DOI: 10.1002/cmdc.201000374
Synthesis and Bioactivity of Carbohydrate Derivatives of
Indigo, Its Isomers and Heteroanalogues
Gnuni Karapetyan,[a] Kuheli Chakrabarty,[a] Martin Hein,[a] and Peter Langer*[a, b]
Dedicated to Professor H. M. R. Hoffmann on the occasion of his 75th birthday
Introduction
Indigo, indirubin, and isoindigo contain a bis-indole framework
and can be found in a number of natural products (Figure 1).
Indigo and its dibromo derivative are well-known dyes, which
Figure 2. Akashin A.
Figure 1. Indigo and its isomers.
have been used for a long time. They have technical applications, and are of considerable theoretical interest as well.[1,2]
The pigment can be obtained from various higher plants and
fungi such as Baphicacanthus cusia (Acanthaceae), Calanthe veratrifolia (Orchidaceae), Isatis tinctoria (Brassicaceae), Polygonum tinctorium (Polygonaceae), Schizophyllum commune, and
Agaricus campester through a process that involves its formation from precursors such as indican and isatan. Numerous derivatives of indigo have also been synthesized for commercial
purposes.
Until 2002, only three naturally occurring substituted indigos
were known: the well-known Tyrian purple (isolated from
purple snail)[3] and two other brominated indigos.[4] In 2002,
Laatsch et al. reported the isolation of the akashines A, B, and
C from terrestric Streptomyces spp.[5] (Figure 2). Besides the
5,5’-dichloro-substituted indigo moiety, the akashines contain
an N-glycosidic 4-amino-4,6-didesoxyglucose (akashine A) or a
4-acetamido-4,6-didesoxyglucose moiety (akashine B). They exhibit considerable growth inhibitory activity toward various
human tumor cell lines, in contrast to the pharmacologically
inactive non-glycosylated indigo.[5]
Indirubin, the red isomer of indigo,[6] is the active ingredient
of the traditional Chinese medicinal recipe Danggui Longhui
Wan, which has been used for the treatment of myelocytic leukemia.[7] This substance and its substituted derivatives are
ChemMedChem 2011, 6, 25 – 37
potent inhibitors of several kinases such as glycogen synthase
kinase-3 (GSK-3) and cyclin-dependent kinases (CDKs).[8, 9] Phosphorylation of serine, threonine, and tyrosine residues by cellular protein kinases plays an important role in the regulation of
various cellular processes.[10] Protein kinases constitute the largest family of human enzymes and are considered to be the
largest class amenable to therapeutic intervention by smallmolecule drugs.[11] CDKs and GSK-3 play key roles in a large
number of cellular processes. They are involved in various diseases, including certain cancers, Alzheimer’s disease, Parkinson’s disease, and cardiovascular diseases, inflammation, and
AIDS among others.[12, 13] Both families of kinases have been
used extensively as targets to identify small-molecular-weight
pharmaceutical inhibitors of potential therapeutic interest.
Among these inhibitors, the bis-indole indirubin and its analogues have gathered considerable attention, as they were discovered to inhibit CDKs and GSK-3. For example, 5-substituted
indirubins display high inhibitory potency toward various CDKs
and GSK-3b.[8, 9, 14] Among indirubin isomers isolated from
marine organisms, the natural product 6-bromoindirubin and
its synthetic, more cell-permeable derivative 6-bromoindirubin3’-oxime also show enhanced selective inhibition of GSK-3
versus CDKs.[15, 16] The high inhibitory potency of 5-nitroindirubin-3’-oxime led various research groups to synthesize disubstituted indirubins, namely at positions 5 and 7, thereby possibly combining selectivity and high activity.[17] Moreover, the
[a] Dr. G. Karapetyan, Dr. K. Chakrabarty, Dr. M. Hein, Prof. Dr. P. Langer
Institute of Organic Chemistry, University of Rostock
Albert-Einstein-Str. 3a, 18059 Rostock (Germany)
Fax: (+ 49) 381-498-6428
E-mail: [email protected]
[b] Prof. Dr. P. Langer
Leibniz Institute of Catalysis e.V., 18059 Rostock (Germany)
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high selectivity of 6-bromoindirubin and 6-bromoindirubin-3’oxime toward GSK-3 prompted various researchers in this field
to extensively investigate the role of an N substituent at position 6. With the aim of improving the pharmacological properties of this promising indigoid scaffold, Beauchard et al. synthesized a new series of 5-substituted-7-bromoindirubins, 6-substituted indirubins, and isoindigos.[18] These synthetic compounds
were also screened for potential kinase inhibitory activity.
Hçssel et al. reported that as potent inhibitors of various CDKs,
indirubin and its analogues competitively inhibit ATP binding
in the catalytic domain of CDK enzymes.[19, 20] The parent compound shows poor solubility and absorption, and so several
analogues have been synthesized in an effort to improve these
characteristics.[21] It was found that the 5’- and 3’-positions are
amenable to molecular permutations for improved potency.[22]
Among others, indirubin-3’-monoxime as well as its derivatives
bearing substituents at the 5- and/or 5’-positions were shown
to be as active as indirubin in several tumor models.[23, 24] Most
indirubin derivatives (IRDs) are also poorly soluble in water and
show limited biological activity. Because most physiological
fluids are aqueous, the pharmaceutically active indigoid bisindoles should be soluble in water or a water-miscible solvent;
the latter, of course, must be physiologically acceptable in
small concentrations. On the other hand, an important factor
for the antitumor activity of indigoid bis-indoles is their ability
to penetrate tumor cell membranes.[15a, 25] Cellular membranes
are composed of lipid bilayers, a rather nonpolar medium.
