
R
lm
I
o
P
st
T
universal choice in multiple related syntheses
15 16
(
with occasional exceptions using KOt-Bu or a phosphazene ).
We recently reported a particular nitroCBI 10 (Scheme 1)
6
We successfully scaled up the NaH method (to about 0.5 mol,
requiring 25 g of a 60% dispersion of NaH in oil), but later found
that it was operationally much simpler to employ K CO as the
with outstanding anticancer activity. This includes combination
with radiotherapy to eliminate all detectable colony-forming cells
in some cervical carcinoma xenografts, and combination with
chemotherapy (gemcitabine) to cause marked regression of all
treated ovarian tumour xenografts. These effects were observed
2
3
base, which gave quantitative reaction to 13 in DMF at 70-80 °C.
The indoline ring of 14 is formed in a radical-mediated
at well-tolerated doses and in some cases gave curative activity
cyclisation reaction. Our previously reported procedure using
6
(tumour-free mice 100 days after treatment). Our reported
Bu SnH and azobisisobutyronitrile (AIBN) initiator worked well
3
3
synthesis of 10 proceeds in 10 steps and gives an overall yield of
only 4.4% (Scheme 1). 2-Naphthoic acid 6 is converted to the
on a large scale (23 g of 13), but removal of tin residues required
3
purification steps (chromatography, crystallisation from
3,10
key intermediate 7 in 7 steps, followed by sequential addition
MeOH, and recrystallisation from CH Cl /petroleum ether)
2
2
4
11
of each of the side chains 8 and 9, and a final deprotection step
which resulted in a yield of 74%. We modified the procedure by
reducing the amount of AIBN used and changing the solvent
from benzene to toluene, but the major change was in the
purification method. Most of the tin residues were removed by
exposure to solid KF (which converts tributyltin halides to
6
to give the desired product. We have carefully re-examined each
of these steps and have improved the overall yield to >40%
(Scheme 2) while reducing the number of chromatography-based
purifications from 6 to 2. We have also substantially improved
the synthesis of each of the side chains (Scheme 3), and used the
optimised method to prepare multi-gram quantities of 10. The
details of these improvements are described herein, some of
17
insoluble polymeric tributyltin fluoride ), followed by filtration
through a short column of neutral alumina. The crude product
1
still contained some tin residues (5-10% by H NMR) but these
12
which have been disclosed in patent form.
were removed after deprotecting the Boc group using methanolic
HCl and repeatedly washing the methanol solution with
petroleum ether. In this way the hydrochloride salt 14 was
obtained in 96% yield starting from nearly 50 g of 13.
The indoline nitrogen was next protected to give
trifluoroacetamide 15 as
a substrate for the following
chlorosulfonylation reaction. This step was the lowest-yielding of
the original synthesis since the reaction, conducted in neat
chlorosulfonic acid at 60 °C, gave a mixture of 6- and 7-SO Cl
2
isomers, with the desired 7-isomer 16 being the minor component
3
and isolated in only 25% yield after chromatography. We had
previously found that the isomer distribution after acetylation of
the same substrate 15 was very sensitive to the reaction
conditions, favoring the 6-acetyl product when conducted in CS2
at 70 °C, but the 7-acetyl product in nitrobenzene at room
3
temperature. Therefore we explored alternative conditions for
the chlorosulfonylation reaction, and found that using just 1.2
equivalents of ClSO H in CH Cl at 3-4 °C produced almost
3
2
2
exclusively the desired 7-isomer. At this temperature the reaction
requires several hours to consume the starting material and
during this time an insoluble product appears. On the assumption
that this was the corresponding sulfonic acid the mixture was
treated with DMF and oxalyl chloride to convert this material
back to the desired sulfonyl chloride, a method that eventually
produced 16 in up to 97% yield. Occasionally, significant
Scheme 1: Overview of the published synthesis of nitroCBI hypoxia-
activated prodrug 10.
amounts of the 6-SO Cl isomer were still produced, requiring
2
. Results and Discussion
2
purification by column chromatography, or by trituration with
EtOAc, but the overall yield was always considerably higher than
that previously obtained.
The synthesis begins with the conversion of 6 to 11 using
diphenylphosphoryl azide (DPPA) in t-BuOH (Scheme 2). The
procedure was conducted on a much larger scale than previously
10
Nitration was achieved using KNO3 in H SO , which
2
4
reported (100 g versus 1.5 g ), with minimal excess reagents and
solvent, and the crude product isolated in practically quantitative
yield without chromatography. This material could be further
purified by recrystallisation (75% yield on a 25 g scale), but we
found this to be unnecessary to attain high yields in the following
steps.
performed well on a 20 g scale to give the key intermediate 7. As
well as the desired 5-NO isomer small amounts of the 9-NO
2
2
product were also formed, but this less polar byproduct was
easily removed by crystallisation of the crude material from
CH Cl /i-Pr O. Side chain 8 was introduced via a high-yielding
2
2
2
sulfonamide formation, with excess base added to complete
removal of the trifluoroacetamide protecting group to generate
17. Side chain 9 was then appended via amide formation using N-
Bromination was achieved simply by exposure to N-
bromosuccinimide (NBS) at 0 °C in CH CN and the product 12
3
was precipitated from the reaction mixture in quantitative yield.
ethyl-N’-(3-dimethylaminopropyl)carbodiimide
hydrochloride
3
For this step we had found that it was not necessary to use either
(EDCI) as the coupling reagent under weakly acidic conditions.
