Improved synthesis of indirubin derivatives by sequential build-up of the indoxyl unit: First preparation of fluorescent indirubins

Article abstract of DOI:10.1002/hlca.201200042

Indirubin, present in extracts of Isatis tinctoria and some other plant species, has promising cytotoxicity against a variety of cell lines by inhibition of cyclin-dependent kinases. Chemical synthesis of its derivatives relies on the combination of isatins and 2,3-dihydro-1H-indol-3-one ('indoxyl') derivatives and usually yields indigo as well as other by-products. Inspection of the hydrolysis of the long-known condensation products of 2-thioxothiazolidin-4-one with isatins gave useful hints for an improved synthesis of indirubins: this reaction does not yield quinoline derivatives but 2-(2,3-dihydro-2-oxo-1H-indol-3-ylidene)-2-sulfanyl acetic acids. By substitution of the sulfanyl group in this oxindoles with anilines and straightforward cyclization under Nazarov conditions, a broad variety of indirubins substituted in the indoxyl ring system are thus available, usually in very good purity and yield. Use of naphthylamines in this reaction sequence yields various fluorescent substances with λ_fl at ca. 630 nm. Copyright

Full text of DOI:10.1002/hlca.201200042

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Improved Synthesis of Indirubin Derivatives by Sequential Build-Up of the
Indoxyl Unit: First Preparation of Fluorescent Indirubins
by Herbert M. Riepl*a) and Corinna Urmannb)
a) Hochschule Weihenstephan-Triesdorf, Fachgebiet Organische & Analytische Chemie, Schulgasse 16,
DE-94315 Straubing (phone: þ 49-9421-187-120; fax þ 49-9421-187-111; e-mail: herbert.riepl@hswt.de)
b) Lehrstuhl fꢀr Rohstoff- & Energietechnologie, Schulgasse 16, DE-94315 Straubing
Indirubin, present in extracts of Isatis tinctoria and some other plant species, has promising
cytotoxicity against a variety of cell lines by inhibition of cyclin-dependent kinases. Chemical synthesis of
its derivatives relies on the combination of isatins and 2,3-dihydro-1H-indol-3-one (ꢁindoxylꢂ) derivatives
and usually yields indigo as well as other by-products. Inspection of the hydrolysis of the long-known
condensation products of 2-thioxothiazolidin-4-one with isatins gave useful hints for an improved
synthesis of indirubins: this reaction does not yield quinoline derivatives but 2-(2,3-dihydro-2-oxo-1H-
indol-3-ylidene)-2-sulfanyl acetic acids. By substitution of the sulfanyl group in this oxindoles with
anilines and straightforward cyclization under Nazarov conditions, a broad variety of indirubins
substituted in the indoxyl ring system are thus available, usually in very good purity and yield. Use of
naphthylamines in this reaction sequence yields various fluorescent substances with lfl at ca. 630 nm.
Introduction. – Since recently, indirubin (1a) is under examination as an
antileukemic agent. This is the result of inspecting the use of indigo dyes in the folk
medicines of India, south-east Asia, and China [1]. Most prominently, this purple
substance is found in the indigo dyeing material obtained from Isatis plant species, but
also it occurs in various quantities in the aqueous extracts of several other plant species
used as medicines like Strobilanthes- and Polygonum spp.
Indirubin is formed as a by-product of the oxidative dimerization of 2,3-dihydro-
1H-indol-3-one (ꢁindoxylꢂ; 2) to yield the blue indigo dye in fermented leaves from the
respective plant species, whereby 2, in the presence of isatin (3a), undergoes
condensation to indirubin (1a; see Scheme 1).
Scheme 1. Indirubin (1a) Formation by Condensation of Indoxyl 2 with Isatin 3a
ꢃ 2012 Verlag Helvetica Chimica Acta AG, Zꢀrich

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Indirubin (1a) has promising cytotoxicity against a variety of cell lines where it
reverses leukaemic transformation by inhibition of cyclin dependent kinases [2], and it
has shown a reduced toxicity in animal experiments. It is essentially insoluble in
aqueous media, a finding, which obscures the elucidation of its biological effects. For
obtaining sound SAR data of the various cytotoxic effects, it will be necessary to
examine a far greater number of derivatives not available at present. These derivatives
should have a far better solubility. Additionally, a future aim will be to examine the
inhibitory activity against protein tyrosin kinases according to an example [3],
dependent on the substituents at C(4’) and C(7’) of the indol-3-one part.
Results and Discussion. – There is only one systematic access to indirubin,
originating from Baeyer. 2-Nitrobenzaldehyde is oxidized in the presence of acetone
which yield – among many side-products – indoxyl (2). The established way to obtain
indirubins (Scheme 1) would then be to condense them with appropriate isatin
derivatives. Additionally, 2 can be prepared in situ by hydrolysis of indoxyl sulfate or
acetate in aqueous base solution. As a by-product, always blue indigo is formed. Access
to other indoxyls apart form the unsubstituted case or the common 5-chloro or 5-
bromoindoxyl is difficult but realizable [4]. These procedures cannot be applied
rigorously to obtain other derivatives. To illustrate the necessity of new methods to
synthesize indirubins, an example of a microbial oxidation procedure [5] can be
mentioned. This, being not selective at all as a disadvantage, also fails to give
reasonable amounts of substance.
We hereby describe a systematic method which allows the preparation of indirubins
substituted at C(4’) to C(7’) of the indol-3-one part, avoiding thereby the preparation of
indoxyls, but using the large variety of commercially available anilines. There is some
older literature describing the synthesis of several differently subsituted isatins.
Together with this new procedure, a large library of indirubins should be avail-
able.
The key step of the method comprises the use of compounds 7, substituted
phenylglycines, capable to undergo a ring closure (Scheme 2) to indirubins 1a – 1j, in
analogy to the old reaction of Heumann, who used the cyclization of N-phenylglycine as
a source of free indoxyl. In this way, formation of free indoxyl and any coupling
products resulting from it is avoided.
This concept was realized with 2-(2,3-dihydro-2-oxo-1H-indol-3-ylidene)-2-sulfanyl-
acetic acids 6, a distinct and stable class of compounds, which were substituted at their
S-atom with anilines to easily yield the desired indirubin precursors 7 (Scheme 3).
2-Thioxothiazolidin-4-one (ꢁrhodaninꢂ, 4) and isatine (3a) reacted in boiling AcOH
or by some other means to give (3Z)-3-(4-oxo-2-thioxo-1,3-thiazolidin-5-ylidene)-1,3-
dihydro-2H-indol-2-one (5a; Scheme 3), which belongs to the class of ꢁindigoidsꢂ, a
generally and easily available class of dark colored compounds. Indigoid 5a was
originally reported to give during hydrolysis either 6a or 2-hydroxy-3-sulfanylquino-
line-4-carboxylic acid (8a) [7][8].
The formation of 6a was questioned for several reasons [6]. After reduction, a
quinoline-2-one derivative was obtained, and so the original condensation product was
claimed to be 8a in its tautomeric 2-one form. Substitution of the S-atom in the claimed
quinolone by refluxing it with aniline was reported to occur and subsequently should

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Scheme 2. A New Synthesis of Indirubins from (2Z)-2-(1,2-Dihydro-2-oxo-3H-indol-3-ylidene)-2-
(phenylamino)ethanoic Acids 7 by a Ring Closure under Nazarov Conditions (polyphosphoric acid
(PPA), 1108)
have given a substance assumed to be 2-hydroxy-3-(phenylamino)quinoline-4-carbox-
ylic acid (8b) in its tautomeric form.
We tried to use this putative 8b from the early report (Scheme 3) for a desired
synthesis of dibenzo[b,f][1,7]naphthyridine-6,12-dione [9]. Cyclization in polyphos-
phoric acid (PPA) surprisingly yielded only indirubine 1a in excellent yield. Various
promoters of the Nazarov-type reaction such as MsOH/phosphorous oxide suspension
were less effective, but do work. Triflic acid was effective even at room temperature.
This could hardly be explained. It would be possible only if the structure 7a, i.e.,
((2Z)-(1,2-dihydro-2-oxo-3H-indol-3-ylidene)(phenylamino)ethanoic acid, is correct
for this compound and not 8b.
In contrast, real compounds 8b could be obtained independently by an alternative
route from the reaction of N,N’-diphenylhydrazines and acetylenedicarboxylates,
followed by rearrangement. Refluxing the acid chloride of 8b with Lewis acids gave
dibenzo[b,f][1,7]naphthyridine-6,12-dione [9], and thus establishing that it is the real
8b. Not even a trace of indirubin was detected in this reaction.
Another evidence in favor of structure 7a is that NMR spectra of compounds of this
type exhibit frequently two distinct sets of signals, often in a ratio of ca. 1:1, e.g.,
compound 7h (R ¼ 5-Cl, R’ ¼ 4’-Cl (1:1)) and 7i (R ¼ 7-Cl, R’ ¼ 2’,3’-Me2, (1:1.5)).
These isomeric mixtures 7 did not contained any 8. Data of the latter are known
accurately from pure samples synthesized independently [9].
Inspection of the 1H/13C-NMR signals of 7 indicate substantially different
electronic environments in some pairs of isomers, so in 7i, other pairs of signal are
only shifted, as in 7h. Whether these spectra really correspond to (Z)/(E) pairs or
represent an admixture of structures like 13 has to be clarified (Scheme 4).

