Preparation and properties of clickable amino analogues of the duocarmycins: Factors that affect the efficiency of their fluorescent labelling of DNA

Article abstract of DOI:10.1002/cmdc.201402169

Herein we report the synthesis of three DNA-alkylating amino analogues of the duocarmycins that carry an alkyne functional group suitable for copper-catalysed click chemistry. The alkyne-containing substituents are connected via a side chain position whic

Full text of DOI:10.1002/cmdc.201402169

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DOI: 10.1002/cmdc.201402169
Preparation and Properties of Clickable Amino Analogues
of the Duocarmycins: Factors That Affect the Efficiency of
Their Fluorescent Labelling of DNA
[
a]
Moana Tercel,* Sarah P. McManaway, H. D. Sarath Liyanage, and Frederik B. Pruijn
Herein we report the synthesis of three DNA-alkylating amino
analogues of the duocarmycins that carry an alkyne functional
group suitable for copper-catalysed click chemistry. The
alkyne-containing substituents are connected via a side chain
position which projects away from the minor groove, and have
only a small effect on DNA alkylation and cytotoxicity. The effi-
ciency of click reactions with fluorophore azides was studied
using alkylated ctDNA by analysing the adenine adducts pro-
duced after thermal depurination. Click reactions “on DNA”
were sensitive to steric effects (tether length to the alkyne)
and, surprisingly, to the nature of the fluorophore azide. With
the best combination of click partners and reagents, adducts
could be detected in the nuclei of treated cells by microscopy
or flow cytometry, provided that an appropriate detergent
(Triton X-100 and not Tween 20) was used for permeabilisation.
The method is sensitive enough to detect adducts at physio-
logically relevant concentrations, and could have application in
the development of nitro analogues of the duocarmycins as
hypoxia-activated anticancer prodrugs.
Introduction
Duocarmycin SA (1, Figure 1) is an example of a small group of
natural products that have attracted attention because of their
extreme cytotoxicity and their potential for development into
[
1]
antitumour agents. These compounds bind in the minor
groove of DNA at AT-rich sites and alkylate via their cyclopro-
pane ring at N3 of adenine at the base of the groove. This ring
can be formed in situ from a seco precursor, as in the CBI var-
iant of the alkylating subunit 2, which has been widely studied
because it is relatively easy to prepare and of similar toxicity to
[2]
the natural products. Our own interest in this area centres on
[
3]
amino analogues of the duocarmycins, for example aminoCBI
, which we have shown to share many of the properties ex-
hibited by the phenol-based analogues. These include very
3
[
4]
similar DNA sequence selectivity, toxicity (for the racemic
[
3]
form and individual enantiomers), and exclusive DNA alkyla-
[
5]
tion at N3 of adenine. The last was demonstrated by reaction
of 3 (in which R is the trimethoxyindole side chain common to
the duocarmycins) with calf thymus DNA (ctDNA) and subse-
quent thermal depurination, leading to the isolation, in practi-
cally quantitative yield, of adenine adduct 4.
We have further shown that aminoCBIs can be formed by
oxygen-sensitive bioreductive metabolism of a nitroCBI precur-
sor, which is substantially less cytotoxic, presumably because it
Figure 1. Structures of the natural product duocarmycin SA and the synthet-
ic alkylating subunits CBI and aminoCBI (both in the seco form); isolation of
the N3 adenine adduct of aminoCBI (where R=TMI) following thermal depu-
rination of alkylated DNA; structures of hypoxia-activated prodrugs nitroCBIs
5 and 7, and their corresponding aminoCBI toxic metabolites 6 and 8.
[
a] Dr. M. Tercel, S. P. McManaway, H. D. S. Liyanage, Dr. F. B. Pruijn
Auckland Cancer Society Research Centre
Faculty of Medical and Health Sciences
The University of Auckland
Private Bag 92019, Auckland 1142 (New Zealand)
Fax: (+64)9373-7502
E-mail: m.tercel@auckland.ac.nz
[6]
is unable to alkylate DNA. This opens the possibility of using
nitroCBIs as hypoxia-activated prodrugs, that is, to selectively
treat the therapy-resistant and highly malignant cell popula-
tion that exists at low oxygen concentrations as commonly
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402169.
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[
7]
found in solid tumours. High hypoxia-selective cytotoxicity of
nitroCBIs is achieved when the alkylating unit is substituted
with an electron-withdrawing sulfonamide or carboxamide
group, and the minor groove binding side chain contains
[
6a,b]
a basic substituent.
The particular example 5, when admin-
istered as a water-soluble phosphate pre-prodrug, was found
to selectively eliminate radiation-resistant cells in human
[
8]
tumour xenografts in mice. Substantial cell killing was ob-
served at nontoxic doses (in particular for the “natural” S enan-
[
9]
tiomer), whereas the corresponding aminoCBI 6 was tolerat-
ed only at much lower doses and, using systemic administra-
tion, was completely inactive against hypoxic tumour cells. No-
tably, in combination with fractionated radiation, 5 (as the
phosphate pre-prodrug) significantly prolonged the life of
[8]
tumour-bearing mice relative to radiation treatment alone.
To further develop nitroCBIs as hypoxia-activated prodrugs,
and to study their mechanism of action in cell culture and in
animal models, we considered that it would be very useful to
develop “clickable” analogues bearing a terminal alkyne func-
tional group. Ideally, the small alkyne group and tether would
not affect bioreductive metabolism or subsequent DNA alkyla-
tion, but would allow Cu-catalysed azide–alkyne cycloaddition
[10]
(CuAAC, click reaction) with, for example, fluorophore azides.
This could be used to establish the cellular or tissue localisa-
tion of aminoCBI adducts and their distribution relative to re-
gions of hypoxia. Some precedents exist for the fluorescent la-
belling of DNA using clickable compounds in cells: the use of
5-ethynyl-2-deoxyuridine (EdU) to detect DNA synthesis, where
the newly incorporated alkyne tag sits within the major
Scheme 1. Synthesis of clickable CBIs 18 and 19. Reagents and conditions:
[
11]
groove;
a platinum acridine anticancer agent bearing an
a) Boc
chloroethyl)morpholine hydrochloride, Cs
d) TFA, CHCl
2
O, DMAP, THF, RT, 18 h; b) H
2
, 10% Pd/C, MeOH, EtOAc, 17 h; c) 4-(2-
CO , acetone, reflux, 3 days;
reflux, 45 min!RT overnight; then NaHCO ; e) MnO , THF, RT,
h; f) DIAD, propargyl alcohol, PPh , THF, RT, 19 h; g) KOH, MeOH, H O, RT,
1 h; then HCl, dioxane; h) EDCI, TsOH, DMA, RT, 3.5 h; i) HCl, MeOH, RT,
.5 h; j) Zn, NH Cl, DMF, acetone, H O, RT, 30 min. Abbreviations: diisopropyl
azide tag which demonstrated mainly nuclear fluorescence
2
3
[
12]
after click reaction with a fluorophore alkyne;
and, most
3
3
2
1
2
1
3
2
closely related to the current work, three phenolic CBI alkynes,
where subcellular fluorescence distribution was related to the
4
2
[13]
presence or absence of a minor groove-binding side chain.
Here we investigate the synthesis and properties of clickable
azodicarboxylate (DIAD), dimethylacetamide (DMA), 4-dimethylaminopyri-
dine (DMAP), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDCI), tert-butyldimethylsilyl (TBDMS), trifluoroacetic acid (TFA), 4-toluene-
sulfonic acid (TsOH).
[
14]
nitro- and aminoCBIs based on 7 and 8, which differ from 5
and 6 only in the choice of the basic side chain substituent
(morpholine in place of dimethylamine). In all cases the alkyne
group is attached at the 7-position of the indole side chain,
chosen because this substituent projects away from the minor
Alkylation of this diphenol with one equivalent of (chloroe-
thyl)morpholine proceeded exclusively at the 5-position, pre-
sumably because of steric hindrance at the 7-OH group, giving
[
1]
groove and was thus considered least likely to affect DNA al-
kylation and subsequent cytotoxicity.
12 in 92% yield. Attempts to alkylate the 7-OH with propargyl
bromide gave mostly morpholine quaternisation, while Mitsu-
nobu reactions on 12 returned recovered starting material. In-
stead the Boc protecting group was removed with TFA, and
the indoline reoxidised to the indole 13, which proceeded in
good yield (65% over two steps) provided that the intermedi-
ate indoline was converted into the free base before the oxida-
tion step. This indole was then a suitable substrate for Mitsu-
nobu reaction with propargyl alcohol to give 14 in moderate
Results and Discussion
Synthesis of alkoxy-linked clickable CBIs 18 and 19
Our first choice for connecting an alkyne to the 7-position of
the indole side chain was via an ether linker, as found in the
duocarmycins and related natural products. A clickable amino-
CBI with a 7-propynyloxy indole substituent was prepared as
shown in Scheme 1. The known 5,7-dibenzyloxyindole carbox-
yield. The ester was hydrolysed, and acid 15 was coupled with
[15]
[14]
ylate 9, prepared as described via a Hemetsberger synthesis,
the previously described nitroCBI subunit 16.
Finally, the
was protected with Boc on the indole nitrogen atom. Hydroge-
nolysis removed both benzyl protecting groups and also unex-
pectedly reduced the indole to the corresponding indoline 11.
TBDMS protecting group was removed to give 18, and the
nitro group was reduced with Zn and NH Cl. Despite experi-
4
mentation, the aminoCBI 19 produced was always found to
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contain a small quantity of the corresponding alkene from par-
tial reduction of the terminal alkyne. This route proved superi-
or to alternative approaches in which the two alkoxy substitu-
ents were differentiated prior to indole ring formation. For ex-
ample, a free phenol was found incompatible with the nitrene
insertion step of the Hemetsberger synthesis, whereas a mono(-
benzyloxy)-mono(morpholinoethoxy) Hemetsberger substrate
gave an inseparable mixture of indole 5,7-isomers after cyclisa-
tion.
The cytotoxicity of the same compounds and their nitro pre-
cursors was measured by clonogenic assay in the human cervi-
cal carcinoma cell line SiHa after exposure for 4 h under oxic
or hypoxic conditions (Table 1). Rather than measuring inhibi-
tion of proliferation (as in an IC50 determination) this assay
measures the ability of the compounds to eliminate colony-
forming cells (clonogens) by counting the colonies produced,
relative to controls, 14 days after compound exposure. There
was no notable effect of the 7-propynyloxy substituent on the
toxicity of the aminoCBIs (19 vs. 8), or on the hypoxic selectivi-
ty of the nitroCBIs (18 vs. 7). Based on the C10 values (concen-
tration required to produce a clonogen surviving fraction of
Biological properties of 18 and 19
10%) both 18 and 7 are >400-fold more toxic to hypoxic than
The effect of the alkyne tag of 19 on DNA binding and alkyla-
tion was investigated by studying the reactions of 8 and 19
with ctDNA, in a similar manner to our previous characterisa-
tion of 4, and the formation of related adenine adducts (those
oxic SiHa cells. In other words, the 7-propynyloxy tag does not
appear to affect nitroCBI bioreductive metabolism, interaction
of the toxic aminoCBI metabolite with DNA, or downstream
biological consequences leading to cell death.
[
3e]
[16]
of aminoCI
and a phenolCBI dimer ). Both compounds
were incubated with a 250-fold base pair excess of ctDNA, and
samples were taken at intervals and the DNA precipitated.
Analysis of the supernatant by LC–MS (Figure 2) showed a grad-
ual loss of aminoCBI, matched by the appearance of the corre-
sponding adenine adducts 20 and 21, which were released
from the DNA after thermal depurination (for characterisation
of these products see Supporting Information Figures S1 and
S2). Clearly the presence of the 7-propynyloxy side chain had
no significant effect on the rate of DNA alkylation.