The addition of a second batch of EDCI drove the reaction to
completion and gave an excellent yield of 18. The final step also
proceeded in excellent yield, using TFA to remove the t-Bu ester
protecting groups from the phosphate, leading to the isolation of
an acid catalyst or DMAP to facilitate the reaction, in surprising
contrast to related duocarmycin analogues bearing an additional
13,14
and activating benzyloxy substituent.
For the subsequent
chloroallylation step our original method employed NaH as the

3
ACCEPTED MANUSCRIPT
Scheme 2: Optimised synthesis of 10.
1
0 as the trifluoroacetate salt. Single batches of up to 3.6 g of 10
material to support further efficacy and toxicology evaluation.
Some of the modifications may well be applicable to the
synthesis of duocarmycin analogues in general, which are of
current interest in targeted anticancer therapies such as antibody-
drug conjugates.
have been made following this procedure, with HPLC purity
routinely >97%.
Significant improvements in the synthesis of the side
chains 8 and 9 are detailed in Scheme 3. For 8 the key was to
optimize the initial phosphorylation reaction to prepare larger
4
quantities of 20. We modified our original procedure by
reducing
the
excess
of
di-tert-butyl
N,N-di-iso-
propylphosphoramidite and tetrazole used, finding the best order
for addition of reagents, optimising the reaction time, and by
swapping from 70% to 30% H O for the oxidation step. These
2
2
changes gave a crude product from which the desired material
could be obtained by crystallisation rather than chromatography.
The product 20 is a low mp solid (43-45 °C) and a seed crystal
was found to be very useful in inducing the crystallisation
process. Even with these carefully optimized conditions, some
1
loss of t-Bu groups was occasionally observed by H NMR
analysis of the crude product. This could represent contamination
18
with a phosphonate byproduct. Switching the acidic activator
from tetrazole to a combination of imidazole and imidazole
18
hydrochloride proved to be a useful alternative, providing 20 in
up to 92% yield on a multi-gram scale. Hydrogenation then
afforded side chain 8.
The synthesis of side chain 9 proceeds in 4 steps from 4-
11
nitrophenol 21 (Scheme 3). The original method used a 2-part
procedure for the first step, isolating the potassium salt of 21 and
then reacting this with 4-(2-chloroethyl)morpholine in toluene to
give 22 in 57% yield. By using K CO as the base in DMF this
2
3
step can be conducted in a one-pot procedure to isolate 22 in 90%
yield without chromatography. Following hydrogenation to the
aniline 23, a Japp–Klingemann-type Fischer indole synthesis
produced 24, isolated in 86% yield on a 35 g scale. The final step
is a simple ester hydrolysis, for which a minor modification aided
purification on a large scale – the product was precipitated and
washed in the zwitterionic form before conversion to the
hydrochloride salt, allowing the isolation of 9 in high yield, again
without recourse to chromatography.
Scheme 3: Optimised synthesis of side chains 8 and 9.
3
. Conclusions
Overall these modifications constitute
a
significantly
improved synthesis of a highly promising anticancer prodrug,
and provide a practical route for the synthesis of sufficient

4
Tetrahedron
1
4
. Experimental Section
evaporation) to give 13 as an oil (66.2 g, 100%), suitable ( H
ACCEPTED MANUSCRIPT
NMR) for the following reactions; δ [(CD ) SO] (mixture of
H
3 2
4
.1. General Experimental
rotamers and E and Z forms) 8.23 (d, J = 8.4 Hz, 1 H), 8.05-7.97
m, 2 H), 7.74-7.62 (m, 2 H), 7.50, 7.45 (2 d, J = 8.6 Hz, 1 H),
(
Solvents were distilled prior to use by common laboratory
6
4
.41-6.30 (m, 1 H), 6.18-6.02 (m, 1 H), 4.57-4.49, 4.42-4.19,
.10-3.97 (3 m, 2 H), 1.49, 1.26 (2 s, 9 H); consistent with that
methods. t-BuOH was dried over CaH . Petroleum ether refers to
the fraction with a bp = 40-60 °C. Melting points were
determined on an Electrothermal 2300 Melting Point Apparatus.
2
3
reported.
NMR spectra were obtained on
spectrometer at 400 MHz for H spectra and 100 MHz for
a Bruker Avance 400
4
.2.4. 1-(Chloromethyl)-1,2-dihydro-3H-benzo[e]indole
1
13
C
hydrochloride (14)
spectra. HRMS was performed on an Agilent 6530B Accurate
Mass Q-TOF mass spectrometer. Combustion analyses were
carried out in the Campbell Microanalytical Laboratory,
University of Otago, Dunedin, New Zealand.
