Rapid cascade synthesis of poly-heterocyclic architectures from indigo

Article abstract of DOI:10.1021/jo401210r

The base-induced propargylation of the dye indigo results in the rapid and unprecedented one-pot synthesis of highly functionalized representatives of the pyrazino[1,2-a:4,3-a′]diindole, pyrido[1,2-a:3,4-b′]diindole and benzo[b]indolo[1,2-h]naphthyridine heterocyclic systems, with the last two reflecting the core skeleton of the anticancer/antiplasmodial marine natural products fascaplysin and homofascaplysins and a ring B-homologue, respectively. The polycyclic compounds 6-8, whose structures were confirmed through single-crystal X-ray crystallographic analysis, arise from sequential inter/intramolecular substitution-addition reactions, and in some cases, ring rearrangement reactions. Preliminary studies on controlling the reaction path selectivity, and the potential reaction mechanisms, are also described. Initial biological activity studies with these new heterocyclic derivatives indicated promising in vitro antiplasmodial activity as well as good anticancer activity. The chemistry described is new for the indigo moiety and cascade reactions from this readily available and cheap starting material should be more broadly applicable in the synthesis of additional new heterocyclic systems difficult to access by other means. Published 2013 by the American Chemical Society.

Full text of DOI:10.1021/jo401210r

Article
pubs.acs.org/joc
Rapid Cascade Synthesis of Poly-Heterocyclic Architectures from
Indigo
Alireza Shakoori,† John B. Bremner,† Anthony C. Willis,‡ Rachada Haritakun,§ and Paul A. Keller*,†
†School of Chemistry, University of Wollongong, Wollongong, NSW, 2522, Australia
‡School of Chemistry, The Australian National University, Canberra, ACT 0200, Australia
§National Centre for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency
(NSTDA), 113 Phaholyothin Road, Klong1, Klong Luang, Pathumanthani 12120 Thailand
S
* Supporting Information
ABSTRACT: The base-induced propargylation of the dye indigo
results in the rapid and unprecedented one-pot synthesis of highly
functionalized representatives of the pyrazino[1,2-a:4,3-a′]-
diindole, pyrido[1,2-a:3,4-b′]diindole and benzo[b]indolo[1,2-h]-
naphthyridine heterocyclic systems, with the last two reflecting the
core skeleton of the anticancer/antiplasmodial marine natural
products fascaplysin and homofascaplysins and a ring B-
homologue, respectively. The polycyclic compounds 6−8, whose
structures were confirmed through single-crystal X-ray crystallo-
graphic analysis, arise from sequential inter/intramolecular substitution−addition reactions, and in some cases, ring
rearrangement reactions. Preliminary studies on controlling the reaction path selectivity, and the potential reaction mechanisms,
are also described. Initial biological activity studies with these new heterocyclic derivatives indicated promising in vitro
antiplasmodial activity as well as good anticancer activity. The chemistry described is new for the indigo moiety and cascade
reactions from this readily available and cheap starting material should be more broadly applicable in the synthesis of additional
new heterocyclic systems difficult to access by other means.
Scheme 1. Allylation of Indigo
INTRODUCTION
■
One of the current goals in organic synthesis is the controlled
construction of complex molecules, in particular, through the
use of cascade reaction sequences.1 Such molecular explora-
tions, yielding novel architectures, are of particular interest for
the investigation of new bioactive agents with possible new
modes of action, which could be subsequently elaborated in
medicinal chemistry programs.2 Approaches to the realization
of these synthetic goals have often been explored in the context
of complex multistep syntheses of natural product targets.3 Our
focus has been on the smaller, though versatile, canvas, of the
abundant and cheap natural product, indigo 1 (Scheme 1).
There is significant advantage in starting with a readily
available advanced precursor such as indigo: its reported
chemistry is very limited despite the presence of an array of
closely positioned functionality in the 2,2′-diindolic unit which
allows for cascade reaction paths. It also provides a unique
opportunity in providing an advanced starting material for the
potential rapid synthesis of the diindolic system of natural
products analogs. Such biologically active natural products (e.g.,
with anticancer or antimicrobial activity) include fascaplysin,4
homofascaplysin,5 iheyamines,6 staurosporine,7 and rebeccamy-
cin8 (Figure 1).
compounds represent new ring systems with functionality
suitable for further elaboration of molecular complexity.
In an extension of this work and in an investigation of
reaction directing elements, particularly those modulating
nucleophilic−electrophilic reactivities, we have studied the
base-induced reactions of indigo with the simple alkyne analog,
To explore our synthetic approach, we initially investigated
the base-mediated reactions of indigo with allylic halides, which
in the case of allylic bromide resulted in the rapid synthesis of
the multicyclic compounds 2 and 3 (Scheme 1).9 These
Received: June 5, 2013
Published XXXX by the American Chemical
Society
A
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The pyrazinodiindole 5 (Scheme 2) was isolated in 21% yield
1
as a red−burgundy solid. The H NMR spectrum showed
singlets at 5.04 and 5.38 ppm, assigned to the exocyclic
methylene protons whereas the 13C NMR spectrum showed 2
peaks at 179.7 and 180.8 ppm, assigned to two nonequivalent
carbonyls. Data from the HRESI mass spectrum was supportive
of the molecular formula of 5 and was indicative of the addition
of one propargyl unit.
The UV−vis spectrum of 5 had a strong adsorption band
with a maximum at 324 nm (ε = 13 088). The burgundy color
was suggestive that the central double bond of indigo remained
intact, with all other indigo derivatives in which this bond had
been converted to a single bond appearing as yellow
compounds. Simple modeling studies (Spartan, Wave function)
indicated that compound 5, while planar through the indigo
moiety, positioned the hydrogen atoms of the endocyclic
methylene group above and below the plane of the molecule
with the exocyclic methylene twisted out of the molecule
plane.10 This product 5 can also be synthesized in high yield
(98%) by the reaction of 4 in DMF in the presence of Cs2CO3
(Scheme 3, Step B).