Therefore, although substitution with highly polar groups improves the water solubility of a given antitumor compound, it
hinders its entry into tumor cells. As a result, antitumor-active
substances that show good activity under certain in vitro conditions must often be rejected owing to their inactivity in tests
with intact cells or in vivo. In this context, there is a need for
appropriate compounds with optimized bioactivities. For this
purpose, indigoid bis-indoles can be coupled to hydrophilic
moieties, and carbohydrates, among other groups, might be a
good choice. One way to attach carbohydrate groups to indirubins is by the introduction of a 3’-oxime functionality and
subsequent glycosylation of the oxime hydroxy group with the
corresponding glycosyl donor (indirubin-O-glycosides).[26] Not
only the O-glycosides, but also the N-glycosides of indigoid
bis-indoles have been studied extensively for their potency as
carcinostatic agents. Most of the compounds studied so far exhibit higher anti-proliferative activity toward human cancer
cells in vitro than their non-glycosylated analogues.[27] Notably,
both deprotected and protected N-glycosides are pharmacologically relevant. For example, the biological activity of the socalled Natura, that is, acetyl-protected b-d-xylopyranosyl-N-isoindigo, was reported to be higher than that of its deprotected
analogue.[28] Moreover, it has already been reported that several other glycosylated indoles and bis-indoles are of remarkable
pharmacological relevance. Prominent carcinostatic derivatives
include the natural products staurosporine, K-252d, rebeccamycin, and the tjipanazoles.[29]
In recent years, there has been dramatic renewed interest in
the synthesis of various indigoid derivatives, including their
sugar conjugates. Herein we provide an overview of the syn-
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thesis and bioactivity of the glycosides of indigo, indirubin,
and isoindigo, as well as heteroanalogues with particular focus
on synthetic methods.
N-Glycosides of Indigo and Its Isomers
N-Glycosides of indigo
A few years ago, Langer and co-workers reported the synthesis
of indigo-N-glycosides (blue sugars).[30] This type of core structure is present in akashines A–C (Figure 2), which were isolated
by Laatsch et al. from Streptomyces sp. GW48/1497.[5] In continuation of their search for new anticancer agents, Langer and
colleagues reported the first synthetic approach to N-indigo
glycosides.[30a] The synthesis of heterocyclic N-glycosides is not
straightforward in many cases. For example, van Vranken et al.
reported that the direct glycosylation of fully unsaturated bisindoles failed; eventually, the glycosylation of 2,2’-indolylindolines and subsequent oxidation led to the desired product.[31]
A strategy for the synthesis of indigo-N-glycosides reported
by Langer et al. is depicted in Scheme 1. N-Benzylindigo (2),
which shows good solubility in many organic solvents, was
prepared by reaction of 1 with sodium hydride and benzyl bromide in DMF.[32] The TMSOTf-mediated reaction of 2 with tri-Opivaloyl-a-l-rhamnosyl trichloroacetimidate (3)[33] afforded the
O-indigo glycoside 4. Interestingly, extension of the reaction
time resulted in the rearrangement of 4 into the desired
Scheme 1. Synthesis of indigo-N-glycosides: a) 1) NaH (1.0 equiv), DMF,
20 8C, 1 h, 2) BnBr (1.2 equiv), 20 8C, 1 h, 30 %; b) TMSOTf, 4 MS, CH2Cl2,
20 8C, 1.5 h; c) TMSOTf, 4 MS, CH2Cl2, 20 8C, 8–12 h, 35 % (based on 3);
d) O2, AcOH, 100 8C, 2 h, 90 %.
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Carbohydrate Derivatives of Indigo
N-indigo glycoside 5 (O!N rearrangement). Oxidative debenzylation of 5 afforded 6 in high yield.
However, the success of the key step of this approach
(Scheme 1), the O!N rearrangement, depends significantly on
the type of carbohydrate moiety and also on the protecting
groups. In fact, the application of this strategy proved effective
only with the rhamnosyl group as glycosyl donor and pivaloyl
as the protecting group. Furthermore, all attempts to remove
the pivaloyl protecting groups failed. As a part of these ongoing studies concerning the synthesis of indigo-N-glycosides,
Langer and colleagues reported the first synthesis of deprotected indigo-N-glycosides[30b] (Scheme 2). The success was due
Scheme 2. Synthesis of indigo glycoside 7: a) KMnO4, AcOH, high-power stirring (12 000 rpm), 20 8C, 3–4 h; b) pyridine/toluene (1:2), 70 8C, 1 h; c) 1) 10,
CH2Cl2, 2) Me3SiI, 20 8C, 30 min, 3) 3, 0 8C, 30 min, 4) nPrSH, 0!20 8C, 1 h,
5) Ac2O/pyridine (3:1), KHF2, 70 8C, 3 h; d) NaOtBu (15 mol %), MeOH, 20 8C,
4 h.
to a new synthetic strategy based on the addition of a glycosyl
iodide to dehydroindigo. Dehydroindigo (9)[34] was prepared in
high yield by reaction of indigo (1) with potassium permanganate in the presence of acetic acid (to give diacetate 8) and
subsequent base-mediated elimination of acetic acid. The reaction of dehydroindigo (9) with TMS-protected l-rhamnosyl
iodide generated in situ by conversion of tetra-O-trimethylsilyll-rhamnopyranose (10) with TMSI, reduction, and subsequent
acetolysis afforded the N-(2,3,4-tri-O-acetyl-l-rhamnosyl)indigo
11 (a/b = 2:1). An analytically pure sample of the a anomer
was isolated by repeated crystallization from MeOH. Treatment
of a methanol solution of 11 with NaOtBu (5–15 mol %) afforded the desired deprotected indigo glycoside 7 (a/b = 2:1). The
use of catalytic quantities of NaOtBu proved to be important,
as stoichiometric amounts, or the use of other reagents (such
as potassium carbonate, methanol) resulted in decomposition.
ChemMedChem 2011, 6, 25 – 37
Notably, this strategy allows the monoglycosylation of indigo
without the need for a nitrogen protecting group.
In contrast to the anti-proliferative properties of the akashins, the synthesized indigo-N-glycosides showed no significant activity against human cancer cell lines.