A solution of 13 (48.8 g, 123 mmol) in toluene (150 mL) was
stirred at 20 °C under nitrogen for 20 min, then Bu SnH (34.2
3
mL, 123 mmol) and AIBN (60.7 mg, 0.37 mmol) were added.
The reaction mixture was stirred at 85-90 °C (bath temperature)
under nitrogen for 1 h and then cooled to 20 °C. Powdered
anhydrous KF (35.7 g, 615 mmol) was added and the mixture
was stirred at 20 °C for 14 h. EtOAc (150 mL) and petroleum
ether (300 mL) were added and the mixture stirred for a further 1
h 30 min. The mixture was filtered through a pad of neutral
alumina (300 g), washing with EtOAc/petroleum ether (1:2) (10
× 50 mL). The combined filtrates were washed successively with
4
4
.2. Synthesis
.2.1. tert-Butyl 2-naphthylcarbamate (11)
Et N (89.2 mL, 638 mmol) was added to solid 2-naphthoic
3
acid (6) (100 g, 581 mmol) and the mixture was stirred at 20 °C
for 10 min. After this time the mixture solidified and was allowed
to stand at 20 °C for a further 20 min. DPPA (freshly opened
bottle, 131.6 mL, 609 mmol) was added, followed by dry t-
BuOH (277.2 mL, 2.90 mol), causing the internal temperature to
rise to 35 °C. The reaction flask was placed in an oil bath and the
bath temperature was raised from 20 °C to 86-87 °C over 1 h 20
min, then held at this temperature for 2 h. The reaction mixture
was cooled briefly and poured into ice-water (ca. 2.5 L) with
vigorous stirring at 20 °C, causing a sticky solid mass to separate.
The mixture was left to stand at 20 °C overnight (15 h) to give a
loose suspended solid. The mixture was stirred again for 5 h. The
fine solid was filtered off, washed with water (4 × 300 mL) and
dried in a vacuum oven at 40-45 °C to constant weight, to give 11
as a beige solid (140 g, 99%); mp 85-87 °C [lit. mp 92-93 °C
water (300 mL), aqueous Na
mL), and then dried (Na SO
CO
) and evaporated to give a yellow
4
(5%, 300 mL) and water (300
2
3
2
1
solid (74 g). H NMR analysis showed this material contained
about 5-10% tin residues. The solid was dissolved in MeOH (150
mL) and HCl gas was bubbled into the solution at room
temperature over 25 min. The acidic mixture was stirred for a
further 10 min, then diluted with petroleum ether (300 mL). The
mixture was stirred at 20 °C for 15 min, then the petroleum ether
layer was separated and discarded. The petroleum ether
procedure was repeated three more times. The acidic MeOH
solution was then evaporated under reduced pressure at 35-40 °C
(bath temperature) to give 14 as a greenish foamy solid (30.1 g,
19
1
(
hexane) ], suitable ( H NMR) for the following reactions. A
96%); mp 180 °C (dec.); δ [(CD ) SO] ∼ 10.5 (v br, 1 H), 8.04-
H 3 2
portion was recrystallised from CH Cl /petroleum ether to give a
7.94 (m, 3 H), 7.66-7.60 (m, 1 H), 7.56-7.51 (m, 1 H), 7.45 (d, J
= 8.7 Hz, 1 H), 4.43-4.36 (m, 1 H), 4.08 (dd, J = 11.0, 3.5 Hz, 1
H), 3.96 (d, J = 11.2 Hz, 1 H), 3.93 (d, J = 11.2 Hz, 1 H), 3.84
(dd, J = 11.5, 2.5 Hz, 1 H). This material contained only traces of
tin residues (about 1%). On a smaller scale (3.0 g, Boc
deprotection conducted using 4 M HCl in dioxane) repeated
washes with petroleum ether (4 × 100 mL) were sufficient to
2
2
cream solid; δ (CDCl ) 7.98 (s, 1 H), 7.75 (apparent d, J = 8.6
H
3
Hz, 3 H), 7.45-7.41 (m, 1 H), 7.38-7.31 (m, 2 H), 6.60 (br s, 1 H),
10,19
1
.56 (s, 9 H); consistent with that reported.
4
.2.2. tert-Butyl 1-bromo-2-naphthylcarbamate (12)
A stirred solution of 11 (92 g, 0.38 mol) in acetonitrile (680
1
mL) at 0 °C was treated portionwise over 2 h with solid NBS (81
g, 0.45 mol). After the addition was complete the reaction
mixture was stirred for a further 45 min at 0 °C. To the mixture
remove all traces of tin residues (as detected by H NMR) to give
an analytically pure product; δ [(CD ) SO] 137.1, 132.3, 130.5,
C
3 2
1
29.1, 129.0, 127.8, 126.1, 123.8, 116.7, 48.7, 46.3, 43.3 (one
aromatic quaternary carbon not observed); [Found: C, 59.71; H,
.36; N, 5.32. C H ClNꢀHClꢀ½H O requires C, 59.33; H, 5.36;
was added a cold solution of aqueous NaHCO (0.2N, 2 L) and
3
the mixture was stirred at 0 °C for 1 h. The solid was filtered off
and washed with water (5 × 200 mL), then dried in vacuum over
solid KOH overnight to give 12 as a beige solid (121 g, 100%);
5
13
12
2
+,
N, 5.32]; HRMS (ESI) MH found 218.0727. C H ClN requires
13
13
2
18.0731.