Table 1. Cytotoxicity of aminoCBIs and nitroCBIs in the human cervical
carcinoma cell line SiHa, as determined by clonogenic cell killing.
[a]
[b]
[c]
Oxic
Hypoxic
C10 [mm]
[
d]
[e]
[d]
[e]
C10 [mm]
C1 [mm]
C1 [mm]
aminoCBIs
8
[
f]
ND
ND
0.27
0.22
1.3
ND
ND
0.38
0.36
0.28
0.90
1
3
9
6
0.53
nitroCBIs
7
1
3
>100
>100
>100
>100
>100
>100
0.23
0.25
1.6
0.60
0.60
>4.0
8
5
[
a] Stirred cell suspensions were exposed to test compounds for 4 h
under the specified gas phase and then plated; after 14 days incubation,
the number of colonies relative to untreated cells was used to determine
the surviving fraction. [b] Incubated under 95% air and 5% CO
2
. [c] Incu-
bated in an anaerobic chamber (<0.001% O ). [d] Interpolated concentra-
2
tion for 10% surviving fraction. [e] Interpolated concentration for 1% sur-
viving fraction. [f] Not determined, less than the lowest concentration
tested.
Click reactions with 19 on ctDNA and in cells
Given the favourable biological properties of 19, we proceeded
to test its ability to fluorescently label intracellular targets by
click reaction. SiHa cells were grown on coverslips and treated
with various concentrations of 19. The cells were fixed and
permeabilised (using paraformaldehyde and Tween 20) and ex-
posed to a click cocktail containing CuSO , sodium ascorbate,
4
[17]
the ligand tris(benzyltriazolylmethyl)amine (TBTA), and Alexa
Fluor 488 azide (Figure 3). Confocal fluorescence microscopy
revealed selective labelling of cells treated with 19 rather than
Figure 2. Alkylation of ctDNA by 8 and 19 and formation of adenine adducts
2
0 and 21. Solutions of 8 (
250 mm in base pairs) in Tris buffer (pH 7.4) at 378C. At various times sam-
ples were taken, and the DNA was precipitated by the addition of CH CN.
&
) or 19 ( ) at 1 mm were incubated with ctDNA
&
(
8
(used as a measure of background staining), but the fluores-
3
cent signal was only detectable at relatively high concentra-
tions of 19 (3 mm exposure for 2 h) and faded after a few hours
to background levels (Figure S3a). Moreover, the signal was cy-
toplasmic (Figure 4a) rather than nuclear, and appeared to co-
localise with lysosomes (Figure S3b,c; co-localisation with mi-
tochondria was not observed, data not shown). These observa-
The amount of aminoCBI remaining (squares) was determined by LC–MS
analysis of the supernatant, and the amount of adenine adduct formed (cir-
cles) was determined after thermal depurination of the DNA pellet, releasing
2
0 (*) or 21 (*) into solution. A: area of identified peaks by HPLC analysis at
74 nm. See the Supporting Information for absorbance spectra and MS
characterisation. Lines are fitted curves for the loss of 19 (t =2.4 h) and for-
3
1
=
2
1
=
2
mation of 21 (t =2.9 h).
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adducts (see Figure 5a for a schematic representation of this
part of the experiment). Both methods suggested that very
little click reaction had occurred; there was no difference in
fluorescence of solutions derived from DNA treated with 19
relative to 8 (Figure S4a), and only traces, if any, of a clicked
adenine adduct were detected. These conclusions held true
when various other click reaction conditions were tried [for ex-
ample, longer reaction times, use of CuOAc, or of the tris(hy-
[17]
droxypropyltriazolylmethyl)amine
(THPTA) ligand]. To pre-
pare an authentic sample of the clicked adenine adduct, an al-
ternative approach was also investigated, that is, click first and
then alkylate DNA. This showed that the click reaction in solu-
tion proceeds without difficulty, giving ~40% reaction of 19
with Alexa Fluor 488 azide under the conditions used for the
click in cell experiments, and higher conversion by other meth-
ods (see Supporting Information). This experiment also
showed that: 1) the clicked product can alkylate ctDNA, giving
a 450-fold increase in the fluorescence of the DNA solution rel-
ative to a control in which 8 was mock-clicked (Figure S4b),
and 2) thermal depurination releases the expected product
Figure 3. Structures of fluorophore azides used in this work. The structure of
Alexa Fluor 647 azide is proprietary.
(
Figures S2 and S5) which is stable to heat treatment. The con-
clusion from these experiments is that the click reaction with
Alexa Fluor 488 azide does not proceed to any useful extent
once 19 has alkylated DNA.
Because samples of 19 pre-clicked with Alexa Fluor 488
azide were readily available we also treated permeabilised cells
with this reaction product and examined the cells by fluores-
cence microscopy (Figure 4b). Clear nuclear localisation of the
fluorescent signal was observed, which was specific to 19 and
not 8 (Figure S6). Although there are some differences be-
tween the experiments used to generate the images in Fig-
ure 4a and 4b (different click reaction conditions and confocal
versus wide-field fluorescence microscopy), the contrast be-
tween the two is dramatic. It seems reasonable to conclude
that the nuclei in Figure 4a are not fluorescent not because
the clickable tag is absent there, but—at least in part—be-
cause the click reaction with 19 is inefficient on DNA.
Figure 4. Cellular localisation of click signal from the reaction between 19
and Alexa Fluor 488 azide. a) SiHa cells grown on coverslips were exposed
to 19 (3 mm, 2 h) then fixed and permeabilised (Tween 20) and clicked with
4
Alexa Fluor 488 azide (10 mm) using CuSO /TBTA. Cells were imaged by con-
focal fluorescence microscopy at 60ꢁ magnification. b) SiHa cells grown on
coverslips were permeabilised (Tween 20) and exposed to the click product
from the reaction of 19 (1 mm) with Alexa Fluor 488 azide (20 mm) using
CuOAc/THPTA. Imaging was performed by wide-field fluorescence microsco-
py at 63ꢁ magnification. In both experiments nuclei were also visualised by
Hoechst counterstaining; see Figures S3 and S6.
Although much work has been described on click reactions
[18]
involving DNA, only a few reports address factors that affect
click efficiency. For EdU it is known that standard click condi-
tions (CuSO , sodium ascorbate) give incomplete reaction, as
4
a second incubation with a different fluorophore azide gives
[
11]
tions suggested that the fluorescent signal was associated
with the accumulation of weakly basic 19 in acidic lysosomes,
rather than representing the cellular site of alkylation.
further coincident labelling.
This effect was attributed to
steric hindrance, even though the alkyne tag is positioned in
the major groove. When the same alkyne-modified base was
incorporated into DNA at high density by PCR, click efficiency
was observed to be higher for single-stranded than double-
stranded product, also attributed to steric hindrance in the
One possible explanation for the failure to detect a nuclear
signal is that the click reaction may be inefficient once 19 has
alkylated DNA, perhaps due to steric hindrance around the
minor groove. To test the efficiency of the click reaction “on
DNA”, a further experiment using ctDNA was designed. The al-
kylation reaction with 19 was allowed to proceed to comple-
tion (overnight incubation, complete reaction established by
the method shown in Figure 2), then click reagents were
added. After 30 min the DNA was precipitated and washed,
and the extent of clicking determined by two methods: by
measuring the fluorescence of re-dissolved DNA samples, or by
thermal depurination and HPLC analysis of released adenine
[19]
major groove. Improved click efficiency was achieved by in-
creasing the fluorophore azide concentration, or lengthening
the tether to the alkyne by incorporating a hexynyl spacer. The
latter spacer has become widely used in DNA click applica-
[18]
tions, and, for example, enabled complete click reaction of
[20]
six consecutive modified bases in a 16-mer oligonucleotide.
These observations suggested to us that alternative ways of
tethering the alkyne tag to the indole 7-position should be in-
vestigated.
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which, although not used in cy-
totoxicity testing, was assessed
for click chemistry applications
(
see below).
Bromoindole 25 was also sub-
jected to Sonogashira reaction
with trimethylsilylocta-1,7-diyne
(
Scheme 3). After a similar se-
quence of reactions to those de-
scribed above (silyl group re-
moval, ester hydrolysis, amide
formation) TBDMS-protected ni-
troCBI 34 was produced. In this
case the TBDMS group was
cleaved using TFA instead of
HCl, using an alkaline work-up
to hydrolyse a small amount of
trifluoroacetate formed during
the cleavage step. Subsequent
reduction of the nitro group pro-
ceeded cleanly to give the ami-
noCBI 36. Clonogenic assay
(Table 1) showed that the longer
Figure 5. Determining the efficiency of the click reaction ‘on DNA’. a) Schematic representation (for ease of illus-
tration the alkylated CBI is drawn adjacent to rather than within the minor groove). The extent of click reaction
with a fluorophore azide (grey oval) is determined after thermal depurination to release soluble adenine adducts.
b) Illustrative chromatogram of depurination products; A: absorbance at 374 nm. In this example ctDNA was alky-
lated with 36 and clicked with TAMRA azide, using the standard conditions described below. Of the total adenine
adducts 51% had undergone click reaction with TAMRA azide (two isomers), based on relative peak areas. c) Com-
parison of click efficiency (E). Click reactions were conducted using the specified fluorophore azide (10 mm),
CuOAc (100 mm), THPTA (500 mm), and sodium ascorbate (5 mm) for 2 h at 378C in Tris buffer (pH 7.4) containing
alkyne
substituent
caused
10% DMF (nt: not tested, fl: <1%).
a small loss of potency for the
aminoCBI (36 is two- to fivefold
Synthesis of alkynyl-linked clickable CBIs
less toxic than 8), but the nitroCBI 35 retained good hypoxia-
selective activity.
For the preparation of clickable CBI analogues with various
tether lengths, we first considered Mitsunobu reaction of 13
with other alkyne-containing alcohols. However, reaction with
Relative efficiency of click reaction on ctDNA
4
-pentyn-1-ol, under the same conditions as used for the prep-
With three different aminoCBI alkynes in hand, we sought to
compare their relative click reaction efficiencies using the
ctDNA model. As it is known that different fluorophores and
aration of 14, was unsuccessful, returning only recovered 13.
Instead, we prepared bromoindole 25 and appended alkyne-
containing
[23]
7-substituents
by
Sonogashira
chemistry
fluorophore azides distribute quite differently within cells,
(
Schemes 2 and 3). The known compound 22 was prepared by
we also expanded our set of fluorophore azides to include
TAMRA azide (close analogues of which evenly distribute
throughout the cytosol and nucleus of unpermeabilised
nitration of 3-bromophenol, and alkylated with 4-(2-chloroe-
thyl)morpholine to give 23. Reduction of the nitro group gave
2
[23]
4 as a substrate for Fischer indolisation, which proceeded in
cells),
and the commercially available Azide Fluor 585
4
0% yield despite the reported propensity of anilines with
(Figure 3), which features a more lipophilic chromophore than
Alexa Fluor 488 azide, and a triethylene glycol tether to the
azide. To compare the various click reaction partners we set-
tled on standard click conditions using CuOAc and THPTA that
gave nearly quantitative click reaction in solution (see Fig-
ure S7 for example) and applied these to samples of ctDNA al-
kylated with each of the clickable aminoCBIs (Figure 5). In each
case the adenine adducts released after thermal depurination
were identified by HPLC or LC–MS analysis (Figures S2 and S8).
Overall, the click efficiency varied widely (Figure 5c), with gen-
erally better click reaction with a longer tether to the alkyne
(36>19>31), which presumably allows the alkyne to project
further from the minor groove. However, there was also an un-
expectedly strong dependence of click efficiency on the nature
of the fluorophore azide, with TAMRA azide being far superior
to the other two, even though in all cases the azide is at the
end of a long flexible side chain. For the worst-performing flu-
orophore azide (Alexa Fluor 488 azide) we also examined the
[21]
ortho substituents to undergo ‘abnormal’ Fischer reactions.