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Scheme 3. Isatin (3a) and 2-thioxothiazolidin-4-one (rhodanine; 4) in Pyridine Formed an Indigoid 5a
that Could Hydrolyze in Aqueous Basic Solution to Yield 2-Hydroxy-3-sulfanylquinoline-4-carboxylic
Acid (8a) or (2Z)-2-(1,2-Dihydro-2-oxo-3H-indol-3-ylidene)-2-(sulfanyl)ethanoic Acid (6a). It was
concluded from the reaction data that 8a was not formed. Reaction of 6a with aniline in DME under
catalysis with anh. ZnCl2 and refluxing for 8 h or microwave heating for minutes yielded 7a, which
subsequently could undergo ring closure to indirubin (1a). If any 8b would have been formed, cyclization
to benzonaphtyridines [9] should have been observed, which was not the case.
Eventually, the question arises whether the (E)-form of 7, cyclized differently from
(Z)-7, or whether a disfavored isomer for indirubine exists. Close inspection of the
obtained dyes revealed no hint for a large amount of such a material, but traces of so far
unknown substances were detected, which would be an interesting topic for future
research. The possibility of easy rotation in 13 would favor the cyclization to the
ordinary indirubin, which is stabilized by the six-membered ring formed by a H-bridge
between NH of one part and the CO group of the other of 1 (Scheme 4).
The substances 7 are also very different from compounds 8 with respect to solubility
in organic media. For example, 8a is sparingly soluble in MeOH, whereas 7a is well

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1
Scheme 4. Besides the Possibility of a (Z)/(E) Mixture of 7a on the Basis of H- and 13C-NMR Signal
Sets Observed, Other Tautomers 13 May Be Responsible for an Equilibration Mechanism and the
Resulting Complete Cyclization of Both Isomers to Indirubin 1a with Its Favourable Configuration by
Involvement of H-Brigdes
soluble. Compounds 7 display particular good H2O solubility, rendering sometimes
problems for their recovery from aqueous solutions.
Existence and good stability of compounds 7 against transformation to 8 is a strong
evidence to assume also structure 6a to be correct and not 8a. Arylation of the SH
group by using aryldiazonium tetrafluoroborates 9 and cuprous salts in MeCN
according to a published procedure [10] would then yield 2-(phenylsulfanyl)acetic
acids 11a and 11b (Scheme 5).
As proof of concept, cyclization of 11 to thioindirubins 12 should be achieved.
Unfortunately, these arylations proved difficult. They give a multitude of products and
frequently fail. But in support of the general structure 6 proposed, the acids available so
far, i.e., 11a (R ¼ 5-Me R’ ¼ 4’-Me) and 11b (R ¼ 5-SO3H, R’ ¼ 4’-Me), do cyclize in
PPA to give thioindirubines 12a (R ¼ 5-Me, R’ ¼ 5’-Me) and 12b (R ¼ 5-SO3H, R’ ¼ 5’-
Me), respectively, in high yield and purity. Usually, compounds 6 are obtained as single
isomers by preferential crystallization. These isomers 6 are stable at room temperature
and react presumably without (E)/(Z)-isomerization to 11.
It is not clear so far under which circumstances 7a would be transformed to 8a, but
only prolonged treatment of 6a or 7a at high pH values seems to cleave the NꢀC(2)
bond of the indolone part according to isatin itself. Ring-opened isatin salts condense
with (phenylsulfanyl)acetone to give indeed 3-(phenylsulfanyl)quinolin-4-carboxylic
acids [11].

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Scheme 5. By Reaction of 6 (6g, R ¼ 5-Me; or 6h, R ¼ 5-SO3H) with Phenyldiazonium Salt 9 (R’ ¼ 4-
Me) in H2O at pH 6 – 7, S-Arylation Could Be Observed, Catalyzed by Cu2þ. Products 11 (11a, R ¼ 5-Me;
R’ ¼ 4’-Me; 11b, R ¼ 5-SO3H, R’ ¼ 4’-Me) yielded thioindirubins 12 (12a, R ¼ 5-Me; R’ ¼ 5’-Me; 12b,
R ¼ 5-SO3H, R’ ¼ 5’-Me) by applying Nazarov conditions (polyphosphoric acid (PPA), 1108).
These observations and the assumption that the original structure 6a is correct, thus
opens a systematic route to indirubins. For practical use as a synthetic method, several
experimental improvements in this concepts had to be found. Notably, it was found that
rhodanin (4) can be condensed with isatines in pyridine when a small quantitiy of H2O
is added, avoiding the long-lasting procedure reported earlier. This method rarely fails.
One of these cases is the reaction of 4-methyl-7-methoxyisatin. In these cases, also the
synthesis of the indigoid in AcOH is difficult.
Second, the substitution of the SH group in compounds 6 to yield derivatives 7 was
practicable only in the case of 7a, because aniline itself was used as a solvent, and 7a
was sparingly soluble therein. This works sometimes for single cases even of more
sensitive compound 7c. Nevertheless, a stoichiometric variant was desirable. The yields
generally being ca. 30 – 40%, assistance by using a thiophilic transition metal salt was
found to be helpful. Use of basic Pb salts indeed results in lead sulfide formation, but
the yield remained low. Cuprous salts resulted in complex decompositions, but use of
anhydrous Zn salts gave promising results. In combination with solvents such as 1,2-
dimethoxyethane (DME), the yield of compounds 7 could be improved up to 70% in a
clean reaction, without additional chromatography of the products. The form of the
starting material is crucial; the reaction does not proceed with sodium salts of 6. For
materials containing such salts, the yield was proportional to the content of free acid. It
was found that protonation by CF3COOH is helpful. In conclusion, a systematic
combination of a variety of commercially available isatins 3 with anilines would lead to
new and unusual indirubins. Examples are 1c and 1d of phenolic character or indirubin-
carboxylic acids 1f and 1g. These indirubins have particularly good H2O solubilities. Of

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great help was the application of microwave heating. Often, indirubins 1 can be
prepared without purification of the intermediates 7. By treating a derivative 6 and a
suitable aniline in a microwave oven, followed by simple extraction with base and
reprecipitation in acid, a material is obtained of sufficient purity because of very short
reaction times, i.e., 8 min instead of 8 h. In this manner, also the reactions with
naphthalenamines and 6d (R ¼ 5-Br), 6e (R ¼ 5-F) were carried out (Scheme 6), which
yielded remarkable ꢁnaphthindirubinsꢂ. It could be shown, by abriged isolation of 7k, 7l,
and 7m, preparation of ꢁnapthindirubinsꢂ from 6d (R ¼ 5-Br) and 6e (R ¼ 5-F) could be
accomplished in one day.
Scheme 6. Cyclization of 7l (R ¼ F) Derived from Naphthalen-1-amine, Interestingly Yielded Two
Isomers, the Major Isomer 15 (R ¼ F) Being a Benzo[de]quinolin-3(2H)-one and 1l, a Regular
ꢀNapthindirubinꢁ (benzo[g]indol-3(2H)-one). Whereas with 7l (R ¼ F) 15 is the major isomer, the Br
derivative 7k predominantly yielded the non-fluorescent 1k. Reaction of naphthalen-2-amine derivative
7m (R ¼ F) did only yield the non-fluorescent ꢁnaphthindirubinꢂ 1m ((Z)-2-(5-fluoro-2,3-dihydro-2-oxo-
1H-indol-3-ylidene)-2,3-dihydro-1H-benzo[e]indol-1-one).
The cyclization of 7l (R ¼ 5-F), derived from naphthalen-1-amine, interestingly
yielded two isomers. The UV-VIS and fluorescence spectra of the major isomer 15
(R ¼ 5-F), which exhibited red-orange fluorescence, are shown in the Figure.
As an impurity, a non-fluorescent minor isomer 1l (R ¼ 5-F) was present,
characterized by a better solubility. Separation was easily achieved by column
chromatography. By the same treatment, 7k, the Br derivative (R ¼ 5-Br), gave the
non-fluorescent isomer 1k (R ¼ 5-Br) in 22% yield and the fluorescent 14 (R ¼ 5-Br) in
a minor amount (1k/14 ca. 3 :1). In case of reaction of 6e with naphthalen-2-amine, also
two isomers were expected (Scheme 6), but only one non-flourescent product was

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Figure. UV/VIS and fluorescence spectrum of (2Z)-2-(5-Fluoro-1,2-dihydro-2-oxo-3H-indol-3-ylidene)-
1,2-dihydro-3H-benzo[de]quinolin-3-one (15). Solvent, acetone; ambient temp. Compound shines
orange-red (629 nm) with a fluorescence quantum yield of 34%.
detected, which was identified as (2Z)-2-(5-fluoro-1,2-dihydro-2-oxo-3H-indol-3-
ylidene)-2,3-dihydro-1H-benzo[e]indol-1-one (1m), the occurrence of a naphthindir-
ubin 1n with the other orientation being considered as less probable due to the
enhanced reactivity of the a-position. This finding raises the question if one of the
isomers in case of naphthalen-1-amine, specifically the fluorescent one, is really an
indirubin-like structure or should be rather formulated as 1,2-dihydro-3H-benzo[de]-
quinolin-3-one 14 (R ¼ Br) and 15 (R ¼ F). Indeed, 1H-benzo[de]quinoline-2,3-diones
show a yellow fluorescence, but not the corresponding naphthisatines (¼1H-
benz[g]indole-2,3-diones). Using 2-methylnaphthalen-1-amine, only a single fluores-
cent compound was formed. With 2-methylnaphthalen-1-amine, no indirubin-like
compound could be obtained since the 2-position was blocked.
Therefore, it was concluded that it is thus the 1H-1-azaphenalen-3-one element is
the source of fluorescence.
Whereas the problem to obtain indirubins substituted in the indoxyl nucleus has
been solved, thus allowing preparation of a wide variety of them, the method is still
dependent on isatins. At present, only nitro-isatins did not lead to any indirubins, since
internal reduction and disappearance of sulfur seems to take place in these cases.
Experimental Part
1
General. IR: Thermo Electron Nicolet 380 (usually in KBr). H- and 13C-NMR: Bruker AMX400.
MS: Finnigan MAT 70, EI: 70 eV, CI: Isobutane. Elemental analyses were performed in the
Mikroanalytisches Labor, Institut fꢀr Anorganische Chemie, Technische Universitꢄt Mꢀnchen. Micro-
wave-asisted syntheses were carried out with CEM DiscoverS class1) single-mode synthesis system
1
)
For further information, see http://www.cem.com

