A cascade synthetic route to new bioactive spiroindolinepyrido[1,2-a]indolediones from indirubin

Article abstract of DOI:10.1016/j.tet.2015.08.012

The allylation of indirubin produced the expected indolic N′-allylindirubin and N,N′-diallylindirubin derivatives in moderate yields, together with the corresponding N-substituted isatin products. At higher temperatures, the base-initiated reaction with a

Full text of DOI:10.1016/j.tet.2015.08.012

Accepted Manuscript
A Cascade Synthetic Route to New Bioactive Spiroindolinepyrido[1,2-a]indolediones
from Indirubin
Alexander M. Sele, John B. Bremner, Anthony C. Willis, Rachada Haritakun, Renate
Griffith, Paul A. Keller
PII:
S0040-4020(15)01189-8
10.1016/j.tet.2015.08.012
TET 27034
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 19 June 2015
Revised Date: 24 July 2015
Accepted Date: 3 August 2015
Please cite this article as: Sele AM, Bremner JB, Willis AC, Haritakun R, Griffith R, Keller PA, A Cascade
Synthetic Route to New Bioactive Spiroindolinepyrido[1,2-a]indolediones from Indirubin, Tetrahedron
(2015), doi: 10.1016/j.tet.2015.08.012.
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A Cascade Synthetic Route to New Bioactive
Spiroindolinepyrido[1,2-a]indolediones from
Indirubin
Leave this area blank for abstract info.
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Alexander M. Sele, John B. Bremner, Anthony C. Willis, Rachada Haritakun, Renate Griffith*, and Paul A.
Keller*
School of Chemistry, University of Wollongong, Wollongong 2522, New South Wales, Australia. Research
School of Chemistry, Australian National University, Canberra 2601, Australian Capital Territory, Australia.
School of Medical Sciences, UNSW Australia, Sydney 2052, New South Wales, Australia. National Center for
Genetic Engineering and Biotechnology (BIOTEC), Bioresources Research Unit (BRU), Pathumthani 12120,
Thailand.

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Tetrahedron
journal homepage: www.elsevier.com
A Cascade Synthetic Route to New Bioactive Spiroindolinepyrido[1,2-a]indolediones from
Indirubin
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1 Alexander M. Sele , John B. Bremner , Anthony C. Willis , Rachada Haritakun , Renate Griffith *, and
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Paul A. Keller *
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School of Chemistry, University of Wollongong, Wollongong 2522, New South Wales, Australia
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Research School of Chemistry, Australian National University, Canberra 0200, Australian Capital Territory, Australia
National Center for Genetic Engineering and Biotechnology (BIOTEC), Bioresources Research Unit (BRU), Pathumthani 12120, Thailand
School of Medical Sciences, UNSW Australia, Sydney 2052, New South Wales, Australia

Corresponding author. Tel.: +61-2-4221-4692; fax: +61-2-4221-4687; e-mail: keller@uow.edu.au
1 ARTICLE INFO
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ABSTRACT
Article history:
Received
Received in revised form
Accepted
Available online
The allylation of indirubin produced the expected indolic N'-allylindirubin and N,N'-
diallylindirubin derivatives in moderate yields, together with the corresponding N-substituted
isatin products. At higher temperatures, the base-initiated reaction with allylic halides yielded
spiroindolinepyrido[1,2-a]indolediones in a one-pot cascade reaction sequence with yields of up
to 70%. These readily accessed, new spiro compounds represent the first reported examples of
indirubin participating in cascade reactions. Preliminary in vitro biological testing of some of the
products indicated promising activity against some cancer cell lines and against Plasmodium
falciparum for two spiro derivatives. Computational methods were used to gain a greater
understanding of the UV/Vis spectroscopic data for the N'-substituted and N,N'-disubstituted
indirubin derivatives.
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Keywords:
Indirubin
cascade reactions
spiroindolinepyrido[1,2-a]indolediones
antimalarial
2
009 Elsevier Ltd. All rights reserved.
anticancer
7
1
. Introduction
inhibition of cyclin-dependent kinases and glycogen synthase
8
kinase-3, the latter being linked to Alzheimer‟s disease.
The search for new molecular entities, especially easily
8 accessible complex heterocycles, is of significant interest in the
9 exploration of new bioactive compounds and in materials
0 chemistry. Often, cascade reactions are used to realize these
synthetic targets starting with small molecules and utilizing them
in complex multistep reactions. Our recent contributions to this
field have focused on a notable variation to this strategy in that
we started with a larger, but abundant and cheap molecule, the
dye indigo 1 (Figure 1). Indigo afforded a unique opportunity in
providing an advanced starting material for the potential rapid
synthesis of the diindolic system of natural product analogs. To
this end, we recently reported the first cascade reactions of indigo
via base-initiated allylation and propargylation, producing
unexpected polyheterocyclic compounds (Figure 1) in multistep
reactions. This surprising result revealed a new arena in the
previously unreported chemistry of indigo, allowing access to
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9
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1-3
unique, relatively complex heterocycles in one pot.
The structural isomers of indigo 1, indirubin 2 and isoindigo 3
4
5 (Figure 2), are well known, but detailed knowledge of their
6 chemistry is still somewhat limited. Indirubin is perhaps the most
7 well studied due to the reported biological activity of various
8 derivatives, which are known to suppress the metastatic ability of
5
human head and neck cancer cells and to reduce the invasion of
9
0
1
2
3
4
5
6
glioma cells both in vitro and in vivo. Further examples include
Figure 1: Selected examples of heterocycles produced via the cascade
reactions of indigo using either allyl bromide or propargyl bromide and base.

2
Tetrahedron
Indirubin also inhibits intersegmental vAessCelCgEroPwTthEaDnd MAstaNbiUlizSinCg RHI··P·OTC H-bond once all the NH functionality is
9
1,2
induces cellular apoptosis in an in vivo zebrafish model while
other indirubin derivatives or analogues,
glycosides, show antiproliferative activity against a number of
human cancer cell lines.
derivatives include anti-protozoal activity as well as inhibition
of lipoxygenase. Isoindigo, its derivatives and analogues, also
display a suite of useful biological effects including anti-
substituted. Also, in contrast to the that observed with indigo,
1
0
including N-
no final O-allylated indirubin derivatives were observed. Further,
no indirubin remained at the end of the reactions suggesting that
compared to indigo, the initial alkylation is easier (to the non-H-
bonded N'H) but the resulting molecules are less reactive than
indigo to subsequent cascade reactions.
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7
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9
1
1,12
Other effects of indirubin or
13
1
4
1
1
5,16
17
The H NMR spectrum of 9 indicated peaks at 4.46 ppm (d, J
cancer
and anti- protozoal activity.
= 5.5 Hz), 5.22 ppm (s), 5.25 ppm (d, J = 5.0 Hz) and 5.86-5.92
ppm (m), assigned to a single N-allyl substituent, as quantified by
its relative integration to the indirubin aromatic protons. A single
signal at 10.53 ppm was assigned to the NH, positioned
downfield due to its H-bonding with the indirubin carbonyl. This
also indicated that the allyl substituent was positioned on the N'-
atom. Measurement of the HRMS (ESI) gave 303.1143,
indicative of a molecular formula of C19H N O , providing
evidence for the presence of only a single allyl moiety. Analysis
of the H NMR spectra of 14 showed an absence of a peak in the
~10.5 ppm region suggesting the loss of an NH moiety. The
integration of the peaks assigned to the allyl substituent (e.g. 4.43
ppm (2H) and 4.82 ppm (2H) relative to the total number of
peaks assigned to the indirubin H-atoms indicated a ratio of
indirubin:allyl substituents to be 1:2. Further, the measured
HRMS (ESI) of 14 relayed a molecular formula of C H N O .
In other base-induced N-alkylation reactions of indirubin by
simple alkyl halides, preferential alkylation of the oxindolic
nitrogen was also observed.
Equally important is the basic chemistry of indirubin, including
reaction at the carbonyl group, e.g. oxime formation, which
often leads to an increase in biological activity, attack by
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0 Grignard reagents and substitution reactions at the amide
20
1 functionality. An isoindigo derivative has also been shown to
2 provide an advanced starting template for total synthesis, e.g. the
3 use of the 2,2'-double bond in N-tosylisoindigo as a dienophile in
4 a key early Diels˗Alder reaction to build up molecular
5 complexity with stereochemical control in synthetic studies
15 2 2
1
2
1
toward the anti-leukemic marine alkaloid communesin F.
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Figure 2. Indigo 1 and its structural isomers indirubin 2 and isoindigo 3.
Therefore, given the recent understanding of cascade reactions
of indigo 1, we investigated the possibility of analogous cascade
reactions of indirubin 2 and their potential to generate new
heterocycles in one pot. These results are disclosed in this paper
0 together with some initial in vitro antiplasmodial,
1 antimycobacterial, and cell-based anticancer activity data.
2
2
. Results and Discussion
3
4
5
6
1
H NMR shifts
(ppm)
Calculated distances and angles
2
.1. Synthesis and Product Identification
NH'···O=C
A and α
H4'··O=C
B and β
2.21
ΔE
kJ/mol
Parameter
Dist. (Å)
angle (º)
Dist. (Å)
angle (º)
Dist.(Å)
c
NH
ArH4'
The initial reactions with indirubin were performed as
1
2.04
124.3
2.03
124.2
-
7 reported for the allylation of indigo. In a typical reaction, a
8 solution of indirubin in DMF in the presence of caesium
9 carbonate was sonicated for 30 minutes to aid solubility and the
0 formation of the anion. The reaction was heated to 70 °C
1 followed by the addition of the allyl bromide and heating,
a^
a
2
9
57.7
61.6
36.1
11.00
8.76
120.3
2.21
b
b
10.53
-
8.89
120.1
2.15
initially at 70 °C for 16 hours. The outcomes of these reactions
are summarized in Scheme 1. The use of the parent allyl bromide
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8.63
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angle (º)
-
117.7
resulted in a 10% yield of the known oxidative cleavage
product N-allylisatin (Type A), together with the pink mono
oxindolic N-allylated indirubin 9 (Type B) and the deep blue
diallylindirubin 14 (Type C) in 24% and 19% yields,
respectively. Even from this initial reaction, it was clear that
indirubin is significantly less reactive than the corresponding
indigo. The use of substituted allyl bromides continued the trend
with the pink mono allylated derivatives (Type B) being
produced in yields from 20-49% in 16 hour reactions (entries 1-
a
b
c
in DMSO-d
6
;
in CDCl
3
; ΔE: Energy difference between E and Z isomers;
^ both NH signals are assigned to the broad peak at 11.00 ppm
Figure 3. The Z-isomer of indirubin (2), N'-allylindirubin (9) and, N,N'-
diallylindirubin (14): Illustrated on 9 – the calculated angles are indicated by
dashed lines, distances are indicated by arrows. NH1--O=C2' distances and
angles as well as ArH4'--O=C3 distances and angles and the corresponding
1
H NMR shifts of indirubin (2), N'-allylindirubin (9) and N,N'-diallylindirubin
(14). Calculated data are obtained using Hartree-Fock theory after
4
), with an extended 20 hour reaction required to produce the N'-
optimization at the 6-31G* level.
cinnamylindirubin 13 (21%). The diallylated indirubins (Type C)
were also produced in the same reactions in yields of 3-30%
although these overall ratios are somewhat misleading due to the
variable stability of the Type C compounds under the reaction
conditions. For example with cinnamyl bromide and a 4 hour
reaction time the N,N'-dicinnamylindirubin 18 was isolated in an
In order to assess whether either the mono- or di-allylated
products had undergone isomerization during the reaction
process to form the 2,3'-E products (with a syn disposition of the
carbonyl groups), and to support the product identifications, we
undertook a series of computational experiments to characterize
the monallylindirubin 9 and the diallylindirubin 14.
1
1% yield (entry 6), but in a 20 hour reaction, the major outcome
was the Type B N'-cinnamylindirubin 13. The instability of the
Type C structures is presumed to arise from the removal of the