Sonogashira coupling with trimethylsilylacetylene, followed by
removal of the silyl group, gave 26, and the ester was hydro-
lysed to give acid 27. Subsequent steps to the targets 30 and
3
1 were complicated by the reactivity of the aryl acetylene; al-
though EDCI-mediated coupling with 16 gave 29 without
problem, cleavage of the TBDMS group with HCl caused com-
petitive addition of HCl to the alkyne. This problem was avoid-
[
8]
ed by using the free alcohol 28 in the coupling step, which,
in this case, proceeded in good yield. Reduction of the nitro
group was more problematic however, because even under
very mild conditions (considerably less forcing than those re-
[
22]
ported for aryl acetylene reduction by this method), compet-
itive alkyne and nitro group reduction was observed. The
cleanest product was obtained after exposure to Zn and NH Cl
4
at RT for 7 min (giving material containing 10% alkene, and
~
5% hydroxylamine from incomplete nitro group reduction)
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Scheme 3. Synthesis of clickable CBIs 35 and 36. Reagents and conditions:
a) trimethylsilylocta-1,7-diyne, CuI, Pd(PPh
b) TBAF, THF, RT, 8 min; c) KOH, EtOH, H O, 508C, 15 min; then HCl, dioxane;
d) 16, EDCI, TsOH, DMA, RT, 2 h; e) TFA, CH Cl , RT, 6 h; then aq Na CO
f) Zn, NH Cl, DMF, acetone, H O, RT, 20 min.
3 2 2 3 3
) Cl , Et N, CH CN, reflux, 2 h;
2
2
2
2
3
;
4
2
Scheme 2. Synthesis of clickable CBIs 30 and 31. Reagents and conditions:
a) 4-(2-chloroethyl)morpholine hydrochloride, K
b) Fe, HOAc, H O, EtOH, reflux, 30 min; c) NaNO
ethyl 2-methylacetoacetate, NaOAc, EtOH, 08C!RT 1.5 h; d) TsOH, toluene,
reflux, 25 min; e) trimethylsilylacetylene, CuI, Pd(PPh Cl , Et N, CH CN,
reflux, 2 h; f) TBAF, THF, RT, 15 min; g) KOH, EtOH, H O, reflux, 15 min; then
HCl, dioxane; h) EDCI, TsOH, DMA, RT, 80 min for 29 or 5.5 h for 30; i) HCl,
MeOH, RT, 23 h; j) Zn, NH Cl, DMF, acetone, H O, RT, 7 min. Abbreviations:
tetrabutylammonium fluoride (TBAF).
2
CO
3
, DMF, 558C!708C 3 h;
2
2
, HCl, 08C, 15 min; then
3
)
2
2
3
3
2
4
2
effect of increasing the azide concentration, and providing
a boost of fresh sodium ascorbate during the click reaction
(
Figure S9). With a 10-fold higher concentration of Alexa Fluor
88 azide (200 mm, that is, 20-fold excess over aminoCBI) the
efficiency of the click reaction with 36 on DNA increased from
–9% to 30–35%. Finally, using the best conditions for click re-
4
7
action on DNA (36, 200 mm fluorophore azide, ascorbate
boost) we revisited the microscopy experiment, but once
again observed cytoplasmic rather than nuclear staining. This
is shown for AlexaFluor 488 azide in Figure 6a, and similar re-
sults were also obtained for TAMRA azide, where the increased
fluorophore azide concentrations also led to high background
staining (data not shown).
Figure 6. Effect of permeabilisation agent on nuclear click signal. SiHa cells
grown on coverslips were exposed to 36 (1 mm, 20 h) then fixed and per-
meabilised using either a) Tween 20 or b) Triton X-100. The cells were clicked
with Alexa Fluor 488 azide [either a) 200 mm or b) 50 mm] using CuOAc/
THPTA. Imaging was performed by confocal fluorescence microscopy at 60ꢁ
magnification, and nuclei were visualised by Hoechst counterstaining.
Triton X-100, but under otherwise identical conditions (apart
from a lower Alexa Fluor 488 azide concentration of 50 mm),
a strong fluorescence signal was observed in the nucleus (Fig-
ure 6b). Clearly Tween 20 can permeabilise the cytoplasmic
membrane to click reagents (Figure 4a and 6a), and the nucle-
Effect of permeabilisation agent on nuclear click signal
After further experimentation we found that altering the deter-
gent used in the permeabilisation step had a profound effect
on the nuclear click signal. By switching from Tween 20 to
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ar membrane to pre-clicked aminoCBIs (Figure 4b, similar re-
sults were found for 19 pre-clicked with both TAMRA azide
and Azide Fluor 585, Figure S10), but Triton X-100 is required
to facilitate efficient click reaction within the nucleus of these
cells. We used a standard 1% Triton X-100, but similar results
were obtained with 0.5% and 0.2%. With any of these concen-
trations higher fluorescence was generally observed at the
edge of the nucleus (distinct from the granular staining ob-
served with Hoechst), suggesting that even with Triton X-100
nuclear penetration of some component of the click reaction is
still limiting.
(Figure S11). This value is quite similar to the click efficiency
found for ctDNA alkylated with 36 at 10 mm, which was 16%
at same fluorophore concentration (Figure S9). For Alexa Fluor
647 azide even cells exposed at 0.01 mm could be distinguish-
ed from blank cells (Figure 7a, median fluorescence ratio 2.2),
establishing this as the more sensitive fluorophore which was
used for subsequent experiments. Alternative conditions were
also explored (fixing with 70% EtOH either alone or in combi-
nation with Triton X-100, permeabilisation under high salt con-
ditions, or increased fluorophore azide concentrations up to
200 mm) without further improving the selectivity of labelling.
The three aminoCBI alkynes were then compared by flow cy-
tometry, confirming the superiority of 36 (Figure 7b). The most
sensitive click chemistry combination (i.e., 36 with Alexa Fluor
647 azide) was finally checked by microscopy (Figure S12)
which confirmed nuclear localisation of the fluorescent signal.
These same optimised conditions were used in our recent
Detergents for cell permeabilisation have been developed
primarily for immunocytochemistry applications, and Tween 20
and Triton X-100—both non-ionic detergents containing poly-
[24]
ethylene glycol side chains—are widely used in this context.
It is known that Tween 20 is generally less effective than Triton
X-100 at solubilising protein and lipid components of cell
[
25]
[16]
membranes.
When used in conjunction with alkynyl fatty
study to show nuclear localisation of a phenolic CBI alkyne.
acids as clickable reporter molecules, Tween 20 permeabilisa-
tion generated cells that were more fluorescent than those
treated with Triton X-100, which was interpreted as better sol-
ubilisation of tagged lipids by the latter detergent, allowing se-
Click detection following hypoxia-selective nitroCBI
activation
[26]
lective fluorescent detection of fatty-acylated proteins. The
precedents described above for fluorescent click labelling of
The ultimate goal of this work was to develop a click chemistry
method that could detect hypoxia-dependent labelling of cells
by nitroCBIs. This was investigated using 35 by both microsco-
py and flow cytometry methods. For microscopy experiments
[
11,12]
[13]
DNA in cells employed either Triton X-100
permeabilisation.
or saponin
Flow cytometry
The applicability of our opti-
mised click protocol to flow cy-
tometry was also tested, using
Alexa Fluor 488 azide, and also
Alexa Fluor 647 azide, which are
suitable for the 488 and 633 nm
laser lines commonly found on
flow cytometry instruments.
Single-cell suspensions were ex-
posed to various concentrations
of 36 for 20 h, and then harvest-
ed, fixed and permeabilised
(
(
Triton X-100), and clicked
CuOAc/THPTA). With Alexa Fluor
4
0
88 azide, cells exposed to
.1 mm 36 could be readily dis-
tinguished from blank cells (not
exposed to aminoCBI but
clicked, Figure 7a), while at 1 mm
36 the treated cells were ~30-
fold more fluorescent than blank
cells. Samples of cells treated
with 1 mm 36 were also subject-
Figure 7. Flow cytometry analysis of aminoCBI click labelling in cells. a) Sensitivity of detection with 36. SiHa cells
were exposed to 36 (0.01, 0.1, or 1 mm for 20 h) then fixed and permeabilised (Triton X-100) and clicked with
Alexa Fluor 488 azide (left) or Alexa Fluor 647 azide (right) (50 mm) using CuOAc/THPTA. b) Comparison of labelling
ed to thermal depurination and by three clickable aminoCBIs. SiHa cells were exposed to 31 (left), 19 (middle), or 36 (right) (0.1 and 1 mm for
2
0 h) then fixed and permeabilised (Triton X-100) and clicked with Alexa Fluor 647 azide (50 mm) using CuOAc/
HPLC analysis, as described for
the ctDNA work, and of the total
THPTA. Unstained cells (white) were not exposed to the click cocktail, and background samples (lightest grey)
were not exposed to aminoCBI but were clicked. The other histograms were treated as marked, with the number
representing the ratio of median fluorescence relative to the appropriate background comparison (for those ex-
identified
adenine
adducts
~
20% were found to be clicked amples where the ratio is >1.5ꢁ).
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Figure 8. Hypoxia-dependent click labelling of cells exposed to nitroCBI. SiHa cells were exposed to 35 (10 mm, 4 h) under hypoxic or oxic conditions, then
seeded onto coverslips. After overnight attachment the cells were fixed and permeabilised (Triton X-100) and clicked with Alexa Fluor 647 azide (50 mm)
using CuOAc/THPTA. Imaging was done by confocal fluorescence microscopy at 60ꢁ magnification. Blank cells were not exposed to nitroCBI, but were clicked
and imaged under the same conditions.
it was technically easier to expose single-cell suspensions to
5 under oxic and hypoxic conditions, and then seed the cells
onto coverslips, which were clicked and imaged the following
day. Even with an overnight delay between exposure and click
reaction, it was possible to distinguish hypoxic and oxic cells
azides. Overall, we conclude that care must be exercised in in-
terpreting sites of intracellular probe molecule localisation on
the basis of a click fluorescent readout, especially where multi-
ple diverse targets are possible, and even more so when some
of these targets may be within the nucleus.
3
(
Figure 8), although with a relatively high nitroCBI concentra-
In the context of reporter molecules for investigating nitro-
CBIs as hypoxia-activated anticancer agents, the method devel-
oped here shows considerable promise. With a suitable length
tether to the alkyne tag, as in nitroCBI 35 and aminoCBI 36,
only minimal impact on toxicity and hypoxic selectivity is ob-
served. The same tether permits click reactions in the nucleus
that are efficient enough to detect aminoCBI adducts after ex-
posure of cells to physiologically relevant concentrations of 36.
Importantly, the method can also distinguish cells that have
been exposed to 35 under oxic versus hypoxic conditions, as
demonstrated by both microscopy and flow cytometry experi-
ments. Nevertheless, the approach would be improved by a fur-
ther increase in sensitivity, particularly if it is to be used to
detect aminoCBI adducts in tumour samples or other tissues
from animal models.
tion (10 mm for 4 h, sixfold higher than hypoxic C10 value; see
Table 1). When the analysis was performed by flow cytometry
immediately after nitroCBI exposure and click reaction, hypoxic
and oxic cells could be differentiated using a lower concentra-
tion of 35 (1 mm for 2 h, Figure S13). The fluorescence ratio
under these conditions between hypoxic and oxic cells was
2
.6-fold, or 12.3-fold if background staining is subtracted.