Helvetica Chimica Acta – Vol. 95 (2012)
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(www.cem.com) interfaced with a laptop pc running CEM synergy software monitoring the reaction. The
temp. was checked by an external IR sensor on the floor of the cavity. Once the target temp. was reached,
the microwave system automatically started to count down the hold time1). For reactions, CEM vials
(10 ml) with snap-on caps were used. The pressure was monitored by a sensor outside the snap-on caps.
The upper pressure limit was set to 17 bar. Temp./pressure recording were attached to CEM synergy
reaction files.
Isatins apart from commercial samples, were obtained according to literature, see, e.g.: isatincarbox-
ylic acids [12 – 14], aminoisatins [15], methoxyisatins [16][17], benzoisatins [18][19]. Compounds 6
rarely provide clean anal. data or melting points – except for 6h. As S-Me derivatives 6a’ – 6g’, they gave
good crystals with sharp melting points. Spectral data usually refer to this material if not indicated
otherwise. These derivatives were usually prepared by shaking weakly alkaline solns. in H2O/EtOH
mixtures with MeI according to [7]. In several cases, the crude compounds 7 were cyclized to the
indirubins directly without further purification, so in cases of 1e, 1f, 1h, 1k, 1l, and 1m. Certain indirubins
were sparingly soluble in nearly every solvent. Good COSY spectra could be obtained only by prolonged
sampling times of ca. 24 h.
General Procedure for the Synthesis of 2-Sulfanyl-2-(2-oxoindolin-3-ylidene)acetic Acids (6). A
quantity of desired isatin was dissolved in pyridine in a three-necked vessel with attached frit. Slightly
more than 1 equiv. of 2-thioxothiazolidin-4-one (4; rhodanine, Mr 133.19) in pyridine was added, and
after several min. of mixing and dissolving, H2O (10 ml) was added. The dark red suspension became
warm, while residual solid dissolved. Soon afterwards crystallization started. Some THF was added, and
the mixture was heated to reflux for 1 h. When cool, after standing in the fridge, excess pyridine and
solvent were sucked over the frit. Then, the vessel was gently evacuated while standing in a warm H2O
bath. Hereby, the bright red solid lost excess pyridine and decayed into a powder. From the mother
liquor, by standing in the fridge, additional material could be obtained. Dil. aq. KOH soln. was prepared
and added to the powder, whereby the color faded. After stirring for 1 h at ca. 508, the soln. was
neutralized with aq. HCl at 08 in an ice bath. Half of the acid was slowly added until the soln. became
turbid, the rest was added then quickly to yield a deep orange or red preciptiate. Otherwise, a yellow
material of different chemical structure was obtained. To obtain sufficiently pure material, neutralization
in the cold was essential. Otherwise, red oils were obtained. In this case, stirring of the decanted oil in
freshly dist. H2O at pH ca. 6 for some h yielded a crystalline material. Half the way of to neutrality, often
a thick yellow salt appeared. This could be isolated by sucction and used successfully to prepare the S-Me
derivatives.
Otherwise, an aliquot of the alkaline soln. of the salt was diluted with 20% aq. EtOH, excess MeI was
added, and the mixture was stirred overnight whereby yellow crystals formed. After standing overnight,
they were collected and recrystallized from MeOH.
(2Z)-2-(1,2-Dihydro-2-oxo-3H-indol-3-ylidene)-2-(sulfanyl)ethanoic Acid (6a). From 28 g of
(0.2 mol) isatin and 26.6 g of (0.2 mol ) 4, 80 ml of pyridine, and 10 ml of H2O. Hydrolysis with 45 g
KOH in 400 ml H2O. Acidification with 110 ml conc. HCl. Yield: 24 g (64%). Intense red powder.
(2Z)-(1,2-Dihydro-2-oxo-3H-indol-3-ylidene)-2-(methylsulfanyl)ethanoic acid (6a’). Yellow needles.
IR (KBr): 3552m (br.), 1675s, 1653s, 1574vs, 1473w, 1465m, 1431m, 1417m, 1370s, 1337m, 1295m, 1273w,
1229m, 1187m, 1099w, 1081m, 1011m, 883w, 789m, 744m, 734m, 711w, 670w, 624m, 594m. 1H-NMR
(CD3OD): 7.68 (d, J ¼ 7.5, 1 H); 7.12 (t-like, J ¼ 8.2, 1 H); 6.97 (t-like, J ¼ 8.2, 1 H); 6.83 (d, J ¼ 7.5, 1 H);
2.6 (s, 3 H). 13C-NMR (CDCl3): 165.9; 163.8; 161.3; 139.9; 126.2; 124.4; 122.5; 120.5; 111.4; 108.7; 14.7.
2-(1,2-Dihydro-2-oxo-3H-indol-3-ylidene)-2-(phenylamino)ethanoic Acid (7a). Compound 6a
(2.2 g, 0.01 mol) and aniline (10 ml) were mixed to homogeneity. A slightly exothermic reaction and
evolution of H2S started. Under N2, the temp. was raised to 1408. After 30 min, the green liquid was
cooled, whereby a yellow crystalline precipitate appeared. Before reaching r.t., the yellow solid was
isolated by suction. The greenish material was washed with ice cold MeOH. After drying, the material
was dissolved in 10% aq. NaOH (20 ml) and filtered. The filtrate was acidified by pouring it into dil. aq.
HCl (20 ml), whereby a yellow powder precipitated (0.84 g). The aniline residue could be worked up as
well by complete sublimation of the excess aniline in vacuo to yield 1 g of material. Yield: 1.84 g (64%).
Yellow needles. Recrystallization from hot MeOH. IR: 3425s (br.), 3047w, 2921w, 2851w, 2606m (br.),
2361w, 1661s, 1620vs, 1595s, 1577s, 1496m, 1463m, 1385m, 1337m, 1325m, 1264w, 1226m, 1197m, 1102w,

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1027w, 997w, 742m, 731m, 690m, 640w, 619w, 589w, 544w, 502w, 500m. 1H-NMR ((D8)THF, (E/Z)
mixture): 11.4 (s, 1 H); 10.6 (s, 1 H); 7.4 (t-like, J ¼ 8.0, 2 H); 7.2 (t-like, J ¼ 8.0, 3 H); 7.15 (t-like, J ¼ 7.0,
2 H); 7.0 (t-like, J ¼ 7.0, 1 H); 7.0 – 6.9 (m, 4 H). 13C-NMR ((D8)THF): 171.1 (COOH); 164.75 (C(2) ¼
O); 146.8; 139.0; 137.6; 130.0; 126.8; 125.5; 125.2; 122.3; 121.2; 118.6; 110.1; 96.4. CI-MS: 105.1 (4),
144.1 (32), 165.0 (2), 236 (100), 280.2 (12).
2-(1,2-Dihydro-2-oxo-3H-indol-3-ylidene)-2-[(4-methylphenyl)amino]ethanoic Acid (7b). Com-
pound 6a (2.2 g, 0.01 mol) was mixed with p-toluidine (10 g). The yellow mass was heated for 20 min
at 1408 under Ar at normal pressure in a Kugelrohr distillation apparatus or in some glassware which
allows distillation of the easily crystallizing anilines. Then, pressure was lowered, and the excess p-
toluidine was sublimed off. The dry green material was dissolved in aq. 10% NaOH soln. (20 ml) and
filtered. The filtrate was acidified by pouring it into 20 ml of dil. aq. HCl whereby a yellow powder
precipitates that was collected and air-dried. Yield: 1.98 g (68%). Recrystallization from hot MeOH. IR:
3281s, 3229s, 3065w, 3026w, 2927w, 2862w, 1710s, 1612vs, 1516s, 1475w, 1458w, 1403m, 1322s, 1248s, 1222s,
1197s, 1103s, 1020m, 1000m, 969w, 874m, 847m, 833m, 802m, 781m, 745m, 728m, 685m, 641m, 600m,
567m, 518s, 486m, 473w, 440w. 1H-NMR ((D8)THF, (E/Z) mixture): 11.8 (s, 1 H); 9.9 (s, 1 H); 7.44 (d, J ¼
7.5, 1 H); 7.2 – 7.14 (m, 4 H); 7.0 (t-like, J ¼ 7.0, 1 H); 6.9 – 6.8 (m, 2 H); 2.35 (s, 3 H). 13C-NMR
((D8)THF): 171.6 (COOH); 164.5 (NCO); 148.0 (ArꢀNR); 137.4 (ArNCO); 137.1 (NC); 135.8; 130.6 (2
arom. C); 130.5; 127.4; 125.6; 122.15 (2 arom. C); 121.8; 121.75; 114.0; 111.25; 96.0; 20.75 (Me). CI-MS:
294.3 (14), 250.3 (100), 150.2 (18), 133.1 (23), 118.1 (16).
General Procedure for the Synthesis of Indirubin Derivatives. Compound 7 (ca. 300 – 500 mg) was
suspended in polyphosphoric acid (PPA; 10 g) and heated in an oil bath to ca. 808 by stirring. The temp.
was then raised to 1208 (internal) and kept for 1 h. A dark color appears. The hot melt was poured into
200 ml of dist. H2O and stirred for 2 h. A purple solid was collected by filtration and washed with dist.
H2O to neutral pH. After drying in air, the filter paper keeping the dark solid was extracted in a Soxhlet
apparatus with 200 ml acetone for some h. The solvent was stripped off (ca. 80%), then the liquid was left
for evaporation in air. Intense purple needles.
5’-Methylindirubin (¼(3Z)-1,3-Dihydro-3-(1,3-dihydro-5-methyl-3-oxo-2H-indol-2-ylidene)-2H-in-
dol-2-one; 1b). From 7b (300 mg). Yield: 260 mg (92%). IR: 3444s (br.), 3303s, 3186s (br.), 3053m
(sh), 2918m, 2875m (sh), 1710s, 1663vs, 1619vs, 1684vs, 1492vs, 1464m, 1385w, 1334m, 1292m, 1197vs,
1
1193vs, 1128s, 1043w, 1022m, 961w, 813w, 777w, 744w, 711w, 618w, 583w, 565w. H-NMR ((D6)DMSO):
11.15 (s, 1 H); 11.1 (s, 1 H); 8.63 (d, J ¼ 7.5, 1 H); 7.32 (s, 1 H); 7.27 (d, J ¼ 8.2, 1 H); 7.18 (d, J ¼ 8.2, 1 H);
7.12 (t-like, J ¼ 7.5, 1 H); 6.89 (t-like, J ¼ 7.5, 1 H); 6.78 (d, J ¼ 7.5, 1 H); 2.32 (s, 3 H). 13C-NMR
((D6)DMSO): 189.4; 171.7; 151.3; 141.4; 139.4; 138.7; 131.2; 129.8; 125.3; 124.9; 124.8; 122.2; 121.9; 119.8;
113.9; 110.2; 106.7; 20.8.
(2Z)-2-(1,2-Dihydro-2-oxo-3H-indol-3-ylidene)-2-[(2-methoxy-4-methylphenyl)amino]ethanoic
Acid (7c). 6a (1 g, 4.5 mmol) was mixed with 5 g of 2-methoxy-4-methylanilin and treated as described
for 7b. After complete stripping of excess 2-methyl-4-methoxyaniline, the residue was dissolved in
MeOH and submitted to column chromatography CC Kieselgel G60 (60 g), column diameter, 3 cm;
height, 20 cm; eluent, MeOH/acetone gradient). Yield: 300 mg (21%). IR: 3443s (br.), 2997m, 2828m,
1645vs, 1616s, 1587s, 1504m, 1455w, 1386w, 1347w, 1229w, 1267w, 1216m, 1163w, 1102w, 1047m, 1000w,
973w, 875w, 856w, 793m, 745w, 679w, 610m, 562w, 518w. 1H-NMR ((D6)acetone): 7.3 (d, J ¼ 7.6, 1 H); 7.23
(d, J ¼ 8.8, 1 H); 7.0 (dt-like, J ¼ 7.5, 1.7, 1 H); 6.95 (d, J ¼ 7.4, 1 H); 6.85 (dt-like, J ¼ 7.4, 1.5, 1 H); 6.82 (d,
J ¼ 3.2, 1 H); 6.75 (dd, J ¼ 8.5, 3.2, 1 H); 3.76 (s, 3 H); 2.73 (s, 3 H). 13C-NMR ((D6)acetone): 173.3
(COOH); 158.6 (CONH); 137.4 (ArN); 136.0 (ArO); 131.0 (ArN); 125.4; 124.9; 124.7; 123.4; 121.25;
118.8; 116.7; 116.2; 112.2; 112.0; 109.8; 55.5 (MeO); 55.29 (Me). CI-MS: 324.4 (16), 280.4 (100), 265.3
(18), 247.4 (5.8), 239 (3.0), 219.3 (2.5).
5’-Methoxy-7’-methylindirubin (¼(3Z)-1,3-Dihydro-3-(1,3-dihydro-7-methoxy-5-methyl-3-oxo-2H-
indol-2-ylidene)-2H-indol-2-one; 1c). Cyclization of 0.147 g 7c in 10 g of PPA. Reaction seemed to be
complete after 30 min at 1108. The melt was dissolved in 200 ml of dist. H2O. Compound 1c was soluble in
H2O at r.t., so KCl (15 g) was added. After standing overnight, a dark blue solid was collected by
filtration. Yield: 69 mg (50%). The blue filtrate was treated with 10 g XAD 1600 N resin (Rohm & Haas)
by stirring for 2 h to extract a second crop of 1c. After elution of the dry resin with MeOH, 30 mg of 1c
was obtained from this solid phase. IR: 3222s (br.), 3055m, 2923s, 2848s, 1664vs, 1615s, 1595m, 1496vs,