19
3
mp 88-89 [lit. mp 88-89 °C;
90-91 °C (MeOH) ]; δH
4
.2.5. 1-(Chloromethyl)-3-(trifluoroacetyl)-1,2-dihydro-3H-
[
(CD ) SO] 8.78 (s, 1 H), 8.15 (d, J = 8.6 Hz, 1 H), 7.99-7.92 (m,
3
2
benz[e]indole (15)
To a stirred mixture of 14 (30.1 g, 118 mmol) in dioxane (100
mL) at 0 °C was added trifluoroacetic anhydride (33.4 mL, 236
2
1
H), 7.71 (d, J = 8.8 Hz, 1 H), 7.69-7.64 (m, 1 H), 7.58-7.53 (m,
H), 1.49 (s, 9 H); consistent with that reported.
3
4
.2.3. tert-Butyl 1-bromo-2-naphthyl-(3-chloro-2-propen-1-
mmol), followed by Et N (49.2 mL, 354 mmol). The mixture was
3
yl)carbamate (13)
stirred at 0 °C for 10 min, at 20 °C for another 50 min, and then
poured into ice-water (500 mL). Conc. HCl (40 mL) was added
and the mixture was stirred at 20 °C for 20 min. The precipitated
solid was filtered off, washed successively with dilute HCl (1N, 4
A mixture of 12 (53.7 g, 167 mmol) and dry K CO (55.2 g,
2
3
4
00 mmol) in dry DMF (500 mL) was stirred at 20 °C for 5 min,
then 1,3-dichloropropene (23.1 mL, 251 mmol, mixed isomers)
was added. The mixture was stirred at 70-80 °C (bath
temperature) under a dry atmosphere for 4 h. The mixture was
cooled and then partitioned between petroleum ether (500 mL)
and water (500 mL). The petroleum ether layer was separated and
×
20 mL), water (10 × 50 mL), petroleum ether (5 × 25 mL), and
dried to give 15 as a pale yellow solid (37.0 g, 100%) suitable for
the following reactions; δ [(CD ) SO] 8.32 (d, J = 9.0 Hz, 1 H),
H
3 2
8
.07-7.97 (m, 3 H), 7.65-7.59 (m, 1 H), 7.56-7.51 (m, 1 H), 4.60-
washed with water (3 × 200 mL), then dried (Na SO ) and
2
4
4.53 (m, 1 H), 4.50-4.40 (m, 2 H), 4.15 (dd, J = 11.3, 3.1 Hz, 1
H), 4.03 (dd, J = 11.3, 5.9 Hz, 1 H); consistent with that
allowed to stand at 5 °C overnight. The precipitated orange solid
was filtered off and the filtrate was evaporated to give an amber
oil (67.5 g). This was again dissolved in petroleum ether and the
procedure repeated (precipitation of an orange solid, filtration,
3
reported.

5
4
.2.6. 1-(Chloromethyl)-3-(trifluoroacetyl)-1,2-dihydro-3H-
product-containing fractions were evaporated to give 17 as a
ACCEPTED MANUSCRIPT
benzo[e]indole-7-sulfonyl chloride (16)