3
Calculated distances (NH1···O=C2') and AanCgleCs E(NP-TH·E··DO) MA7)N, aUrisSinCg fRroImPtThe introduction of three allylic moieties; at 70
1
between the amine group and the proximal carbonyl oxygen in
indirubin (2) and its N'-allylated (9) and N,N'-diallylated (14)
derivatives highlight the hydrogen bond character (Figure 3).
Additionally, the distance (H4···O=C3) and angle (C-H···O)
between the aromatic hydrogen H4' and the proximal carbonyl
°C, 19 (see also Figure 4) was isolated in only 2% yield. The H
NMR of 19 showed a ddd centred at 2.06 ppm with J = 18.3, 5.3
and 1.8 Hz, assigned to the sp alkenyl H8' - the corresponding
H8' was assigned to the dt (J = 18.3, 1.8 Hz) at 3.00 ppm. The
signals assigned to H6' and H7' are contained within multiplets at
7.00 - 7.06 and 5.02 - 5.41 ppm respectively. The signal in the
C NMR spectra at 48.0 ppm was assigned to the spiro C3, and
2
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9
a
b
1
group are in accordance with the remarkable H NMR downfield
shift of the aromatic hydrogen H4' and indicate the strong
1
3
interaction (“H-bond”) of H4' with the proximal carbonyl group.
the C2 amide carbonyl was assigned to the peak at 175.6 ppm
whereas the peak at 199.1 ppm was attributed to the C10'
carbonyl. Confirmation of the molecular formula of 19 was
provided by analysis of the HR ESI mass spectrum which
The initial reaction product results at 70 °C clearly indicated a
lower reactivity of indirubin to subsequent cyclisation. Therefore,
in an attempt to force potential cascade reactions, the reactions
+
indicated an exact mass of 383.1751, assigned to the [M + H]
1
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0 were repeated at 110 °C (Scheme 1, entries 7-12). This gave rise
1 to the new spiroindolinepyrido[1,2-a]indoledione heterocycles of
2 the type D in up to 70% yield in the case of allyl bromide (entry
ion of C H N O .
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Temp Time
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Entry
1
R
R
R
A
B
C
D
Y
°C
h
70
16
H
H
H
4 (10%) 9 (24%)
14 (19%) 19 (2%)
2
3
4
5
6
70
70
70
70
70
16
16
16
20
4
Me
H
H
H
H
5 (3%)
10 (20%) 15 (30%)
Me
6 (trace) 11 (49%) 16 (9%)
H
Me Me 7 (trace) 12 (29%) 17 (3%)
H
Ph
Ph
H
H
13 (21%)
H
18 (11%)
7
8
110
110
6
6
H
H
H
H
H
19 (70%)
20 (37%)
Me
10 (10%)
12 (10%)
†
9
110
110
110
6
6
6
H
H
H
Me
H
21 (24%)
1
1
0
1
Me Me
Ph
Ph
H
H
8 (9%)
22 (8%)
1
2
110
24
H
22 (13%)
Scheme 1. The products of the reactions of indirubin 2 with a series of allyl bromides: Type A - isatin derivatives (4 - 8), Type B
N'-substituted indirubin derivatives (9 - 13), Type C - N,N'-disubstituted indirubin derivatives (14 - 18) and Type D -
-
†
spiroindolinepyrido[1,2-a]indoledione derivatives (19 - 22). Note: C-allyl substituent is not crotyl but has rearranged – see
Figure 4 for details of structure. Trace indicates detected by TLC analysis at the completion of the reaction. # 3 Å sieves were
additionally added to procedure B reactions.

4
Tetrahedron
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Figure 4. Crystal structures of the spiroindolinepyrido[1,2-a]indolediones 19, 20 and 21.
1
9 Analysis of the H NMR spectra of 21 revealed a dd at 5.03 ppm,
0 assigned to the H7' olefin and multiplets at 7.08-7.16 and 3.40-
1 3.47 ppm which were assigned to H6' and H8' within these peaks
2 respectively (Figure 4). The terminal methyl group of the N-1-
3 butenyl substituent was assigned to the doublet at 1.72 ppm (J =
4 7.3 Hz) whereas the methyl substituent of the N-2-butenyl
5 substituent was assigned to the doublet at 0.52 ppm (J = 8.0 Hz).
6 The terminal olefinic methylene of this substituent was assigned
the extra bulk of the substituted allyl moieties sterically impeding
the spiro cyclisation. Further, in the 110 °C reactions (entries 7-
12), there was no evidence of the presence of Type C products
with Type B compounds probably reacting further at the higher
temperature to the Type D products. The N-allylisatins were only
detected in the 110 °C reactions in the case of the cinnamyl
moiety (9%) at 6 h (entry 11). It is likely that the bulky cinnamyl
unit lead to a slower reaction, allowing time for the oxidative
cleavage to occur. The longer reaction times at higher
temperatures probably resulted in further degradation in the
reaction mixture.
to the doublet of doublets at 4.64 ppm (J = 10.0, 1.8 Hz) and 4.86
ppm (J = 17.0, 2.1 Hz) with the former in a cis orientation to the
adjacent CH (multiplet at 5.72-5.89 ppm) and the latter in the
relative trans orientation. These assignments indicated the
presence of the C-methylallyl substituent, possibly formed by a
rearrangement process after O-alkylation as discussed in the
mechanistic section.
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Along with these major products, minor fragmentation
products were isolated including the anthranilate esters allyl 2-
(allylamino)benzoate
and
2-methylallyl
2-((2-
methylallyl)amino)benzoate in very low yield from the respective
allylation reactions at 110 °C and 70 °C. Structural elucidations
of these esters arose from analysis of spectral data, with a
diagnostic absorption band for the ester carbonyl group being
The structures of the spiroindolinepyrido[1,2-a]indolediones
9, 20 and 21 were confirmed by single crystal X-ray
1
6 crystallographic analysis, corroborating the C-allylic substituents
7 and relative stereochemistry with the syn disposition of the C-
8 allylic substituent and the 3,8' C-C bond in each case (Figure 4).
9
-
1
-1
apparent at 1673 cm for the former and at 1682 cm for the
latter.
2
4,25
3
There are reports
of the synthesis of molecules with the
For comparison purposes with the indigo substrate, reaction
0
1
2
3
4
5
6
same spiro heterocyclic skeleton to that in 19-22, however these
arose from reaction of skatole with 2-methylindole, followed by
acylation in polar solvents – subsequent examination of the by-
products revealed a spiro-type system, however, there was
limited spectral evidence to support the proposed structures.
of propargyl bromide with indirubin in the presence of base at 70
°C for 16 hours only yielded N,N'-dipropargylindirubin 23 in low
yield (13%) plus some (8%) of the isatin-derived anthranilate
ester, prop-2-yn-1-yl 2-(prop-2-yn-1-ylamino)benzoate.
2.2. Mechanistic Proposals
The yields for the spiroindolinepyrido[1,2-a]indoledione
The proposed mechanism for the 70 °C reactions proceeds
with N-allylation on the oxindole ring producing the Type B
compounds with a simple second allylation affording the Type C
compounds (Scheme 2). In the 110 °C reactions, the
diallylindirubin (Type C) upon O-protonation (tentatively with
the weakly acidic bicarbonate ion as the proton source) and
subsequent electron redistribution yields the intermediate ion X,
which under base-catalysed conditions, undergoes cyclisation to
form the spiro moiety (intermediate Y), with the C3' position
activated by the conjugated protonation nitrogen atom.
Subsequent O-allylation would generate the intermediate Z, a
molecule not yet isolated from this reaction, as it is presumably
converted rapidly to the isolated Type D spiro compounds. A
number of possibilities exist for the mechanism converting Z to
Type D compounds: a 1,3-Claisen rearrangement would yield the
7 systems from indirubin decrease with substitution of the allyl
bromide, e.g. the yield drops to 37% with the additional methyl
substituent at C2 of the allyl moiety (entry 8) and down to 13%
with the use of cinnamyl bromide (entry 12), this latter reaction
requiring significantly longer reaction times (24 h) to achieve
even this yield. With a very bulky terminal gem-dimethyl group
in the allylic bromide, no spiro-cyclization was observed. This
trend is likely to arise from the steric impedance of the
orthogonal spiro scaffold blocking the approach of the substituted
allyl units during the installation of the C-allyl substituent (see
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Section
2.2
Mechanistic
Proposals).
Although
the
spiroindolinepyrido[1,2-a]indoledione 19, arising from addition
of allyl bromide, was also observed in the reaction performed at
0 °C in a 2% yield (entry 1), no other examples were detected in
reactions at this temperature (entries 2-6), probably indicative of
7