Helvetica Chimica Acta – Vol. 95 (2012)
1471
1461s, 1421m, 1382w, 1331s, 1288s, 1265s, 1246m, 1207m, 1195m, 1173s, 1146m, 1100w, 1064w, 1041s,
996m, 959m, 932m, 875w, 816m, 775m, 760w, 731w, 699w, 676w, 608m, 599m, 542w. 1H-NMR
((D6)DMSO): 10.9 (s, 1 H); 10.3 (s, 1 H); 8.68 (d, J ¼ 7.4, 1 H); 7.25 (t-like, J ¼ 7.4, 1 H); 7.11 (s, 1 H);
7.04 – 7.33 (m, 2 H); 6.92 (d, J ¼ 7.6, 1 H); 2.26 (s, 3 H); 1.35 (s, 3 H). 13C-NMR ((D6)DMSO): 188.3;
171.5; 156.0; 154.7; 145.4; 140.7; 139.3; 129.4; 125.9; 122.8; 121.5; 121.2; 119.1; 109.8; 106.5; 104.5; 26.3;
14.9.
(2Z)-(1,2-Dihydro-2-oxo-3 H-indol-3-ylidene)[(3,4-dimethoxyphenyl)amino]ethanoic Acid (7d).
Compound 6a (0.63 g, 2.8 mmol) and 3,4-dimethoxyaniline (0.9 g, 5.8 mmol) was dissolved in 1,2-
dimethoxyethane (DME; 5 ml). After addition of a few drops CF3COOH and dry ZnCl2 (0.8 g,
5.8 mmol), the red soln. was refluxed 6 to 8 h (8 min microwave). The soln. was worked up by pouring it
into 15% dil. aq. HCl (50 ml), isolation of the solids, dissolving it in dil. aq. NaOH (5 ml), and
repreciptiation with 15% dil. aq. HCl. Losses because of good H2O solubility. Yield: 0.5 g (50%) (with
1
microwave: 62%). Yellow powder H-NMR ((D6)DMSO): 11.3 (s, 1 H); 10.7 (s, 1 H); 7.18 (d, J ¼ 7.4,
1 H); 7.02 (t-like, J ¼ 7.4, 1 H); 6.95 (d, J ¼ 8.6, 1 H); 6.93 – 6.85 (m, 3 H); 6.78 (dd, J ¼ 2.5, 8.6, 1 H); 3.75
(s, 3 H); 3.73 (s, 3 H). 13C-NMR ((D6)DMSO): 170.5; 164.2; 149.2; 147.1; 146.7; 136.9; 131.7; 124.3; 122.0;
120.5; 117.7; 113.4; 112.4; 109.4; 106.7; 94.8; 55.8; 55.5. CI-MS: 340 (2), 339 (5), 298 (39), 296 (36), 246
(7), 207 (10), 206 (100), 154 (61), 153 (38).
5’,6’-Dimethoxyindirubin (¼(3Z)-1,3-Dihydro-3-(1,3-dihydro-5,6-dimethoxy-3-oxo-2H-indol-2-yl-
idene)-2H-indol-2-one; 1d). Cylization of 0.26 g of 7d in 5 g of PPA. Workup in 200 ml of dist. H2O.
Dark powder, separated from a red soln. Yield: 108 mg (43%). An additional crop of 43 mg could be
extracted from the soln. by application of 10 g of XAD 1600N resin (Rohm & Haas). Compound seems
to be sensitive to air. Extraction of the dark powder with acetone in a Soxhlet extractor left considerable
dark residue insoluble in acetone. TLC of the extract (toluol/acetone 5 :2) showed also presence of a little
amount of the other possible regioisomer. Purification by CC Kieselgel G 60 (60 g), cyclohexane/acetone
3 :1; column diameter, 3 cm; height, 20 cm). Yellow material (minor isomer eluating first) was discarded.
The main fraction was collected, and the solvent was stripped off in vacuo until precipitation occured.
The dark flakes were collected by centrifugation, and the clear supernatant was discarded. Yield: 20 mg.
IR: 2961 (sh), 2924m, 2856w, 1663s, 1620s, 1596m, 1490s, 1464m, 1352w, 1311m, 1290m, 1269w, 1244w,
1208s, 1156m, 1133m, 1099w, 1022s, 956w, 856m, 773w, 743w, 669w. 1H-NMR ((D6)DMSO): 10.8 (s, 1 H);
10.6 (s, 1 H); 8.8 (d, J ¼ 7.4, 1 H); 7.21 (t-like, J ¼ 7.4, 1 H); 7.08 (s, 1 H); 7.07 (s, 1 H); 6.99 (t-like, J ¼ 7.4,
1 H); 6.88 (d, J ¼ 7.4, 1 H); 3.85 (s, 3 H); 3.75 (s, 3 H). 13C-NMR ((D6)DMSO): 187.7; 172.3; 159.0; 151.5;
146.0; 142.0; 141.2; 130.3; 126.0; 122.7; 122.6; 111.4; 110.9; 107.5; 107.0; 98.1; 57.4; 57.2. CI-MS: 322 (3),
321 (2), 306 (3), 305 (2), 292 (2), 291 (2), 278 (17), 248 (20), 247 (14), 166 (42), 165 (58), 164 (14), 151
(13), 135 (13), 134 (100), 133 (51).
6’,7’-Dimethylindirubin (¼(3Z)-1,3-Dihydro-3-(1,3-dihydro-6,7-dimethyl-3-oxo-2H-indol-2-yl-
idene)-2H-indol-2-one; 1e). Compound 6a (2.0 g, 10 mmol) was mixed with 10 g 2,3-dimethylaniline
and treated as described for 7b. After evaporation to dryness in a high vacuum, the material was directly
subjected to 20 g of PPA and cyclized for 1 h at 1108. By workup in 500 ml of dist. H2O, dark violet
material separated. After filtration, drying in air, and extraction of the solid with acetone, a dark powder
was obtained. Yield: 1.9 g (72%). IR: 3432s (br.), 1698w, 1664vs, 1615vs, 1592s, 1492w, 1462m, 1434m,
1382w, 1316w, 1293s, 1270w, 1209s, 1180m, 1150w, 1082m, 1030m, 994m, 964w, 801w, 771m, 746m, 698w,
673w. 1H-NMR ((D6)DMSO): 10.95 (s, 1 H); 10.5 (s, 1 H); 8.68 (d, J ¼ 8.6, 1 H); 7.43 (d, J ¼ 7.4, 1 H); 7.25
(t-like, J ¼ 7.4, 1 H); 7.0 (t, J ¼ 7.4, 1 H); 6.9 – 6.88 (m, 2 H); 2.29 (s, 3 H); 2.16 (s, 3 H). 13C-NMR
((D6)DMSO): 187.9; 171.6; 150.5; 147.0; 140.7; 139.5; 129.3; 124.5; 123.6; 121.9; 121.4; 121.2; 119.5; 117.3;
109.8; 106.3; 20.1; 11.6.
Indirubin-5-carboxylic Acid (¼(2Z)-2,3-Dihydro-2-(1,2-dihydro-2-oxo-3H-indol-3-ylidene)-3-oxo-
1H-indole-5-carboxylic Acid; 1f). Compound 6a (0.663 g, 3 mmol) was dissolved in 5 ml of DME and 1 g
of ethyl 4-aminobenzoate was added. After addition of three drops of CF3COOH and 408 mg of ZnCl2,
the mixture was refluxed for 24 h and worked up with 50 ml of 5% aq. NaOH. Precipitation occurred by
acidification. Yield: 0.23 g. Another crop of 100 mg could be obtained by repeated extraction of the solid
residue from the initial workup with 5% aq. NaOH.
A part of the dried material 1f (70 mg) was cyclized in 15 g of PPA at a prolonged reaction time of
1.5 h. Workup in 150 ml of H2O. The product had considerable solubility in H2O. The purple acid was