The pyridodiindole 6 (Scheme 2) was isolated in 17% yield
as yellow−orange crystals. Analysis of the 1H NMR indicated a
peak at 9.62 ppm assigned to the aldehyde proton, and a
doublet at 4.79 ppm (J = 6.5 Hz) assigned to the terminal
protons of the allene moiety and a singlet at 2.33 assigned to
the terminal alkyne proton. The 13C NMR spectrum showed
peaks at 90.4, 208.4, and 80.4 ppm, assigned sequentially to the
three allene carbons from the CH. A peak at 195.7 ppm was
assigned to the aldehyde. The HRESI mass spectrum of 6 was
consistent with a molecular formula of C25H16N2O2. The
molecular structure was confirmed as 6 by X-ray crystallo-
graphic analysis, together with the disposition of the allene
group over the pyrido ring rather than pointing away from it
(Scheme 2). There is one stereogenic carbon present at C12a,
but in the absence of any chiral element during the reaction, the
stereochemical outcome was a racemic mixture, as confirmed
by optical rotation analysis. The structure of 6 poses interesting
mechanistic questions. The addition of three propargyl units is
evidenced by the presence of the allene, the N-propargyl unit
and the three-carbon moiety encompassing the aldehyde, and
C6 and C7. Interestingly, this three-carbon unit is attached to
the indigoyl N at the middle carbon, and not through the
propargyl bromide methylene or the terminal alkynic positions.
The major product of the reaction was the benzoindolo-
naphthyridinone 7 (Scheme 2) isolated in 31% yield as a yellow
solid which was also highly fluorescent in CH2Cl2 solution with
a brilliant yellow color under UV light (365 nm). The 1H NMR
showed two pairs of doublets at 8.01 and 7.33 ppm (J = 7.4 Hz)
assigned to H6 and H7, respectively. Two singlets at 2.14 and
2.30 ppm were assigned to the terminal acetylenic protons with
the corresponding propargyl methylenes assigned to the
doublets at 4.74 and 5.49 ppm (J = 2.4 Hz). A comparative
analysis of the NMR and mass spectra indicated that although
two substituent propargyl units were present, the molecular ion
indicated the addition of three propargyl units, the last being
incorporated into a ring (Scheme 2, 7, blue). The structure of 7
was confirmed by X-ray crystallographic analysis. The ring
expansion of the indigo 5-membered ring into a 6-membered
ring is new chemistry, with the three additional carbons of the
indolo[1,2-h][1,7]naphthyridine parent structure being sourced
from the additional propargyl unit. Retrosynthetically, sourcing
the C6−C7−C7a moiety from a propargyl unit with the
Figure 1. Some biologically active natural products with an embedded
2,2′-diindolic unit.
propargyl bromide. New facile routes to the core heterocyclic
system of fascaplysin, homofascaplysin, and the B-ring
homologue of this system were discovered and the results are
reported in this paper together with some initial in vitro
antiplasmodial and anticancer activity data.
RESULTS AND DISCUSSION
■
Synthesis and Structural Elucidation. Our previous
experience in the chemistry of indigo9 revealed that relatively
small changes to reaction conditions could have major impacts
on the product outcome. Therefore, our attempts at the
propargylation of indigo paid particular attention to stringent
and repeatable reactions conditions. In this context, a solution
of indigo in DMF was generated through sonication for 30 min
at room temperature and then transferred to a septum-
equipped flask which contained predried molecular sieves and
cesium carbonate under an inert atmosphere. The flask was
then plunged into a preheated oil-bath (strictly 85−87 °C) and
stirred for 30 min, followed by the addition of propargyl
bromide and the reaction mixture heated at this temperature for
5 min. Following quenching, a sequence of separations yielded
the five new products 4−8 in a combined yield of 81% (Scheme
2).
The expected mono N-substituted indigo derivative (4) was
isolated as a deep blue, papery solid in 11% yield after silica-gel
column chromatography. Data from the HRESI mass spectrum
was consistent with the molecular formula of 4 and was
indicative of the addition of one propargyl unit. 1H NMR NOE
analysis also confirmed the presence of the expected trans
isomer. In a separate reaction, under analogous conditions but
with a very short reaction time (<1 min) and a stoichiometric
quantity of propargyl bromide, compound 4 could be isolated
in a much improved 93% yield (Scheme 3, Step A). The
increased solubility in a range of organic solvents (e.g., THF,
CH2Cl2) enables this monopropargylated material to be used as
a starting material for subsequent reactions, including
cyclizations. This increased flexibility in the use of solvents
allows for a greater variation in reaction conditions to be used.
B
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Scheme 2. Base-Mediated Propargylation of Indigo to Produce the Heterocyclic Compounds 4−8 (Adjacent to Each Structure
Is Illustrated a Solution, Highlighting the Color)
Color is an important qualitative element in the structural
elucidation of these polycyclic compounds. The disappearance
of the blue and emergence of yellow appears to indicate the loss
of H-bonding between the indigo carbonyl and the NH, along
with loss of unsaturation in the indigo central bond and the
presence of either sp3 hybridized carbon atoms (e.g., 6), or
extended fused ring systems, e.g. heterocycles 7 or 8. The
mono-N-propargylated 4, with both structural elements still
present, maintains the deep blue intensity, whereas the cyclized
structure 5, which still contains the central double bond but has
lost the H-bonding, is a burgundy color (Scheme 2).
Scheme 3. Synthesis of 4 and Subsequent Conversion to 5
Mechanistic and Reaction Discussion. The proposed
mechanisms for the propargylation of indigo are summarized in
Schemes 4−6 and involve five key pathways. The pyrazino-
diindole 5 is derived from the monopropargylated indigo 4
(blue, Path I, Scheme 4, and Scheme 3) after N-deprotonation,
followed by delocalization allowing rotation around the central
bond to the cisoid conformation. Subsequent intramolecular
nucleophilic addition to the propargyl C2 position yields 5
(Scheme 4).
remaining skeleton from indigo is not intuitive and highlights
the novelty of this new chemistry of indigo. The final product,
isolated in very low yield (<1%), was the yellow benzoindo-
lonaphthyridinone 8 (Scheme 2), with the structure confirmed
by single-crystal X-ray analysis.