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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
Scheme 2. Proposed mechanism towards the formation of the spiroindolinepyrido[1,2-a]indolediones 19 - 22.
observed outcome for spirocycles 19 and 20, but not for 21 and
2. Similarly, a route via enolate anion formation followed by a
and then addition to the C3 carbonyl, followed by fragmentation
with loss of carbon dioxide and propene would give the minor
anthranilate ester observed.
2
direct C9a allylation process, while satisfactory for most
examples, does not explain outcome 21. A possible unifying
mechanism that explains all results is a thermally-induced
homolytic cleavage radical cleavage of the allyloxy group in Z to
produce two highly stabilised radical species, which could then
2.3. Colour Properties
Indirubin (2) as well as its N'-substituted derivatives (9-13) are
red compounds with UV/Vis absorption maxima of about 536 nm
and 533 nm in methanol whereas the N,N'-disubstituted
derivatives (14-18) without an NH…OC hydrogen bond are blue
and have a UV/Vis absorption maximum of about 575 nm in
methanol. Selected data is given in Table 1.
2
react through attack at C9a to afford 21. With the comparable
cinnamyl product 22 it is likely that steric factors mitigate against
the cinnamyl radical at the carbon bearing the phenyl group.
1
3
The C NMR spectra for these heterocycles showed no
duplication in peaks, consistent with the formation of a single
enantiomeric pair out of the four possible diastereomers. The
proposed mechanism is in accordance with this observation with
the initial cyclisation to form the spiro atom likely to produce
both possible stereoisomers as approach from either face would
be equal. The generation of the second stereogenic atom occurs
during the final rearrangement step and here, there is a clear
facial discrimination with the approach of the allyl unit (probably
an allylic radical species) from the less hindered carbonyl side.
The higher temperature of 110 °C is required to drive the reaction
through to the Type D spiro compounds, while at 70 °C, N-
monoallylation and N,N'-diallyation products predominate. In a
separate experiment, reaction of the N-monoallylated indirubin
Calculated UV/Vis absorption maxima differ significantly
from the experimental values but the increase in λmax from the N'-
allylated to the N,N'-diallylated indirubin is predicted correctly
with the Hartree Fock and DFT methodology. However, the
calculated wavelengths using B3LYP theory are closer to the
experimental wavelengths in comparison to Hartree-Fock theory.
Taking into account the solvent effect, which leads to a
bathochromic shift of the absorption maximum wavelengths the
more polar the solvent used, the B3LYP results are acceptable as
the calculated values are in vacuo (Table 2). In contrast to the
indirubins, the spiroindolinepyrido[1,2-a]indoledione (19) is
yellow, reflecting the absence of the 2,3'-double bond and the
consequent decrease in conjugation. In order to examine this
further, the calculated UV/Vis absorption spectra in vacuo and
experimentally obtained UV/Vis spectra of 19 were compared
(9) with excess allyl bromide at 110 °C for 20 h, only produced a
complex mixture with no indication of the
spiroindolinepyridoindole heterocycle (14), presumably due to
further reaction of this compound under the conditions. We
observed that N,N'-disubstituted indirubins were not stable in
solution at room temperature under light exposure. When N,N'-
diallylindirubin was kept in dichloromethane for several days, its
colour gradually changed from dark blue to reddish-yellow. It is
known that indigo is stable against photoinduced Z -> E
isomerisation due to its stabilizing NH hydrogen bonding, but
substitutions on both nitrogens results in a loss of hydrogen
(
Table 1). Again, density functional theory (B3LYP) correctly
predicted the observed large decrease in λmax for 19 compared
with 14. The predictive power of DFT has been well established
by Jacquemin et al for indirubin, isoindigo and derivatives
including the observation that better fits between theory and
experiment occur when bulk solvent effects are included in the
calculations. Further investigations are required to more fully
examine this with compounds 9 and 14.
2
5,26
2
6
The loss of a hydrogen bond should lead in theory to an
increased energy difference between the ground state and the
excited state and therefore to an UV/Vis absorption maximum at
bonding and leads to phototropic behaviour. The same loss of
H-bonding may be the reason for the low stability of the
2 disubstituted indirubins (c.f. Fig. 3): the Z configuration of
3 indirubin and its N'-substituted derivatives exhibit stabilizing
4 NH-mediated hydrogen bonds and are therefore inert against Z to
5 E isomerisation, while this is not the case for the disubstituted
6 indirubins and photoinduced Z to E isomerization occurs.
2
9,30
lower wavelengths.
However, the opposite is observed with
the calculated values (2 to 9). This bathochromic shift can be
explained by referring to the „cross-conjugated‟ system („H-
chromophore‟), which was found to be an important feature to
3
1
explain (although other explanations have been proposed ), the
7
32,33
The formation of the isatin based products can be rationalised
in terms of oxidative cleavage with adventitious oxygen.
Subsequent nucleophilic attack by allyl alcohol (formed from the
bromide in the presence of carbonate) on the isatin C2-carbonyl
colour of indigoid molecules.
It consists of a C=C double
8
9
0
1
2
3
4
5
22
bond substituted with two electron donor groups (EDGs; in an
anti configuration to each other) and two electron withdrawing
groups (EWGs; in an anti configuration to each other). In

6
Tetrahedron
indirubin and the N-allylated derivatives suchAaCchCroEmoPpThoEreDis MAN
T
U
abl
S
e
C
2.
R
A
I
nt
P
i-cancer, anti-tubercular, and anti-plasmodial activity of
T
present with the two carbonyl moieties as EWGs and an amino
group and a fused aryl ring as electron donor groups.
Calculations of the UV/Vis absorption maxima using B3LYP
compounds against three different cancer cell lines small cell lung cancer
(NCI-H187), KB-oral cavity and MCF 7 breast cancer, all tested using a
resazurin microplate assay (REMA), as well as anti-TB (Mycobacterium
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
(
after optimization at the 6-31G* level) on 2, 9 and 14, as well as
tuberculosis, H37Ra strain), tested using a green fluorescent protein
cut down oxopyrrolidinylideneindolinone theoretical model
systems lacking one fused benzene ring, supported the
significance of the H-chromophore in determining the colour of
these systems (see Supplementary Information for the structures
of the model systems and the full results).
microplate assay (GFPMA) and anti-plasmodial activity (Plasmodium
falciparum, K1 strain), tested using a microculture radioisotope technique.
Lung
Cancer
IC50
Oral
Cancer
IC50
Breast
Cancer
IC50
Anti-
Anti-
Plasmodial
IC50
TB
IC50
Table 1. Summary of experimentally obtained UV/Vis absorption
maxima (spectra are measured in methanol) and calculated UV/Vis
absorption maxima (spectra are calculated in vacuo) using B3LYP and
Hartree Fock theory after optimization at the 6-31G* level. The numbers in
between rows are the differences between absorption maxima for the
respective compound pairs.
μg/mL
μg/mL
(µM)
μg/mL
(µM)
μg/mL
(µM)
(µM)
μM
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
4
9.37
(50.1)
2.9
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
5
-
-
-
-
-
-
Colour
UV/Vis absorption maxima (λmax, nm)
(
14.4)
5.86
Experimental
(CH Cl
B3LYP
Hartree
Fock
9
Compound
(solid)
red
2
2
)
(19.4)
indirubin (2)
536
474
+16
311
10
11
12.50
-3
+6
(39.6)
N'-allylindirubin (9)
red
blue
533
490
+22
317
335
335
7.52
+
44
+18
0
(23.8)
0.66
1
1
1
1
1
2
3
5
6
7
3 N,N'-diallylindirubin
4 (14)
5
(2.0)
33.78
577
426
512
43.37
(114.7)
12.37
-151
-127
6
(89.4)
7 spiroindolinepyrido[1,2-
6.46
3.94
8 a]indoledione (19)
9
0
yellow
385
5
0.00
(17.5)
20.69
(33.4)
(10.6)
4.82
2
.4. Biological Activity
As indirubin and its derivatives are known to be active against
-
-
-
-
-
-
-
1
2
(55.9)
(13.0)
20.72
15.19
(38.1)
29.98
(88.7)
18.94
(47.8)
3 various cancer cell lines, we screened our derivatives against a
4 small number of cancer cell lines, namely small cell lung cancer
5 (NCI-H187), KB-oral cavity cancer and MCF-7 breast cancer, as
6 well as testing for in vitro anti-TB (Mycobacterium tuberculosis,
7 H37Ra strain) and anti-plasmodial (Plasmodium falciparum, K1
8 strain) activity. The results are summarized in Table 2.
9
-
-
(52.1)
23
33.15
(98.1)
1
9
9.82
3.69
(9.3)
-
-
-
-
-
-
-
-
(24.8)
The isatin derivatives 4 and 5 showed modest cytotoxic
,
0
1
2
3
4
5
6
20
activity against the small cell lung cancer cell line but not against
the other tested cancer cell lines, nor the mycobacterial or
plasmodial strains. The N'-substituted and N,N'-disubstituted
indirubins show activities against the lung cancer cell line with
the most potent compound being the mono diamethylallyl
substituted derivative 12, with an IC50 value of 0.66 μg/mL (2.0
µM) comparable to the positive controls ellipticine (1.47 μg/mL;
5
-
-
-
-
-
-
2
1
22
7.94
11.07
(26.1)
0.737
3.54
(8.3)
(18.7)
Ellipticine
1.47
7
8
9
.97 µM) and doxorubicin (0.077 μg/mL; 0.147 µM). A little
surprisingly, compound 12 was inactive against the other two
cancer cell lines. The most promising compound in the N,N'-
Doxorubicin 0.077
Tamoxifen
0.504
7.97
0 disubstituted indirubin series was the 2-methylallyl derivative 15,
1 with activity against all three cancer cell lines, and being
2 somewhat better than the positive controls against the rather
3 refractory MCF-7 cell line; modest anti-TB activity in vitro was
4 also apparent with this compound, while the disubstituted
5 indirubin 16 was active in the anti-plasmodial assay. Although
9.47
Rifampicin
0.025
0.625
1
3
6 indirubin derivatives have been shown
to inhibit the
Streptomycin
Mefloquine
7 apicomplexan protozoal parasite Toxoplasma gondii, to the best
8 of our knowledge anti-plasmodial activity of N,N'-disubstituted
9 indirubins involving the apicomplexan Plasmodium falciparum
(0.0303)
has not been reported previously. However, derivatives with the
0
1
2
3
4
5
-
= inactive.