red-orange foam (3.60 g, 95%); δ (CDCl ) 8.90 (d, J = 1.7 Hz, 1
H 3
A solution of chlorosulfonic acid (1.77 mL, 26.5 mmol) in dry
CH Cl (20 mL) at 20 °C was added dropwise over 1 h to a
H), 7.90 (dd, J = 8.9, 1.8 Hz, 1 H), 7.78 (d, J = 8.9 Hz, 1 H), 7.72
(s, 1 H), 5.73 (t, J = 5.8 Hz, 1 H), 4.43 (s, 1 H), 4.14-3.94 (m, 5
H), 3.77 (dd, J = 11.2, 3.7 Hz, 1 H), 3.59 (dd, J = 11.0, 10.0 Hz,
1 H), 3.32-3.24 (m, 2 H), 1.46 (s, 9 H), 1.45 (s, 9 H).
2
2
stirred mixture of 15 (6.92 g, 22.1 mmol) and CH Cl (60 mL) in
2
2
an ice bath, maintaining the internal temperature at 3-4 °C
throughout. The mixture was stirred for a further 2 h 30 min at
the same temperature. Another batch of chlorosulfonic acid (1.77
mL, 26.5 mmol) in dry CH Cl (10 mL) was added dropwise over
4
.2.9. Di-tert-butyl 2-(1-(chloromethyl)-3-(5-(2-
morpholinoethoxy)-1H-indole-2-carbonyl)-5-nitro-2,3-dihydro-
2
2
1
H-benzo[e]indole-7-sulfonamido)ethyl phosphate (18)
4
5 min and the reaction was stirred in the ice bath for a further 1
To a stirred mixture of 17 (2.91 g, 5.03 mmol) and 9 (2.06 g,
h 30 min. At this point TLC (EtOAc:petroleum ether 1:4)
indicated complete consumption of the starting material. Dry
DMF (10 mL, sufficient to dissolve all suspended solid) was
added, followed by oxalyl chloride (5.55 mL, 63.6 mmol). The
reaction mixture was stirred in the ice bath for 30 min and then
left to stand at 0-5 °C overnight. The solvent was evaporated
under reduced pressure at 20 °C to give an amber solid. Cold
water (100 mL) was added and the mixture was stirred at 0 °C
6
.30 mmol) in DMA (80 mL) was added EDCI (1.94 g, 10.0
mmol) and anhydrous TsOH (175 mg, 1.00 mmol). The mixture
was stirred at 20 °C for 2 h 30 min. Another batch of EDCI (1.94
g, 10.0 mmol) and anhydrous TsOH (88 mg, 0.50 mmol) were
added and the mixture was stirred for a further 3 h 30 min. The
reaction flask was cooled in an ice-bath and cold aqueous
NaHCO (5%, 160 mL) was added, followed by cold water (160
3
mL). After stirring at 0 °C for 5 min the precipitated solid was
filtered off, washed with cold water several times, and dried to
(bath temperature) for 15 min. The solid was filtered off, washed
with cold water (5 × 20 mL), and dried to give 16 as a pale
3
give 18 as a yellowish orange solid (4.06 g, 95 %); δ
(CDCl )
H 3
yellow solid (8.8 g, 97%); mp 187-190 °C (lit. mp 189-192 °C );
9
.41 (br s, 1 H), 9.33 (s, 1 H), 8.99 (d, J = 1.5 Hz, 1 H), 8.06 (dd,
δH (CDCl ) 8.68-8.62 (m, 2 H), 8.10 (d, J = 9.0 Hz, 1 H), 8.09
3
J = 8.9, 1.7 Hz, 1 H), 7.97 (d, J = 8.9 Hz, 1 H), 7.38 (d, J = 8.9
Hz, 1 H), 7.14 (d, J = 2.2 Hz, 1 H), 7.08-7.03 (m, 2 H), 6.11-6.03
(dd, J = 9.0, 2.0 Hz, 1 H), 8.00 (d, J = 9.0 Hz, 1 H), 4.68 (d, J =
1
1.6 Hz, 1 H), 4.50 (dd, J = 11.5, 8.6 Hz, 1 H), 4.30-4.24 (m, 1
(
m, 1 H), 4.92 (dd, J = 10.8, 2.2 Hz, 1 H), 4.83 (apparent t, J =
9.8 Hz, 1 H), 4.39-4.32 (m, 1 H), 4.19 (t, J = 5.7 Hz, 2 H), 4.11-
.04 (m, 2 H), 3.97 (dd, J = 11.5, 3.4 Hz, 1 H), 3.80-3.74 (m, 4
H), 3.94 (dd, J = 11.6, 3.4 Hz, 1 H), 3.63 (dd, J = 11.6, 8.8 Hz, 1
3
H); consistent with that reported.
4
4
3
.2.7. 1-(Chloromethyl)-5-nitro-3-(trifluoroacetyl)-1,2-dihydro-
H-benzo[e]indole-7-sulfonyl chloride (7)
H), 3.66 (dd, J = 11.5, 9.1 Hz, 1 H), 3.38-3.31 (m, 2 H), 2.86 (t, J
= 5.7 Hz, 2 H), 2.66-2.58 (m, 4 H), 1.46 (s, 9 H), 1.44 (s, 9 H).
To a stirred solution of 16 (22.1 g, 53.6 mmol) in conc. H SO4
2
4
.2.10. 2-(1-(Chloromethyl)-3-(5-(2-morpholinoethoxy)-1H-
(
200 mL) at 0 °C was added solid KNO (5.96 g, 59.0 mmol)
3
indole-2-carbonyl)-5-nitro-2,3-dihydro-1H-benzo[e]indole-7-
sulfonamido)ethyl dihydrogen phosphate trifluoroacetate (10)
portionwise over 2 h. After the addition was complete the
mixture was stirred at 0 °C and reaction progress was monitored
by TLC (EtOAc:petroleum ether 2:3). After 5 min it was found
that significant starting material was still present, thus another
A filtered solution of 18 (3.78 g, 4.45 mmol) in CH Cl (150
2
2