Path II starts with the identical key intermediate 4 (blue,
Scheme 5) undergoing prototropic tautomerism (A) which
subsequently N-alkylates (B). Deprotonation of the N-
C
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“P” and “B” heteroatom ylid.14 Therefore the postulated cyclic
allene intermediate C (Scheme 5) is reasonable.
Scheme 4. Proposed Mechanism for Formation of 4 and 5
(Colored Structures Indicate Common Intermediates in the
Overall Mechanism and Are the Same within Schemes 5 and
6
The cyclic allene could then undergo a ring-expansion
reaction, to produce the benzo[b]indolo[1,2-h][1,7]-
naphthyridin-8-(13H)-one ring structure D (Scheme 5). The
proposed driving force behind this ring-expansion is relief of
ring strain of the 7-membered allenic ringtherefore, there is a
favorable energy balance between the 7-membered allenic ring
formation, and its subsequent role in providing a driving force
for ring expansion. An additional crucial component of this step
is the presence of an electrophile (E)the major product
arising from the reaction, 7, requires E = H+, whereas the minor
product 8 requires E = CO2, probably generated, on the basis of
the results noted in Table 1, from carbonic acid decomposition,
the acid in turn resulting ultimately from the Cs2CO3 base via
bicarbonate (see below for a greater discussion). Once the
carboxylate unit is incorporated, a further propargyl moiety
could be added via nucleophilic displacement to produce the
ester substituent of 8. Subsequent dehydrogenation, followed
by base-induced aromatization allows for O-propargylation in a
cascade process, yielding the final products 7 (Path III) and 8
(Path IV)(Scheme 5).
methylene generates a stabilized ylid, which allows cyclization
onto the carbonyl generating an activated cyclic allene
intermediate (C). Under standard conditions, an “alkyne
nucleophile” is insufficiently strong to attack an electrophilic
carbonyl in the absence of a metal (e.g., Au, Ru) or an
activating influence. In this instance, the anion from the ylid
serves as a formal negative charge allowing this cyclization to
the 7-ring allene to occur. A comprehensive review on allenes
from 198911 reported the isolation of an eight-membered
carbocyclic allene, however, the corresponding six-membered
rings have been plausibly demonstrated as reactive intermedi-
ates.12 Further, with the seven-membered carbocyclic allene,
isodesmic reaction energy calculations indicate13 an allene
strain component of 13.5−14.3 kcal/mol, consistent with its
ready preparation and trapping. Heterocyclic allenes have also
been isolated as small as eight-membered rings, with a mixed
The addition of a 1-carbon unit is novel and imposes the
question as to the origin of this carbon, even though 8 is
isolated in very low yield. Two possibilities arise and involve
either the well-known degradation of DMF15 to produce a
“CO” fragment that could be incorporated, or it could arise
from the generated bicarbonate anion that is present in the
solution. Table 1 summarizes experiments to determine the
source of the additional carbon atom in 8. Entry 1 is the
standard reaction as previously outlined, whereas entry 2
describes replacing the DMF solvent with DMSOthis
resulted in an increase from <1% to a 5% yield suggesting
that DMF was not the source of the carboxylate of 8. Bubbling
CO2 gas through a standard reaction (entry 3) resulted in a 6%
yield of 8, however, the most significant outcome from this
reaction is the notably reduced yields of 4, 5, 6, and 7, and a
Scheme 5. Proposed Mechanism for Formation of 7 and 8
D
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Table 1. Experiments to Probe the Source of the Ester Moiety 8
entry solvent other reaction conditions 4 yield % 5 yield % 6 yield % 7 yield % 8 yield % baseline material (%mass) recovered indigo (%mass)
1
2
3
4
5
DMF
DMSO
DMF
DMF
DMF
Cs2CO3, N2
Cs2CO3, N2
Cs2CO3, CO2
K3PO4, N2
11
21
17
12
7
31
13
10
1
5
6
11
65
61
13
21
10
60
a
Cs2CO3, Ar
15
14
28
1
a
Not isolated.
Scheme 6. Proposed Mechanism for Formation of 6
dramatic increase in the production of noncharacterizable
baseline material. This suggests that the presence of significant
quantities of electrophiles could be reacting with different
indigo-based nucleophiles as they are being generated resulting
in mixtures of products. Entry 4 describes the experiment
replacing the Cs2CO3 with K3PO4 as base, to eliminate the
presence of a bicarbonate source. However, the lack of
solubility of this base in DMF is the likely reason for the
outcome of mostly unreacted indigo being isolated from the
reaction.
Path V (Scheme 6) describes a possible mechanism to the
allene 6 and diverges from the same intermediate B (red,
Scheme 5). In this instance it is the iminium indigo moiety that
activates the adjacent carbonyl, allowing sufficient electro-
philicity to attract the relatively weak nucleophilic alkyne to
undergo a cyclization reaction, promoted by the initial attack of
the other indigo nitrogen lone electron pair onto the propargyl
C2 in a concerted process yielding the strained fused-aziridine
E. Carbonate could then act as a nucleophile in a ring-opening
of the aziridine and subsequent aromatization of the central
pyridinyl ring to give F. O-Propargylation of F could then afford
intermediate G. The acidic proton in the CH2OH group α to
the iminium ion in G may then be removed under the influence
of base and further OH proton loss would yield the aldehyde
moiety in the intermediate H. A Claisen rearrangement of the
propargyloxy group with the indolic C2−C3 bond could then
give rise to product 6.