7
isomeric isoindigo skeleton do show such iAnhCibiCtoEryPaTctEivDity MAsaNmpUleSs CusiRngIPa TNicolet Avatar 360 FT-IR spectrometer fitted
1
7
against P. falciparum.
with a Smart Omni-Sampler germanium crystal accessory. IR
data is recorded in nanometers (nm) with peak intensity assigned
as weak (w), medium (m) or strong (s). Low resolution mass
spectra were obtained by electrospray ionisation (ESI) mass
spectroscopy on a Micromass Platform LCZ spectrometer by
injecting the samples as a solution in methanol. In some cases 1%
HCOOH was added to suppress dimerization and/or aid in
protonation. Alternatively, electron impact (EI) mass spectra
were performed using a Shimadzu QP-5050 spectrometer. High
resolution mass spectrometry (HRMS) was performed using
electrospray ionisation technique on a Waters QTOF Xevo
spectrometer or electron impact technique on a Fison/VG
Autospec-TOF spectrometer at 70 eV with a source temperature
of 250 °C. Ion mass charge (m/z) values of molecular ions (M),
fragment peaks and adducts are stated with their relative
abundances in parentheses. For compounds with more than one
major isotope all significant isotopic peaks are reported. Proton
Of the four spiroindolinepyrido[1,2-a]indolediones tested,
anti-proliferative activity was seen only with compounds 19 and
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
2
2, although no clear SAR outcomes can be drawn from this
limited series. Promising anti-plasmodial activity in the low
micromolar range was also seen with 19 and 22, and these
compounds could serve as useful new leads for potential
antimalarial drug development, with the incorporation of a spiro
3
4,35
element in the framework of particular interest.
. Conclusions
We report here for the first time cascade reactions of indirubin
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
through the reaction with allylic bromides in the presence of
base. The initial reaction can be controlled to produce the pink-
red N-monoallylated products, but the subsequent synthesis of
the deep blue N,N'-diallylated indirubin was less successful due
to the instability of the products. The cascade process begins
when the reaction is heated to 110 °C – these are higher
temperatures to induce the ongoing reaction process when
compared to the reactions of the related indigo where the cascade
1
13
( H) and carbon ( C) nuclear magnetic resonance (NMR) spectra
were recorded at 500 and 125 MHz respectively on a Varian
Inova 500 MHz spectrometer or a VNMRS PS54 500 MHz
spectrometer. Alternatively, H and C NMR spectra were
recorded at 300 and 75 MHz respectively on a Varian Mercury
1
13
process completely consumes the starting material after 1 h at 88
2
0 °C. Therefore, the indirubin molecule is more stable than indigo.
1 The overall result of the cascade allylation of indirubin is the
2 unexpected one-pot production of spiroindolinepyrido[1,2-
3 a]indoledione heterocycles. This method provides simple access
4 to these relatively complex structures with good
5 diastereoselectivity. Importantly, this chemistry provides a facile
6 route to the spirooxindole skeleton, a motif that is becoming
7 increasingly important with the emergence of numerous
3
00 MHz spectrometer. All NMR shifts are reported in parts per
million (ppm) and the δ values reported relative to a standardised
value (standard in brackets). NMR data was acquired in CDCl
3
1
13
(TMS, H: δ = 0 ppm; CDCl , C: δ = 77.0 ppm), (CD ) SO
(DMSO, H: δ = 2.50 ppm; (CD ) SO, C: δ = 39.5 ppm),
CD OD (methanol, H: δ = 3.31 ppm; CD OD, C: δ = 49.0
ppm) and (CD ) CO (acetone, H: δ = 2.05 ppm; (CD ) CO, C:
3
3 2
1
13
3
2
1
13
3
3
1
13
3
2
3
2
δ = 29.8 ppm). Coupling constants (J) are reported in Hertz (Hz)
and refer to coupling between hydrogen nuclei over three bonds
if one coupling constant is reported. If two coupling constants are
reported, the smaller of the two refers to coupling between
hydrogen nuclei over four bonds. Multiplicities are reported as
singlet (s), broad singlet (bs), doublet (d), doublet of doublet
(dd), triplet (t), doublet of triplet (dt), quartet (q), pentet (p),
sextet, septet and multiplet (m). Assignment of carbons and
protons for all compounds are based on the 1D and 2D NMR
spectroscopic experiments APT, gCOSY, gHSQC, gHMBC and
NOESY unless otherwise stated.
3
6-38
8 examples both from nature and with biological activity.
9 results reported here highlight this importance with examples of
The
derivatives which show significant activity as lead compounds
against P. falciparum and micromolar activity against 3 different
cancer cell lines. Therefore, this new chemistry of indirubin
presents fresh possibilities to explore heterocyclic chemical space
in a compact one-pot synthetic regime, which should be
amenable to further generalisation.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
3
.1. Experimental section
.2. General Methods
UV/Vis spectra were obtained on dichloromethane solutions
using a double beam UV-Vis-NIR spectrophotometer (Cary 500)
operating between 300 – 1400 nm. All solutions were
appropriately diluted with dichloromethane prior to analysis to fit
within the absorbance limits of the detector, and placed into 1 cm
pathlength quartz cuvettes. All spectra were collected at room
temperature. Sample sizes were between 1.000-2.000 mg,
weighed with an accuracy of 6 decimal places using the seven
figure balance CAHN C-35. Monosubstituted indirubin
derivatives were diluted to 50.0 mL and all other indirubin
DMF for dry reactions was obtained from a solvent
purification system. Dichloromethane for extractions and column
chromatography was distilled in air prior to use or HPLC grade
dichloromethane was used, while other solvents were purchased
reagent grade and used without further purification. Indirubin (2)
was synthesized according to a literature procedure from isatin
10
and 3-iodo-(1H)-indole. Reactions were carried out under
nitrogen which was dried by passage through a 20 cm tube filled
with CaCl or silica based drying agent, unless otherwise stated.
2
-
derivatives to 25.0 mL The ε values reported have the units M
Solvents were removed in vacuo on a rotary evaporator and
products dried under high vacuum (~1 mbar) at room
temperature. Flash column chromatography was performed using
1
-1
cm .
X-ray Structure Determination – images were measured on a
Nonius Kappa CCD diffractometer (MoKα, graphite
monochromator, λ = 0.71073 Å) and data extracted using the
DENZO package. Structure solution was by direct methods
SIR92). The structures were refined using the CRYSTALS
program package. Atomic coordinates, bond lengths and angles,
and displacement parameters for compounds 19, 20 and 21 have
been deposited at the Cambridge Crystallographic Data Centre
CCDC 1048189-1048191). These data can be obtained free-of-
charge via www.ccdc.cam.ac.uk/data_request/cif, by emailing
data_request@ccdc.cam.ac.uk, or by contacting The Cambridge
1 Merck flash silica gel 60 (63-200 mesh), particle size 40-63 μm,
2 under a positive pressure of air. Preparative TLC (PTLC) was
3 performed using Merck silica gel F254 pre-coated glass plates
4 (20 x 20 cm) with a layer thickness of 500, 1000, 1500 or 2000
5 μm.
39
4
0
(
41
6
7
8
9
Compounds were visualized under UV light (254 and 365 nm)
using Merck silica gel F254 precoated aluminium plates. Melting
points (Mp) were determined using a Gallenkamp (Griffin)
(
melting point apparatus, are uncorrected and stated in degrees
Celsius (°C). Infrared (IR) spectra were recorded with neat
0
1
2
3
4
5

8
Tetrahedron