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Helvetica Chimica Acta – Vol. 95 (2012)
isolated by collection of the solid phase (5 g of Amberlite XAD 1600 N, Rohm & Haas) after 1 d stirring.
The acid was isolated by elution with acetone. Yield: 60 mg. IR: 3428s (br.), 3200 (sh), 2918m, 2849w,
1680s, 1667s, 1617s, 1538w, 1494m, 1463m, 1384w, 1331w, 1284w, 1262m, 1209m, 1176m, 1117w, 961w.
1H-NMR ((D6)DMSO): 11.27 (s, 1 H); 10.9 (s, 1 H); 8.77 (d, J ¼ 7.4, 1 H); 8.2 – 8.1 (m, 2 H); 7.47 (d, J ¼
8.6, 1 H); 7.27 (t-like, J ¼ 7.4, 1 H); 7.02 (t-like, J ¼ 6.4, 1 H); 6.9 (d, J ¼ 7.3, 1 H).
(3Z)-3-[Carboxy(sulfanyl)methylidene]-2,3-dihydro-2-oxo-1H-indole-7-carboxylic Acid (6b). From
isatin-7-carboxylic acid (¼2,3-dihydro-2,3-dioxo-1H-indole-7-carboxylic acid; 5.8 g, 0.03 mol) and 3.99 g
rhodanine in 40 ml pyridine and 5 ml of dist. H2O. When few crystals appeared, 50 ml of THF was added.
Reflux for 1 h. Isolation of the red compound after standing in the fridge. Hydrolyis in 6 g NaOH/100 ml
H2O. After 1 h/508 stirring, neutralization with 45 ml 17% aq. HCl. Red powder, yield 6.7 g (83%).
(3Z)-3-[Carboxy(methylsulfanyl)methylidene]-2,3-dihydro-2-oxo-1H-indole-7-carboxylic Acid
(6bꢀ). Yellow needles. IR: 3421s (br.), 1669s, 1601s, 1558s, 1507w, 1476m, 1436m, 1375s, 1334m,
1293m, 1241m, 1188m, 1158w, 1084w, 860w, 782m, 750w, 733m, 712m, 668w, 628w. 1H-NMR
((D6)DMSO): 6.9 (d, J ¼ 7.6, 1 H); 6.8 (d, J ¼ 7.6, 1 H); 6.17 (t-like, J ¼ 7.6, 1 H); 1.58 (s, 3 H).
13C-NMR ((D6)DMSO): 173.6; 169.0; 166.7; 157.3; 139.7; 128.0; 125.8; 123.7; 121.2; 117.4; 113.7; 14.2.
Indirubin-7-carboxylic Acid (¼(3Z)-2,3-Dihydro-2-oxo-3-(1,3-dihydro-3-oxo-2H-indol-2-ylidene)-
1H-indole-7-carboxylic Acid; 1g). Compound 6b (0.5 g) was suspended in 5 ml of DME together with 3 –
4 of drops of CF3COOH, and 0.35 g (2 equiv.) freshly dist. aniline was added. A yellow precipitate
formed, and 0.5 g of ZnCl2 was added. The mixture was properly closed with plastic septum and placed in
a microwave oven (heating 2 min). The temp. reached 1708 and 7 bar. After cooling, the green liquid and
black solid were dissolved in 25 ml of H2O with 0.8 g of NaOH. After standing for 10 min and filtering,
the dark soln. was acidified with 17% aq. HCl. A beige precipitate formed, which was isolated by suction
and dried. Yield 0.28 g. This material was cyclized with 5 g of PPA at 1108 for 45 min. The hot melt was
poured into 100 ml of dist. H2O. A red precipitate was isolated, which was collected by filtration and
purified by dissolving in dil. aq. NaOH, and reprecipitated with 17% aq. HCl. Yield: 210 mg. IR: 3397s,
3322s, 1686s, 1654m, 1637m, 1607vs, 1585s, 1480m, 1466s, 1442m, 1393w, 1348w, 1311s, 1267m, 1219m,
1178vs, 1156vs, 1093w, 992s, 876w, 797w, 775w, 749m, 709w, 686w, 637w, 602w, 576w. 1H-NMR
((D6)DMSO): 11.06 (s, 1 H); 10.27 (s, 1 H); 8.94 (d, J ¼ 8.2, HꢀC(4’)); 7.74 (d, J ¼ 8.9, HꢀC(6’)); 7.65 (d,
J ¼ 7.5, HꢀC(4)); 7.57 (t-like, J ¼ 8.2, HꢀC(6)); 7.36 (d, J ¼ 7.5, HꢀC(7)); 7.11 (t-like, J ¼ 7.5, HꢀC(5’));
7.02 (t-like, J ¼ 7.5, HꢀC(5)). 13C-NMR ((D6)DMSO): 189.5; 171.3; 167.8; 153.0; 141.9; 140.0; 138.2
(C(5)); 130.3 (C(6’)); 128.9 (C(4’)); 125.3 (C(7)); 123.3 (C(6)); 122.5 (C(5’)); 121.8; 119.6; 114.2 (C(4));
112.1; 105.16. CI-MS: 307 (8), 306 (39), 288 (27), 263 (11), 262 (62), 261 (6), 260 (23), 248 (3), 234 (34),
206 (13), 205 (25), 204 (19), 192 (10), 191 (78), 164 (7), 163 (61), 162 (12), 145 (50), 144 (67), 135 (16),
119 (100), 118 (12), 117 (50), 116 (17).
The residue of this acid/base treatment, recrystallized from acetone, was methyl (3Z)-2-oxo-3-(3-
oxo-1,3-dihydro-2H-indol-2-ylidene)-2,3-dihydro-1H-indole-7-carboxylate (1gꢀ). 1H-NMR ((D6)DMSO):
11.1 (s, 1 H); 10.52 (s, 1 H); 8.96 (d, J ¼ 7.35, 1 H); 7.73 (d, J ¼ 8.6, 1 H); 7.62 (d, J ¼ 7.4, 1 H); 7.55 (t-like,
J ¼ 8.6, 1 H); 7.39 (d, J ¼ 8.6, 1 H); 7.11 (t-like, J ¼ 7.4, 1 H); 7.01 (d, J ¼ 7.4, 1 H); 3.85 (s, 3 H). CI-MS:
321 (19), 320 (100), 289 (11), 288 (38), 263 (12), 262 (59), 261 (10), 260 (26), 234 (21), 205 (13.4).
(2Z)-2-(5-Chloro-1,2-dihydro-2-oxo-3H-indol-3-ylidene)-2-(sulfanyl)ethanoic Acid (6c). From 5-
chloroisatine (4.5 g, 0.024 mol), 3.6 g of rhodanine, in 40 ml of pyridine, as described for 6a. Reflux with
50 ml of THF, hydrolysis with 5.66 g of KOH in 75 ml of dist H2O, and then neutralization by dropwise
addition of 12 – 15% aq. HCl (ca. 45 – 50 ml) afforded the product. Sometimes sticky material was
isolated from the mother liquid. This was stirred for some h in neutral dist. H2O, whereby crystalization
took place. Yield: 5.12 g (81%). Red powder. S-Me derivative (6cꢀ): IR: 3418 (br.), 1684s, 1568vs, 1469s,
1429w, 1364s, 1300s, 1257w, 1228m, 1192m, 1174m, 1109w, 1085s, 957w, 923w, 853w, 804m, 778m, 724w,
685w, 659m, 617s, 592w, 559w. 1H-NMR ((D6)DMSO): 10.23 (s, 1 H); 7.49 (d, J ¼ 1.9, 1 H); 7.1 (dd, J ¼
1.9, 8.3, 1 H); 6.75 (d, J ¼ 8.3, 1 H); 2.46 (s, 3 H). 13C-NMR ((D6)DMSO): 165.6; 163.8; 163.2; 138.2;
126.2; 125.7; 124.3; 121.9; 110.9; 109.9; 14.9. FAB-MS: 270 (0.83), 271 (0.17), 272 (0.36), 273 (0.18). CI-
MS: 241 (9), 240 (9), 239 (22), 228 (18), 227 (40), 226 (54), 225 (100), 224 (3), 210 (12), 206 (6), 196 (3),
194 (11), 192 (27), 180 (20), 179 (4), 178 (34), 164 (18), 163 (2).
(2Z)-(5-Chloro-1,2-dihydro-2-oxo-3H-indol-3-ylidene)[(4-chlorophenyl)amino]ethanoic Acid (7h).
From 6c (1.2 g) in 10 ml of DME, adding 2 – 3 drops CF3COOH, 1.27 g of p-chloraniline and 0.68 g of