The outcomes from this propargylation reaction are
repeatable and preliminary investigations also indicate reliable
scalability up to at least double quantities. Further, our
mechanistic proposals for the formation of 6−8 all start from
N,N-dipropargylated intermediates, rather than an intermediate
that had cyclized initially from a N-monopropargylated
molecule. In support of this proposal were the outcomes
from an experiment where a DMF solution of 4 was dripped
into a mixture of Cs2CO3 and propargyl bromide in DMF over
3 min before quenching after 2 min. The result was formation
of 5 (<3%), 6 (11%), 7 (25%), and 8 (<1%) with complete
consumption of the starting material. The poor return of 5 with
respect to 6−8 suggests that the second N-propargylation is a
more competitive reaction than cyclization, and lends support
to our proposed dipropargylated compounds as intermediates.
It is also relevant to compare the mechanistic outcomes of
this propargylation reaction with the outcomes of the
corresponding allylation reaction (Scheme 1).9 Although
cyclization onto the indigo carbonyl of the unsaturated moiety
occurred in both instances, reaction onto the indigo C2
position to form a spiro compound only occurs in the case of
the alkene. There was no evidence suggesting an equivalent
mechanism pathway in the presence of the alkyne. Presumably,
the linear alkyne is not able to approach the indigo C2 position
whereas the “bent” nature of the alkene makes this cyclization
reasonable. Further, a significant byproduct of the allylation
reaction (Scheme 1) was N-allylisatin (structure not
E
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illustrated), derived by oxidative cleavage of the central indigo
double bond. Under propargylation conditions, there was no N-
propargylisatin present, as evidenced by TLC analysis of the
reaction mixture against an authentic sample.
the cascade reaction, the reactivity of different sites on the
indigo moiety changes, and additionally, different types of
reactivity can occur at different times, including sites acting as
nucleophiles or electrophiles, with bond breakage leading to
ring expansion and dehydration (Figure 3). A key element to
We have also undertaken some simple experiments to
ascertain that the reactions are likely to proceed through
nucleophilic mechanisms rather than through radical-based
sequences. Previous studies have shown that radical reactions
with indigo will proceed exceptionally slowly at room
temperature and in 4 h at 100 °C in the presence of oxygen
and with irradiation.16 In contrast, we have repeated our
propargylation reaction (as shown in Scheme 2) in the absence
of oxygen and light (under argon) with these conditions
realizing the same product outcome after 5 min of reaction at
86 °C. Radical reactions are unlikely to proceed under these
conditions, let alone to produce a total yield of 84% of products
in 5 min of reaction time.
Figure 3. Graphical representation of the changing reactivity of the
indigo skeleton with sites of nucleophilicity, electrophilicity,
dehydration, and bond breakage illustrated. The degree of reactivity
at each of these points changes as the molecule(s) progress through
the cascade reaction.
The heterocycles 7 and 8 are analogs of the marine natural
products fascaplysin and homofascaplysin (Figure 2), initially
the reactivity is the ability of the indigo heterocycle to
tautomerize under different circumstances (e.g., upon gen-
eration of the N-centered anion with base) to shift reactivity to
a different site. This constantly changing reactivity leads to the
variety of outcomes as demonstrated. Further, this range of
different chemistry all occurs in a tight cluster in the center of
the indigo structure and this combination of properties is the
key to the variety of unusual heterocycles that arises. Further,
the unexpected outcomes of this initial study not only shows us
that there is highly unusual and significant chemistry of indigo,
but that there is much yet to be explored in this area.
Biological Testing. As an initial screen for biological
activity, compounds 4−7 were subjected to in vitro
antiplasmodial and anticancer testing (Table 2). All four
compounds revealed notable antiplasmodial20 activity against a
drug-resistant strain in the micromolar range, sufficient for all
to be considered lead compounds for further development.
More significantly, 4 (antiplasmodial IC50 0.33 μg/mL, 1.1
μM) and 5 (antiplasmodial IC50 0.256 μg/mL, 0.85 μM) are an
order of magnitude more potent than compounds 6 and 7. In
the case of 4, there was also a significant difference between the
level of mammalian (Vero) cell toxicity (IC50 16.26 μg/mL)
and antiplasmodial activity (Table 1). With respect to the
activity against cancer cells,20 compounds 4−7 showed notable
activity against the NCI-H187 lung cancer and KB-oral cavity
cell lines. In particular, 7 was equipotent with the positive
control ellipticine. Cytotoxicity20 studies were particularly
interesting for 5 and 6, with the latter being noncytotoxic to
normal mammalian cells but the former being toxic to these
cells. Clearly, significant medicinal chemistry studies would
need to be undertaken to decrease the toxicity to normal
mammalian cells and increase the required selectivity. However,
as a random screening process, all these compounds showed
activity confirming that they could be considered as new lead
compounds for a variety of targets.
Figure 2. Representation of compounds 6 and 7 as analogs of
fascaplysin/homofascaplysin and fascaplysin/homofascaplysin ring B-
homologue, respectively.
isolated from the sponge Fascaplysinopsis reticulata with
fascaplysin noted as an ATP-competitive inhibitor of Cdk4/
D1 (IC50 = 0.35 μM)3,17 activity. There have been reported
syntheses of these natural products including the silver-
catalyzed cascade synthesis of their parent compound and
analogs, with this approach involving the initial intermolecular
reaction of two components of similar molecular complexity,
however, this necessitated the prior synthesis of these two
components.18 The B-ring homologue of fascaplysin has also
been synthesized starting from 4(1H-indol-1-yl)butanoic acid
in a 2 step synthesis in a best yield of 42%.19
We report here a facile synthesis of the benzoindolo[1,2-
h][1,7]naphthyridine (fascaplysin) heterocyclic skeleton from
indigo in the presence of propargyl bromide and base in a 17%
unoptimized yield. This provides direct access to this skeleton
starting from exceptionally cheap starting materials and
reagents, and provides a convenient access to this system for
further elaboration in structure−activity studies.