Crystallographic Data Centre, 12 Union RoadA, CCamCbEriPdgTe ECBD2 MA(CN7'aU),S1C42R.4I(PCT2), 154.4 (C7a), 166.8 (C2'), 188.5 (C3). MS
+
+
1
EZ, UK; fax: +44 1223 336033.
(EI): m/z 342 (M , 100%), 301 (M -C H , 52). MS (ESI): 343
3
5
+
(M+H , 100%). HRMS (ESI): calculated for C H N O :
22 19 2 2
Computational Methods
3
43.1447, found 343.1425.
Further elution of the SiO flash column provided N-allylisatin
1
2
3
4
5
6
7
8
9
Geometry Optimisations. The calculations were performed in
2
vacuo throughout using the Spartan package 2010 (V1.1.0) as the
calculation tool and the geometry of the molecule in the ground
state was optimized using Hartree-Fock (HF) theory and the 6-
1
(4) (29.0 mg, 10%) as an orange solid. Mp 87-88 ºC (Lit. 89-91
-1
°
C). UV/Vis: λmax (ε) 410 (435) nm. IR [cm ]: ν 2959 (w), 2919
(w), 2847 (w), 1722 (s), 1601 (s), 1464 (s), 1348 (m), 1183 (w),
3
1G* basis set.
1
1085 (w), 925 (w), 760 (s). H NMR [500 MHz]: δ 4.37 (2H, d, J
= 5.5 Hz, H3'), 5.29-5.35 (2H, m, H1'), 5.82-5.88 (1H, m, H2'),
6.90 (1H, d, J = 8.0 Hz, H7), 7.13 (1H, t, J = 8.0 Hz, H5), 7.57
UV/Vis spectra. The Spartan package 2010 (V1.1.0) was used
as the tool to calculate the spectra of the molecules in the ground
state using either density functional theory and the hybrid
0 functional B3LYP or HF theory after geometry optimisation with
1
3
(1H, t, J = 8.0 Hz, H6), 7.62 (1H, d, J = 7.5 Hz, H4). C NMR
[125 MHz]: δ 42.7 (C3'), 111.1 (C7), 117.8 (C3a), 118.9 (C1'),
124.0 (C5), 125.6 (C4), 130.6 (C2'), 138.5 (C6), 151.1 (C7a),
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
1 the 6-31G* basis set.
+
2
158.1 (C2), 183.5 (C3). MS (ESI): m/z 188 (M+H , 100%).
3
.3. Synthesis
3
4
5
6
7
8
9
0
1
2
3
4
5
6
3
.3.1.2. 2-Methylallyl 2-((2-
Synthetic Note: All potentially chiral compounds in this work
were isolated or used as racemates.
methylallyl)amino)benzoate; (Z)-1'-(2-Methylallyl)-
[2,3'-biindolinylidene] -2',3-dione (10); (Z)-1,1'-
bis(2-methylallyl)-[2,3'-biindolinylidene] -2',3-
dione (15); 1-allylindoline-2,3-dione (5).
3
.3.1. General Procedure A.
A suspension of indirubin (1.0 eq.) in anhydrous DMF (20
mL) was sonicated for 5 min. To the stirred solution was added
caesium carbonate (2.4 eq.) and the reaction mixture was heated
to 70 °C. Allylic bromide (or derivative) (2.9 eq.) was added and
the reaction mixture was stirred for the specified time at 70 °C.
The mixture was filtered while hot, washed with a little DMF and
the filtrate concentrated under reduced pressure. The crude
residue was subjected to flash silica gel column chromatography
Following the general procedure A, a suspension of indirubin
(201 mg, 767 μmol) in DMF (20 mL) was heated with caesium
carbonate (600 mg, 1.84 mmol) and 3-bromo-2-methylpropene
(224 μL, 2.22 mmol) for 16 h to obtain 2-methylallyl 2-((2-
methylallyl)amino)benzoate as a colourless oil (12.8 mg, 7%). IR
[cm ]: ν 3371 (w), 2919 (m), 2845 (w), 1682 (s), 1222 (s). H
NMR [500 MHz]: δ 1.79 (3H, s, H1''), 1.84 (3H, s, H1'''), 3.78
(2H, s, H3), 4.70 (s, 2H, H3'), 4.89 (1H, s, H1 ), 4.98 (2H, s, H1 ',
-
1
1
(
100% petroleum spirit to CH Cl /petroleum spirit 4:1).
2 2
a
a
H1 ), 5.07 (1H, s, H1 '), 6.59 (1H, t, J = 8.0 Hz, ArH3), 6.63 (1H,
d, J = 8.5 Hz, ArH5), 7.33 (1H, t, J = 8.0 Hz, ArH4), 7.97 (1H, d,
J = 8.0 Hz, ArH2). C NMR [125 MHz]: δ 19.8 (C1''), 20.6
b
b
7 3.3.1.1. N'-Allylindirubin (9); N,N'-diallylindirubin
8 (14); N-allylisatin (4).
9
0
13
(
(
C1'''), 49.0 (C3), 67.6 (C3'), 110.0 (ArC1), 111.2 (C1''), 111.9
ArC5), 112.8 (C1'''), 114.8 (ArC3), 131.7 (ArC2), 134.8 (ArC4),
Following the general procedure A, a suspension of indirubin
202 mg, 771 μmol) in DMF (20 mL) was heated with caesium
carbonate (603 mg, 1.85 mmol) and allyl bromide (193 μL, 2.24
mmol) for 16 h to obtain N'-allylindirubin (9) (56.1 mg, 24%) as
(
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
140.5 (C2''), 142.0 (C2'''), 151.6 (ArC6), 166.2 (C=O). MS (EI)
m/z: 245 (M , 63), 172 (100%). HRMS (EI) m/z: calculated for
C H NO : 245.1416; found 245.1415.
+
a red solid. Mp 177-179 °C. UV/Vis: λ (ε) 535 (10184) nm. IR
15 19
2
max
-1
[
cm ]: ν 3306 (NH, w), 1690 (w), 1654 (s), 1610 (s), 1463 (s),
Further elution of the SiO
methylallyl)-[2,3'-biindolinylidene]-2',3-dione (10) (48.5 mg,
0%) as a red solid. Mp 154-156 °C. UV/Vis: λ (ε) 536
flash column provided (Z)-1'-(2-
1
2
1
4
5
321(s), 1174 (s), 1103 (m), 1036 (m). H NMR [500 MHz]: δ
.46 (2H, d, J = 5.5 Hz, H3''), 5.22 (1H, s, H1'' ), 5.25 (1H, d, J =
a
2
max
.0 Hz, H1'' ), 5.86-5.92 (1H, m, H2''), 6.86 (1H, d, J = 8.0 Hz,
-1
b
(10339) nm. IR [cm ]: ν 3314 (w), 2915 (w), 2845 (w), 1645 (s),
H7'), 6.96 (1H, d, J = 8.0 Hz, H7), 7.00 (1H, t, J = 8.0 Hz, H5),
.12 (1H, t, J = 8.0 Hz, H5'), 7.28 (1H, t, J = 8.0 Hz, H6'), 7.49
1H, t, J = 7.5 Hz, H6), 7.73 (1H, d, J = 7.5 Hz, H4), 8.89 (1H, d,
1
1
604 (s), 1464 (s), 1316 (s), 1168 (s), 1082 (s). H NMR [500
7
(
MHz]: δ 1.76 (3H, s, H1'''), 4.38 (2Η, s, H3''), 4.87 (1H, s, H1''a),
4
8
.95 (1H, s, H1'' ), 6.86 (1H, d, J = 8.0 Hz, H7'), 6.96 (1H, d, J =
13
b
J = 8.0 Hz, H4'), 10.53 (1H, s, NH). C NMR [125 MHz]: δ 42.4
.0 Hz, H7), 7.00 (1H, t, J = 7.0 Hz, H5), 7.12 (t, J = 8.0 Hz,
(
(
(
(
C3''), 106.6 (C3'), 108.8 (C7'), 112.1 (C7), 117.7 (C1''), 120.3
C3a), 121.4 (C2), 121.8 (C5), 122.9 (C5'), 125.5 (C4), 125.8
C4'), 129.4 (C6'), 131.7 (C2''), 137.1 (C6), 139.6 (C3'a), 141.4
H5'), 7.26 (1H, t, J = 8.0 Hz, H6'), 7.49 (1H, t, J = 7.5 Hz, H6),
.72 (1H, d, J = 7.0 Hz, H4), 8.89 (1H, d, J = 7.5 Hz, H4'), 10.54
7
13
(1H, s, NH). C NMR [125 MHz]: δ 20.2 (C1'''), 46.0 (C3''),
C7'a), 151.9 (C7a), 170.6 (C2'), 188.5 (C3). MS (EI): m/z 302
1
1
1
1
06.6 (C3'), 109.0 (C7'), 112.1 (C7), 112.8 (C1''), 120.3 (C3a),
21.3 (C3'a), 121.8 (C5), 122.9 (C5'), 125.5 (C4), 125.7 (C4'),
29.4 (C6), 134.4 (C2), 137.1 (C6), 139.5 (C2''), 141.6 (C7'a),
+
6 (M , 100%). HRMS (ESI): calculated for C H N O : 303.1134,
7 found 303.1143.
1
9
15
2
2
8
+
51.9 (C7a), 170.8 (C2'), 188.5 (C3). MS (EI): m/z 316 (M ,
Further elution of the SiO2 flash column provided N,N'-
diallylindirubin (14) (50.1 mg, 19%) as a blue solid. Mp 82-84
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
100%). HRMS (EI): calculated for C20H N O : 316.1212, found
3
16 2 2
42
-1
16.1218.
°
(
4
5
C. UV/Vis: λmax (ε) 577 (4660 ) nm. IR [cm ]: ν 1670 (s), 1603
1
s), 1461 (s), 1328 (s), 1173(s), 748.3 (s). H NMR [500 MHz]: δ
Further elution of the SiO
flash column provided (Z)-1,1'-
2
.43 (2H, d, J = 5.0 Hz, H3''), 4.82 (2H, d, J = 6.0 Hz, H3'''),
.19-5.23 (3H, m, H1'' , H1'' , H1''' ), 5.28 (1H, d, J = 10.0 Hz,
bis(2-methylallyl)-[2,3'-biindolinylidene]-2',3-dione (15) (85.1
mg, 30%) as a blue solid. Mp 88-90 °C. UV/Vis: λ (ε) 580
a
b
a
max
H1''' ), 5.84-5.89 (1H, m, H2''), 5.94-5.97 (1H, m, H2'''), 6.82
-1
b
(4957) nm. IR [cm ]: ν 1666 (s), 1596 (s), 1464 (s), 1324 (s),
(
1H, d, J = 8.0 Hz, H7'), 7.02-7.09 (2H, m, H5, H5'), 7.12 (1H, d,
1
1
168 (s), 1078 (s), 741 (s). H NMR [500 MHz]: δ 1.50 (3H, s,
J = 8.5 Hz, H7), 7.25 (1H, t, J = 7.5 Hz, H6'), 7.53 (1H, t, J = 7.5
Hz, H6), 7.72 (1H, d, J = 7.5 Hz, H4), 8.63 (1H, d, J = 7.5 Hz,
H1''''), 1.75 (3H, s, H1'''''), 4.35 (2H, s, H3'''), 4.81-4.91 (4H, m,
H1'' , H1'' , H1''' , H1''' ), 4.91 (2H, s, H3''), 6.80 (1H, d, J = 7.5
Hz, H7'), 7.03-7.06 (2H, m, H5, H5'), 7.08 (1H, d, J = 8.5 Hz,
H7), 7.23 (1H, t, J = 7.5 Hz, H6'), 7.51 (1H, t, J = 8.0 Hz, H6),
13
a
b
a
b
H4'). C NMR [125 MHz]: δ 42.5 (C3''), 52.5 (C3'''), 108.7
(
(
(
C7'), 110.9 (C3'), 112.5 (C7), 117.4 (C1''), 118.6 (C1'''), 121.8
C3'a), 122.0 (C5'), 122.4 (C3a), 122.6 (C5), 125.0 (C4), 125.6
C4'), 129.7 (C5'), 131.9 (C2''), 133.8 (C2'''), 136.6 (C6), 142.0
13
7
.72 (1H, d, J = 7.5 Hz, H4), 8.66 (1H, d, J = 8.0 Hz, H4').
C