Indirubin-N-glycosides
The first synthesis of deprotected indirubin-N-glycosides reported by Langer et al. is depicted in Scheme 3.[35] A central
building block of this reaction sequence is an isatin-N-glycoside, and Scheme 3 shows the complete synthesis. The reac-
Scheme 3. Synthesis of N-(b-l-rhamnopyranosyl)indirubin 16a(b): a) PhNH2,
EtOH, RT, 12 h; b) Ac2O, pyridine, 0!4 8C, 8–12 h; c) oxalyl chloride, AlCl3,
55 8C, 1.5 h; d) Na2CO3, MeOH, RT, 4 h.
tion of free sugars with various anilines and subsequent acetylation afforded N-glycosyl anilines. The aluminum trichloride
mediated cyclization of acetyl-protected N-glycosyl anilines
with oxalyl chloride afforded the corresponding N-glycosyl
isatin. Following this procedure, N-(2,3,4-tri-O-acetyl-a,b-lrhamnopyranosyl)isatin (15 a,b) was prepared from l-rhamnose
(12) as an anomeric mixture that was difficult to separate by
column chromatography.[36]
Simple indirubins were prepared previously by reaction of a
methanolic solution of indoxyl acetate with isatins.[17b] Reaction
of the pure b anomer of the N-glycosyl isatin 15 b with indoxyl
acetate resulted in the formation of the desired deprotected
N-(b-l-rhamnopyranosyl)indirubin 16 a(b) in up to 77 % yield
(Scheme 3). During the optimization of this reaction, the use of
excess sodium carbonate in step d) proved important for complete cleavage of the acetyl protecting groups from the sugar
moiety. Following this procedure, the anomerically pure indiru-
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bin glycosides of d-glucose (17 b), d-ribose (18 b), d-galactose
(19 b), and d-mannose (20 b) were successfully prepared in
good yields starting from the corresponding N-glycosyl isatins
(Figure 3). In the case of l-rhamnose, both anomeric glycosyl
chloro derivative of tetra-O-acetylated indirubin-N-rhamnoside
22 b(b) in 63 % yield (Scheme 4). The acetylation in step b) was
applied for convenient structure elucidation, because a mixture
Scheme 4. Synthesis of the chloro derivative of indirubin-N-rhamnoside
16 c(b) and synthesis of 16 a(a) and 16 c(a): a) Na2CO3, MeOH, 20 8C, 2 h;
b) Ac2O, pyridine, 0!4 8C, 12 h; c) KOtBu, MeOH, 20 8C, 12 h.
Figure 3. N-Glycosylated indirubins with various substituents.
isatins could be isolated after cyclization of 14 a,b with oxalyl
chloride. In contrast, only small amounts of the a anomers
were detected for all other sugars. In these cases, isomerically
enriched b-anomers (b/a > 5:1) of the corresponding acetylated N-glycosyl anilines were used in the cyclization reaction.
However, the b/a ratio seemed to vary slightly during the
course of the reaction; otherwise, higher amounts of the aanomeric N-glycosyl isatins would have been detected after
the reaction.
The strategy allows the synthesis of unsubstituted indirubinN-glycosides as well as derivatives that contain different substitution patterns at both aromatic moieties (the oxindole and
the indoxyl portions) of indirubin. The substitution pattern of
the oxindole part seems to be limited to donor substituents
because the cyclization reaction (Scheme 3, step c), which provides the isatin-N-glycosides, does not work well with acceptor-substituted aniline glycosides.
The sodium carbonate mediated reaction of isatin-N-rhamnoside 15 b with the chlorinated indoxyl acetate 21 b[37] and
subsequent acetylation of the crude product afforded the
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of partly deprotected products was formed during the condensation. Stirring of a methanol solution of 22 b(b) in the presence of a catalytic amount of KOtBu (0.06 equiv) gave the deprotected rhamnoside 16 c(b) in 72 % yield. The analogous reaction of isatin-N-rhamnoside 15 a with indoxyl acetates 21 a
and 22 b and subsequent acetylation gave the tetra-O-acetylated indirubin-N-rhamnosides 22 a(a) and 22 b(a), respectively
(Scheme 4). The KOtBu-catalyzed deacetylation of the latter afforded the deprotected rhamnosides 16 a(a) and 16 c(a). Furthermore, the rhamnosylated indirubin-3’-oxime 24 b was prepared by reaction of the O-acetylated indirubin-N-glycoside
22 a(b) with hydroxylamine hydrochloride (to give 23 b) and
subsequent deprotection (Scheme 5). Product 24 b was synthesized to compare its anti-proliferative activity with that of the
corresponding aglycon.
The anti-proliferative activities of indirubin-N-glycosides 16 a,
16 b, 17, 19, 20, 16 c and oxime 24 against four adherent
human cancer cell lines [bladder (5637), small-cell lung (A-427),
esophageal (Kyse-70), and breast (MCF-7)] were studied.[27]
Most of the compounds exhibit significant anti-proliferative activity against various human cancer cell lines. Good results
were observed for the indirubin-N-mannoside, which was
shown to have medium to high anti-proliferative activity
against all cell lines investigated. The highest activity and selectivity against the MCF-7 breast cancer cell line were observed for the anomeric indirubin-N-rhamnosides. The experiments also revealed that the rhamnosides containing a substituted indirubin moiety show lower activity than rhamnosides
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ChemMedChem 2011, 6, 25 – 37
Carbohydrate Derivatives of Indigo
nitrogen atom (Figure 4).[38] The synthetic strategy relies on the
synthesis of previously unknown indoxyl-N-glycosides and
their condensation with isatins (Scheme 6). Indirubin-N’-glyco-
Scheme 5. Synthesis of the glycosylated indirubin-3-monoxim 24b:
a) NH2OH·HCl, pyridine, 90 8C, 7 h; b) KOtBu, MeOH, 20 8C, 12 h.
that contain a non-substituted indirubin moiety. In most cases,
the anti-proliferative activity of the indirubin-N-glycosides was
shown to be higher than that of the corresponding aglycons.[9]
In particular, the activity of rhamnosides 16 a(a) and 16 a(b)
against the human breast cancer cell line MCF-7 is much
higher (10- to 100-fold) than that of the non-glycosylated indirubins tested before.