Helvetica Chimica Acta – Vol. 95 (2012)
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1
anh. ZnCl2. Reflux for 8 h and workup yielded 0.68 g (40%) of 7h (1.5 :1 (Z)/(E)-mixture). H-NMR
((D6)DMSO; major isomer): 11.5 (s, 1 H); 10.8 (s, 1 H); 7.45 (d, J ¼ 7.4, 2 H); 7.27 (d, J ¼ 7.4, 2 H); 7.19 (s,
1 H); 7.08 (d, J ¼ 7.3, 1 H); 6.9 (d, J ¼ 7.3, 1 H). 13C-NMR ((D6)DMSO): 170.7 (C¼O, major isomer);
164.2 (C¼O, minor isomer); 149.2 (NC¼O, major isomer); 145.2 (NC¼O, minor isomer); 137.7 (major
isomer); 135.8 (minor isomer); from DEPT-90: 129.7 (2 arom. CH, major isomer); 129.0 (2 arom. CH,
minor isomer); 125.1; 124.2; 123.0 (2 arom. CH, major isomer); 121.3; 117.9; 117.0 (2 arom. CH, minor
isomer); 111.0 (olef. C, major isomer); 105.0 (olef. C, minor isomer); 95.2 (olef. C, major isomer); 94.8
(olef. C, minor isomer).
5,5’-Dichloroindirubin (¼(3Z)-5-Chloro-3-(5-Chloro-1,3-dihydro-3-oxo-2H-indol-2-ylidene)-1,3-di-
hydro-2H-indol-2-one; 1h). From 7h (150 mg) in 5 g of PPA, heating for 30 min at 1308. Workup in 100 ml
of dist. H2O. Precipitate was collected, dried, and extracted with acetone. Yield: 146 mg (97%). Pure
material, intense purple powder. IR: 3432s (br.), 3336s, 3300s, 2921s, 2851m, 1674vs, 1663vs, 1616s,
1592m, 1539w, 1468s, 1382w, 1293m, 1278m, 1215m, 1172m, 1125w, 1112w, 1018w, 983w, 889w, 823m,
816m, 775w, 714w, 668w, 653m, 592m. 1H-NMR ((D6)DMSO): 11.1 (s, 1 H); 11.0 (s, 1 H); 8.8 (s, 1 H); 7.7
(s, 1 H); 7.6 (dd, J ¼ 2.5, 8.6, 1 H); 7.45 (d, J ¼ 8.6, 1 H); 7.3 (dd, J ¼ 2.5, 8.6, 1 H); 6.9 (d, J ¼ 8.6, 1 H).
13C-NMR ((D6)DMSO): 187.7; 170.4; 151.1; 139.7; 139.0; 136.6; 128.7; 125.7; 125.1; 123.9; 123.7; 122.8;
120.2; 115.3; 110.8; 106.0. CI-MS: 333 (9), 332 (8), 331 (12), 330 (8), 308 (6), 307 (20), 306 (26), 305 (31),
304 (34), 229 (5), 228 (37), 227 (31), 226 (100), 225 (57), 224 (4).
(2Z)-2-(7-Chloro-1,2-dihydro-2-oxo-3H-indol-3-ylidene)-2-(sulfanyl)ethanoic Acid (6d). 7-Chloro-
isatine (9 g, 0.05 mol), rhodanin (7.2 g, 0.054 mol), 60 ml of pyridine, 10 ml of dist. H2O. 180 ml of THF:
crystalline precipitate (9 g). From the mother liquor another 4.5 g; total yield: 13.5 g (91%). Hydrolysis:
5.7 g of NaOH in 25 ml of dist. H2O with 9 g precipitate. At 50 – 608 after 1 h, a greenish solid appeared.
Addition of 5 ml of dist. H2O, the solid dissolved leaving a dark yellow liquid. After standing in the
fridge, a mass of needles crystallized: the sodium salt of 6d. It was directly used to prepare (2Z)-(7-
chloro-1,2-dihydro-2-oxo-3H-indol-3-ylidene)-2-(methylsulfanyl)ethanoic acid (6d’). Yellow needles. IR
(KBr): 3437 (br.), 1690w, 1568m, 1445w, 1368w, 1330m, 1317m, 1294w, 1240w, 1170m, 1144m, 1084w,
1053w, 955w, 788w, 749w, 722m, 637w, 600w, 534m. 1H-NMR ((D6)DMSO): 10.5 (s, 1 H); 7.5 (d, J ¼ 7.5,
1 H); 7.11 (d, J ¼ 8.2, 1 H); 6.93 (t-like, J ¼ 8.2, 1 H); 2.47 (s, 3 H). 13C-NMR ((D6)DMSO): 165.6; 164.3;
163.0; 137.1; 126.3; 125.8; 121.7; 120.9; 113.1; 111.2; 14.9. CI-MS: 268 (3), 270 (1), 241 (14), 240 (19), 239
(32), 228 (20), 227 (40), 226 (64), 225 (100), 210 (11), 192 (33), 180 (18), 164 (13).
(2Z)-2-(7-Chloro-1,2-dihydro-2-oxo-3H-indol-3-ylidene)-2-[(2,3-dimethylphenyl)amino]ethanoic
Acid (7i). (2Z)-(7-Chloro-1,2-dihydro-2-oxo-3H-indol-3-ylidene)-2-(methylsulfanyl)ethanoic acid (6d’;
1 g, 3.7 mmol) was suspended with 2,3-dimethylanilin (0.94 g, 7.8 mmol) in 8 ml of AcOH and heated to
reflux. After complete dissolution of all solid matter, a yellow precipitate appeared after 2 h. When cold,
the liquid was subjected to suction, and the yellow solid was washed with EtOH, and dried. Yield: 760 mg
1
(57%). Yellow powder, H-NMR ((D6)DMSO; 2 :1 mixture of isomers): 11.71 (s, 1 H, major isomer);
10.6 (s, 1 H, minor isomer); 7.6 (d, J ¼ 8.2, 1 H, major isomer); 7.54 (d, J ¼ 7.5, 1 H, minor isomer); 7.38 (d,
J ¼ 7.5, 1 H, major isomer); 7.24 (d, J ¼ 8.2, 1 H, minor isomer); 7.05 – 6.98 (m, ca. 3 H); 6.94 (d, J ¼ 7.5,
1 H, minor isomer); 6.91 (d, J ¼ 8.2, 1 H, major isomer); 6.81 (dd, J ¼ 7.5, 7.5, 1 H, major isomer); 2.49 –
2.51 (m, ca. 6 H); 2.25 (s, 3 H, major isomer); 2.22 (s, 3 H, minor isomer). 13C-NMR ((D6)DMSO;
2 :1 mixture of isomers): 171.2 (C¼O, major isomer); 165.0 (C¼O, minor isomer); 163.8 (C¼O, major
isomer); 158.9 (C¼O, minor isomer); 171.2; 163.8; 137.85; 137.0; 132.3; 127.1; 126.7; 126.1; 125.9; 122.4;
121.8; 121.3; 120.9; 118.8; 116.8; 112.85; 20.4; 13.5. CI-MS: 342 (38), 343 (12), 344 (15), 345 (3), 346 (1),
300 (24), 299 (23), 298 (100), 281 (6), 192 (12), 178 (36), 167 (20).
7-Chloro-6’7’-dimethylindirubin (¼(2Z)-2-(7-Chloro-1,2-dihydro-2-oxo-3H-indol-3-ylidene)-2-
[(2,3-dimethylphenyl)amino]ethanoic Acid; 1i). Compound 7i (342 mg, 1 mmol) cyclized in 27 g of
PPA at 1208 for 1 h. After workup in 100 ml of dist. H2O, the precipitate was collected, dried, and
extracted with acetone. Yield: 270 mg (83%). Dark red powder. 1H-NMR ((D6)DMSO): 11.32 (s, 1 H);
10.58 (s, 1 H); 8.62 (d, J ¼ 7.8, 1 H); 7.42 (d, J ¼ 7.8, 1 H); 7.28 (d, J ¼ 7.8, 1 H); 7.0 (d, J ¼ 7.8, 1 H); 6.92 (t-
like, J ¼ 8.2, 1 H); 2.3 (s, 3 H); 2.17 (s, 3 H).
(2Z)-2-(5-Bromo-1,2-dihydro-2-oxo-3H-indol-3-ylidene)-2-(sulfanyl)ethanoic Acid (6e). a) Bromo-
isatin (13.5 g, 0.06 mol), 7.98 g of rhodanin (0.061 mol) in 70 ml pyridine, induced by adding 3.5 ml of dist.
H2O. When the liquid separated solids (after 30 min), 50 ml of THF was added, and the mixture was