9
NMR [125 MHz]: δ 19.9 (C1''''), 20.2 (C1''''')A, 4C6.0C(ECP3''T), E54D.5 MAmNetUhySlbCutR-2IP-eTn-1-yl)-[2,3'-biindolinylidene] -2',3-
(C3'''), 108.8 (C7'), 111.0 (C3'), 112.3 (C1''), 112.5 (C7), 114.9
(C1'''), 121.5 (C3'a), 121.9 (C5'), 122.3 (C3a), 122.5 (C5), 125.1
(C4), 125.6 (C4'), 129.8 (C6'), 136.6 (C6), 139.1 (C2'''), 139.7
(C2''), 142.0 (C2), 142.3 (C7'a), 154.5 (C7a), 167.0 (C2'), 188.6
(
dione (17).
Following the general procedure A, a suspension of indirubin
209 mg, 798 μmol) in DMF (20 mL) was heated with caesium
carbonate (624 mg, 1.91 mmol) and 3,3-dimethylallyl bromide
267 μL, 2.31 mmol) for 16 h to obtain (Z)-1'-(3-methylbut-2-en-
-yl)-[2,3'-biindolinylidene]-2',3-dione (12) as a red solid (76.4
mg, 29%). Mp 184-186 °C. UV/Vis: λ (ε) 535 (10777) nm. IR
1
2
3
4
5
6
7
8
(
+
C3). MS (EI): m/z 370 (M , 100%). HRMS (EI): calculated for
(
1
C H N O : 370.1681, found 370.1686.
2
4
22
2
2
Further elution of the SiO2 flash column provided 1-(2-
methylallyl)indoline-2,3-dione (5) (8.0 mg, 3%) as an orange
max
-
1
[
(
cm ]: ν 3316 (w), 2920 (w), 1641 (s), 1600 (s), 1464 (s), 1350
s), 1318 (s), 1166 (s), 1068 (s), 1036 (s), 742 (s). H NMR [300
3
b
1
1
solid. Mp 87-88 ºC (Lit. Mp 87-88 °C). H NMR [500 MHz]: δ
1.77 (3H, s, H1''), 4.30 (2H, s, H3'), 4.98 (2H, d, J = 19.5 Hz,
H1'), 6.89 (1H, d, J = 8.0 Hz, H7), 7.12 (1H, t, J = 7.5 Hz, H5),
MHz]: δ 1.73 (3H, s, H1''), 1.86 (3H, s, H1'''), 4.41 (2H, d, J = 6.3
Hz, H4''), 5.22 (1H, t, J = 6.6 Hz, H3''), 6.83 (1H, d, J = 8.1 Hz,
H7'), 6.93-7.00 (2H, m, H5, H7), 7.10 (1H, t, J = 7.5 Hz, H5'),
7.47 (1H, t, J = 7.2 Hz, H6), 7.71 (1H, d, J = 7.5 Hz, H4), 8.86
(1H, d, J = 7.5 Hz, H4'), 10.53 (1H, s, NH). C NMR [75 MHz]:
δ 18.5 (C1''), 26.0 (C1'''), 38.2 (C4''), 107.1 (C3'), 108.7 (C7'),
112.2 (C7), 118.8 (C2''), 120.4 (C3a), 121.6 (C3'a), 121.8 (C5),
122.8 (C5'), 125.5 (C4), 125.9 (C4'), 129.4 (C6'), 137.0 (C2''),
137.2 (C6), 139.6 (C2), 141.7 (C7'a), 152.0 (C7a), 170.5 (C2'),
9
1
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
0 7.56 (1H, t, J = 7.5 Hz, H6), 7.62 (1H, d, J = 7.5 Hz, H4).
C
1 NMR [125 MHz]: δ 20.1 (C1''), 46.3 (C3'), 111.3 (C7), 113.7
1
3
2 (C1'), 117.7 (C3a), 124.0 (C5), 125.5 (C4), 138.3 (C2'), 138.6
+
3 (C6), 151.3 (C7a), 158.3 (C2), 183.4 (C3). MS (EI): m/z 201 (M ,
4 100%).
5
3
.3.1.3. (Z)-1'-(But-2-en-1-yl)-[2,3'-
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
biindolinylidene] -2',3-dione (11); (Z)-1,1'-di(but-2-
en-1-yl)-[2,3'-biindolinylidene] -2',3-dione (16).
+
+
188.6 (C3). MS (EI): m/z 330 (M , 80), 262 (M -C
HRMS (ESI): calculated for C21 : 331.1447, found
331.1425.
H , 100%).
5
8
H N O
19 2 2
Following the general procedure A, a suspension of indirubin
(
201 mg, 767 μmol) in DMF (20 mL) was heated with caesium
carbonate (600 mg, 1.84 mmol) and crotyl bromide (229 μL, 2.22
mmol) for 16 to obtain (Z)-1'-(but-2-en-1-yl)-[2,3'-
Further elution of the SiO flash column provided (Z)-1,1'-
bis(3-methylbut-2-en-1-yl)-[2,3'-biindolinylidene]-2',3-dione (17)
9.53 mg, 3%) as a blue solid. Mp: 92-94 °C. UV/Vis: λ (ε)
2
h
(
5
max
biindolinylidene]-2',3-dione (11) with a 2.3:1.0 ratio mixture of
E:Z in the N'-side chain (119 mg, 49 %) as a red solid. Mp 190-
1
-1
78 (5892) nm. IR [cm ]: ν 2958 (w), 2917 (m), 2875 (w), 1673
1
-
1
(s), 1603 (s), 1460 (s), 1325 (s), 1163 (s), 1071 (s), 742 (s). H
NMR [500 MHz]: δ 1.66 (3H, s, H1'''), 1.70 (3H, s, H1'''''), 1.71
(3H, s, H1''), 1.84 (3H, s, H1''''), 4.41 (2H, d, J = 7.0 Hz, H4''),
.82 (2H, d, J = 6.0 Hz, H4'''), 5.18-5.24 (2H, m, H3'', H3'''), 6.90
(1H, d, J = 7.5 Hz, H7'), 7.02-7.06 (3H, m, H5, H5', H7), 7.25
(1H, t, J = 7.5 Hz, H6'), 7.51 (1H, t, J = 7.0 Hz, H6), 7.70 (1H, d,
J = 7.0 Hz, H4), 8.61 (1H, d, J = 7.5 Hz, H4'). C NMR [125
MHz]: δ 18.4 (C1'''), 18.5 (CH '''), 25.8 (C1''), 25.9 (CH ''), 38.3
(C4'''), 48.1 (C4''), 108.5 (C7'), 108.7 (C3'), 112.5 (C7), 119.1
(C3''), 120.2 (C3'''), 121.8 (C5'), 122.0 (C3'a), 122.4 (C5), 122.6
(C3a), 124.9 (C4), 125.6 (C4'), 129.6 (C6'), 136.4 (C2'''), 136.5
92 °C. UV/Vis: λmax (ε) 535 (10711) nm. IR [cm ]: ν 3306 (w),
2
923 (w), 1646 (s), 1601 (s), 1459 (s), 1317 (s), 1165 (s), 1103
1
(s), 1072 (s), 743 (s). H NMR [300 MHz]: δ 1.69 (3H, d, J = 6.0
4
Hz, H1''), 4.39 (2H, d, 5.5 Hz, H4''), 5.40-5.55 (1H, m, H3''),
.69-5.77 (1H, m, H2''), 6.89 (1H, d, J = 7.8 Hz, H7'), 6.96-7.03
2H, m, H5, H7), 7.13 (1H, t, J = 7.5 Hz, H5'), 7.29 (1H, t, J =
.0 Hz, H6'), 7.50 (1H, t, 7.4 Hz, H6), 7.73 (1H, d, J = 7.5 Hz,
5
(
8
13
13
3
3
H4), 8.89 (1H, d, J = 8.1 Hz, H4'), 10.56 (1H, s, NH). C NMR
75 MHz]: δ 17.9 (C1''), 41.8 (C4''), 108.6 (C3), 108.9 (C7'),
[
1
1
1
1
12.2 (C7), 120.4 (C3'a), 121.5 (C3a), 121.9 (C5), 122.9 (C5'),
24.6 (C3''), 125.5 (C4), 125.8 (C4'), 128.7 (C2''), 129.4 (C6'),
37.2 (C6), 139.6 (C5), 141.6 (C3'a), 151.6 (C3a), 170.6 (C2'),
(C6), 136.6 (C2''), 142.1 (C7'a), 142.6 (C2), 154.5 (C7a), 166.6
+
+
+
(C2'), 188.9 (C3). MS (EI): m/z 398 (M , 90), 329 (M+-C
5
H
9
,
88.6 (C3). MS (EI): m/z 316 (M , 100%), 262 (M -C H , 40).
+
4
6
5
7), 262 (M -C H , 100%). HRMS (ESI): calculated for
10 16
HRMS (ESI): calculated for C H N O : 317. 1290, found
3
2
0
17
2
2
C H N O : 399.2073, found 399.2060.
26 27
2
2
17.1303.
3
2
.3.1.5. (Z)-1'-Cinnamyl-[2,3'-biindolinylidene] -
',3-dione (13).
Further elution of the SiO flash column provided (Z)-1,1'-
2
di(but-2-en-1-yl)-[2,3'-biindolinylidene]-2',3-dione (16) with a
.5:1.0 ratio mixture of E:Z in the N-side chain and a 2.0:1.0 ratio
mixture of E:Z in the N'-side chain (25.5 mg, 9%) as a blue solid.
5
Following the general procedure A, a suspension of indirubin
(
204 mg, 779 μmol) in DMF (20 mL) was heated with caesium
carbonate (609 mg, 1.87 mmol) and cinnamyl bromide (450 mg,
2.26 mmol) for 16 to obtain (Z)-1'-cinnamyl-[2,3'-
biindolinylidene]-2',3-dione (13) (61.8 mg, 21%) as a red solid.
Mp 180-182 °C. UV/Vis: λmax (ε) 535 (10159) nm. IR [cm ]: ν
3303 (w), 2920 (w), 2847 (w), 1638 (s), 1600 (s), 1464 (s), 1356
s), 1315 (s), 1169 (s), 1074 (s), 742 (s). H NMR [300 MHz]: δ
.58 (2H, d, J = 5.7 Hz, H3''), 6.22 (1H, dt, J = 15.9 Hz, J = 5.7
Hz, H2''), 6.59 (1H, d, J = 15.9 Hz, H1''), 6.87-6.99 (3H, m, H5,
H7, H7'), 7.09 (1H, t, J = 7.8 Hz, H5'), 7.20-7.32 (6H, m, ArH2,
ArH3, ArH4, ArH5, ArH6, H6'), 7.45 (1H, t, J = 7.5 Hz, H6),
7.69 (1H, d, J = 7.2 Hz, H7'), 8.86 (1H, d, J = 7.5 Hz, H4'), 10.51
1H, s, NH). C NMR [75 MHz]: δ 42.1 (H3''), 106.8 (C3'),
08.9 (C7), 112.2 (C7'), 120.4 (C3a), 121.5 (C3'a), 121.9 (C5),
123.0 (C2''), 123.3 (C5'), 125.6 (C4), 125.9 (C4'), 126.8 (ArC2,
ArC6), 128.2 (C6'), 128.9 (ArC3, ArC5), 129.5 (Ar4), 133.1
-
1
Mp 88-90 °C. IR [cm ]: ν 2952 (w), 2914 (m), 2844 (w), 1705
1
(
s), 1606 (s), 1467 (s), 1356 (m), 1176 (m), 1081 (m), 752 (s). H
NMR [300 MHz]: δ 1.63-1.66 (6H, m, H1'', H1'''), 4.36 (2H, d, J
5.7 Hz, H4''), 4.76 (2H, d, J = 6.0 Hz, H4'''), 5.41-5.74 (4H, m,
H3'', H3''', H2'', H2'''), 6.82 (1H, d, J = 8.7 Hz, H7'), 6.70-7.08
2H, m, H5, H5'), 7.16 (1H, d, J = 8.1 Hz, H7), 7.25 (1H, t, J =
.8 Hz, H6'), 7.52 (1H, t, J = 8.1 Hz, H6), 7.70 (1H, d, J = 7.5
h
-
1
=
1
(
7
(
4
13
Hz, H4), 8.62 (1H, d, J = 7.5 Hz, H4'). C NMR [75 MHz]: δ
1
1
1
1
1
3
7.9 (H1'''), 18.1 (H1''), 41.9 (H4'''), 52.0 (H4''), 108.7 (C7'),
10.9 (C3), 112.7 (C7), 121.8 (C5'), 122.0 (C3a), 122.48 (C5'),
22.53 (C3'a), 124.8 (C4), 125.0 (C3''), 125.7 (H4'), 126.3 (C3'''),
26.4 (C2'''), 129.7 (C6'), 130.4 (C2''), 136.6 (C6), 142.2 (C7'a),
42.5 (C2), 154.6 (C7a), 166.8 (C2'), 188.7 (C3'). MS (EI): m/z
13
(
1
+
70 (M , 100%), 315 (M+-C H , 56). HRMS (ESI): calculated
4
7
for C H N O : 371.1760, found 371.1744.
2
4
23
2
2
(
(
(
C1''), 136.5 (ArC1), 137.2 (C6), 139.7 (C2), 141.4 (C7'a), 151.9
C7a), 170.7 (C2'), 188.5 (C3). MS (EI): m/z 378 (M , 86), 263
3
.3.1.4. (Z)-1'-(3-Methylbut-2-en-1-yl)-[2,3'-
+
biindolinylidene] -2',3-dione (12); (Z)-1,1'-bis(3-
+
M -C H , 100%). HRMS (ESI): calculated for C H N O :
9
7
25 19
2
2
3
79.1447, found 379.1454.