Conclusions
An important conclusion from this work is that intracellular
CuAAC click reactions between alkyne-containing probe mole-
cules and fluorophore azides cannot always be assumed to
proceed with high efficiency. The click reaction is influenced by
steric effects, as previously reported for some click reactions
on DNA, and as found here with the adducts of three amino-
CBI alkynes. Surprisingly, however, the click reaction is also
strongly affected by the structure of the fluorophore azide.
Presumably these factors would be equally important if the
click partners were reversed, that is, in a reaction between an
azide-containing probe molecule and a fluorophore alkyne
click partner. If the probe molecule was bound to proteins or
other biomolecules besides DNA, it seems likely that the partic-
ular environment in the vicinity of the alkyne or azide tag
could affect click efficiency just as it does on DNA. A further
unexpected observation was that intranuclear click reactions
are highly sensitive to the detergent used for membrane per-
meabilisation, to the extent that nuclear staining can be com-
pletely absent (Tween 20) or the most dominant intracellular
signal (Triton X-100). The failure of Tween 20 to permeabilise
the nuclear membrane to click reagents was particularly sur-
prising, as this detergent allowed strong nuclear staining by
aminoCBI alkynes pre-clicked with the same fluorophore
Experimental Section
Synthesis
General: Solvents were distilled prior to use by common laborato-
ry methods. Petroleum ether refers to the fraction with a bp in the
range 40–608C. Organic solutions were dried over anhydrous
Na SO or MgSO . Thin-layer chromatography was carried out on
2
4
4
aluminum-backed silica gel plates (Merck 60 F254) with visualisation
of components by UV light (l 254 nm) or exposure to I . Column
2
chromatography was carried out on silica gel (Merck 230–
400 mesh) unless otherwise stated. Melting points were deter-
mined on an Electrothermal 2300 Melting Point Apparatus. NMR
spectra were obtained on a Bruker Avance 400 spectrometer.
Chemical shifts are listed in ppm referenced to SiMe as an internal
4
standard. HRMS was performed with a Bruker micrOTOF-QII mass
spectrometer. Combustion analyses were carried out in the Camp-
bell Microanalytical Laboratory, University of Otago, Dunedin, New
Zealand. The following abbreviations are used: DIAD=diisopropyl
azodicarboxylate, DMA=dimethylacetamide, DMAP=4-dimethyla-
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minopyridine,
mide hydrochloride,
EDCI=1-ethyl-3-(3-dimethylaminopropyl)carbodii-
TBAF=tetrabutylammonium fluoride,
Methyl-7-hydroxy-5-(2-morpholinoethoxy)-1H-indole-2-carboxyl-
ate (13): TFA (3.3 mL, 43 mmol) was added to a solution of 12
TBDMS=tert-butyldimethylsilyl, TFA=trifluoroacetic acid, TsOH=4-
(1.81 g, 4.28 mmol) in CHCl (10 mL) and the mixture was stirred at
3
toluenesulfonic acid.
reflux for 45 min and then allowed to cool to RT overnight. The
THF was evaporated and the residue was dissolved in EtOAc. This
1
-tert-Butyl-2-methyl-5,7-bis(benzyloxy)-1H-indole-1,2-dicarbox-
solution was washed with aq NaHCO and the aqueous layer was
3
ylate (10): A solution of methyl 5,7-bis(benzyloxy)-1H-indole-2-car-
extracted repeatedly with EtOAc (ꢁ13). The combined EtOAc layers
were dried and evaporated to give crude Boc-deprotected indoline
[15]
boxylate 9 (5.78 g, 14.9 mmol), di-tert-butyl dicarbonate (6.5 g,
0 mmol), and DMAP (91 mg, 0.75 mmol) in THF (100 mL) was
3
(
(
methyl-7-hydroxy-5-(2-morpholinoethoxy)indoline-2-carboxylate)
1.26 g, 91%).
stirred at RT for 18 h. The THF was evaporated at 308C and the re-
sidual oil was crystallised from EtOAc/petroleum ether to give 10
as a cream solid (6.89 g, 95%). A sample was recrystallised from
MnO (85% ‘activated’, 1.20 g, 11.7 mmol) was added to a solution
2
EtOAc/petroleum ether to give a white solid: mp: 123–1268C;
of this crude material (1.26 g, 3.91 mmol) and the mixture was
stirred at RT for 1 h. The mixture was filtered through Celite, wash-
ing with THF, and the filtrates were evaporated. The remaining
1
H NMR (400 MHz, CDCl ): d=7.45–7.28 (m, 10H), 7.10 (s, 1H), 6.69
3
(
d, J=2.0 Hz, 1H), 6.55 (d, J=2.0 Hz, 1H), 3.90 (s, 3H), 1.46 ppm (s,
9
7
H); Anal. calcd for C H NO : C 71.44, H 6.00, N 2.87, found: C
1.63, H 6.07; N, 2.89.
solid was triturated with CH
(0.71 g, 57%). The remaining CH
2
Cl
2
(100 mL) to give 13 as a tan solid
Cl solution was evaporated onto
29
29
6
2
2
silica gel and purified by column chromatography (EtOAc then
EtOAc/MeOH 50:1) to give more 13 (0.18 g, 14%). A sample was
1
-tert-Butyl-2-methyl-5,7-dihydroxyindoline-1,2-dicarboxylate
(11): Pd/C (10%, 0.22 g) was added to a solution of 10 (1.27 g,
recrystallised from MeOH as a light-brown solid: mp: 211–2148C;
2
.60 mmol) in EtOAc (40 mL) and MeOH (40 mL) and the mixture
1
H NMR (400 MHz, [D ]DMSO): d=11.25 (brs, 1H), 9.57 (brs, 1H),
6
was hydrogenated at 50 psi for 17 h. The suspension was filtered
through Celite, washing with EtOAc, and the combined filtrates
were evaporated to give 11 as a grey foam (0.81 g, 100%) suitable
for the next step. A sample was crystallised from EtOAc/petroleum
6
.99 (s, 1H), 6.59 (d, J=2.1 Hz, 1H), 6.29 (d, J=2.1 Hz, 1H), 4.02 (t,
J=5.8 Hz, 2H), 3.84 (s, 3H), 3.60–3.56 (m, 4H), 2.68 (t, J=5.8 Hz,
2
6
H), 2.50–2.45 ppm (m, 4H); Anal. calcd for C H N O : C 59.99, H
16 20 2 5
.29, N 8.75, found: C 60.01, H 6.39; N, 8.83.
1
ether to give a white solid: mp: 1438C (dec); H NMR (400 MHz,
[
D ]DMSO): d=11.03 (s, 1H), 9.19 (s, 1H), 6.11 (d, J=2.3 Hz, 1H),
6
The yield in the MnO
2
oxidation was considerably lower (32%) if
6
3
1
.06 (d, J=2.4 Hz, 1H), 4.91 (dd, J=11.5, 3.9 Hz, 1H), 3.71 (s, 3H),
.46 (dd, J=16.8, 11.5 Hz, 1H), 2.95 (dd, J=16.9, 4.1 Hz, 1H),
.43 ppm (s, 9H); Anal. calcd for C H NO : C 58.25, H 6.19, N 4.53,
conducted on the TFA salt rather than the free base following Boc
deprotection. Compound 13 appeared somewhat unstable to fur-
ther oxidation, with several impurities appearing in the mother
liquor during MeOH recrystallisation.
15
19
6
found: C 58.22, H 6.25; N, 4.50.
When the hydrogenation was conducted with 5% Pd/C for 1 h,
TLC analysis showed a mixture of 10, 11, and a component of in-
termediate polarity, while LRMS analysis showed peaks correspond-
ing to 10, 11, and an indoline-monobenzyl ether. This suggests it is
not possible to remove both benzyl ethers by this method without
concomitant reduction of the indole.
Methyl-5-(2-morpholinoethoxy)-7-(prop-2-yn-1-yloxy)-1H-indole-
2-carboxylate (14): DIAD (95%, 0.22 mL, 1.1 mmol) was added
slowly to a mixture of 13 (283 mg, 0.88 mmol), propargyl alcohol
(62 mL, 1.1 mmol), and PPh (278 mg, 1.1 mmol) in THF (6 mL),
3
causing slight warming and the dissolution of all suspended solid.
The solution was stirred at RT for 19 h and the solvent was evapo-
rated. The residue was dissolved in EtOAc and the solution was ex-
tracted with aq HCl (2n, ꢁ3). The combined extracts were basified
1
-tert-Butyl-2-methyl-7-hydroxy-5-(2-morpholinoethoxy)indo-
line-1,2-dicarboxylate (12): 4-(2-Chloroethyl)morpholine hydro-
with NH OH (to pH~8) and then extracted with EtOAc (ꢁ2). The
4
chloride (1.07 g, 5.75 mmol) and Cs CO (5.12 g, 15.7 mmol) were
2
3
extracts were dried and evaporated and the residue was purified
by column chromatography (EtOAc) to give 14 as a colourless oil
added to a solution of 11 (1.62 g, 5.24 mmol) in acetone (100 mL)
and the mixture was stirred at reflux for three days. The acetone
(175 mg, 55%). A sample was crystallised from EtOAc/petroleum
was evaporated and the residue was diluted with H O and extract-
2
ether at low temperature to give a very pale-yellow solid: mp: 60–
ed with EtOAc (ꢁ2). The extracts were washed with aq NaCl and
then dried and evaporated. Purification of the residue by column
1
6
2
38C; H NMR (400 MHz, CDCl ): d=8.97 (brs, 1H), 7.09 (d, J=
3
.3 Hz, 1H), 6.70 (d, J=1.6 Hz, 1H), 6.56 (d, J=2.0 Hz, 1H), 4.81 (d,
chromatography (EtOAc then EtOAc/MeOH 50:1) gave 12 as a very
J=2.4 Hz, 2H), 4.14 (t, J=5.7 Hz, 2H), 3.93 (s, 3H), 3.77–3.73 (m,
H), 2.83 (t, J=5.7 Hz, 2H), 2.62–2.57 ppm (m, 5H); Anal. calcd for
C H N O : C 63.68, H 6.19, N 7.82, found: C 63.40, H 6.35; N, 7.83.
1
pale-yellow oil (2.03 g, 92%): H NMR (400 MHz, [D ]DMSO): d=
6
4
1
1
1.14 (s, 1H, OH), 6.32 (d, J=2.4 Hz, 1H, H-4), 6.23 (d, J=2.5 Hz,
H, H-6), 4.94 (dd, J=11.5, 3.9 Hz, 1H, H-2), 3.99 (t, J=5.7 Hz, 2H,
OCH ), 3.71 (s, 3H, CO Me), 3.58–3.55 (m, 4H, OCH ), 3.51 (dd, J=
19
22
2
5
N-(2-((tert-Butyldimethylsilyl)oxy)ethyl)-1-(chloromethyl)-3-(5-(2-
morpholinoethoxy)-7-(prop-2-yn-1-yloxy)-1H-indole-2-carbonyl)-
5-nitro-2,3-dihydro-1H-benzo[e]indole-7-sulfonamide (17): A so-
2
2
2
1
7.0, 11.6 Hz, 1H, H-3), 2.99 (dd, J=17.0, 3.9 Hz, 1H, H-3), 2.63 (t,
J=5.8 Hz, 2H, NCH ), 2.46–2.42 (m, 4H, NCH ), 1.44 ppm (s, 9H,
2
2
1
3
Boc); C NMR (100 MHz, [D ]DMSO): d=171.5 (CO Me), 157.0 (C5),
lution of KOH (198 mg, 3.6 mmol) in H O (4 mL) was added to a so-
2
6
2
1
53.8 (Boc carbonyl), 144.3 (C7), 132.3 (C3a), 120.3 (C7a), 102.51
lution of 14 (172 mg, 0.48 mmol) in MeOH (5 mL) and the mixture
was stirred at RT for 21 h. The MeOH was evaporated and the
aqueous layer was washed with EtOAc. The aqueous layer was
acidified with HCl (2n) to pH~5 and then extracted with EtOAc (ꢁ
6). The combined extracts were dried and evaporated, and the resi-
due was mixed with HCl (4m in dioxane, 4 mL). Over several hours
a green colour developed and a solid separated. After 17 h the
solid was filtered off, washed with dioxane, and dried to give 5-(2-
morpholinoethoxy)-7-(prop-2-yn-1-yloxy)-1H-indole-2-carboxylic
acid hydrochloride (15) as a pale-green solid (102 mg, 56%);
and 102.34 (C4 and C6), 83.2 (tBu), 66.0 (OCH ), 65.6 (OCH ), 60.4
2
2
(
C2), 56.8 (NCH ), 53.4 (NCH ), 52.3 (OMe), 32.1 (C3), 27.5 ppm
2 2
+
(tBu); HRMS-FAB: m/z [M+H] calcd for C H N O : 423.21313,
21 31 2 7
found: 423.21303.