The reaction of indigo with propargyl bromide in the
presence of base produces new complex heterocycles via a
multistep, one-pot, cascade-type series of reactions. This is new
science for the indigo moiety and represents an exciting
untapped area of heterocyclic chemistry. Our proposed
mechanisms (Schemes 4−6) reveal that at different stages of
CONCLUSION
■
We have reported here new cascade reactions of indigo via
base-mediated propargylation. The resulting shifting of
reactivity during each step in the process generates, and then
propagates, a cascade reaction, producing a range of poly-
F
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Table 2. Anti-Plasmodial and Anti-Cancer Activity of the Indigo-Derived Heterocyles 4−7
a
Plasmodium falciparum
NCI-H187 lung anticancer
KB-oral cavity anticancer
IC50 μg/mL (μM)
cytotoxicity vero cell IC50
entry
compound
antiplasmodial IC50 μg/mL (μM)
IC50 μg/mL (μM)
μg/mL (μM)
1
2
3
4
5
4
5
6
7
0.33 (1.1)
0.26 (0.85)
3.34 (8.9)
3.75 (9.9)
0.24/0.03
4.79 (15.9)
1.88 (6.24)
4.79 (12.7)
17.3 (46.0)
8.95 (29.8)
1.31 (4.36)
7.81 (20.7)
8.44 (22.34)
16.26 (54.2)
1.93 (6.43)
b
16.4 (43.6)
dihydroartemisinine/
mefloquine
6
ellipticine/doxorubicin
1.21/0.108
1.04/0.674
a
b
Drug resistant K1 strain. Noncytotoxic.
consecutively with 4 different solvent mixtures: 1, 70:30 CH2Cl2/
petroleum spirit (250 mL); 2, CH2Cl2; 3, 50:50 CH2Cl2/EtOAc; and
4, 95:5 EtOAc/MeOH (250 mL). Four fractions were collected from
the four different elutions. Fraction 1 was concentrated and slowly
recrystallized in 9:1 petroleum spirit/EtOAc then filtered to yield 1-
(prop-2-yn-1-yl)-[2,2′-biindolinylidene]-3,3′-dione 4 (33.00 mg
11%) as a blue, papery solid. Rf (7:3 CH2Cl2/petroleum spirit) =
0.53, mp 267−269 °C; λmax/nm (ε, M−1cm−1) 291 (10447), 634
(6381). IR (neat) υmax 3278 (m), 1605 (s), 1463 (s), 1297 (s), 1066
(s), 1027 (s), 927 (m), 743 (m) cm−1. 1H NMR δ 2.17 (1H, s, H3″),
5.41 (1H, s, H1″), 6.96 (1H, t, J = 7.1 Hz, H5′), 6.99 (1H, d, J = 8.1
Hz, H7′), 7.09 (1H, t, J = 7.1 Hz, H5), 7.22 (1H, d, J = 8.1 Hz, H7),
7.48 (1H, t, J = 7.5 Hz, H6), 7.60 (1H, t, J = 7.5 Hz, H6′), 7.69 (1H, d,
J = 7.5 Hz, H4′), 7.77 (1H, d, J = 7.5 Hz, H4) 10.60 (1H, s, H1). 13C
NMR (CDCl3) δ 37.1 (C1″), 72.5 (C3″), 78.4 (C2″), 111.5 (C7′),
111.9 (C7), 120.4 (C3′a), 120.9 (C5′), 121.5 (C5), 122.0 (C2′), 122.4
(C2), 125.0 (C4′), 125.1 (C4), 126.2 (C3a), 135.9 (C6), 136.4 (C6),
151.8 (C7′a), 152.6 (C7a), 187.6 (C3), 189.6 (C3′). MS (EI) m/z:
300 (100%, M+), 271 (53), 262 (32). HRMS (ESI) [M + H]+ calcd for
C19H13N2O2, 301.0977; found, 301.0964.
The mother liquors from fraction 1 were combined with fraction 2
and then subjected to a silica gel column and elution with 7:3 CH2Cl2/
petroleum spirit gave 13-oxo-12-(prop-2-yn-1-yl)-12b (propa-1,2-
dien-1-yl)-12b,13-dihydro-12H-pyrido[1,2-a:3,4-b′]diindole-6-car-
baldehyde 6 as yellow−orange crystals (63.92 mg, 17%). X-ray quality
crystals were grown through slow crystallization from petroleum
spirit/EtOAc (5:3). Rf (CH2Cl2) = 0.26, mp 229−231 °C; UV−vis
(CH2Cl2) λmax/nm (ε, M−1cm−1) 386 (12825), 244 (34151). IR
(neat) υmax 3278 (m), 1604 (s), 1463 (s), 1297 (m), 1065 (s), 1027
(s), 1015 (s), 743 (s) cm−1. 1H NMR δ 2.33 (1H, s, H3′), 4.79 (2H, d,
J = 6.5 Hz, H3″), 5.42 (1H, t, J = 6.5 Hz, H1″), 5.51−5.66 (2H, m,
H1′), 6.82 (1H, d, J = 8.4 Hz, H11), 6.94 (1H, t, J = 7.2 Hz, H3), 7.27
(1H, t, J = 7.6 Hz, H2), 7.35 (1H, t, J = 8.0 Hz, H9), 7.49 (1H, t, J =
8.0 Hz, H8), 7.54 (1H, d, J = 8.4 Hz, H10), 7.64−7.68 (2H, m, H1,
H4), 7.65 (1H, s, H7), 9.62 (1H, s, 1′″). 13C NMR (CDCl3) δ 36.2
(C1′), 71.3 (C12b), 73.6 (C3′), 77.8 (C2′), 80.4 (C3″), 90.4 (C1″),
110.7 (C7a), 111.1 (C13a), 111.5 (C9), 113.6 (C1), 117.2 (C12a),
118.6 (C11), 121.1 (C3), 122.6 (C8), 124.1 (C10), 124.5 (C11a),
124.9 (C4), 131.8 (C7), 137.7 (C7b), 138.2 (C4a), 138.5 (C2), 158.6
(C6), 185.3 (C1′″), 195.7 (C13), 208.4 (C2″). MS (EI) m/z 376 (5,
M+), 371 (100%), 298 (24). HRMS (ESI) [M + H]+ calcd for
C25H17N2O2, 377.1298; found, 377.1285.