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refluxed 2 h. From time to time, 10 ml of THF were added (total 100 ml). After cooling overnight, the red
solid was isolated by suction and dried in air. After hydrolysis in 100 ml of dist. H2O and 9.6 g of NaOH,
the dark soln. was kept at 608 for 1 h, whereby a yellow soln. appeared, which was neutralized and
acidified with 17% HCl at 08 to pH 1. A bright red powder apperars. Yield: 15.5g (86%).
b) Pyridinium Salt of 6e. Hydrolysis of 70 g of indigoid (obtained from refluxing equimolar amounts
of 5-bromoisatin and rhodanin in AcOH for 8 h) in 120 ml of H2O and 16 g NaOH at 608 gave a yellow
precipitate, which was isolated by suction. Yield: 19 g. 1H-NMR (D2O): 8.77 (d, J ¼ 2.0, 1 H); 8.38 (d, J ¼
4.1, 2 H); 7.73 (t-like, J ¼ 4.1, 1 H); 7.4 (s, 1 H); 7.31 (t-like, J ¼ 4.1, 2 H); 7.15 (dd, J ¼ 2.0, 8.1, 1 H); 6.7 (d,
J ¼ 8.0, 1 H). 13C-NMR (D2O): 184.8; 179.6; 167.9; 148.9; 138.0; 136.9; 128.9; 127.4; 124.8; 124.1; 114.0;
113.6; 110.7.
(2Z)-2-(5-Bromo-1,2-dihydro-2-oxo-3H-indol-3-ylidene)-2-[(4-methylphenyl)amino]ethanoic Acid
(7j). Compound 6e (5.22 g, 0.017 mol) was mixed with 10 g of molten 4-methylaniline and treated as
described for 7a: 3.6 g (55%) of 7j. Dull yellow powder. IR: 3376s (br.), 3246s (br.), 2919m, 2857m,
1710s, 1620vs, 1577m, 1517m, 1474m, 1384w, 1308m, 1240m, 1225s, 1192m, 1116w, 1064w, 1019w, 876w,
809m, 747w, 676w, 594w, 551w. 1H-NMR ((D8)THF, ca. 2 :1 mixture of isomers): 11.7 (s, 1 H, major
isomer); 11.6 (minor isomer); 9.8 (s, 1 H, major isomer); 9.7 (minor isomer); 7.4 (s, 1 H, major isomer);
7.3 (major isomer); 7.15 (m, 5 H); 7.12 (minor isomer); 7.0 (minor isomer); 6.85, 6.8 (d, 1 H, major
isomer); 6.7 (minor isomer); 2.3 (s, 3 H, major isomer); 2.2 (minor isomer). 13C-NMR (D2O):172.0
(minor isomer); 171.6; 164.8 (minor isomer); 164.5; 148.1; 146.8 (minor isomer); 137.3 (minor isomer);
137.1 (minor isomer); 135.7; 130.6; 130.5; 130.2; 127.3 (minor isomer); 125.6; 125.0; 122.1; 121.8 (minor
isomer); 121.7; 121.2; 119.3; 114.0; 111.2; 109.8; 97.5 (minor isomer); 96.0; 30.5 (minor isomer); 24.1. CI-
MS: 374 (1), 330 (17), 294 (3), 284 (10), 213 (25), 211 (23), 183 (6), 147 (14), 107 (19).
(2Z)-2-(5-Bromo-1,2-dihydro-2-oxo-3H-indol-3-ylidene)-1,2-dihydro-3H-benzo[g]indol-3-one (1k).
Obtained from 6e (852 mg) and naphthalen-1-amine (858 mg) in 10 ml of DME/402 mg of ZnCl2, after
18 h reflux. After workup with 50 ml of dist. H2O/0.5 g NaOH and acidification, yellow powder (662 mg)
was obtained. Alternatively, a mixture of above mentioned compounds was properly shaken or stirred in
an appropriate reagent tube closed with a plastic septum and subjected to microwave conditions for
2 min. Temp. reached 1708 at 7 bar. Yield was identical compared to thermal treatment. This material
without further workup was cyclized in 20 g of PPA at 1208. The product contained two isomers in a ratio
of ca. 3 :1. TLC showed a non-fluorescent bluish major isomer at Rf 0.3, another compound 14 at Rf 0.1,
which was fluorescent in an orange tone. The precipitate resulting from the workup in H2O after drying in
air was extracted with acetone for 1 h, only to exploit bad solubility of the minor isomer. Then, extraction
was terminated, and the solvent was stripped off: pure 1k. Yield: 132 mg. Dark powder. IR: 3433m,
2922m, 2851m, 1692sh, 1665vs, 1631s, 1618s, 1577s, 1529s, 1464s, 1445s, 1421m, 1389m, 1324m, 1284m,
1201s, 1175s, 1140w, 1116w, 1007w, 979w, 878w, 805w, 761m, 711w, 672w, 647w, 540w. 1H-NMR
((D6)DMSO): 11.12 (s, 1 H); 11.08 (s, 1 H); 9.0 (s, 1 H); 8.25 (d, J ¼ 8.6, 1 H); 8.0 (d, J ¼ 8.6, 1 H); 7.74 (t-
like, J ¼ 7.4, 1 H); 7.66 (t-like, J ¼ 7.4, 1 H); 7.62 (d, J ¼ 7.4, 1 H); 7. 52 (d, J ¼ 7.4, 1 H); 7.45 (d, J ¼ 7.4,
1 H); 6.89 (d, J ¼ 7.4, 1 H). 13C-NMR ((D6)DMSO): 187.2; 170.8; 152.3; 140.4; 139.3; 137.8; 132.1; 130.6;
128.6; 127.1; 126.9; 122.6; 121.6; 119.6; 119.4; 113.7; 113.4; 111.6; 110.8; 107.6.
(2Z)-2-(5-Fluoro-1,2-dihydro-2-oxo-3H-indol-3-ylidene)-2-(sulfanyl)ethanoic Acid (6f). Obtained
from 5-fluorisatin (2.57 g, 0.015 mol) with rhodanin (2.0 g) in 10 ml of pyridine, and 10 drops dist. H2O.
After solidification, addition of 15 ml of THF, and reflux for 1 h, all solvents were evaporated in vacuo.
The solid was hydrolyzed in 55 ml of H2O/2.4 g of NaOH. After stirring for 1 h, the soln. was neutralized
with 50 ml of dil. HCl at 08. The red solid was hygroscopic and oily, and needed to be dried in vacuo.
Otherwise, stirring the sticky solid overnight in 20 ml dist. H2O resulted in a red powder. Yield: 3.28 g
(91%).
(2Z)-(5-Fluoro-1,2-dihydro-2-oxo-3H-indol-3-ylidene)-2-(methylsulfanyl)ethanoic Acid (6f’). IR:
3418 (br.), 1684s, 1568vs, 1469s, 1429w, 1364s, 1300s, 1257w, 1228m, 1192m, 1174m, 1109w, 1085s, 957w,
923w, 853w, 804m, 778m, 724w, 685w, 659m, 617s, 592w, 559w. 1H-NMR ((D6)DMSO): 10.23 (s, 1 H); 7.49
5
3
4
(dd, 3J(H,F) ¼ 9.8, J(H,H) ¼ 2.5, 1 H); 7.1 (dt, 3J(H,F) ¼ 8.7, J(H,H) ¼ 8.7, J(H,H) ¼ 2.5, 1 H); 6.75
(dd, 4J(H,F) ¼ 4.9, 3J(H,H) ¼ 8.6, 1 H); 2.46 (s, 3 H).
(2Z)-2-(5-Fluoro-1,2-dihydro-2-oxo-3H-indol-3-ylidene)-1,2-dihydro-3H-benzo[de]quinolin-3-one
(15). Obtained from 6f (930 mg), naphthalen-1-amine (1.1 g), and ZnCl2 (0.52 g) in 5 ml of DME. The

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mixture of the above mentioned compounds was properly shaken or stirred in an appropriate reagent
tube closed with a plastic septum and subjected to microwave conditions for 2 min. The temp. reached
1708 at 7 bar. Workup in 50 ml of H2O/0.5 g NaOH and neutralization with dil. HCl at 08 yielded the
crude material: 1.2 g. This material (738 mg) was dissolved in 15 ml of H2O/1 g of NaOH, filtered, and
reprecipitated by addition of dil. HCl. Yield: 440 mg. This material was cyclized in 10 g of PPA. After
workup in 250 ml of H2O, the dark insoluble residual material was collected by filtration. After drying,
the filter was extracted in a Soxhlet with acetone. Yield: 390 mg. TLC showed presence of a minor isomer.
Chromatography of 50 mg of this material with 80 g of Kieselgel G60 with hexane/acetone 3 :1 as eluent
provided a deep red eluate, which was concentrated and subjected to prep. HPLC (Kieselgel G60, column
diameter, 2.5 cm; length, 25 cm; eluent: heptane acetone 1:1; at 12 ml/min; lmonitor at 400 and 500 nm).
An impurity was eluted at 6.5 min and discarded. The minor non-fluorescent isomer 1l eluted at 8.9 min
and the major product at 14 min. Fractions were collected and evaporated in vacuo. Yield of fluorescent
15: 38 mg. IR: 3477s (br.), 3366s, 3250m, 2926w, 1645s, 1626s, 1573vs, 1555w, 1528m, 1502w, 1479w, 1467w,
1399m, 1365w, 1349w, 1310m, 1279m, 1260s, 1184w, 1133w, 969w, 893w, 802w, 787w, 749w, 683w, 624w,
585w. 1H-NMR ((D6)DMSO): 12.14 (s, 1 H); 11.36 (s, 1 H); 10.25 (d, J ¼ 8.6, 1 H); 9.39 (dd, 3J(H,F) ¼
13.5, 5J(H,H) ¼ 2.5, 1 H); 8.85 (d, J ¼ 8.6, 1 H); 8.76 (d, J ¼ 7.4, 1 H); 7.8 (t-like, J ¼ 8.6, 1 H); 7.62 (d, J ¼
7.4, 1 H); 7.39 (dd, 4J(H,F) ¼ 4.9, 3J(H,H) ¼ 8.6, 1 H); 7.33 (ddd, 3J(H,F) ¼ 8.6, 3J(H,H) ¼ 8.6, 4J(H,H) ¼
2.45, 1 H); 6.85 (d, J ¼ 8.6, 1 H). CI-MS: 302 (97), 301 (3).
(2Z)-2-(5-Fluoro-1,2-dihydro-2-oxo-3H-indol-3-ylidene)-2,3-dihydro-1H-benz[e]indol-1-one (1m).
Obtained from 6f (418 mg, 1.75 mmol) and naphthalen-2-amine (500 mg, 3.5 mmol) in 5 ml of DME,
traces of CF3COOH, and 470 mg of ZnCl2. After heating under microwave condition (1608, 2 min) or 8 h
conventional reflux the soln. was added to 50 ml of dist. H2O/600 mg of NaOH. The solid residue was
extracted once more with fresh base. After filtration from the residue, the filtrate was acidified: Yellow
amorphous solid. Yield: ca. 220 mg. This solid was cyclized in 15 g of PPA at 1108/1h. The melt was
dissolved in 150 ml of H2O, whereby brown solid remained. After filtration, the dry filter paper was
washed with acetone to give a pink soln. Yield of the amorphous product: 200 mg. According to TLC,
there was a certain amount of naphthalen-2-amine in the solid. Chromatography with 90 g of Kieselgel
G60 (column, 4 cm diameter; eluted with hexane/acetone 1:1). Up to 140 ml of eluate, traces of
unknown substances, and naphthalen-2-amine, and then 1m were eluated. This material was chromato-
graphed once more to remove traces of naphthalen-2-amine. Yield: 30 mg. IR: 3440m, 3325m, 3223m,
3050m, 2919m, 2851m, 1697m, 1665s, 1680vs, 1630m, 1580vs, 1535s, 1465s, 1452s, 1391w, 1331w, 1285w,
1
1256m, 1236s, 1188m, 1139m, 1085m, 1059w, 993w, 950m, 807m, 778w. H-NMR ((D6)DMSO): 11.1 (s,
1 H); 10.85 (s, 1 H); 8.76 (dd, 3J(H,F) ¼ 10.9, 5J(H,H) ¼ 3.4, 1 H); 8.66 (d, J ¼ 8.0, 1 H); 8.16 (d, J ¼ 9.6,
1 H); 7.91 (d, J ¼ 8.0, 1 H); 7.69 – 7.65 (m, 2 H); 7.4 (t, J ¼ 9.6, 1 H); 7.13 (ddd, 3J(H,F) ¼ 2.9, 3J(H,H) ¼
4
9.04, 4J(H,H) ¼ 3.9, 1 H); 6.88 (dd, J(H,F) ¼ 4.0, 3J(H,H) ¼ 9.0, 1 H).
(2Z)-2-(1,2-Dihydro-5-methyl-2-oxo-3H-indol-3-ylidene)-2-(sulfanyl)ethanoic Acid (6g). To 5-meth-
ylsatin (7.36 g, 0.045 mol) and rhodanin (7.2 g, 0.05 mol), dissolved in 60 ml of pyridine at 508, 20 ml of
dist. H2O and 100 ml of THF were added, and the soln. was heated at 658 for 2 h. Crystallization
overnight at 48: yield: 13 g. Often hydrolysis with 9.5 g of NaOH in 40 ml of warm H2O (ca. 508, 1 h), the
soln. was stared for a couple of h in a fridge at 08. From time to time crystals were isolated. Yield: 8 g
(67%).
(2Z)-2-(1,2-Dihydro-5-methyl-2-oxo-3H-indol-3-ylidene)-2-(methylsulfanyl)ethanoic acid (6g’). Yel-
low needles. IR: 3415s (br.), 1684s, 1591s, 1575vs, 1484m, 1420w, 1365m, 1311m, 1274w, 1236w, 1210m,
1150m, 1121m, 1080m, 803m, 752w, 731w, 670w, 618m, 588m. 1H-NMR ((D6)DMSO): 10.02 (s, 1 H); 7.37
(s, 1 H); 6.86 (d, J ¼ 7.5, 1 H); 6.65 (d, J ¼ 7.5, 1 H); 2.44 (s, 3 H); 2.25 (s, 3 H). 13C-NMR ((D6)DMSO):
166.02; 163.73; 161.0; 137.7; 128.9; 126.5; 124.6; 123.3; 111.25; 108.35; 21.2; 14.7. CI-MS: 221 (3), 220 (7),
219 (34), 206 (10), 205 (100), 204 (15), 190 (30), 186 (33), 172 (57), 162 (13), 160 (11).
(3Z)-1,3-Dihydro-5-methyl-3-(5-methyl-3-oxo-1-benzothiophen-2(3H)-ylidene)-2H-indol-2-one
(12a). Compound (6g, 1.07 g) was dissolved together with 3 g of AcONa in 25 ml of dist. H2O. At ca. 108,
a soln. of 4-methylphenyldiazonium acetate, prepared from 0.7 g of p-toluidine in 5 ml of AcOH, and
0.55 g of butyl nitrite, at 08 was added. The pH of the soln. dropped from 8 to ca. 4 – 5. After stirring the
precipitate, some grains of CuCl2 · 2 H2O, dissolved in 2 ml of dist. H2O were added. The ice bath was
removed, and from time to time K2CO3 soln. (total 1 g in 10 ml of H2O) was added, followed by 5 ml of