The NMR spectra were assigned on the basis of 1D and 2D experi-
ments (DEPT, HSQC, HMBC, COSY, and ROESY) which allowed un-
ambiguous assignment of the C5 phenol as the site of alkylation.
For example, ROESY cross-peaks were observed from OH to Boc,
and from OCH to both C4 and C6.
2
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1
H NMR (400 MHz, [D ]DMSO): d=12.76 (vbrs, 1H), 11.63 (s, 1H),
diagnostic peaks: d=6.16–6.06 (m, 1H), 5.62 (dq, J=17.2, 2.0 Hz,
1H), 5.29 (dq, J=10.6, 1.8 Hz, 1H).
6
1
(
3
(
0.67 (brs, 1H), 7.00 (d, J=2.2 Hz, 1H), 6.83 (d, J=1.7 Hz, 1H), 6.62
d, J=2.0 Hz, 1H), 4.92 (d, J=2.4 Hz, 2H), 4.43–4.35 (m, 2H), 4.02–
.95 (m, 2H), 3.85–3.75 (m, 2H), 3.62 (t, J=2.3 Hz, 1H), 3.56–3.48
m, 2H), 3.27–3.16 ppm (m, 4H).
3
-Bromo-4-nitrophenol (22): An initial attempt to nitrate 3-bromo-
phenol using KNO in H SO at 5–108C gave multiple products. In-
3
2
4
stead the nitration was conducted as follows: conc HNO (10.3 mL,
3
[14]
A mixture of 16 (112 mg, 0.22 mmol), 15 (102 mg, 0.27 mmol),
EDCI (172 mg, 0.88 mmol), and anhyd TsOH (24 mg, 0.13 mmol) in
DMA (3 mL) was stirred at RT for 3.5 h then cooled to 08C. Ice-cold
170 mmol) was cooled in an ice bath. NaNO (1.96 g, 28 mmol) was
2
added, followed by
a solution of 3-bromophenol (24.6 g,
142 mmol) in CH Cl2 (50 mL) added dropwise over 15 min while
2
aq NaHCO (diluted to 20% from saturated) was added, and after
maintaining the internal temperature <208C. The mixture was
3
a few min the resulting solid was filtered off, washed with cold
dilute aq NaHCO and H O and then dried. The solid was triturated
stirred in the ice bath for 25 min, then diluted with H O and ex-
2
tracted with CH Cl (ꢁ3). The combined extracts were washed with
3
2
2
2
with EtOAc to give 17 as a yellow solid (120 mg, 65%): mp: 211–
H O, dried, and evaporated, and the residue was purified by
2
1
2
8
1
138C; H NMR (400 MHz, [D ]DMSO): d=11.58 (s, 1H), 9.21 (s, 1H),
.86 (d, J=1.6 Hz, 1H), 8.43 (d, J=8.9 Hz, 1H), 8.02 (dd, J=8.9,
.7 Hz, 1H), 7.99 (t, J=5.7 Hz, 1H), 7.14 (d, J=2.1 Hz, 1H), 6.80 (d,
column chromatography (EtOAc/petroleum ether 1:10 then 1:5
then 2:3, repeating for mixed fractions) to give 5-bromo-2-nitro-
phenol as a yellow solid (6.93 g, 22%), recovered 3-bromophenol
6
1
J=1.8 Hz, 1H), 6.60 (d, J=1.9 Hz, 1H), 4.97 (d, J=2.3 Hz, 2H), 4.93
(4.33 g, 18%), and 22 as a yellow solid (11.97 g, 39%): H NMR as
[27]
(
(
t, J=10.1 Hz, 1H), 4.65–4.58 (m, 2H), 4.17–4.06 (m, 4H), 3.64–3.59
m, 5H), 3.56 (t, J=6.2 Hz, 2H), 2.93 (q, J=5.9 Hz, 2H), 2.73 (t, J=
reported. A sample was recrystallised from Et O/petroleum ether
2
[28]
as yellow–orange needles: mp: 129–1318C (lit. mp: 130–1318C).
5
ꢀ
.8 Hz, 2H), ~2.5 (4H obscured by DMSO peak), 0.79 (s, 9H),
0.05 ppm (s, 6H); Anal. calcd for C H ClN O SSi· = H O: C 56.07,
39 48 5 9 2 2
1
4-(2-(3-Bromo-4-nitrophenoxy)ethyl)morpholine (23): A mixture
of 22 (7.26 g, 33.3 mmol), 4-(2-chloroethyl)morpholine hydrochlo-
H 5.91, N 8.38, found: C 55.80, H 5.82; N, 8.28.
ride (6.20 g, 33.3 mmol), and K CO3 (13.8 g, 100 mmol) in DMF
2
1
7
1
-(Chloromethyl)-N-(2-hydroxyethyl)-3-(5-(2-morpholinoethoxy)-
-(prop-2-yn-1-yloxy)-1H-indole-2-carbonyl)-5-nitro-2,3-dihydro-
H-benzo[e]indole-7-sulfonamide hydrochloride (18): HCl (1.25m
(50 mL) was stirred at 558C for 2.5 h and then allowed to cool to
RT. The mixture was diluted with aq NaCl and extracted with
EtOAc (ꢁ3). The extracts were washed with aq NaCl, dried, and
evaporated, and the residue was purified by column chromatogra-
phy (EtOAc/petroleum ether 1:1 then 3:1 then EtOAc then EtOAc/
MeOH 50:1) to give 23 as a pale-yellow oil that crystallised on
in MeOH, 1 mL) was added to
a
solution of 17 (108 mg,
0
1
.13 mmol) in CH Cl (12 mL) and the mixture was stirred at RT for
.5 h. The precipitated solid was filtered off, washed with CH Cl ,
2
2
2
2
and dried to give 18 as a yellow–orange solid (94 mg, 96%): mp>
standing (9.68 g, 88%). A sample was recrystallised from MeOH:
1
1
3
1
8
2
008C; H NMR (400 MHz, [D ]DMSO): d=11.71 (s, 1H), 10.54 (brs,
mp: 81–828C; H NMR (400 MHz, CDCl ): d=7.97 (d, J=9.1 Hz, 1H),
6
3
H), 9.19 (s, 1H), 8.86 (d, J=1.7 Hz, 1H), 8.43 (d, J=8.9 Hz, 1H),
.03 (dd, J=8.9, 1.7 Hz, 1H), 7.93 (t, J=5.9 Hz, 1H), 7.14 (d, J=
.0 Hz, 1H), 6.90 (s, 1H), 6.68 (s, 1H), 4.98 (d, J=2.3 Hz, 2H), 4.92 (t,
7.25 (d, J=2.6 Hz, 1H), 6.92 (dd, J=9.1, 2.7 Hz, 1H), 4.17 (t, J=
5.6 Hz, 2H), 3.75–3.71 (m, 4H), 2.82 (t, J=5.6 Hz, 2H), 2.59–
+
79
2.50 ppm (m, 4H); HRMS-CI: m/z [M+H] calcd for C H BrN O :
12
16
2
4
81
J=10.6 Hz, 1H), 4.73–4.58 (m, 3H, 2H after D O exchange), 4.45–
331.02934, found: 331.02912; calcd for C H BrN O : 333.02730,
12 16 2 4
2
4
2
2
2
.39 (m, 2H), 4.17–4.07 (m, 2H), 4.04–3.97 (m, 2H), 3.85–3.75 (m,
H), 3.65 (t, J=2.3 Hz, 1H), 3.64–3.51 (m, 4H), 3.39 (t, J=6.2 Hz,
H), ~3.3 (2H obscured by water peak), 2.87 ppm (q, J=6.2 Hz,
H); Anal. calcd for C H ClN O S·HCl·H O: C 51.70, H 4.87, N 9.14,
found: 333.02797; Anal. calcd for C H BrN O : C 43.52, H 4.57, N
12 15 2 4
8.46, found: C 43.58, H 4.53; N, 8.40.
2
-Bromo-4-(2-morpholinoethoxy)aniline (24): H O (50 mL) then
2
33
34
5
9
2
HOAc (10 mL) and then Fe powder (4.22 g, 76 mmol, washed with
n HCl and then H O) were added to a stirred solution of 23
found: C 51.49, H 4.68; N, 9.01.
2
2
5
-Amino-1-(chloromethyl)-N-(2-hydroxyethyl)-3-(5-(2-morpholi-
(6.26 g, 18.9 mmol) in EtOH (70 mL) at reflux. The mixture was
stirred at reflux for a further 30 min and then allowed to cool to
noethoxy)-7-(prop-2-yn-1-yloxy)-1H-indole-2-carbonyl)-2,3-dihy-
dro-1H-benzo[e]indole-7-sulfonamide (19): solution of 18
47 mg, 0.063 mmol) in DMF (2 mL) and acetone (2 mL) was stirred
in a water bath. H O (2 mL), NH Cl (168 mg, 3.1 mmol), and Zn
A
RT. Conc NH OH (30 mL) was added and the resulting mixture was
4
(
filtered through Celite, washing with EtOAc. The filtrate was ex-
tracted with EtOAc (ꢁ3) and the combined extracts were washed
2
4
powder (123 mg, 1.9 mmol) were added. The yellow–green mixture
was stirred at RT for 30 min and then diluted with CH Cl and aq
with aq NaCl and then dried and evaporated to give 24 as a light-
1
brown oil (5.46 g, 96%): H NMR (400 MHz, CDCl ): d=7.03 (d, J=
2
2
3
Na CO (2%). The organic layer was separated and the aqueous
2.6 Hz, 1H), 6.75 (dd, J=8.7, 2.6 Hz, 1H), 6.70 (d, J=8.6 Hz, 1H),
4.02 (t, J=5.7 Hz, 2H), 3.79 (s, 2H), 3.75–3.71 (m, 4H), 2.75 (t, J=
2
3
layer was extracted with more CH Cl (ꢁ2). The combined extracts
2
2
+
were dried and evaporated to give a yellow glass. Trituration with
5.7 Hz, 2H), 2.58–2.54 ppm (m, 4H); HRMS-ESI: m/z [M] calcd for
1
79
EtOAc gave 19 as a pale-yellow solid (38 mg, 80%): H NMR
C H BrN O : 300.04734, found: 300.04650; calcd for
12
17
2
2
81
(
400 MHz, [D ]DMSO): d=11.37 (s, 1H), 8.53 (d, J=1.6 Hz, 1H), 7.92
C H BrN O : 302.04529, found: 302.04512.