Indirubin-N’-glycosides
Indirubin derivatives contain an amine- and an amide-type nitrogen atom. The synthesis of indirubin-N-glycosides (red
sugars) containing a carbohydrate moiety located at the amide
type nitrogen atom was described in the preceding section
(Figure 4). In this section, we discuss indirubin glycosides with
the sugar moiety at the amine-type nitrogen atom.
Scheme 6. Synthesis of glycosylated indoxyl-3-acetates 29 a(b) and 29 b(b):
a) EtOH, 20 8C, 12 h; b) DDQ, dioxane, 20 8C, 12 h; c) BnBr (for 27 a(b)) or MeI
(for 27 b(b)), NaH, DMF, 0 8C!4 8C, 12 h; d) I2, NaOH, DMF, 20 8C, 1 h;
e) AgOAc, AcOH, 80 8C, 4 h.
sides have been synthesized from N-glycosylated indoxyl-3acetates 29 a(b) and 29 b(b). The 3-acetyl group in 29 a(b) and
29 b(b) was selectively removed under slightly basic and reducing conditions (sodium sulfate, dioxane, water).[39] The crude
deacetylated materials were used directly for the next reaction
step, resulting in the formation of the purple solids 30 a(b) and
30 b(b), respectively. All attempts to deprotect the methylated
derivative 30 b(b) were unsuccessful. In contrast, treatment of
benzyl-protected derivative 30 a(b) with boron tribromide resulted in formation of the desired deprotected indirubin-N’-glycoside 31 a(b), which was isolated as a purple solid in up to
67 % yield (Scheme 7). The preliminary results of that report
suggest that different isatins and N-glycosylated indoxyls can
be successfully employed.
Initial anti-proliferation tests of the indirubin-N’-glycosides
30 a(b), 30 b(b), and 31 b toward various malignant melanoma
cell lines did not show significantly high activities for these
compounds.
Figure 4. Structure of N- and N’-glycosylated indirubins (sug = glycosyl
moiety).
Isoindigo-N-glycosides
Thorough investigations into the pharmacological properties
of the indirubins revealed that they are ATP-competitive inhibitors of CDK2. This discovery places indirubin and its analogues
in the larger class of ATP-competitive CDK inhibitors of the oxindole family.
In continuation of their search for effective CDK inhibitors,
Langer et al. reported the first synthesis of indirubin-N’-glycosides that contain a sugar moiety attached to the amine-type
ChemMedChem 2011, 6, 25 – 37
Isoindigo-N-glycosides are also an important class of indigoid
bis-indoles with significant biological activity against various
human cancer cell lines. Interestingly, both deprotected and
protected isoindigo-N-glycosides are pharmacologically relevant. For example, the biological activity of Natura (the acetylprotected b-d-xylopyranosyl-N-isoindigo mentioned above),
was reported to be higher than that of its deprotected analogue.[28]
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Scheme 7. Synthesis of the deprotected indirubin-N’-glycoside 31 b:
a) Na2SO3, dioxane, H2O (for 30 a(b): 110 8C, 2 days; for 30 b(b): 80 8C); b) piperidine, benzene, 80 8C, 2 h; c) BBr3, CH2Cl2, 78 8C, 3.5 h.
The synthesis of Natura was described by Wang et al. in
2003.[28] They prepared acetyl-protected isatin-N-glycosides by
reaction of N-glycosylated anilines with oxalyl chloride (see
also Scheme 3). The products were converted by reaction with
oxindoles under acidic conditions into different isoindigo-Nglycosides. Later, Sassatelli et al. described the preparation of
isoindigo-N-glycosides that possess considerable anti-proliferative activity toward various tumor cell types as well as kinase
inhibitory potency.[40] They synthesized glycosylated isoindigo
derivatives diversely substituted at one of the aromatic rings
with either electron donor or acceptor substituents.[40a] The
preparation started from different substituted isatin-N-glycosides, which were synthesized in four steps from the corresponding commercially available indoline.[41] To obtain the corresponding N-glycosylated isoindigo derivatives, compounds
32 a–32 c were treated in an acidic medium in the presence of
oxindole.[42] Debenzylation of the glycosyl moiety was performed by reaction of derivatives 33 a–33 c with boron tribromide to give compounds 34 a–34 c (Scheme 8).
Over the course of their studies related to the development
of potentially bioactive indigoid derivatives, the same research
group also reported the synthesis of isoindigo glycosides substituted at the 5- or 5’-positions of both aromatic rings (Figure 5).[40b]
Figure 5. Diversely substituted glycosylated isoindigos.
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Scheme 8. Synthesis of glycosylated isoindigo-N-glycosides with substituents
at one aromatic ring.
The in vitro anti-proliferative activities of compounds 33 and
34 and of the derivatives shown in Figure 5 were evaluated
against a panel of human solid cancer cell lines (PC3, DLD-1,
MCF-7, M4Beu, A549, PA 1), a murine cell line (L929), and
human fibroblast primary culture. The most potent compound
was the one bearing a 4-oxobutanoic acid side chain at the 5’position (Figure 5), which is cytotoxic toward all the cell lines
tested. In contrast, compound 33 a, which lacks this substitution, was completely inactive against the cell lines tested. Compounds bearing a hydrogen or bromine atom at the 5-position
(R = H or Br in Figure 5) had similar inhibition profiles, except
for the PC3 cells, against which the 5-brominated derivative
was slightly more active than its non-brominated analogue.
They were both active on PA 1, L929, DLD-1, and MCF-7 cells.
In contrast to the compound with a 5’-4-oxobutanoic acid substituent, they show selectivity between normal and tumor cell
lines. They are both inactive against healthy human fibroblasts.