heterocyclic compounds in unprecedented outcomes. Some of
these compounds also hold promise as leads for the
development of new antimalarial and anticancer agents. The
scope for this reaction currently remains untapped, however,
these initial studies suggest that the chemistry of indigo has
significant potential for further development toward novel
polycyclic compounds. The exploration of a variety of reaction
types, inclusive of cascade processes, using indigo as a starting
material remains an additional exciting area of synthetic
chemistry to investigate. Interestingly, the possibility of using
indirubin and isoindigo, as indigo analogs, in comparable
cascade reactions remains unexplored and these starting
materials also offer possibilities for the generation of new
heterocycles in a one-pot process. These studies are currently
underway in our laboratories.
EXPERIMENTAL SECTION
■
General Experimental Information. Reagents and solvents were
purchased reagent grade and used without further purification unless
otherwise stated. All reactions were performed in standard oven-dried
glassware under a nitrogen atmosphere unless otherwise stated.
Melting point temperatures are expressed in degrees Celsius (°C) and
1
are uncorrected. H and 13C NMR spectra (CHCl3) solutions) were
recorded at 500 and 125 MHz with chemical shifts (δ) reported in
parts per million relative to TMS (δ = 0 ppm) or CDCl3 (δ = 77.0
ppm) as internal standards. Coupling constants (J) are reported in
Hertz (Hz). Multiplicities are reported as singlet (s), broad singlet
(bs), doublet of doublets (dd), or multiplet (m). Electron impact (EI)
mass spectra (MS) and electrospray (ESI single quadrupole) mass
spectra have their ion mass to charge (m/z) values stated with their
relative abundances as a percentage in parentheses. Peaks assigned to
the molecular ion are denoted by M+ or M + 1. Infrared (IR) spectra
were recorded on neat samples. UV−visible spectra were recorded
with solutions of samples in CH2Cl2. Images from crystals were
captured using stereo microscopes. All the images were provided from
X-ray quality single crystals. Optical rotations were measured in
CH2Cl2 solution at 25 °C. Thin layer chromatography (TLC) was
performed using silica gel F254 aluminum sheets. Column
chromatography was performed under gravity using silica gel 60
(0.063−0.200 mm). Eluents are in volume to volume (v:v)
proportions. Solvent extracts or chromatographic fractions were
concentrated by rotary evaporation in vacuo. Indigo (dye content
95%) was used without further purification. Petroleum spirit had a bp
range of 40−60 °C.
Products from the Propargylation of Indigo. A suspension of
powdered indigo (262 mg, 1.0 mmol) in anhydrous DMF (40 mL)
was sonicated for 30 min and stirred vigorously under N2 overnight.
The resulting suspension was added to predried anhydrous cesium
carbonate (1.20 g, 3.4 mmol) and molecular sieves (4 Å) while being
stirred and warmed to 80−85 °C under a N2 atmosphere. After 30 min
propargyl bromide (0.595 mg 5.0 mmol) was added and the reaction
mixture was heated at 82−85 °C for 5 min. The mixture was filtered
hot and then the solution was concentrated by rotary evaporation and
the residue applied to a short plug of silica gel/celite (1:1) and washed
Fraction 3 was recrystallized from CH2Cl2/petroleum spirit (1:9)
giving 13-(prop-2-yn-1-yl)-14-(prop-2-yn-1-yloxy)benzo[b]indolo-
[1,2-h][1,7]naphthyridin-8-(13H)-one 7 (116.6 mg, 31%) as orange
crystals. X-ray quality crystals were grown through slow crystallization
from petroleum spirit/ethyl acetate (4:3). Rf (9:1 CH2Cl2/EtOAc) =
0.52, mp 218−220 °C; UV−vis (CH2Cl2) λmax/nm (ε, M−1cm−1) 324
(27663), 441 (10360). IR (neat) υmax 3205 (w), 1588 (m), 1485 (m),
1349 (m), 1260 (m), 1059 (m), 725 (s) cm−1. 1H NMR δ 2.14 (1H, t,
J = 2.4 Hz, H3′), 2.30 (1H, t, J = 2.4 Hz, H3″″), 4.74 (2H, d, J = 2.4
Hz, H1′), 5.49 (2H, d, J = 2.4 Hz, H1″), 7.33 (1H, d, J = 7.4 Hz, H7),
7.45 (3H, m, H1, H2, H3), 7.74−7.77 (1H, t, J = 7.8 Hz, H11), 7.85−
7.90 (3H, m, H4, H10, H12), 8.01 (1H, d, J = 7.4 Hz, H6), 8.71 (1H,
dd, J = 8.1, 1.4 Hz, H9). 13C NMR (CDCl3) δ 44.0 (C1″), 63.7 (C1′),
74.5 (C3′), 76.5 (C3″), 78.5 (C2″), 78.6 (C2′), 104.0 (C7), 110.9
G
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The Journal of Organic Chemistry
Article
(C3), 117.9 (C7a), 118.6 (C12), 118.9 (C13b), 119.1 (C10), 119.9
(C6), 121.6 (C4a), 123.3 (C2), 123.9 (2C, C1, C4), 126.4 (C8a),
126.9 (C9), 129.3 (C14a), 132.6 (C11), 133.2 (C14), 139.1 (C13a),
143.5 (C12a), 175.8 (C8). MS (EI), m/z 376 (6, M+), 337 (100%),
309 (20), 298 (75). HRMS (ESI) [M + H]+ calcd for C25H17N2O2,
377.1285; found, 377.1302.