1
0
Tetrahedron
3
.3.1.6. (Z)-1,1'-Dicinnamyl-[2,3'-
A suspension of indirubin (1.0 eq.) in dry DMF (20 mL for
ACCEPTED MANUSCRIPT
biindolinylidene] -2',3-dione (18).
763 μmol) was sonicated for 5 min and then 3 Ǻ molecular sieves
40.0 g for 763 μmol) were added. To the stirred solution was
(
Following the general procedure A, a suspension of indirubin
added caesium carbonate (2.4 eq.) and the reaction mixture was
flushed with N and heated to 70 °C. The allyl bromide or an
analogous derivative (4.7 eq.) was added and the reaction mixture
was heated to 110 °C and stirred at this temperature for 6-24 h
under an atmosphere of N . The mixture was filtered while hot,
washed with a little DMF and the filtrate concentrated under
reduced pressure. The crude residue was subjected to flash silica
gel column chromatography (gradient to CH Cl /petroleum spirit
1
2
3
4
5
6
7
8
9
(
201 mg, 767 μmol) in DMF (20 mL) was heated with caesium
carbonate (600 mg, 1.84 mmol) and cinnamyl bromide (443 mg,
.22 mmol) for to obtain (Z)-1,1'-dicinnamyl-[2,3'-
2
2
4 h
biindolinylidene]-2',3-dione (18) with a 1.0:1.1 ratio mixture of
E:Z in the N-side chain and a 1.0:1.5 ratio mixture of E:Z in the
N'-side chain (41.7 mg, 11%) as a blue solid. Mp 89-93 °C. IR
2
-
1
[
(
cm ]: ν 2917 (m), 1673 (s), 1603 (s), 1464 (s), 1325 (m), 1173
s), 1084 (s), 739 (s). H NMR [300 MHz]: δ 2.81 (2H, d, J = 7.2
1
2
2
4
:1).
Hz, H3'') 4.61 (2H, d, J = 6.0 Hz, H3'''), 6.21 (1H, dt, J = 15.9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
0 Hz, 5.7 Hz, H2''), 6.36 (1H, dt, J = 15.9 Hz, 5.7 Hz, H2'''), 6.59
1 (1H, d, J = 15.9 Hz, H1''), 6.63 (1H, d, J = 16.2 Hz, H1'''), 6.88
2 (1H, d, J = 7.8 Hz, H7'), 7.04 (1H, t, J = 8.1 Hz, H5'), 7.10-7.32
3 (12H, m, ArH2, ArH2', ArH3, ArH3', ArH4, ArH4', ArH5,
4 ArH5', ArH6, ArH6', H6', H7), 7.49 (1H, t, J = 7.8 Hz, H6), 7.73
3.3.2.1. Allyl 2-(allylamino)benzoate; 1,9a'-Diallyl-
8'H-spiro[indoline-3,9'-pyrido[1,2-a]indole]-
2
,10'(9a'H)-dione (19).
Following the general procedure B, a suspension of indirubin
1
3
(206 mg, 786 μmol) in DMF (20 mL) was heated with caesium
carbonate (615 mg, 1.89 mmol) and allyl bromide (319 μL, 3.69
mmol) for 6 h to obtain allyl 2-(allylamino)benzoate as a
5 (1H, d, J = 7.8 Hz, H4), 8.65 (1H, d, J = 8.1 Hz, H4'). C NMR
6 [75 MHz]: δ 40.8 (C3''), 42.2 (C3'''), 108.9 (C7'), 111.0 (ArC),
7 112.7 (ArC), 121.9 (ArC), 122.1 (C5), 122.5 (ArC), 122.8 (C5'),
8 123.5 (C2'''), 123.7 (C2''), 124.1 (ArC), 125.1 (C4), 125.8 (C4''),
-1
colourless oil (11.6 mg, 7%). IR [cm ]: ν 3364 (w), 3266 (m),
1
2
914 (w), 2848 (w), 1673 (m), 1232 (s), 1103 (s). H NMR [500
1
1
26.5 (2 x ArC), 126.6 (ArC), 126.76 (2 x ArC), 127.5 (ArC),
28.3 (ArC), 128.8 (2 x ArC), 129.9 (ArC), 131.4 (ArC), 132.8
9
0
1
2
3
4
5
MHz]: δ 3.87 (2H, t, J = 5.5 Hz, H3), 4.78 (2H, d, J = 6.0 Hz,
H3'), 5.18 (1H, d, J = 10.5 Hz, H1 ), 5.27 (1H, d, J = 9.0 Hz,
a
(
(
(
(
ArC), 134.1 (ArC), 136.8 (C6), 142.1 (ArC), 143.1 (C7'a), 154.5
C7a), 166.9 (C2), 178.8 (C2'), 188.6 (C3). MS (EI): m/z 494
M , 40), 377 (M+-C H , 33), 362 (M+-C H , 100%). HRMS
ESI): calculated for C H N O : 495.2073, found 495.2080.
H1 ), 5.29 (1H, d, J = 15.3 Hz, H1 '), 5.40 (1H, d, J = 17.5 Hz,
b
a
+
H1
b
'), 5.91-5.99 (1H, m, H2), 5.99-6.06 (1H, m, H2'), 6.59 (1H, t,
9
9
10 12
J = 7.5 Hz, ArH3), 6.66 (1H, d, J = 8.5 Hz, ArH5), 7.34 (1H, t, J
7.8 Hz, ArH4), 7.86 (1H, bs, NH), 7.96 (1H, d, J = 8.0 Hz,
3
4
27
2
2
=
1
3
3
.3.1.7. Prop-2-yn-1-yl 2-(prop-2-yn-1-
ArH2). C NMR [125 MHz]: δ 45.5 (C3), 65.1 (C3'), 111.8
(ArC1), 114.9 (ArC5), 116.3 (C1), 118.0 (C1'), 131.9 (ArC5),
132.8 (C2'), 134.5 (C2), 134.8 (ArC4), 151.3 (ArC6), 168.5
(C=O). MS (ESI) m/z: 217 (M+H , 100%). HRMS (ESI) m/z:
calculated for C H NO : 218.1181; found 218.1181.
6 ylamino)benzoate; (Z)-1,1'-di(prop-2-yn-1-yl)-
7 [2,3'-biindolinylidene] -2',3-dione.
+
8
9
Following the general procedure A, a suspension of indirubin
1
3
16
2
(
205 mg, 767 μmol) in DMF (20 mL) was heated with caesium
carbonate (600 mg, 1.84 mmol) and propargyl bromide (443 mg,
.22 mmol) for 16 h to obtain prop-2-yn-1-yl 2-(prop-2-yn-1-
ylamino)benzoate (13.2 mg, 8%) as a colourless solid. Mp 53-54
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
Further elution of the silica gel flash column provided 1,9a'-
diallyl-8'H-spiro[indoline-3,9'-pyrido[1,2-a]indole]-2,10'(9a'H)-
dione (19) (210 mg, 70%) as a yellow solid. Mp 189-191 °C.
UV/Vis: λmax (ε) 414 (2014). IR [cm ]: ν 1699 (s), 1605 (s), 1472
(m), 1361 (w), 1197 (w), 752 (s). H NMR [300 MHz]: δ 2.06
2
5
a
1
-1
ºC (Lit. 55-56 °C). H NMR [300 MHz]: δ 2.24 (1H, t, J = 2.7
Hz, H1''), 2.50 (1H, t, J = 2.7 Hz, H1'), 4.04 (2H, dd, J = 5.9 Hz,
1
2
.4 Hz, H3''), 4.87 (2H, d, J = 2.4 Hz, H3'), 6.69 (1H, td, J = 7.5
(1Η, ddd, J = 18.3 Hz, 5.3 Hz, 1.8 Hz, H8'
13.7 Hz, 7.2 Hz, H3''' ), 3.00 (1H, dt, J = 18.3 Hz, 1.8 Hz, H8'
3.33 (1H, dd, J = 14.1 Hz, 6.9 Hz, H3''' ), 4.33 (1H, ddt, J = 15.9
Hz, 5.4 Hz, 3.6 Hz, H3'' ), 4.54-4.61 (1H, m, H3'' ), 4.83 (1H, dd,
J = 9.9 Hz, 2.4 Hz, H1''' ), 5.05 (1H, dd, J = 16.8 Hz, 1.8 Hz,
H1''' ), 5.02-5.41 (4H, m, H1''a, H1''b, H2''', H7'), 5.88-6.11 (1H,
), 2.74 (1H, dd, J =
a
Hz, 0.6 Hz, H5), 6.80 (1H, d, J = 8.4 Hz, H3), 7.43 (1H, td, J =
a
),
b
8
1
.0 Hz, 1.5 Hz, H4), 7.85 (1H, bs, NH), 7.97 (1H, dd, J = 8.1 Hz,
.8 Hz, H6). C NMR [75 MHz]: δ 32.7 (C3''), 52.2 (C3'), 71.6
b
13
a
b
(C1''), 75.0 (C1'), 78.2 (C2'), 79.8 (C2''), 110.4 (C1), 111.9 (C3),
a
1
16.1 (C5), 132.1 (C6), 135.3 (C4), 150.4 (C2), 167.8 (C=O).
b
+
MS (ESI), m/z 214 (M+H , 100%). HRMS (ESI): calculated for
C H NO : 214.0868, found 214.0860.
m, H2''), 6.57 (1H, t, J = 7.5 Hz, H5), 6.68 (1H, t, J = 8.3 Hz,
H2'), 6.74 (1H, d, J = 7.5 Hz, H7), 6.83 (1H, d, J = 7.2 Hz, H4),
1
3
12
2
7
7
.00-7.06 (3H, m, H6, H6', H4'), 7.24 (1H, d, J = 8.1 Hz, H1'),
.45 (1H, t, J = 8.4 Hz, H3'). C NMR [75 MHz]: δ 30.1 (C8'),
Further elution of the SiO flash column provided (Z)-1,1'-
13
2
di(prop-2-yn-1-yl)-[2,3'-biindolinylidene]-2',3-dione (33.8 mg,
3%) as a blue solid. Mp 183-185 °C. UV/Vis: λ (ε) 559
35.0 (C3'''), 42.8 (C3''), 48.0 (C3), 70.4 (C9'a), 103.3 (C1''), 107.8
(C4'), 108.9 (C7), 117.5 (C2'''), 119.2 (C2'), 119.4 (C1'''), 121.8
(C5), 122.0 (C10'a), 122.5 (C6'), 124.3 (C1'), 124.7 (C4), 128.2
(C6), 128.9 (C3a), 131.0 (C7'), 131.6 (C2''), 137.3 (C3'), 142.1
(C7a), 156.1 (C4'a), 175.6 (C2), 199.1 (C10'). MS (EI): m/z 382
1
max
-
1
(4292) nm. IR [cm ]: ν 3261 (w), 3234 (w), 1650 (s), 1606 (s),
1
464 (s), 1330 (s), 1165 (s), 1076 (s), 1019 (m), 930 (m), 756 (s).
1
H NMR [500 MHz]: δ 2.24 (1H, t, J = 2.0 Hz, H1''), 2.27 (1H, t,
J = 2.0 Hz, H1'''), 4.62 (2H, d, J = 2.5 Hz, H3''), 5.02 (2H, d, J =
+
+
+
(M , 74), 341 (M -C
(ESI): calculated for C25
H
5
, 100%), 300 (M -C
H10, 51). HRMS
3
6
2
.5 Hz, H3'''), 7.05 (1H, d, J = 8.0 Hz, H7'), 7.09-7.15 (2H, m,
H N O
23 2 2
: 383.1760, found 383.1751.
H5, H5'), 7.27 (1H, d, J = 8.0 Hz, H7), 7.34 (1H, t, J = 7.5 Hz,
H6'), 7.61 (1H, t, J = 8.0 Hz, H6), 7.75 (1H, d, J = 7.0 Hz, H4),
3.3.2.2. 7'-Methyl-1,9a'-bis(2-methylallyl)-8'H-
spiro[indoline-3,9'-pyrido[1,2-a]indole] -
13
8
3
.70 (1H, d, J = 8.0 Hz, H4'). C NMR [125 MHz]: δ 29.5 (C3''),
9.5 (C3'''), 72.3 (C1''), 74.0 (C1'''), 77.2 (C2''), 77.3 (C2'''), 108.9
2
,10'(9a'H)-dione (20).
(
(
(
(
(
C7'),111.5 (C7'a), 112.6 (C7), 122.05 (C3'), 122.07 (C3'a), 122.6
C5), 123.1 (C5'), 125.1 (C4), 126.3 (C4'), 130.3 (C6'), 136.8
C6), 141.2 (C7'a), 142.2 (C2), 153.7 (C7a), 166.3 (C2'), 188.0
C3). MS (EI): m/z 338 (M , 100%), 299 (M -C H , 78). HRMS
ESI): calculated for C H N O : 339.1134, found 339.1127.
Following the general procedure B, a suspension of indirubin
(205 mg, 782 μmol) in DMF (20 mL) was heated with caesium
carbonate (612 mg, 1.88 mmol) and 3-bromo-2-methylpropene
+
+
3
3
(370 μL, 3.68 mmol) for 6 h to obtain 7'-methyl-1,9a'-bis(2-
2
2
15
2
2
methylallyl)-8'H-spiro[indoline-3,9'-pyrido[1,2-a]indole]-
2,10'(9a'H)-dione (20) (123 mg, 37%) as a yellow solid. Mp 212-
3
.3.2. General Procedure B.