As previously reported for Natura,[28] the acetylated derivatives
bearing hydrogen and bromine at the 5-position (R = H and Br
in Figure 5) were more cytotoxic than their benzylated analogues or parent compounds (R = H, R’ = H and R = Br, R’ = H,
respectively, in Figure 5). In contrast, the nitro derivative (R =
NO2 in Figure 5) was inactive. The results obtained so far from
the structure–activity relationship studies with various substituted isoindigo glycosyl derivatives have revealed that the
pharmaceutical profile of this series could be optimized by
substitution of the oxindole portion, which does not contain
the N-glycosyl moiety. The presence of acetyl groups on the
sugar residue also proved to be effective in enhancing the cytotoxicity of isoindigo derivatives. Bouchikhi et al. recently reported the synthesis, kinase inhibitory potencies, and in vitro
anti-proliferative activities of 7’-azaisoindigo derivatives.[43]
The presence of an additional nitrogen atom in the isoindigo glycoside framework seems to be effective for the antitumor activity of these compounds. As a part of these studies,
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Carbohydrate Derivatives of Indigo
Bouchikhi et al. reported their work on the biological activities
of acetylated glycosylated isoindigo and 7’-azaisoindigo derivatives, diversely substituted at the 6- and/or 5’- and/or 6’-positions, to evaluate the influence of a 7’-azaindolin-2-one moiety
instead of an indolin-2-one moiety on the biological activities
of these compounds[43a] (Scheme 9). The key intermediates in
various functionalized alkynyl side chains at the C5’-position of
the upper indolin-2-one or 7’-azaindolin-2-one moiety by Sonogashira cross-coupling (Scheme 10).[43b]
Scheme 9. Synthesis of the substituted isoindigo- and 7’-azaisoindigo-N-glycosides 35.
this synthesis are acetyl-protected glycosyl isatins, which can
be prepared in four steps from the corresponding indoline-Nglycosides. The required diversely substituted indolin-2-one derivatives were either commercially available or prepared by
standard procedures.[43a] In vitro anti-proliferative activities of
isoindigo and compounds 35 a–35 k were tested in triplicate
against the human buccal carcinoma cell line (KB) and human
myeloid leukemia cell lines (K562 and HL60).[43a] The results revealed that isoindigo is active against KB cells, whereas it is
slightly cytotoxic toward HL60 and K562 cell lines. None of the
glycosyl-isoindigo derivatives (compounds 35 a–35 i) exhibited
relevant cytotoxicity toward the cell lines tested. In contrast,
the two compounds bearing a 7’-azaindolin-2-one moiety (35 j
and 35 k) exhibit significant anti-proliferative activities. Compound 35 j inhibited the proliferation of all the cell lines tested
in the 75–80 % range (KB IC50 = 1.6 mm). Compound 35 k suppressed the proliferation of KB cells (IC50 = 13.9 mm), and K562
cells were inhibited by ~ 60 %. This experiment demonstrated
that the presence of an additional nitrogen atom appears to
be favorable for cytotoxicity.
Previous studies have revealed that the most active compound of the isoindigo series is the one with a 4-oxobutanoic
acid side chain at the 5’-position. Moreover, the 7’-azaisoindigo
derivatives were shown to be more cytotoxic than their nonaza counterparts, particularly toward two myeloid leukemia
cell lines (K562 and HL60).
To get insight into the substitution pattern required for optimal biological potency, Bouchikhi et al. synthesized a number
of acetyl-protected isoindigo and azaisoindigo glycosides with
ChemMedChem 2011, 6, 25 – 37
Scheme 10. Synthesis of the substituted isoindigo- and 7’-azaisoindigo-Nglycosides 36 and 37.
Compounds 36 a–36 f and 37 a–37 g were tested in vitro to
evaluate their anti-proliferative activities toward human myeloid leukemia cell lines (K562 and HL60).[43b] At a final concentration of 106 m, none of the tested compounds showed any
cytotoxicity toward the cell lines tested. Only five compounds
(36 a, 36 f, 37 a, 37 c, and 37 e) showed modest anti-proliferative effects when tested at 105 m. The most active compound
in the isoindigo series was compound 36 a, bearing a sugar
moiety and a 5’-iodo substituent. This compound inhibits the
proliferation of both K562 and HL60 cell lines by ~ 50 %. Compound 36 f, which contains a hydroxybut-3-ynyl side chain at
the 5’-position, suppressed the proliferation of K562 cells by
51 %. In the 7’-azaisoindigo series, the most active compound
was 37 a, which contains a sugar moiety and a bromine atom
located at the 5’-position. This compound inhibits the proliferation of the K562 and HL60 cell lines in the range of 60–75 %.
Compounds 37 c and 37 e suppressed the proliferation of K562
cells in the 50–60 % range. In vitro kinase inhibitory potencies
of all these compounds were also tested against CDK5/p25,
GSK-3, CK1, and DyrK1A.[44] Furthermore, Yao et al. described
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the syntheses and medicinal application of a series of 7-azaisoindigo and 7-azaindirubin derivatives, including some glycosylated derivatives.[45]
N-Glycosides of Heteroanalogous Indigos,
Isoindigos, and Indirubins
Heteroanalogous indirubins
For the preparation of heteroanalogous indirubin-N-glycosides,
the same strategy can be applied as for the normal indirubinN-glycosides. Isatin-N-glycosides containing different carbohydrate moieties proved to be the key intermediates for
these syntheses. The acetyl-protected isatin-b-N-glycosides of
l-rhamnose 15 b, d-mannose, d-glucose, and d-galactose were
synthesized by following the same procedure described in the
preceding section (Scheme 3).[35, 36] For the following condensation step, various nucleophiles were used instead of indoxyl
acetate. Only the reaction conditions differ from those reported for the synthesis of normal glycosylated indirubins.