80−85 °C for 30 min and then propargyl bromide (119 mg, 1.00
mmol) was added under a N2 atmosphere. The resulting mixture was
stirred at 80−85 °C for 30 min, poured into ice water, and the
suspension was partitioned between CH2Cl2 (20 mL) and water (20
mL). The aqueous layer was washed with CH2Cl2 (4 × 5 mL), and the
combined organic layers were washed with water (3 × 10 mL), dried
(MgSO4), and concentrated. The residue was recrystallized from
chloroform/hexane (1:6) to give N-propargylisatin (133 mg, 91%) as
The mother liquor from the recrystallization of 7 was concentrated
and then subjected to silica gel column chromatography, and elution
with CH2Cl2/EtOAc (92:8) resulted in prop-2-yn-1-yl 8-oxo-13-
(prop-2-yn-1-yl)-14-(prop-2-yn-1-yloxy)-8,13-dihydrobenzo[b]-
indolo[1,2-h][1,7]naphthyridine-7-carboxylate 8 (2 mg, <1%) as
bright orange crystals. X-ray quality crystals were grown through slow
crystallization from chloroform. Rf (9:1, CH2Cl2/EtOAc) = 0.59, mp
258−260 °C; IR (neat) υmax 3278 (m), 2925 (m), 1718 (s), 1595 (s),
1
an orange solid. H NMR (CDCl3) δ 2.32 (2H, d, J = 1.7 Hz, H3′),
4.54 (2H, d, J = 1.9, H1′), 7.16 (2H, m, H5, H7), 7.66 (2H, m, H4,
H6). 13C NMR (CDCl3) δ 29.8 (C3′), 73.7 (C2′), 76.0 (C1′), 111.4
(C7), 118.0 (C3a), 124.5 (C5), 125.8 (C4), 138.8 (C6), 149.9 (C7a),
157.5 (C2), 182.9 (C3). MS (EI) m/z 185 (85%, M+), 129 (100%)
consistent with literature values.21
1
1482 (m), 1237 (m), 1062 (m), 964 (m), 749 (s), 643 (s) cm−1. H
Antiplasmodial Assay. The compounds and extracts were tested
in vitro against Plasmodium falciparum, K1CB1 (K1), which is a
multidrug resistant (chloroquine and antifolate resistant) strain,
received as a generous gift from Professor Sodsri Thaithong,
Chulalongkorn University, Bangkok, Thailand. The parasites were
maintained in human red-blood cells in RPMI 1640 medium
supplemented with 25 mM HEPES, 0.2% sodium bicarbonate, and
8% human serum at 37 °C in a 3% carbon dioxide gas incubator
(Trager and Jensen, 1976). Samples were made up in DMSO solution
and the in vitro antimalarial activity testing was carried out using the
microdilution radioisotope technique. The test sample (25 μL, in the
culture medium) was placed in triplicate in a 96-well plate where
parasitized erythrocytes (200 μL) with a cell suspension (1.5%) of
parasitemia (0.5−1%) were then added to the wells. The ranges of the
final concentrations of the samples were varied from 2 × 10−5 to 1 ×
10−7 M with 0.1% of the organic solvent. The plates were then
cultured under standard conditions for 24 h after which 3H-
hypoxanthine (25 μL, 0.5 mCi) was added. The culture was incubated
for 18−20 h after which the DNA from the parasite was harvested
from the culture onto glass fiber filters and a liquid scintillation
counter used to determine the amount of 3H-hypoxanthine
incorporation.22 The inhibitory concentration of the sample was
determined from its dose−response curves or by calculation.
Cancer Growth Inhibition and Vero Cell Toxicity Assay.
Cancer growth inhibition assay and the Vero cell assay were performed
using the Resazurin microplate assay (REMA) method as described by
O’Brien et al.23 In brief, cells at a logarithmic growth phase were
harvested and diluted to 2.2 × 104 cells/mL for KB and 3.3 × 104
cells/mL for NCI-H187, in fresh medium. Successively, 5 μL of test
sample diluted in 5% DMSO, and 45 μL of cell suspension were added
to 384-well plates, incubated at 37 °C in 5% CO2 incubator. After the
incubation period (3 days for KB, and 5 days for NCI-H187), 12.5 μL
of 62.5 μg/mL resazurin solution was added to each and the plates
were then incubated at 37 °C for 4 h. Fluorescence signal was
measured using SpectraMax M5 multidetection microplate reader
(Molecular Devices, USA) at the excitation and emission wavelengths
of 530 and 590 nm. Percent inhibition of cell growth was calculated by
the following equation: % Inhibition = [1 − (FUT/ FUC)] × 100
where FUT and FUC are the mean fluorescent unit from treated and
untreated conditions, respectively. Dose−response curves were plotted
from 6 concentrations of 3-fold serially diluted test compounds.
Sample concentrations that inhibited cell growth by 50% (IC50) were
derived using the SOFTMax Pro software (Molecular Devices, USA).
Ellipticine and doxorubicin were used as a positive control, and 0.5%
DMSO and water were used as a negative control.23
NMR δ 2.14 (1H, bs, H3″), 2.30 (1H, bs, H3′″), 2.53 (1H, bs, H3′),
4.76 (2H, d, J = 2.0 Hz, H1″), 5.06 (2H, d, J = 1.7 Hz, H1′″), 5.43
(2H, d, J = 1.7 Hz, H1′), 7.43−7.50 (3H, m, H1, H2, H3), 7.75 (1H, t,
J = 7.8 Hz, H11), 7.82−7.90 (3H, m, H4, H10, H12), 8.01 (1H, s,
H6), 8.48−8.50 (1H, d, J = 7.8, H9). 13C NMR (CDCl3) δ 44.1
(C1′″), 53.4 (C1′), 63.5 (C1″), 74.7 (C3′″), 75.1 (C3′), 76.5 (C3″),
77.9 (C2″), 78.1 (C2′), 78.2 (C2′″), 110.7 (C7a), 110.8 (C7), 112.2
(C3), 115.1 (C13b), 118.1 (C12), 118.7 (C10), 118.8 (C6), 122.0
(C4a), 123.8 (C2), 124.1 (C1), 124.7 (C4), 126.7 (C8a), 126.8 (C9),
130.0 (C14a), 133.6 (C11), 134.5 (C14), 139.2 (C13a), 142.9 (C12a),
167.1 (ester CO) 174.5 (C8). MS (EI), m/z 458 (5, M+), 419
(100%), 380 (10), 375 (5), 337 (20), 298 (45). HRMS (ESI) [M +
H]+ calcd for C29H19N2O4, 459.1345; found, 459.1363.