12 17 2 2
6
(
7
(
(
4
d, J=8.9 Hz, 1H), 7.73 (dd, J=8.9, 1.7 Hz, 1H), 7.71 (s, 1H), 7.46–
.41 (m, 1H), 7.00 (d, J=1.9 Hz, 1H), 6.79 (d, J=1.8 Hz, 1H), 6.57
d, J=2.0 Hz, 1H), 6.28 (s, 2H), 4.96 (d, J=2.3 Hz, 2H), 4.74–4.66
m, 1H), 4.64 (t, J=5.6 Hz, 1H), 4.40 (dd, J=11.0, 1.7 Hz, 1H), 4.16–
.07 (m, 3H), 3.97 (dd, J=10.9, 3.1 Hz, 1H), 3.76 (dd, J=11.0,
.9 Hz, 1H), 3.63–3.58 (m, 5H), 3.39 (q, J=6.1 Hz, 2H), 2.87–2.81
Ethyl-7-bromo-5-(2-morpholinoethoxy)-1H-indole-2-carboxylate
25): A solution of 24 (5.85 g, 19.4 mmol) in H O (20 mL) and conc
(
2
HCl (7.7 mL) was cooled to 08C and a solution of NaNO (1.61 g,
2
2
3.3 mmol) in H O (10 mL) was added dropwise. The mixture was
2
stirred at 08C for 15 min and then was added slowly to a mixture
of ethyl 2-methylacetoacetate (90%, 3.36 mL, 21.3 mmol) and
NaOAc (15.9 g, 194 mmol) in EtOH (60 mL) at 08C. This mixture
was stirred at 08C for 5 min, then the ice bath was removed, and
stirring was continued for another 1.5 h. H O, CH Cl , and Na CO
3
7
(
m, 2H), 2.73 (t, J=5.7 Hz, 2H), ~2.5 ppm (4H obscured by DMSO
+
peak); HRMS-ESI: m/z [M+H] calcd for C H ClN O S: 682.2097,
found: 682.2070. H NMR and HPLC analysis showed this product
contained 10% of an impurity resulting from reduction of the
alkyne to the corresponding alkene: H NMR (400 MHz, [D ]DMSO)
33
37
5
7
1
2
2
2
2
1
(5 g) were added, causing all solid to dissolve and giving a slightly
6
alkaline aqueous phase (pH~8). The CH Cl2 layer was separated
2
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2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1
and the aqueous phase extracted again with CH Cl . The combined
C H N O ·HCl· = H O: C 56.75, H 5.60, N 7.79, found: C 56.40, H
17 18 2 4 2 2
2
2
extracts were washed with aq NaCl and then dried and evaporated
to give the crude hydrazone [ethyl-2-(2-(2-bromo-4-(2-morpholi-
noethoxy)phenyl)hydrazono)propanoate] as an orange–brown oil
5.38; N, 7.55.
N-(2-((tert-Butyldimethylsilyl)oxy)ethyl)-1-(chloromethyl)-3-(7-
ethynyl-5-(2-morpholinoethoxy)-1H-indole-2-carbonyl)-5-nitro-
(
8.63 g).
2
,3-dihydro-1H-benzo[e]indole-7-sulfonamide (29): A mixture of
A solution of TsOH·H O (14.8 g, 77.6 mmol) in toluene (120 mL) was
16 (114 mg, 0.23 mmol), 27 (96 mg, 0.28 mmol), EDCI (175 mg,
0.92 mmol), and anhyd TsOH (53 mg, 0.31 mmol) in DMA (1.5 mL)
was stirred at RT for 80 min then cooled to 08C. EtOAc and ice-
2
stirred at reflux under a Dean–Stark trap until no further water
azeotrope was collected. The dry solution was allowed to cool and
then added to the crude hydrazone. This mixture was stirred at
cold aq NaHCO (diluted to 20% from saturated) were added. The
3
reflux under a CaCl drying tube for 25 min, then cooled, and the
EtOAc layer was separated, dried, and evaporated at RT. The resi-
due was purified by column chromatography (EtOAc then EtOAc/
MeOH 20:1 then 10:1) and the product triturated with EtOAc to
2
toluene evaporated. The residue was mixed with H O and sufficient
2
Na CO (7 g) to give a slightly alkaline aqueous phase, and the mix-
2
3
1
ture was extracted with EtOAc (ꢁ2). The extracts were washed
with aq NaCl, dried and evaporated, and the residue was purified
by column chromatography (EtOAc then EtOAc/MeOH 100:1) to
give 29 as a yellow solid (113 mg, 62%): mp: 2028C (dec); H NMR
(400 MHz, [D ]DMSO): d=11.56 (s, 1H), 9.22 (s, 1H), 8.86 (d, J=
6
1.6 Hz, 1H), 8.44 (d, J=8.9 Hz, 1H), 8.02 (dd, J=8.9, 1.7 Hz, 1H),
8.00 (t, J=5.6 Hz, 1H), 7.32 (d, J=2.3 Hz, 1H), 7.21 (d, J=1.6 Hz,
1H), 7.09 (d, J=2.3 Hz, 1H), 4.96–4.89 (m, 1H), 4.65–4.58 (m, 2H),
4.47 (s, 1H), 4.16–4.06 (m, 4H), 3.62–3.58 (m, 4H), 3.55 (t, J=6.2 Hz,
2H), 2.96–2.90 (m, 2H), 2.72 (t, J=5.8 Hz, 2H), ~2.5 (4H obscured
by DMSO peak), 0.79 (s, 9H), ꢀ0.05 ppm (s, 6H); Anal. calcd for
C H ClN O SSi: C 57.31, H 5.82, N 8.79, found: C 57.18, H 5.99; N,
give an orange–brown oil (4.7 g). Crystallisation from EtOH gave
1
2
5 as a yellow solid (3.08 g, 40%): mp: 80–828C; H NMR
(
400 MHz, [D ]DMSO): d=11.66 (s, 1H), 7.22–7.19 (m, 2H), 7.16 (d,
6
J=1.8 Hz, 1H), 4.34 (q, J=7.1 Hz, 2H), 4.10 (t, J=5.8 Hz, 2H), 3.60–
.55 (m, 4H), 2.69 (t, J=5.7 Hz, 2H), 2.50–2.45 (m, 4H), 1.34 ppm (t,
J=7.1 Hz, 3H); Anal. calcd for C H BrN O : C 51.40, H 5.33, N
3
17
21
2
4
38 46
5
8
7
.05, found: C 51.63, H 5.41; N, 6.99.
8.70.
Ethyl-7-ethynyl-5-(2-morpholinoethoxy)-1H-indole-2-carboxylate
1-(Chloromethyl)-3-(7-ethynyl-5-(2-morpholinoethoxy)-1H-
indole-2-carbonyl)-N-(2-hydroxyethyl)-5-nitro-2,3-dihydro-1H-
benzo[e]indole-7-sulfonamide (30): From 29: Compound 29 was
treated with HCl in MeOH as described above for the preparation
(26): Trimethylsilylacetylene (1.51 mL, 10.7 mmol), CuI (68 mg,
0
.36 mmol), and Pd(PPh ) Cl (251 mg, 0.36 mmol) were added to
3
2
2
a suspension of 25 (2.84 g, 7.14 mmol) in CH CN (30 mL) and Et N
3
3
1
(
5 mL) under nitrogen, and the mixture was stirred at reflux for 2 h.
of 18. After 2.5 h workup and H NMR analysis of the product
The solvents were evaporated and the residue was diluted with aq
NaCl and extracted with EtOAc (ꢁ2). The extracts were washed
with aq NaCl and then dried and evaporated. The residue was puri-
fied by column chromatography (EtOAc) to give crude ethyl-5-(2-
morpholinoethoxy)-7-((trimethylsilyl)ethynyl)-1H-indole-2-carboxyl-
ate as a dark-green–brown solid (3.08 g).
showed incomplete TBDMS deprotection (~10% starting material
remaining), while treatment for a further 23 h gave a product with
a co-eluting 10% impurity resulting from addition of HCl to the
1
alkyne: H NMR (400 MHz, [D ]DMSO) diagnostic peaks: d=6.01 (d,
6
J=2.4 Hz, 1H), 5.90 ppm (d, J=2.4 Hz, 1H).
[8]
From 28: A mixture of 28 (82 mg, 0.21 mmol), 27 (104 mg,
0.29 mmol), EDCI (163 mg, 0.84 mmol), and anhyd TsOH (7.3 mg,
0.04 mmol) in DMA (2.5 mL) was stirred at RT for 5.5 h then cooled
TBAF (1m in THF, 8.6 mL, 8.6 mmol) and THF (30 mL) were added
to this crude product. The mixture was stirred at RT for 15 min and
then diluted with aq NaCl and extracted with EtOAc (ꢁ3). The ex-
tracts were washed with aq NaCl and then dried and evaporated.
The residue was purified by column chromatography (EtOAc/petro-
leum ether 1:1 then 2:1 then EtOAc alone) to give a yellow solid
to 08C. Ice-cold aq NaHCO (diluted to 20% from saturated) were
3
added, and after a few min the resulting solid was filtered off,
washed with more aq NaHCO and H O and dried to give 30 as
3
2
1
a yellow solid (143 mg, 99%): mp>2958C; H NMR (400 MHz,
(
1
1.96 g). Recrystallisation from EtOH gave 26 (1.74 g, 71%): mp:
[D ]DMSO): d=11.56 (s, 1H), 9.22 (s, 1H), 8.87 (d, J=1.6 Hz, 1H),
6
1
46–1498C; H NMR (400 MHz, [D ]DMSO): d=11.70 (s, 1H), 7.26
8.43 (d, J=8.9 Hz, 1H), 8.03 (dd, J=8.9, 1.7 Hz, 1H), 7.93 (t, J=
5.9 Hz, 1H), 7.32 (d, J=2.3 Hz, 1H), 7.21 (d, J=2.0 Hz, 1H), 7.09 (d,
J=2.3 Hz, 1H), 4.96–4.88 (m, 1H), 4.66 (t, J=5.6 Hz, 1H), 4.65–4.60
(m, 2H), 4.47 (s, 1H), 4.17–4.07 (m, 4H), 3.62–3.58 (m, 4H), 3.39 (q,
J=6.0 Hz, 2H), 2.88 (q, J=6.2 Hz, 2H), 2.72 (t, J=5.8 Hz, 2H),
~2.5 ppm (4H obscured by DMSO peak); Anal. calcd for
6
(
d, J=2.3 Hz, 1H), 7.11 (d, J=1.4 Hz, 1H), 7.06 (d, J=2.4 Hz, 1H),
4
3
.43 (s, 1H), 4.33 (q, J=7.1 Hz, 2H), 4.10 (t, J=5.8 Hz, 2H), 3.60–
.56 (m, 4H), 2.70 (t, J=5.8 Hz, 2H), 2.50–2.46 (m, 4H), 1.34 ppm (t,
J=7.1 Hz, 3H); Anal. calcd for C H N O : C 66.65, H 6.48, N 8.18,
19
22
2
4
found: C 66.58, H 6.51; N, 8.14.