1
1
-
1
1
2
2
14 °C. UV/Vis: λmax (ε) 435 (2274) nm. IR [AcmC]C: νE2P96T9E(wD), MA(sN) HUNSMCRR[3I0P0TMHz]: δ 3.17 (1H, ddd, J = 14.1 Hz, 7.8 Hz,
1
923 (w) 1690 (s), 1600 (s), 1476 (s), 1349 (m), 745 (s). H
1.2 Hz, H3''' ), 3.71 (1H, ddd, J = 13.5 Hz, 7.2 Hz, 1.2 Hz, H3''' ),
a b
,
NMR [500 MHz]: δ 1.51 (3H, s, H1''''''), 1.85 (3H, s, H1''''), 1.89
3H, s, H1'''''), 1.89 (1H, d, J = 18.0 Hz, H8' ), 2.68 (1H, d, J =
4.23 (1H, ddd, J = 16.4, 5.7 Hz, 1.8 Hz, H3'' ), 4.52-4.61 (2H, m,
a
(
H3'' , H8'), 5.30 (1H, dd, J = 8.0 Hz, 2.1 Hz, H7'), 5.74-5.85 (2H,
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
a
b
1
1
1
3.5 Hz, H3''' ), 2.98 (1H, d, J = 18.0 Hz, H8' ), 3.29 (1H, d, J =
m, H2''', H2''), 6.23 (1H, d, J = 16.2 Hz, H1'''), 6.45-6.54 (1H, m,
H1''), 6.68 (1H, t, J = 7.2 Hz, H2'), 6.87 (1H, t, J = 6.6 Hz, H5),
6.9-7.30 (21H, m, ArH2, ArH3, ArH4, ArH5, ArH6, ArH2',
ArH3', ArH4', ArH5', ArH6', ArH2'', ArH3'', ArH4'', ArH5'',
ArH6'', H1', H4', H6', H4, H6, H7), 7.46 (1H, t, J = 7.7 Hz, H3').
a
b
4.0 Hz, H3''' ), 4.32 (1H, d, J = 16.0 Hz, H3'' ), 4.41 (1H, d, J =
b
a
6.0 Hz, H3'' ), 4.59 (1H, s, H1''' ), 4.61 (1H, s, H1''' ), 4.97 (1H,
b
a
b
s, H1'' ), 5.02 (1H, s, H1'' ), 6.54 (1H, t, J = 7.5 Hz, H5), 6.60
a
b
(
1H, t, J = 7.5 Hz, H2'), 6.74 (1H, d, J = 8.0 Hz, H7), 6.78 (1H, d,
J = 8.0 Hz, H4), 6.79 (1H, s, H6'), 6.95 (1H, d, J = 8.5 Hz, H4'),
.01 (1H, t, J = 8.0 Hz, H6), 7.20 (d, J = 7.5 Hz, H1'), 7.41 (1H,
1
3
C NMR [75 MHz]: δ 34.8 (C3'''), 42.3 (C3''), 45.2 (C8'), 53.7
7
(C3), 72.3 (C9'a), 107.8 (C7'), 108.3 (C1''), 109.1 (C1'''), 119.9
(C5), 121.6 (C1''), 123.0 (ArC), 123.3 (C2'''), 123.4 (C2''), 123.5
(ArC), 124.9 (ArC), 125.7 (ArC), 126.39 (2 x ArC), 126.44
(ArC), 126.7 (ArC), 126.8 (2 x ArC), 127.3 (ArC), 127.7 (2 x
ArC), 127.8 (ArC), 128.5 (ArC), 128.6 (2 x ArC), 128.7 (2 x
ArC), 128.8 (ArC), 129.9 (2 x ArC), 132.3 (ArC), 134.7 (ArC),
136.8 (ArC), 137.7 (ArC), 138.0 (ArC1), 142.9 (C7a), 156.1
13
t, J = 7.0 Hz, H3'). C NMR [125 MHz]: δ 20.3 (C1''''), 20.4
(
(
(
(
C1'''''), 24.3 (C1''''''), 36.0 (C8'), 38.4 (C3'''), 46.8 (C3''), 49.0
C3), 70.5 (C9'a), 107.8 (C4'), 109.3 (C7), 112.8 (C1''), 113.2
C7'), 116.2 (C1'''), 117.8 (C4), 118.8 (C2'), 122.1 (C5), 122.7
C10'a), 124.5 (C1'), 124.9 (C6'), 128.4 (C6), 129.3 (C3a), 137.4
C3'), 139.4 (C2''), 139.8 (C2'''), 142.7 (C7a), 156.7 (C4'a), 176.1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
(
+
+
+
(C2), 199.6 (C10'). MS (EI): m/z 424 (M , 30), 369 (M -C H ,
(C4a), 175.2 (C2), 199.4 (C10'). MS (EI): m/z 610 (M , 90%),
4
7
+
+
1
4
00%). HRMS (EI): calculated for C H N O : 424.2151, found
24.2150.
493 (M -C H , 100%), 375 (M -C H , 40%). MS (ESI): m/z
2
8
28
2
2
9
9
18 19
+
611 (M+H 100%). HRMS (ESI): calculated for C H N O :
,
43 35
2
2
6
11.2699, found 611.2681.
3
.3.2.3. (E)-1-(But-2-en-1-yl)-9a'-(but-3-en-2-yl)-
8
'-methyl-8'H-spiro[indoline-3,9'-pyrido[1,2-
The second product separated from the above PTLC
procedure was 1-cinnamylindoline-2,3-dione (8) (17.7 mg, 9%),
obtained as an orange solid. Mp 108-110 °C. UV/Vis: λ (ε)
a]indole] -2,10'(9a'H)-dione (21).
max
Following the general procedure B, a suspension of indirubin
-1
4
(
(
24 (569) nm. IR [cm ]: ν 1726 (s), 1601 (s), 1464 (m), 1366
m), 1343 (m), 1165 (m), 762 (s). H NMR [300 MHz]: δ 4.53
2H, d, J = 5.7 Hz, H3'), 6.18 (1H, dt, J = 5.7 Hz, J = 16.2 Hz,
(202 mg, 771 μmol) in DMF (20 mL) was heated with caesium
1
carbonate (603 mg, 1.85 mmol) and crotyl bromide (373 μL, 3.62
mmol) for 6 h to obtain a fraction that was further purified by
PTLC providing a mixture of at least two stereoisomers (arising
H2'), 6.68 (1H, d, J = 16.2 Hz, H1'), 6.96 (1H, d, J = 7.8 Hz, H7),
.12 (1H, t, J = 7.8 Hz, H5), 7.25-7.37 (5H, m, ArH2, ArH3,
ArH4, ArH5, ArH6), 7.56 (1H, t, J = 7.8 Hz, H6), 7.62 (1H, d, J
7
6 from the cis/trans mixture in the double bond of the crotyl
7 bromide starting material) of 1-(but-2-en-1-yl)-9a'-(but-3-en-2-
8 yl)-8'-methyl-8'H-spiro[indoline-3,9'-pyrido[1,2-a]indole]-
9 2,10'(9a'H)-dione (21) (78.5 mg, 24%) as a yellow solid. Mp
13
=
1
7.5 Hz, H4). C NMR [75 MHz]: δ 42.5 (C3'), 111.2 (C7),
18.0 (C3a), 121.8 (C2'), 124.1 (C5), 125.7 (C7), 126.8 (ArC3,
ArC5), 128.5 (ArC4), 129.0 (ArC2, ArC6), 134.4 (C1'), 136.0
-
1
0 154-160 °C. UV/Vis: λmax (ε) 411 (2211) nm. IR [cm ]: ν 1691
(
ArC1), 138.7 (C6), 151.1 (C7a), 158.2 (C2), 183.6 (C3). MS
1
1 (s), 1606 (s), 1470 (m), 1363 (w), 1337 (w), 748 (s). H NMR
+
(EI): m/z 263 (M , 100%). HRMS (ESI): calculated for
2 [300 MHz]: δ 0.52 (3H, d, J = 8.0 Hz, H1''''), 1.53 (3H, d, J = 7.3
3 Hz, H1'''''), 1.72 (3H, d, J = 7.3 Hz, H1''), 3.40-3.47 (2H, m, H8',
4 H3'''), 4.33-4.46 (2H, m, H4''), 4.64 (1H, dd, J = 10.0 Hz, 1.8 Hz,
C H NO : 264.1025, found 264.1012.
1
7
14
2
3
.3.2.5. Attempted allylation starting from N1' -
5 H1'''
), 4.86 (1H, dd, J = 17.0 Hz, 2.1 Hz, H1'''
8.9 Hz, 2.3 Hz, H7'), 5.35-5.48 (1H, m, H2'''), 5.51-5.57 (1H,
m, H3''), 5.72-5.89 (1H, m, H2''), 6.48 (1H, t, J = 8.3 Hz, H5),
.57 (1H, d, J = 7.8 Hz, H4), 6.67-6.76 (2H, m, H2', H4'), 7.01
1H, t, J = 8.3 Hz, H6), 7.08-7.16 (3H, m, H1', H6', H7), 7.45
), 5.03 (1H, dd, J
allylindirubin.
a
b
=
6
7
8
9
0
1
2
3
4
5
6
7
8
Following general procedure B, N1'-indirubin (9) (97.1 mg,
3
21 μmol) in DMF (8 mL) was heated with caesium carbonate
6
(126 mg, 385 μmol) and allyl bromide (74.3 μL, 770 μmol) was
(
(
(
1
3
added before being stirred for 20 h. Workup of the reaction
produced an indefinable mixture of compounds with only the
corresponding N-allylisatin detected by TLC analysis.
1H, t, J = 7.8 Hz, H3'). C NMR [75 MHz]: δ 15.6 (C1'''), 17.9
C1''''), 18.0 (C1''), 34.6 (C8'), 42.6 (C4''), 43.3 (C3'''), 53.7 (C3),
7
1
1
1
2
3
4
3.6 (C9'a), 109.07 (C6'), 109.12 (C4'), 110.9 (C7'), 116.0 (C1'''),
20.1 (C2'), 121.5 (C5), 123.9 (C10'a), 124.5 (C1'), 124.9 (C3''),
25.2 (C7), 125.7 (C4), 127.2 (C3'a), 128.2 (C6), 129.3 (C2''),
36.8 (C3'), 139.6 (C2'''), 143.6 (C7a), 157.5 (C4'a), 175.9 (C2),
Acknowledgements
+
+
Financial and other support from the University of
Wollongong, through the Centre for Medicinal Chemistry and a
University of Wollongong scholarship to A.M.S. is gratefully
acknowledged. Access to equipment and facilities from the
UNSW Australia, the Australian National University, and
BIOTEC (Thailand) is gratefully acknowledged.
00.0 (ArC10'). MS (EI): m/z 424 (M , 26), 369 (M -C H , 75),
4
7
+
15 (M -C H , 100%). HRMS (ESI): calculated for C H N O :
4
6
28 29
2
2
25.2229, found 425.2237.
9 3.3.2.4. 1,9a'-Dicinnamyl-8'-phenyl-8'H-
0 spiro[indoline-3,9'-pyrido[1,2-a]indole] -
2
,10'(9a'H)-dione (22); 1-cinnamylindoline-2,3-
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
Supporting information
dione (8).
Analytical and spectral characterization data for all new
compounds, computational details, selected UV/Vis spectra and
biological testing procedures. This material is available free of
charge via the Internet at http://pubs.acs.org.
Following the general procedure B, a suspension of indirubin
(205 mg, 782 μmol) in DMF (20 mL) was heated with caesium
carbonate (612 mg, 1.88 mmol) and cinnamyl bromide (731 mg,
.68 mmol) for 24 h to obtain a fraction that was further purified
3
by PLC providing two products, the first of which was 1,9a'-
dicinnamyl-8'-phenyl-8'H-spiro[indoline-3,9'-pyrido[1,2-
a]indole]-2,10'(9a'H)-dione (22) (39.2 mg, 8%), obtained as a
4
2
yellow solid. Mp 118-120 °C. UV/Vis: λ (ε) 414 (1659 ) nm.
max
-
1
IR [cm ]: ν 1701(s), 1606 (s), 1470 (s), 1340 (w), 962 (w), 745

1
2
Tetrahedron
B.; Spencer, K. R.; González-Páez, G. E.; Lakshminarayana, S. B.; Goh,
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Supporting Information
A Cascade Synthetic Route to New Bioactive Spiroindolinepyrido[1,2-a]indolediones
from Indirubin
Alexander M. Sele, John B. Bremner, Anthony C. Willis, Rachada Haritakun, Renate Griffith*, and
Paul A. Keller*
School of Chemistry, University of Wollongong, Wollongong 2522, New South Wales, Australia.
Research School of Chemistry, Australian National University, Canberra 0200, Australian Capital
Territory,
Australia. School of Medical Sciences, UNSW Australia, Sydney 2052, New South Wales, Australia.
National Center for Genetic Engineering and Biotechnology (BIOTEC), Bioresources Research Unit
(BRU), Pathumthani 12120, Thailand.
Yet-to-be-synthesised model compound used in the calculation of UV spectra.
NMR Spectra for Type A, B, C and D Compounds Synthesised.
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