mL) was stirred with TFA (6.9 mL, 89 mmol) at 20 °C for 45
min. The mixture was concentrated under reduced pressure at
2
mL) was added and the mixture was stirred at 20 °C for 2 h 30
min. The resulting solid was filtered off, washed with EtOAc
several times, and dried in vacuum over silica gel to give 10 as a
yellowish orange solid (3.60 g, 95%); δ [(CD ) SO] 11.84 (s, 1
batch of KNO (1.19 g, 11.8 mmol) was added over a period of
3
0 °C (bath temperature) to ca. 30 mL total volume. EtOAc (300
2
0 min. After a further 5 min TLC analysis showed that the
reaction was complete. The reaction mixture was poured onto ice
1.5 L) and the product was extracted into EtOAc (1.5 L). The
(
EtOAc layer was washed with cold water, then dried (MgSO4)
and evaporated under reduced pressure at 30 °C to give a brown
oil. To the oil was added CH Cl (50 mL) and diisopropyl ether
H
3 2
H), ca. 10.7 (v br s, 1 H), 9.29 (s, 1 H), 8.87 (d, J = 1.7 Hz, 1 H),
2
2
8
1
7
4
.45 (d, J = 8.9 Hz, 1 H), 8.27-8.20 (m, 1 H), 8.03 (dd, J = 8.9,
.7 Hz, 1 H), 7.44 (d, J = 8.9 Hz, 1 H), 7.26 (d, J = 2.2 Hz, 1 H),
.24 (d, J = 1.8 Hz, 1 H), 7.02 (dd, J = 8.9, 2.3 Hz, 1 H), 5.01-
.94 (m, 1 H), 4.71 (dd, J = 10.8, 2.1 Hz, 1 H), 4.68-4.62 (m, 1
(
100 mL), and the solution was stirred and then left at 5 °C
overnight. The solid that formed was filtered off, washed with
diisopropyl ether several times, and dried to give 7 (17.5 g, 71%)
as a light brown solid. The mother liquor was left at −10 °C to
H), 4.34-4.29 (m, 2 H), 4.19-4.09 (m, 2 H), 3.87-3.76 (m, 6 H),
.26-3.13 (m, 4 H), 3.06-3.00 (m, 2 H), (2 H not observed,
6
give an additional crop (0.48 g, 2%); δH (CDCl ) 9.35 (s, 1 H),
3
3
9
.29 (d, J = 1.7 Hz, 1 H), 8.23 (dd, J = 9.0, 1.8 Hz, 1 H), 8.11 (d,
J = 9.0 Hz, 1 H), 4.74 (d, J = 11.5 Hz, 1 H), 4.59 (dd, J = 11.4,
obscured by water peak δ 3.5-3.3); consistent with that reported.
Large scale batches (>1 g) made by this method had HPLC purity
of 98.2-94.4%, as determined by the previously reported
8
3
.8 Hz, 1 H), 4.42-4.34 (m, 1 H), 3.95 (dd, J = 11.7, 3.5 Hz, 1 H),
3
.74 (dd, J = 11.7, 7.7 Hz, 1 H); consistent with that reported.
6
method.
4
.2.8. Di(tert-butyl) 2-(1-(chloromethyl)-5-nitro-1,2-dihydro-3H-
4
(
.2.11. Benzyl 2-((di(tert-butoxy)phosphoryl)oxy)ethylcarbamate
20)
Using tetrazole: Tetrazole (3 wt% solution in acetonitrile, 468
benzo[e]indole-7-sulfonamido)ethyl phosphate (17)
A cold solution of 8 (1.98 g, 7.8 mmol) and i-Pr NEt (1.22
2
mL, 6.8 mmol) in THF (10 mL) was added to solution of 7 (2.98
g, 6.5 mmol) in THF (30 mL) at 0 °C. The mixture was stirred at
0
mL, 159 mmol) was added gradually over 1 h to a stirred mixture
of di-tert-butyl N,N-di-iso-propylphosphoramidite (95%, 53.0
mL, 159 mmol) and benzyl 2-hydroxyethylcarbamate (19) (26.0
g, 133 mmol) in THF (500 mL) at 20 °C under a nitrogen
atmosphere. After the addition was complete the mixture was
stirred at this temperature for 19 h. The mixture was cooled to
°C for a further 20 min, then solid Cs CO (4.04 g, 12.4 mmol)
2 3
and cold MeOH (20 mL) were added. The mixture was stirred at
°C for a further 10 min and then partitioned between EtOAc
400 mL) and ice-water (400 mL). The organic layer was
0
(
separated and the aqueous layer was further extracted with
EtOAc. The combined EtOAc extracts were washed again with
water, then dried (Na SO ), and evaporated under reduced
pressure at 25 °C (bath temperature). The residue was dissolved
in CH Cl and the solution was filtered through a short silica
0
°C and H O (30% aqueous, 48 mL, 494 mmol) was added.
2 2
After 1.5 h at 0 °C the mixture was poured into ice-water and the
product was extracted into EtOAc. The EtOAc layer was washed
successively with cold aqueous Na CO and water (× 2) and then
2
4
2
3
2
2
dried (Na SO ). The organic solvents were removed under
2
4
column (Merck 230−400 mesh), eluting with CH Cl . The
2
2

6
Tetrahedron
reduced pressure to give a colourless oil. A
A
se
C
ed
C
c
E
ry
P
st
T
al
E
w
as MA4.
D
N
2.1
U
5.