-Ethynyl-5-(2-morpholinoethoxy)-1H-indole-2-carboxylic
3
C H ClN O S· = H O: C 55.25, H 4.85, N 10.07, found: C 55.13, H
32
32
5
8
4
2
7
acid
4
.80; N, 10.09.
hydrochloride (27): A solution of KOH (542 mg, 9.66 mmol) in H O
2
(
10 mL) was added to a suspension of 26 (674 mg, 1.97 mmol) in
5-Amino-1-(chloromethyl)-3-(7-ethynyl-5-(2-morpholinoethoxy)-
1H-indole-2-carbonyl)-N-(2-hydroxyethyl)-2,3-dihydro-1H-ben-
zo[e]indole-7-sulfonamide (31): H O (3 mL), NH Cl (302 mg,
EtOH (20 mL) and the mixture was stirred at reflux. All solid dis-
solved within 5 min, and after 15 min the solution was cooled and
the EtOH was evaporated. The aqueous remainder was neutralised
with HCl (2n, 4.83 mL) and the mixture was evaporated to dryness
to give a brown glass (0.58 g). Dioxane (10 mL) and HCl-saturated
dioxane (6 mL) were added and the mixture was stirred at RT for
2
4
5.5 mmol), and Zn powder (222 mg, 3.3 mmol) were added to
a stirred solution of 30 (77 mg, 0.11 mmol) in DMF (3 mL) and ace-
tone (3 mL), causing slight warming. The mixture was stirred (with-
out a cooling bath) for 7 min, then diluted with CH Cl and aq
2
2
2
h. The resulting solid was filtered off and dried to give 27 as
Na CO3 (2%). The organic layer was separated and the aqueous
2
1
a light-tan solid (574 mg, 83%): mp: 223–2258C; H NMR (400 MHz,
layer was extracted with more CH Cl (ꢁ2). The combined extracts
2
2
[
(
D ]DMSO): d=12.98 (brs, 1H), 11.65 (s, 1H), 10.49 (brs, 1H), 7.33
d, J=2.2 Hz, 1H), 7.15 (d, J=2.3 Hz, 1H), 7.10 (d, J=2.0 Hz, 1H),
were dried and evaporated and the residue was triturated with
EtOAc to give 31 as a yellow solid (42 mg, 57%): H NMR
6
1
4
2
.44 (s, 1H), 4.42–4.38 (m, 2H), 4.02–3.93 (m, 2H), 3.83–3.72 (m,
H), 3.57–3.48 (m, 2H), 3.30–3.15 ppm (m, 4H); Anal. calcd for
(400 MHz, [D ]DMSO): d=11.38 (s, 1H), 8.54 (d, J=1.6 Hz, 1H), 7.93
(d, J=8.9 Hz, 1H), 7.74 (dd, J=8.9, 1.7 Hz, 1H), 7.71 (s, 1H), 7.44 (t,
6
ꢀ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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FULL PAPERS
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J=6.0 Hz, 1H), 7.30 (d, J=2.3 Hz, 1H), 7.07 (d, J=1.9 Hz, 1H), 7.06
(m, 4H), 3.27–3.14 (m, 2H), 2.79 (t, J=2.6 Hz, 1H), 2.53 (t, J=
6.9 Hz, 2H), 2.24 (td, J=6.9, 2.4 Hz, 2H), 1.76–1.67 (m, 2H), 1.66–
(
d, J=2.4 Hz, 1H), 6.30 (s, 2H), 4.67 (dd, J=10.9, 9.0 Hz, 1H), 4.64
t, J=5.6 Hz, 1H), 4.45 (s, 1H), 4.38 (dd, J=11.0, 1.8 Hz, 1H), 4.16–
1
(
1.57 ppm (m, 2H); Anal. calcd for C H N O ·HCl· = H O: C 62.79, H
23
26
2
4
2
2
4
7
6
.09 (m, 3H), 3.98 (dd, J=11.0, 3.2 Hz, 1H), 3.77 (dd, J=11.0,
.7 Hz, 1H), 3.61–3.57 (m, 4H), 3.39 (q, J=6.1 Hz, 2H), 2.84 (q, J=
6.42, N 6.37, found: C 62.43, H 6.33; N, 6.11.
N-(2-((tert-Butyldimethylsilyl)oxy)ethyl)-1-(chloromethyl)-3-(5-(2-
morpholinoethoxy)-7-(octa-1,7-diyn-1-yl)-1H-indole-2-carbonyl)-
5-nitro-2,3-dihydro-1H-benzo[e]indole-7-sulfonamide (34): A mix-
ture of 16 (96 mg, 0.19 mmol), 33 (99 mg, 0.23 mmol), EDCI
.2 Hz, 2H), 2.71 (t, J=5.8 Hz, 2H), ~2.5 ppm (4H obscured by
+
DMSO peak); HRMS-ESI: m/z [M+H] calcd for C H ClN O S:
32
35
5
6
1
6
52.1991, found: 652.1992. H NMR and HPLC analysis showed this
product contained 10% of the corresponding alkene and 5% of
a product tentatively identified as the corresponding hydroxyla-
mine: H NMR (400 MHz, [D ]DMSO) diagnostic peaks for alkene:
d=7.50 (dd, J=17.4, 11.1 Hz, 1H), 5.96 (d, J=17.3 Hz, 1H),
(147 mg, 0.76 mmol), and anhyd TsOH (20 mg, 0.11 mmol) in DMA
1
(1.5 mL) was stirred at RT for 2 h then cooled to 08C. Ice-cold aq
NaHCO3 (diluted to 20% from saturated) was added and after
a few min the precipitated solid was filtered off, washed with more
aq NaHCO and H O, and dried. Trituration with EtOAc gave 34 as
6
5
.37 ppm (d, J=12.0 Hz, 1H); and for the hydroxylamine D O-ex-
2
3
2
changeable peaks at d=9.40 (s, 1H), 8.56 ppm (s, 1H).
1
a yellow solid (110 mg, 65%): mp: 192–1948C; H NMR (400 MHz,
When the reaction was conducted with water bath cooling for
[D ]DMSO): d=11.53 (s, 1H), 9.26 (s, 1H), 8.87 (d, J=1.6 Hz, 1H),
6
2
min the alkene was not observed but the crude product con-
tained 40% hydroxylamine, while under the same conditions for
0 min the hydroxylamine was not observed but the crude prod-
8.44 (d, J=8.9 Hz, 1H), 8.05–8.00 (m, 2H), 7.23 (d, J=2.2 Hz, 1H),
7.20 (d, J=2.1 Hz, 1H), 6.97 (d, J=2.3 Hz, 1H), 4.98–4.90 (m, 1H),
4.67–4.60 (m, 2H), 4.17–4.07 (m, 4H), 3.62–3.57 (m, 4H), 3.54 (t, J=
6.2 Hz, 2H), 2.92 (q, J=6.1 Hz, 2H), 2.78 (t, J=2.6 Hz, 1H), 2.71 (t,
J=5.7 Hz, 2H), 2.55 (t, J=7.0 Hz, 2H), ~2.5 (4H obscured by DMSO
peak), 2.25 (td, J=6.9, 2.6 Hz, 2H), 1.76–1.68 (m, 2H), 1.67–1.59 (m,
9
uct contained ~35% alkene.
Ethyl-5-(2-morpholinoethoxy)-7-(octa-1,7-diyn-1-yl)-1H-indole-2-
carboxylate (32): CuI (50 mg, 0.26 mmol), and Pd(PPh ) Cl
2
3
2
2
H), 0.78 (s, 9H), ꢀ0.05 ppm (s, 6H); Anal. calcd for
(
186 mg, 0.26 mmol) were added to a solution of 25 (1.05 g,
C H ClN O SSi: C 60.29, H 6.21, N 7.99, found: C 60.29, H 6.32; N,
[20]
44 54
5
8
2
.64 mmol) and trimethylsilylocta-1,7-diyne (0.80 g, 4.5 mmol) in
7
.71.
CH CN (10 mL) and Et N (5 mL) under nitrogen, and the mixture
3
3
was stirred at reflux for 2 h. The solvents were evaporated and the
residue was diluted with aq NaCl and extracted with EtOAc (ꢁ3).
The extracts were washed with aq NaCl (ꢁ3) and then dried and
evaporated. The residue was purified by column chromatography
1-(Chloromethyl)-N-(2-hydroxyethyl)-3-(5-(2-morpholinoethoxy)-
7-(octa-1,7-diyn-1-yl)-1H-indole-2-carbonyl)-5-nitro-2,3-dihydro-
1H-benzo[e]indole-7-sulfonamide (35): TFA (0.17 mL, 2.2 mmol)
was added to a solution of 34 (97 mg, 0.11 mmol) in CH Cl (3 mL)
2
2
(
EtOAc) to give crude ethyl-5-(2-morpholinoethoxy)-7-(8-(trimethyl-
silyl)octa-1,7-diyn-1-yl)-1H-indole-2-carboxylate as a dark-brown oil
1.3 g) contaminated with OPPh3.
and the solution was stirred at RT for 6 h and then evaporated to
dryness. The residue was treated with aq Na CO (2%) and extract-
ed with CH Cl (ꢁ3). The extracts were dried and evaporated, and
2 2
2
3
(
the residue was triturated with EtOAc to give 35 as a yellow solid
TBAF (1m in THF, 4.0 mL, 4.0 mmol) was added to a solution of this
crude product in THF (10 mL), and after 8 min the mixture was di-
luted with aq NaCl and extracted with EtOAc (ꢁ2). The extracts
were washed with aq NaCl and then dried and evaporated. The
residue was purified by column chromatography on silica (EtOAc)
and then on neutral alumina (EtOAc/petroleum ether 1:1) to give
1
(
64 mg, 78%): mp: 195–1998C (dec); H NMR (400 MHz, [D ]DMSO):
6
d=11.53 (s, 1H), 9.26 (s, 1H), 8.87 (d, J=1.7 Hz, 1H), 8.44 (d, J=
8.9 Hz, 1H), 8.03 (dd, J=8.9, 1.7 Hz, 1H), 7.95 (t, J=5.8 Hz, 1H),
7.23 (d, J=2.3 Hz, 1H), 7.21 (d, J=2.0 Hz, 1H), 6.97 (d, J=2.3 Hz,
1H), 4.97–4.90 (m, 1H), 4.70 (t, J=5.6 Hz, 1H), 4.66–4.60 (m, 2H),
4.18–4.08 (m, 4H), 3.62–3.57 (m, 4H), 3.39 (q, J=6.0 Hz, 2H), 2.87
a yellow oil. Crystallisation from EtOAc/petroleum ether gave 32 as
(q, J=5.9 Hz, 2H), 2.79 (t, J=2.7 Hz, 1H), 2.71 (t, J=5.8 Hz, 2H),
1
a
pale-yellow solid (736 mg, 66%): mp: 69–718C; H NMR
2
.55 (t, J=7.0 Hz, 2H), ~2.5 (4H obscured by DMSO peak), 2.25 (td,
(
400 MHz, [D ]DMSO): d=11.67 (s, 1H), 7.17 (d, J=2.3 Hz, 1H), 7.08
6
J=6.9, 2.7 Hz, 2H), 1.76–1.68 (m, 2H), 1.67–1.59 ppm (m, 2H);
(
d, J=2.0 Hz, 1H), 6.93 (d, J=2.4 Hz, 1H), 4.34 (q, J=7.1 Hz, 2H),
+
HRMS-ESI: m/z [M+H] calcd for C H ClN O S: 762.2359, found:
38
41
5
8
4
2
2
1
7
.03 (t, J=5.8 Hz, 2H), 3.60–3.55 (m, 4H), 2.78 (t, J=2.7 Hz, 1H),
.68 (t, J=5.8 Hz, 2H), 2.53 (t, J=6.9 Hz, 2H), 2.50–2.45 (m, 4H),
.24 (td, J=6.9, 2.7 Hz, 2H), 1.75–1.67 (m, 2H), 1.66–1.58 (m, 2H),
.34 ppm (t, J=7.1 Hz, 3H); Anal. calcd for C H N O : C 71.07, H
+
7
7
62.2345; [M+Na] calcd for C H ClN NaO S: 784.2178, found:
38 40 5 8
84.2150.