Fraction 4 was purified by preparative TLC using CH2Cl2/EtOAc
(88:12) as the developing solvent and gave 6-methylene-6,7-
dihydropyrazino[1,2-a:4,3-a′]diindole-13,14-dione 5 was isolated
as a dark burgundy powder (63 mg, 21%. Rf (8.5:1.5, CH2Cl2/EtOAc)
= 0.51, mp 280−284 °C. UV−vis (CH2Cl2) λmax/nm (ε, M−1cm−1)
324 (13088), 573 (6430). IR (neat) υmax 1701 (m), 1604 (m), 1470
1
(m), 1298 (m), 1185 (m), 1122 (s), 742 (s) cm−1. H NMR δ 4.41
(2H, s, H7), 5.04 (1H, H1′a), 5.38 (1H, H1′b), 6.95−7.00 (2H, m,
H9, H11), 7.09 (1H, t, J = 7.4 Hz, H2), 7.48−7.55 (3H, m, H3, H4,
H10), 7.73 (1H, d, J = 7.5 Hz, H12), 7.82 (1H, d, J = 7.5 Hz, H1). 13C
NMR (CDCl3) δ 45.9 (C7), 97.9 (C1′), 109.5 (C12), 112.7 (C4),
121.4 (C13a), 121.8 (C11), 122.7 (C2), 123.0 (C12a), 124.6 (C14a),
125.3 (C1), 125.5 (C6), 131.6 (C13b), 135.1 (C10), 135.5 (C3),
147.0 (C4a), 150.0 (C8a), 179.7 (C14), 180.8 (C13). MS (EI), m/z
300 (14), 207 (100%). HRMS (ESI) [M + H]+ calcd for C19H13N2O2,
301.0972; found, 301.0960.
1-(Prop-2-yn-1-yl)-[2,2′-biindolinylidene]-3,3′-dione (4). A
suspension of powdered indigo (262 mg, 1.0 mmol) in anhydrous
DMF (50 mL) was sonicated for 60 min and stirred vigorously under
N2 overnight. The resulting suspension was added to predried
anhydrous cesium carbonate (2.4 g, 7.42 mmol) and the mixture was
stirred and warmed to 80−85 °C under a N2 atmosphere. After 30 min
propargyl bromide (1.90 mg 10.0 mmol) was added and the reaction
mixture was heated at 82−85 °C for 5 s. The mixture was then poured
into ice water and the resulting precipitate was filtered and
recrystallized from petroleum spirit/EtOAc (90:10) to furnish 1-
(prop-2-yn-1-yl)-[2,2′-biindolinylidene]-3,3′-dione 4 (279.00 mg
93%) as a blue fluffy solid.
6-Methylene-6,7-dihydropyrazino[1,2-a:4,3-a′]diindole-
13,14-dione (5). A solution of 4 (100 mg, 0.33 mmol) in anhydrous
DMF (20 mL) was stirred and warmed to 80−85 °C under a N2
atmosphere for 20 min. The solution was then added to predried
anhydrous cesium carbonate (107 mg, 0.33 mmol) and was stirred and
warmed at 80−85 °C under a N2 atmosphere for 10 min. The mixture
was then poured into ice water and the resulting precipitate was
separated and subjected to silica gel short column chromatography and
eluted with CH2Cl2/EtOAc (85:15) to give 6-methylene-6,7-
dihydropyrazino[1,2-a:4,3-a′]diindole-13,14-dione 5 as a dark bur-
gundy powder (98 mg, 98%).
ASSOCIATED CONTENT
■
S
* Supporting Information
1
Copies of H and 13C NMR for compounds 4−8 and UV−vis
spectra for compounds 4−7 , ORTEP plots and CIF files for 6,
7, and 8, images of fluorescence emission for 7 and 8, and a
computed model for 5. This material is available free of charge
via the Internet at http://pubs.acs.org.
Preparation of N-Propargylisatin. To a solution of isatin (147
mg, 1.00 mmol) in dry DMF (40 mL) was added cesium carbonate
(650 mg, 2.00 mmol). The resulting brown suspension was stirred at
H
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The Journal of Organic Chemistry
Article
(21) Radul, O. M.; Zhungietu, G. I.; Rekhter, M. A.; Bukhanyuk, S.
M. Khim. Geterotsikl. Soedin. 1983, 3, 353−355.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +61 2 4221 4692. Fax: +61 2 4221 4287. E-mail: keller@
uow.edu.au
(22) (a) Changsen, C.; Franzblau, S. G..; Palittapongarnpim, P.
Antimicrob. Agents Chemother. 2003, 47, 3682−3687. (b) Desjardins,
R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J. D. Antimicrob. Agents
Chemother. 1979, 16, 710−718.
Notes
(23) O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Eur. J. Biochem.
2000, 267, 5421−5426.
The authors declare no competing financial interests.
ACKNOWLEDGMENTS
■
Financial support from the University of Wollongong, through
the Centre for Medicinal Chemistry and a UPA scholarship to
A.S. is gratefully acknowledged. We also thank Dr. Solomon
Beckman for his help in the preparation of microscopic images
of the crystals, and the Australian National University and
BIOTEC for their support.
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I
dx.doi.org/10.1021/jo401210r | J. Org. Chem. XXXX, XXX, XXX−XXX