S
E
C
thy
R
l 5
I
-(2-morpholinoethoxy)-1H-indole-2-carboxylate
P
T
added and the mixture was allowed to stand at 20 °C for 1 h, then
petroleum ether was added and the mixture was left at 5 °C for
crystallisation to complete. The resulting solid was filtered off,
washed with petroleum ether, and dried to give 20 (24.9 g, 49%)
as a colourless solid; mp 43-45 °C; δH (CDCl ) 7.37-7.28 (m, 5
H), 5.44 (br s, 1 H), 5.11 (s, 2 H), 4.07-4.00 (m, 2 H), 3.49-3.42
(24)
A solution of sodium nitrite (9.95 g, 144 mmol) in water (27
mL) was added to a mixture of 23 (29.0 g, 131 mmol), water
(223 mL) and conc. HCl (65.7 mL, 657 mmol) at 0 °C. The
mixture was stirred for a further 10 min at 0 °C, then added to a
stirred mixture of ethyl 2-methylacetoacetate (19.8 g, 138 mmol),
sodium acetate (112 g, 1.37 mol), EtOH (163 mL), and ice (133
g, freshly added just before mixing) at 0 °C (bath temperature).
The final reaction mixture was stirred at 0 °C for 5 min, then at
3
4
(m, 2 H), 1.47 (s, 18 H); consistent with that reported. The
mother liquor was left at 5 °C to give a second crop (12.9 g,
20
2
5%).
2
0 °C for 1 h 40 min. Solid Na CO was added to give a pH of
2 3
Using imidazole and imidazole hydrochloride: To a stirred
approximately 7-8. The mixture was extracted with CH Cl (2 ×
3
then dried (Na SO ) and evaporated to give a red oil. To the oil
was added EtOH (60 mL) and HCl-saturated EtOH (100 mL).
The mixture was stirred at reflux for 30 min, then cooled and
evaporated under reduced pressure to give a dark solid. Water
2
2
solution of 19 (1.07 g, 5.48 mmol) in DMF (20 mL) under
nitrogen was added imidazole (0.75 g, 10.96 mmol) and
imidazole hydrochloride (1.15 g, 10.96 mmol). Di-tert-butyl
N,N-di-iso-propylphosphoramidite (95%, 2.73 mL, 8.20 mmol)
was added dropwise and the resulting mixture was stirred at room
00 mL) and the extracts were washed with cold water (400 mL),
2
4
temperature for 18 h. The mixture was cooled to 0 °C and H O2
2
(200 mL) was added and the aqueous mixture was basified at
(
30% aqueous, 1.17 mL, 14.96 mmol) was added dropwise. The
0
°C with 10% aqueous Na CO3 solution to pH of
a
2
mixture was stirred at 0 °C for 15 min, then allowed to warm to
room temperature and stirred for a further 3 h. The mixture was
cooled to 0 °C and aqueous Na SO (10%, 25 mL) was added
approximately 7-8. The resulting pale orange solid was filtered
off, washed with water (5 × 80 mL), and dried to give 24 as a
yellow solid (35.9 g, 86%); mp 130-132 °C; δ (CDCl ) 8.78 (br
2
3
H
3
slowly. Water was added to dissolve the precipitated solids and
the aqueous mixture was extracted with EtOAc (3 × 50 mL). The
organic fractions were washed with water (100 mL), dried
s, 1 H), 7.30 (d, J = 8.9 Hz, 1 H), 7.13 (dd, J = 2.1, 0.8 Hz, 1 H),
.08 (d, J = 2.4 Hz, 1 H), 7.01 (dd, J = 8.9, 2.4 Hz, 1 H), 4.40 (q,
J = 7.1 Hz, 2 H), 4.15 (t, J = 5.8 Hz, 2 H), 3.77-3.71 (m, 4 H),
7
(
Na SO ), and the solvent was removed under reduced pressure.
2 4
2
3
.83 (t, J = 5.7 Hz, 2 H), 2.63-2.57 (m, 4 H), 1.41 (t, J = 7.1 Hz,
A seed crystal was added to the colourless oil and the mixture
11
H); consistent with that reported. [Found: C, 63.82; H, 7.23;
was allowed to crystallise at ‒4 °C overnight, giving 20 as a
1
N, 8.91. C17H N O requires C, 64.14; H, 6.97; N, 8.80].
22 2 4
colourless solid (1.95 g, 92.0 %); H NMR (CDCl ) same as that
3
described above.
4.2.16. 5-(2-Morpholinoethoxy)-1H-indole-2-carboxylic acid
hydrochloride (9)
4
.2.12. 2-Aminoethyl di(tert-butyl) phosphate (8)
A solution of 20 (2.32 g, 5.99 mmol) in MeOH (100 mL) with
KOH (19.0 g, 339 mmol) was added to a mixture of ester 24
(35.9 g, 113 mmol), MeOH (250 mL) and water (125 mL) and
Pd/C (5%, 0.46 g) was hydrogenated at 40 psi for 2.5 h. The
mixture was filtered through Celite, washing with MeOH, and
the filtrate was evaporated to give 8 (1.52 g, 100%) as a
colourless oil; δH (CDCl ) 4.01-3.94 (m, 2 H), 2.97-2.91 (m, 2
H), 1.53 (br s, 2 H), 1.50 (s, 18 H); consistent with that reported.
the mixture was stirred at 20 °C for 1 h. The MeOH was
evaporated under reduced pressure at 40 °C (bath temperature).