5-Amino-1-(chloromethyl)-N-(2-hydroxyethyl)-3-(5-(2-morpholi-
noethoxy)-7-(octa-1,7-diyn-1-yl)-1H-indole-2-carbonyl)-2,3-dihy-
25
30
2
4
.16, N 6.63, found: C 70.82, H 7.38; N, 6.56.
dro-1H-benzo[e]indole-7-sulfonamide (36):
39 mg, 0.051 mmol) in DMF (1.5 mL) and acetone (1.5 mL) was
stirred in a water bath. H O (1.5 mL), NH Cl (137 mg, 2.6 mmol),
A solution of 35
5
-(2-Morpholinoethoxy)-7-(octa-1,7-diyn-1-yl)-1H-indole-2-car-
(
boxylic acid hydrochloride (33): A solution of KOH (512 mg,
.1 mmol) in H O (10 mL) was added to a suspension of 32
2
4
9
2
and Zn powder (101 mg, 1.5 mmol) were added and the mixture
was stirred for 20 min. The mixture was diluted with aq Na CO
(
590 mg, 1.40 mmol) in EtOH (15 mL) and the mixture was stirred
2
3
at 508C. All solid dissolved and after 15 min the EtOH was evapo-
rated and the aqueous remainder was neutralised with HCl (2n,
(
2%) and extracted with CH Cl2 (ꢁ3), and the extracts were
2
washed with aq NaCl and then dried and evaporated. The residue
was purified by column chromatography (EtOAc then EtOAc/MeOH
4
.56 mL), causing a yellow oil to separate. The supernatant was re-
moved, and the residue was dissolved in dioxane (6 mL) and treat-
ed with HCl-saturated dioxane (3 mL). Petroleum ether (5 mL) was
added, causing an oil to separate, which solidified on standing at
5
3
0:1 then 20:1) and the product was triturated with EtOAc to give
6 as a yellow solid (24 mg, 64%): mp: 196–2008C (dec); H NMR
1
(
400 MHz, [D ]DMSO): d=11.35 (d, J=1.4 Hz, 1H), 8.54 (d, J=
6
4
8C for 16 h. The solid was filtered off and dried to give 33 as
1
1
6
.5 Hz, 1H), 7.93 (d, J=8.9 Hz, 1H), 7.76–7.71 (m, 1H), 7.48 (t, J=
.0 Hz, 1H), 7.20 (d, J=2.2 Hz, 1H), 7.06 (d, J=2.0 Hz, 1H), 6.94 (d,
a white solid (524 mg, 87%): mp: 186–1888C; H NMR (400 MHz,
[
(
D ]DMSO): d=12.99 (brs, 1H), 11.64 (s, 1H), 10.79 (brs, 1H), 7.23
d, J=2.2 Hz, 1H), 7.07 (d, J=2.0 Hz, 1H), 7.03 (d, J=2.3 Hz, 1H),
6
J=2.3 Hz, 1H), 6.33 (s, 2H), 4.73–4.66 (m, 2H), 4.39 (dd, J=11.0,
.5 Hz, 1H), 4.16–4.09 (m, 3H), 3.98 (dd, J=10.9, 3.0 Hz, 1H), 3.77
1
4
.43–4.37 (m, 2H), 4.01–3.92 (m, 2H), 3.85–3.75 (m, 2H), 3.59–3.46
(dd, J=11.0, 7.6 Hz, 1H), 3.61–3.56 (m, 4H), 3.42–3.35 (m, 2H), 2.83
ꢀ
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(
2
q, J=6.2 Hz, 2H), 2.78 (t, J=2.6 Hz, 1H), 2.70 (t, J=5.7 Hz, 2H),
.54 (t, J=7.0 Hz, 2H), ~2.5 (4H obscured by DMSO peak), 2.24 (td,
ments clicking 19 with Alexa Fluor 488 azide using TBTA and
CuSO4 see Supporting Information): fluorophore azide (10 mm),
CuOAc (100 mm), THPTA (500 mm), sodium ascorbate (5 mm), 378C
in Tris buffer (pH 7.4) containing 10% DMF. After 1 h fresh sodium
ascorbate (5 mm) was added. After 2 h the DNA was precipitated
by the addition of ice-cold EtOH (six volumes) and after centrifuga-
tion (13000 rpm, 10 min) the supernatant was separated. The EtOH
wash step was repeated twice more, and the pellet was resuspend-
ed in Tris buffer and depurinated as described above. The adenine
adducts produced were analysed by LC–MS as described above, or
by HPLC, which was performed using the same conditions apart
from a more concentrated buffer for solvent B (450 mm ammoni-
um formate, pH 4.5). Gradient elutions starting with 10–15% sol-
vent A and changing to 40–90% solvent A over 10–16 min, com-
J=6.9, 2.6 Hz, 2H), 1.76–1.68 (m, 2H), 1.67–1.59 ppm (m, 2H);
Anal. calcd for C H ClN O S: C 62.33, H 5.78, N 9.56, found: C
6
3
8
42
5
6
2.21, H 5.68; N, 9.36.
Alkylation of ctDNA
Solutions of 8, 19, 31, or 36 (1 mm) in 90% Tris buffer (10 mm,
pH 7.4) and 10% DMF containing calf thymus DNA (ctDNA, Sigma,
2
volume: 1–4 mL). For time course experiments using 8 and 19,
samples (300 mL) were taken at 30 min or 1 h intervals up till 6 h
(
50 mm in base pairs) were incubated at 378C (total incubation
and optionally at 24 h) and the DNA was precipitated by the addi-
ꢀ
1
bined with flow rates of 0.3–0.4 mLmin , were sufficient to resolve
the peaks of interest.
tion of six volumes of ice-cold CH CN. The mixture was centrifuged
3
(
13000 rpm, 10 min, 08C) and the supernatant was separated (’su-
pernatant 1’). The DNA pellet was resuspended in Tris buffer
150 mL), heated (1 h, 908C), cooled, and diluted with ice-cold
CH CN (900 mL). The mixture was centrifuged as above to generate
Authentic adduct was made (by click first then alkylate methods)
for 19 with Azide Fluor 585 and TAMRA azide, and for 31 with
Azide Fluor 585. All adenine adduct click products were character-
ised by UV/Vis absorbance, and in some cases by MS (see Support-
ing Information).
(
3
‘
supernatant 2’. Both supernatants were evaporated to dryness and
reconstituted in mobile phase (100 mL of 12% CH CN and 88% am-
3
monium formate buffer, 45 mm, pH 4.5). After centrifuging
5
Microscopy: SiHa cells (50 mL, 1.5ꢁ10 cells per mL) were allowed
(
13000 rpm, 5 min) aliquots (40 mL) were analysed by LC–MS using
to attach to sterile glass coverslips for 2 h at 378C and then incu-
bated in aMEM (containing 5% heat-inactivated fetal calf serum,
2 mL) overnight. The cells were incubated with aminoCBI using the
specified concentrations and times (0.01–3 mm; 2–22 h) then
washed with fresh medium (ꢁ3), fixed (4% paraformaldehyde in
PBS, 15 min, RT), permeabilised (0.3% Tween 20 or 1% Triton X-
a Hewlett Packard series 1100 HPLC coupled with an Agilent 6150
Quadrupole LC–MS under the following conditions: Alltima C
8
ꢀ1
5
mm 2.1ꢁ150 mm column; flow rate 0.3 mLmin ; gradient elu-
tion from 10% solvent A (4:1 CH CN/H O) and 90% solvent B
3
2
(
1
45 mm ammonium formate buffer, pH 4.5) to 60% solvent A over
0 min, then to 80% solvent A over 2 min, and a return over 4 min
1
00 in PBS, 30 min, RT), and washed with PBS (ꢁ3). Cells were ex-
to the starting conditions; electrospray ionisation in positive mode
with m/z scan from 100–1500 amu at a fragmentor voltage of 75 V.
Analysis of supernatants 1 and 2 showed clean conversion from
aminoCBI to adenine adduct, and the absence of hydrolysis prod-
ucts. Absorbance spectra and mass spectra of identified compo-
nents from the alkylation reactions are shown in the Supporting In-
formation.
posed to click cocktail A or B (100 or 500 mL in 10 mm Tris-saline,
pH 7.4) under the following conditions; cocktail A (for Figure 4a
and S3): Alexa Fluor 488 azide (10 mm), CuSO (1 mm), TBTA (1 mm),
4
sodium ascorbate (100 mm) for 30 min at RT; cocktail B (for
Figure 6, 8, and S12): Alexa Fluor 488 azide or Alexa Fluor 647
azide (50 or 200 mm as specified), CuOAc (1 mm), THPTA (5 mm),
sodium ascorbate (100 mm) for 2 h at RT, with addition of a second
portion of sodium ascorbate after 1 h. The click reaction and sub-
sequent steps were conducted while minimising exposure to light.
Cells were washed with PBS (ꢁ3) and treated with anti-fade
mounting medium (Citifluor). For nuclear counterstaining cells
were also exposed to Hoechst 33342 (10 mm in PBS, 15 min, RT)
after the click reaction. For lysosomal or mitochondrial staining
(Figure S3) cells were exposed to Lysotracker Red (50 nm in serum-
free media, 1 h, 378C) or Mitotracker Red (200 nm in media con-
taining 5% FCS, 15 min, 378C) before fixation. For imaging with
In vitro cell culture and cytotoxicity
Cell culture and clonogenic survival assays with SiHa cells were
[9]
performed as previously described. Hypoxic and oxic conditions
refer to the following: hypoxic (95% N , 5% CO ); oxic (95% air,
2
2
5
% CO ), except for Figure 8 and Figure S13 (95% O , 5% CO ).
2 2 2
Click reactions
Click reaction components: Alexa Fluor 488 azide and Alexa Fluor
1
9-preclicked with Alexa Fluor 488 azide (Figure 4b and S6) the
click product was prepared as described in the Supporting Infor-
mation, and incubation (378C, 22 h) was conducted after permea-
bilisation of cells (Tween 20). For imaging with the nitroCBI
6
47 azide were obtained from Life Technologies, Azide Fluor 585
from Click Chemistry Tools, and TAMRA azide was prepared from 5-
and 6)-carboxytetramethylrhodamine succinimidyl esters (see Sup-
(Figure 8) single cell suspensions were exposed to 35 (10 mm, 4 h,
(
oxic or hypoxic conditions) prior to seeding on coverslips, which
were left overnight before fixation, permeabilisation, and clicking.
porting Information). The pure 5-isomer of TAMRA azide is com-
mercially available (Life Technologies). The following stock solu-
tions were used: fluorophore azides (2 mm in DMSO), CuSO and
Imaging was performed using an Olympus FV1000 confocal micro-
scope with a 60ꢁ oil immersion objective, and the following set-
tings: Alexa Fluor 488 (excitation 473 nm, emission band filter 485/
100); TAMRA azide (excitation 559 nm, emission band filter 570/
100); Azide Fluor 585 (excitation 559 nm, emission band filter 570/
100); Alexa Fluor 647 (excitation 635 nm, emission band filter 650/
4
CuOAc (100 mm in H O), TBTA and THPTA (200 mm in DMSO),
2
sodium ascorbate (2.5m in H O). CuOAc and sodium ascorbate
2
stocks were prepared immediately prior to use, and the ascorbate
solution was added last and immediately before addition of the
click cocktail to DNA or cell suspensions.
1
00); Hoechst (excitation 405 nm, emission band filter 425/50); Ly-
Click ‘on DNA’: ctDNA was alkylated with aminoCBIs as described
above, except that 10 mm aminoCBI was used and the incubations
were conducted for 24 h. The resuspended DNA pellet was clicked
under the following standard conditions (for preliminary experi-
sotracker Red (excitation 559 nm, emission band filter 625/100); Mi-
totracker Red (excitation 559 nm, emission band filter 625/100), or
a Zeiss Axioplan2 wide-field fluorescence microscope with a 63ꢁ
ꢀ
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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oil immersion objective, and the following settings: Alexa Fluor
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88 (excitation 472/30 nm, emission 520/35 nm); TAMRA azide (ex-
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1 nm, emission 447/60 nm).
[
[
1
Flow cytometry: SiHa cells were exposed under the specified con-
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2657.
(
Figure S13, 1 mm, 2 h, oxic or hypoxic conditions). Cells were
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Received: May 1, 2014
Published online on July 17, 2014
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2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 2193 – 2206 2206