Oxa-analogous indirubin-N-glycosides were obtained with
the use of 3-coumaranone in the condensation step. Reaction
of the pure b anomer 15 b with 3-coumaranone (38) in the
presence of acetic acid, acetic anhydride, and sodium acetate
afforded a separable mixture of E and Z isomers of 1-(2’’,3’’,4’’tri-O-acetyl-b-l-rhamnopyranosyl)-3-[3’-oxobenzofuran-2’-(Z)-ylidene]oxindole ((E)-41) in up to 55 % yield, and 1-(2’’,3’’,4’’-tri-Oacetyl-b-l-rhamnopyranosyl)-3-[3’-oxobenzofuran-2’-(E)-ylidene]oxindole ((Z)-41) in up to 41 % yield (Scheme 11).[46] The E/Z
ratio depends on the reaction time; it is presumed that the
Scheme 11. Synthesis of the heteroanalogous indirubin-N-glycosides:
a) Ac2O, CH3COOH, NaOAc, 80 8C, 1 h; b) Et3N, EtOH, 20 8C, 12 h; c) DMAP,
Et3N, MsCl, 0 8C!20 8C, 20 h; d) Ac2O, CH3COOH, NaOAc, 80 8C, 1.5 h.
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Z-configured product is the thermodynamically more stable
geometrical isomer. Extension of the reaction time seems to
result in an equilibrium between the two isomers (Z/E ~ 2:1),
but at the expense of yield.
The reaction of the acetylated N-rhamnosyl isatin 15 b with
thiaindan-3-one (39), under analogous acidic conditions, resulted in the exclusive formation of the desired Z isomer 42 in up
to 90 % yield. Derivatives with other sugar moieties could also
be prepared.[47] It was also possible to prepare carba-analogous
indirubin derivatives.[46] Unfortunately, the reaction of 15 b with
1-indanone (40 a), using the acidic conditions used before,
gave only moderate yields of the desired compounds. The reaction of 1-indanone (40 a) with 15 b under basic conditions
(triethylamine in ethanol) resulted in the formation of the diastereomeric mixture (43) with new chiral centers at carbon
atoms C3 and C2’.
To obtain the desired unsaturated product 44 a, compound
43 was converted into its corresponding mesyl derivative.
Using DMAP as a nucleophilic catalyst, mesyl chloride, and triethylamine, the introduction of the mesyl group and the elimination could be carried out in one step. The desired 3-(1’-indanon-2’-(E)-ylidene)-1-(2’’,3’’,4’’-tri-O-acetyl-b-l-rhamnopyranosyl)oxindole (44 a) was formed in up to 94 % yield. Likewise,
the reaction of 5-bromo-1-indanone (40 b) with 15 b gave the
substituted derivative 44 b. (Scheme 11). Derivatives of d-glucose, d-mannose, and d-galactose were also synthesized by
using the same protocol. Deprotection of the acetyl groups in
the case of the sulfur-analogous derivatives could be carried
out with base catalysis (Zmplen conditions; sodium, methanol, room temperature, 3 h) and resulted in formation of the
desired products 45 a(b)–45 d(b) in up to 56–84 % yield
(Figure 6).[47] In contrast, the deprotection was not successful
for the carba- and oxa-analogous derivatives. Reaction of the
carbonyl oxygen atom (at C3’) was observed in the case of the
oxa-analogues.
Figure 6. Thia-analogous indirubin-N-glycosides with various sugar substituents.
2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 25 – 37
Carbohydrate Derivatives of Indigo
The application of acidic deacetylation conditions allowed
deprotection of the carba-analogues and gave the desired 3(1’-indanon-2’-(E)-ylidene)-1-(b-l-rhamnopyranosyl)oxindole
(46 a) in up to 67 % yield, and 3-(5’-bromo-1’-indanon-2’-(E)-ylidene)-1-(b-l-rhamnopyranosyl)oxindole (46 b) in up to 63 %
yield (Scheme 12).[46]
cal and subsequent clinical studies. They not only stop melanoma cell proliferation at concentrations similar to chemotherapeutic agents in current use, but also induce a significant rate
of apoptosis. Finally, these thia analogues of indirubin-N-glycosides interfere with a well-defined intracellular signaling pathway active in melanoma cells.
Heteroanalogous isoindigos
Scheme 12. Synthesis of 3-(1’-indanone-2’-(E)-ylidene)-1-(b-l-rhamnopyranosyl)oxindole (46): a) KOtBu, MeOH, 20 8C, 12 h; b) Na2CO3, MeOH, 20 8C, 12 h;
c) 1 % methanolic HCl, 20 8C, 12 h.
The first synthesis of oxa-analogues of isoindigo-N-glycosides
was reported by Langer et al.[50] As in the cases of indirubin-Nglycosides and their heteroanalogues, N-glycosylated isatins
are the key intermediates. The base-mediated reaction of 15 b
with 2-coumaranone (3H-benzofuran-2-one) was unsuccessful
due to reaction of the lactone moiety. On the other hand, the
reaction of 15 b with 2-coumaranone in the presence of acetic
acid, acetic anhydride, and sodium acetate[46, 51] afforded the
desired oxa-analogous isoindigo-N-glycoside 47 a(b) in up to
44 % yield (Scheme 13).
The synthesized heteroanalogous indirubin-N-glycosides
were investigated with regard to their activity against various
malignant melanoma cell lines. Malignant melanoma is a
highly aggressive tumor with increasing incidence and poor
prognosis in the metastatic stage.[48] There is mounting evidence that oncogenic mutations in intracellular signal transduction pathways play an important role in melanoma development.[49] The anti-proliferative activities of most of the investigated oxa- and carba-analogous indirubin derivatives against
four different melanoma cell lines (SK-MeI-19, SK-MeI-29, SKMeI-103, and SK-MeI-147) were not significant. The highest activity was observed for the thia-analogous indirubin-N-glycosides 45 a(b)–45 d(b).[47] In addition to the effect on melanoma
cell growth, apoptosis was also examined. The IC50 values of
thia-analogous glycosylated indirubins showed slight variations
among the different cell lines, with 45 c(b) and 45 b(b) having
less activity in SK-Mel-19 cells relative to the three other cell
lines tested (Table 1).
Table 1. Inhibition of metastatic melanoma cell lines by various glycosylated thia-analogous indirubins.