The basic aqueous mixture was washed with ether (3 × 200 mL),
filtered, and the filtrate was acidified with conc. HCl at 0 °C to
pH 5-6. The precipitated solid was filtered off, washed with
water many times (until the washings were no longer red), and
dried in a vacuum oven at 50-60 °C for 7 h to give 5-(2-
morpholinoethoxy)-1H-indole-2-carboxylic acid (31.3 g, 96%) as
a pale yellow solid. Dioxane (300 mL) and HCl in dioxane (4M,
3
4
4
.2.13. 4-(2-(4-Nitrophenoxy)ethyl)morpholine (22)
To a stirred mixture of 4-nitrophenol (21) (42 g, 0.30 mol) and
-(2-chloroethyl)morpholine hydrochloride (56 g, 0.30 mol) in
4
anhydrous DMF (400 mL) was added dry K CO (104 g, 0.75
2
3
4
0.5 mL, 162 mmol) were added to this acid (31.3 g, 108 mmol)
mol). The mixture was stirred under nitrogen at 80-90 °C (bath
temperature) for 3 h, then at 20 °C overnight (15 h). The mixture
was poured into ice-water (1.5 L) and stirred at 0 °C for 1 h. The
precipitated solid was filtered off, washed with water (6 × 100
mL) until the washes were colourless, then dried to give 22 as a
pale yellow solid (68.4 g, 90%); mp 79-81 °C [lit. mp 82-83 °C
and the mixture was stirred at 20 °C for 15 h. The solid was
filtered off, washed with EtOAc several times, and dried to give
9
as a grey-white solid (32 g, 91%); mp 251-254 °C; δH
[
(CD ) SO] 12.87 (br s, 1 H), 11.65 (s, 1 H), 11.31 (br s, 1 H),
3 2
7
2
.37 (d, J = 8.9 Hz, 1 H), 7.20 (d, J = 2.4 Hz, 1 H), 7.01 (dd, J =
.0, 0.6 Hz, 1 H), 6.98 (dd, J = 8.9, 2.4 Hz, 1 H), 4.43 (t, J = 4.9
21
(EtOAc/hexane) ]; δ (CDCl ) 8.23-8.17 (m, 2 H), 6.99-6.94 (m,
H 3
Hz, 2 H), 4.03-3.80 (m, 4 H), 3.60-3.45 (m, 4 H), 3.30-3.12 (m, 2
H); consistent with that reported. [Found: C, 55.02; H, 5.89; N,
2
H), 4.20 (t, J = 5.7 Hz, 2 H), 3.76-3.71 (m, 4 H), 2.84 (t, J = 5.7
11
11
Hz, 2 H), 2.62-2.55 (m, 4 H); consistent with that reported.
8
.50. C H N O ꢀHCl requires C, 55.13; H, 5.86; N, 8.57].
[
Found: C, 57.36; H, 6.44; N, 11.25.C H N O requires C,
15 18 2 4
12
16
2
4
5
7.13; H, 6.39; N, 11.10].
.2.14. 4-(2-Morpholinoethoxy)aniline (23)
A solution of 22 (33.3 g, 132 mmol) in MeOH (170 mL) and
5
. Acknowledgements
4
The authors thank Sisira Kumara for HPLC analyses and Dr
Maruta Boyd and Dr Adrian Blaser for NMR spectrometry. This
work was supported by the Foundation for Research, Science and
Technology, the Health Research Council of New Zealand, and
the Auckland Division of the Cancer Society of New Zealand.
THF (50 mL) with Pd/C (5%, ca. 3 g) was hydrogenated at 40 psi
overnight (17 h). The mixture was filtered through Celite and the
Celite plug was washed with CH Cl :MeOH (5:1) several times.
2
2
The combined filtrates were evaporated to give 23 as a pink solid
29.0 g, 99%); δH (CDCl ) 6.77-6.72 (m, 2 H), 6.65-6.60 (m, 2
(
3
6. Notes and references
H), 4.04 (t, J = 5.8 Hz, 2 H), 3.76-3.70 (m, 4 H), 3.41 (br s, 2 H),
.76 (t, J = 5.8 Hz, 2 H), 2.60-2.54 (m, 4 H); consistent with that
2
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reported. [Found: C, 64.94; H, 8.08; N, 12.66. C H N O
requires C, 64.84; H, 8.16; N, 12.60].
12
18
2
2

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H NMR analysis showed that the second crop contained
about 5% of the starting material 19. However this
second crop can still be used to obtain clean product in
the subsequent reaction by proceeding as follows: the
crude product from hydrogenolysis [of the second crop
(
8)
9)
(11.7g)] was dissolved in CH Cl2 and washed
2
successively with aqueous Na CO and water, and then
2
3
(
dried (Na SO ) and evaporated to give pure 8 (6.13 g,
2 4
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Supplementary data associated with this article can be found
in the online version, at https://doi.org/10.1016/j.tet.xxxx.xx.xxx.
These data include H NMR spectra for all compounds reported
in this manuscript.
1
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Synthesis of N-(Tert-Butyloxycarbonyl)-CBI, CBI, CBI-