SK-Mel-19
IC50 [mm]
SK-Mel-29
SK-Mel-103
SK-Mel-147
12.08 1.21
5.81 1.17
24.36 1.52
17.10 1.35
6.29 1.29
3.96 1.25
5.57 1.33
8.39 1.26
7.52 1.14
4.28 1.13
6.27 1.24
4.98 1.17
Compd
45 a(b)
45 b(b)
45 c(b)
45 d(b)
10.38 1.16
6.69 1.17
6.94 1.39
6.06 1.19
In conclusion, evaluation of the biological properties of derivatives 45 a(b)–45 d(b) reveals that these substances are quite
active against malignant melanoma cells in vitro, and that
compounds of this class could be good candidates for precliniChemMedChem 2011, 6, 25 – 37
Scheme 13. Synthesis of acetyl-protected glycosylated heteroanalogous isoindigos 47(b): a) PhNH2, EtOH, 20 8C, 12 h; b) Ac2O, pyridine, 0!4 8C, 8–12 h;
c) oxalyl chloride, AlCl3, 55 8C, 1.5 h; d) AcOH, Ac2O, NaOAc, 90 8C, 6 h for
47 a(b), and AcOH, Ac2O, NaOAc, 130 8C, 2 days for 47 b(b).
However, all attempts to remove the acetyl groups of
47 a(b) failed due to base-mediated side reactions of the lactone moiety. This problem was solved by the use of benzyl
protecting groups (Scheme 14). The reaction of l-rhamnose
with indoline afforded anomerically pure 25 b, which was
transformed into the indole-N-glycoside 26 b by dehydrogenation (DDQ). Benzylation and subsequent oxidation (chromium
trioxide) afforded N-(b-l-rhamnopyranosyl)isatin 48 b (see also
Ref. [41]). The condensation of 48 b with 2-coumaranone, fol-
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P. Langer et al.
target. One of the possibilities to attach carbohydrate moieties
to indirubins is the introduction of a 3’-oxime functionality and
subsequent glycosylation of the oxime hydroxy group with a
suitable glycosyl donor. This strategy was investigated by
Eisenbrand.[26] He reported two general strategies for the synthesis of carbohydrate-containing indirubin-3’-oxime ethers of
type 55 b (Scheme 15). At the beginning, a modular approach
Scheme 14. Synthesis of deprotected 3-(2’-coumaranon-3’-(E)- ylidene)-1-(bl-rhamnopyranosyl)oxindole 50 b: a) indoline, EtOH, 20 8C, 12 h; b) DDQ, dioxane, 20 8C, 12 h; c) NaH, BnBr, DMF, 0!4 8C, 12 h; d) CrO3, acetone, AcOH,
H2O, 20 8C, 1.5 h; e) AcOH, Ac2O, NaOAc, 90 8C, 2 h; f) BBr3, CH2Cl2, 78 8C,
2 h.
lowing the conditions as described for 47 a(b), afforded the
red-colored condensation product 49 b in 40 % yield. Treatment of the latter with boron tribromide resulted in the formation of the desired deprotected oxa-analogous isoindigo-N-glycoside 50 b in 63 % yield which was isolated as an orange–red
solid. The double bond between the coumaranone and the
glycosylated oxindole part of compounds 47 a(b), 49 b, and
50 b was found to have the E configuration. The strategy for
the synthesis of oxa-analogues of isoindigo-N-glycosides is
rather general. It is also possible to synthesize thia analogues
of isoindigo-N-glycosides by using thiaindan-2-one instead of
2-coumaranone.[46, 50] . Other glycosyl derivatives of thia-analogous isoindigos could be prepared as well. The cleavage of the
acetyl protecting groups under basic conditions failed, as in
case of compound 47 a(b).
The bioactivity of some of the synthesized heteroanalogous
isoindigo-N-glycosides was investigated in anti-proliferation
tests on malignant melanoma cell lines (SK-MeI-19, SK-MeI-29,
SK-MeI-103, and SK-MeI-147), but no significant activity could
be detected for any of these compounds.
O-Glycosides of Indigo, Isoindigo, and
Indirubin Derivatives
As discussed in the introduction above, the presence of the
glycosyl moiety and the substitution pattern of the aromatic
ring can improve the solubility of parent indigoid bis-indoles
and, consequently, interaction with the active site of the
34
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Scheme 15. Synthesis of indirubin-3’-(2-b-d-glucopyranosyloxyethyl)oxime
ether (55 b).
was applied for the synthesis of indirubins of type 51,[19] that
is, the condensation of an indoxyl derivative with an isatin,
which was first published by Russell and Kaupp (Scheme 15).[52]
Subsequent condensation of 51 with hydroxylamine hydrochloride furnished the indirubin-3’-oxime 52. Next, haloalkylsubstituted O-glycoside 54 was prepared by boron trifluoride
catalyzed glycosylation of acetyl-protected glycosyl fluoride 53
with chloroethanol.[53] Besides 53, other peracetylated glycosyl
fluorides of mono- and disaccharides were also used as glycosyl donors. The stereochemistry of the glycosylation reaction is
controlled by the neighboring group effect of the acetyl group
located at position O2[54] which results in a b-glycosidic linkage
in the formed haloalkyl glycoside 54. Furthermore, the reaction
of the indirubin-3’-oxime 52 with haloalkyl-O-glycoside 54
under basic conditions provided the monoxime ether 55 b
containing an unprotected sugar moiety. The deprotection of
55 a occurs in one step along with coupling under the basic
conditions employed. Various carbohydrate-containing derivatives of type 55 b were synthesized in low to moderate yields
by application of this strategy.
Notably, the chain length of the hydrocarbon unit between
the sugar and the indirubin moiety can be varied by using the
corresponding haloalcohols. In the second method described
in the same patent application by Eisenbrand,[26] unprotected
haloalkyl-O-glycosides were obtained by direct glycosylation of
carbohydrates with the corresponding haloalcohols and subsequent coupling reaction with the corresponding indirubin-3’-
2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 25 – 37
Carbohydrate Derivatives of Indigo
Scheme 16. Synthesis of indirubin-3’-(2-l-arabinopyranosyloxyethyl)oxime
ether (57).
oximes (Scheme 16). By application of this procedure, a multistep protection–deprotection sequence can be avoided. For
the preparation of 2-haloethyl-b-l-arabinopyranosides 57, a
simplified procedure based on acid-catalyzed high-temperature glycosylation of l-arabinose with 2-haloethanols was applied (Scheme 16).[55] The corresponding 2-chloroethyl-, 2-bromoethyl-, or 2-iodoethyl-b-l-arabinopyranosides were obtained
as crystalline intermediates in 20, 86, and 23 % yields, respectively. The reaction of 56 with 52 under basic conditions provided the b-l-arabinopyranoside 57 in 43 % yield.[26] In another
method, unprotected carbohydrates, such as amino sugars or
corresponding alditols bearing amino groups, were coupled
with indirubin-3’-oxime haloalkyl ethers 58 to provide products
59 (Scheme 17).[15a] However, in the patent application only elemental analyses are given, and no data regarding the structure (possible pyranose or furanose forms, or a or b configuration of the anomeric center) of the corresponding sugars are
provided.
Nam et al. tested the biological activity of various indirubin3’-oximes obtained by the methods of Eisenbrand.[56] They discovered that unbranched, short-chain oxime ethers with one
or two hydroxy groups block the signal transducer and activator of transcription (STAT3) protein and induce apoptosis of
Scheme 17. Synthesis of amino sugar derivatives 59 of indirubin-3’-oxime
(52).
ChemMedChem 2011, 6, 25 – 37
human breast cancer cells. However, inhibitory potency was
impaired or abolished for the compounds bearing sugar or
aminopolyol moieties. Furthermore, it was found that the indirubin derivatives of this series inhibit different CDKs and GSK3b.[17a, 57]
Compound 55 b, indirubin-3’-(2-b-d-glucopyranosyloxyethyl)oxime ether, was tested for its ability to inhibit phosphorylation of the retinoblastoma tumor-suppressor protein (pRb).[58]
It is also noteworthy that 55 b has higher solubility (23 mg L1)
than many other indirubins and indirubin-3’-oxime ethers.[59] It
was found that in contrast to the unsubstituted indirubin,
which is not metabolized to a detectable extent, the incorporation of a methyl or methoxy group at the 5-position of indirubin renders the molecule easily metabolizable.[60] Experiments
in liver microsome preparations from cows, pigs and rats, and
also from a spectrum of human liver samples have shown that
5-methylindirubin is rapidly metabolized, yielding mainly ringhydroxylated indirubin derivatives. In particular, two main metabolites were isolated and structurally characterized, namely
6-hydroxy-5-methyindirubin and 6,7’-dihydroxy-5-methylindirubin.
Furthermore, it was found that the primary metabolite of 5methylindirubin, 6-hydroxy-5-methylindirubin, had increased
solubility in a physiological saline environment (pH 7.4) by a
factor of more than 2000. From a chemical point of view, the
6-hydroxy metabolite is very attractive for further derivatization. Based on the aforementioned information, and keeping
in mind the specificity of glucuronated secondary metabolites,
the synthesis of cell-membrane-penetrating glucuronide indirubin derivatives appeared to be promising. For this purpose, 6hydroxy-5-methylindirubin (63) was synthesized by starting
from 5-amino-2-methylphenol (60) (Scheme 18). In the first
Scheme 18. Synthesis of the glucuronide derivative 66 of 6-hydroxy-5-methylindirubin (63).
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P. Langer et al.
step, the hydroxy and amino groups of 60 were protected
with triisopropylsilyl and Boc groups. Next, the fully protected
derivative 61 was metalated (DOM) and treated with diethyl
oxalate. After workup and holding the residue at reflux in a
mixture of 6 m HCl and ethanol, 6-hydroxy-5-methylisatin 62
was obtained in 19 % overall yield. The coupling of 62 with indoxyl acetate provided the target compound 63. Afterward,
the glycosylation of 63 with the glycosyl bromide 64 under
Koenigs–Knorr conditions provided the acetyl-protected glucuronide 65 in 44 % yield. After removal of the protecting
groups, the glucuronide 66 could be obtained in 77 % yield.
Generally, glucuronated secondary metabolites are water soluble and can be readily excreted through the urinary tract.
However, the glycosidic bond in secondary glucuronide metabolites can be hydrolyzed to some extent with glucuronidases
present either in the gastrointestinal (GI) tract or in the anaerobic regions of solid tumors, providing 6-hydroxy-5-methylindirubin. Thus, an enhanced enrichment of the highly active 6-hydroxy metabolite will take place in the GI tract and from anaerobic tumor tissues, because glucuronidase activity is expressed in poorly oxygenated tumor tissue. In this context, the
glucuronide 66 was suggested as potential drug for the treatment of solid cancers and leukemia and metastases thereof.
Outlook
As discussed in the preceding sections, many carbohydrate derivatives of indigoid bis-indoles (indigo, indirubin, and isoindigo) have been prepared and investigated in recent years. The
studies of their enzyme inhibitory potential and in vitro antitumor activity have shown that some of these glycosides are
interesting candidates for cancer therapy.
Whereas syntheses and properties of carbohydrate derivatives of indirubin and isoindigo are already well studied, glycosides of substituted indigos and related heteroanalogous compounds are described only scarcely in the scientific literature.
To some degree, this could be due to difficulties encountered
in the synthesis of such compounds. Nonetheless, this compound class is an interesting research topic. Furthermore, glycosyl derivatives of the other three isomers of indigo (bismetaindolone, phthalorubin, and phthalaurin),[61] which are the
combinations of either an indoxyl or oxindole fragment with a
phthalimidine unit, may be worthy of detailed investigations.
Acknowledgements
Financial support by the State of Mecklenburg-Western Pommerania, the Deutsche Forschungsgemeinschaft, and the Deutsche
Krebshilfe (Melanomverbund, grant number 108008) is gratefully
acknowledged.
Keywords: antitumor agents · enzyme inhibitors · glycosides ·
indigo · indirubin
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Received: September 3, 2010
Revised: October 14, 2010
Published online on November 24, 2010
2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
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