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Helvetica Chimica Acta – Vol. 95 (2012)
MeOH. Gradually N2 evolved. Then, stirring was continued overnight. After manually removing a layer
of brown resin, the soln. was filtered, acidified and the yellow precipitate of (2Z)-2-(5-methyl-2-oxo-1,2-
dihydro-3H-indol-3-ylidene)-2-[(4-methylphenyl)sulfanyl]ethanoic acid (11a; 800 mg) was isolated by
suction. Yellow powder. This material 11a was cyclized in 10 g of PPA at 958/30 min. Workup in 250 ml of
H2O and isolation by suction afforded 12a. Yield: 85%. Dark red powder. IR: 3432 (br.), 3179 (br.),
2916w, 2855w, 1697s, 1670vs, 1658sh, 1620m, 1604m, 1578w, 1472s, 1323m, 1306m, 1279m, 1237w, 1216w,
1165w, 1083w, 1058s, 1019w, 932m, 906w, 816w, 772m, 671w, 592w. 1H-NMR ((D6)DMSO): 10.98 (s, 1 H);
8.85 (s, 1 H); 7.66 (s, 1 H); 7.62 (d, J ¼ 8.2, 1 H); 7.56 (d, J ¼ 7.8, 1 H); 7.21 (d, J ¼ 8.2, 1 H); 6.82 (d, J ¼
7.8), 2.36 (s, 3 H); 2.32 (s, 3 H). CI-MS: 311 (8), 310 (39), 309 (25), 308 (100), 307 (16), 148 (15).
Sodium (3Z)-3-[Carboxy(sulfanyl)methylidene]-2,3-dihydro-2-oxo-1H-indole-5-sulfonate (6h). Ob-
tained from rhodanin (37.5 g, 0.28 mol), sodium isatinsulfonate (43.5 g, 0.17 mol), 400 ml of pyridine,
15 ml of dist. H2O. Hydrolysis with 200 ml of dist. H2O and 34 g of NaOH gave a soln. from which only a
minor quantitiy of crystals was separated. The mother liquor was diluted with the same volume MeOH
and kept for a week at – 258. Crystals were isolated by suction and dried. Yield: 51 g (65%) of 6h. IR:
3455s, 1674s, 1627s, 1587w, 1547vs, 1467m, 1385m, 1302w, 1230m, 1212m, 1191m, 1128w, 1099m, 1061m,
1029m, 820w, 780w, 720w, 679w, 622m. 1H-NMR (D2O): 9.5 (s, 1 H); 7.44 (d, J ¼ 8.0, 1 H); 6.9 (d, J ¼ 8.0,
1 H). 13C-NMR (D2O):179.8 (COOH); 168.4(CONH); 140.3; 135.4; 127.1; 121.9; 118.9; 116.3; 114.1;
109.3.
(2Z)-2-(1,2-Dihydro-2-oxo-5-sulfo-3H-indol-3-ylidene)-2-[(4-methylphenyl)sulfanyl]ethanoic Acid
(11b). Sodium salt 6h (352 mg, 0.9 mmol) was dissolved in 20 ml of dist. H2O, one or two tiny crystals
of CuCl2 · 2 H2O were added, and the soln. was stirred until it turned greenish and clear again; pH was ca.
8 – 9. Separately, 0.6 g of p-toluidine was dissolved in 5 ml of dist. H2O, mixed with 2.5 ml of 50% HBF4
aq. soln., and cooled to 08. To this soln., a chilled soln. of NaNO2 (450 mg) was added. After dissolution of
all solids, a new mass of solid platelets appears. This p-methylphenyldiazonium tetrafluoroborate was
isolated by suction and dried in a stream of N2 some h (yield: 740 mg). A soln. of 267 mg of this
diazonium salt (1.2 equiv.) in 5 ml of dist. H2O was added to the chilled soln. of 6h prepared above. The
soln. became black, but the color faded immediately, and gas evolution started. Stirring was continued at
r.t. After stirring overnight, some resin developed, which was filtered off. The soln. was made acidic with
3 ml of dil. aq. HCl, and 10 g of a collector resin XAD 1600 N (Rohm & Haas) were added. The resin was
collected, rinsed with dist. H2O to pH 7, and dried. The resin was packed in a small column and eluted
with MeOH. The yellow soln. contained a major fraction of 11b as an oily material which was subjected
to CC. The total amount of formed 11b could not be collected from the mixture by this method, even by
increasing ionic strength by addition of KCl or some more XAD. The pure material was a yellow powder.
Yield: 200 mg (50%). IR: 3402vs, 1664s, 1622vs, 1618vs, 1577s, 1523m, 1498w, 1463w, 1397m, 1351w,
1300w, 1262m, 1215s, 1174m, 1124w, 1097m, 1036m, 1014m, 806w, 722w, 625m, 573w, 545m, 497w.
1H-NMR ((D6)DMSO): 11.8 (s, 1 H); 10.34 (s, 1 H); 7.75 (s, 1 H); 7.28 (d, J ¼ 8.6, 2 H); 7.22 (d, J ¼ 7.4,
1 H); 7.09 (d, J ¼ 8.6, 2 H); 6.7 (d, J ¼ 7.4, 1 H); 2.25 (s, 3 H). 13C-NMR ((D6)DMSO): 171.5; 164.3; 157.1;
140.0; 137.15; 135.0; 132.4; 129.5 (p-Ar); 123.6; 120.1; 119.5 (p-Ar’); 116.15; 106.9; 91.0; 20.4(Me). CI-
MS: 392.9 (3), 391.9 (23), 390.9 (100), 278.9 (20), 246.9 (23).
(3Z)-2,3-Dihydro-3-(5-methyl-3-oxo-1-benzothiophen-2(3H)-ylidene)-2-oxo-1H-indole-5-sulfonic
Acid (12b). Obtained from 11b (250 mg) in PPA (15 g) at 1108. After hydrolysis in 500 ml of dist. H2O,
25 g of NaCl were added, the liquid was then extracted with butan-2-one in a liquidꢀliquid extraction
apparatus. Solvent was evaporated and the residual red dye was crystallized from BuOH by evaporation
in a glass dish. Alternatively, the diluted soln. was subjected to solid-phase extraction with 10 g of XAD
1600 N resin (Rohm & Haas) and strirring for 2 h. The resin was collected by filtration, washed to pH 7,
whereby the red dye leaked out again, so it was stopped. The dried resin was packed in a small column
and extracted with MeOH. The red soln. was evaporated in vacuo. Purification by CC over Kieselgel G 60
(40 g), in a column of 4-cm internal diameter; solvent: MeOH/acetone 1:4. IR: 3371vs (br.), 2923w,
2852w, 1693m, 1658vs, 1613s, 1470m, 1306m, 1198s, 1099s, 1059m, 1030s, 912m, 821w, 770w, 725w, 619m,
539m, 401w. 1H-NMR ((D6)DMSO): 11.12 (s, 1 H); 9.38 (s, 1 H); 7.70 – 7.67 (m, 2 H); 7.6 (d, J ¼ 8.6, 1 H);
7.55 (d, J ¼ 7.4, 1 H); 6.86 (d, J ¼ 8.6, 1 H); 2.38 (s, 3 H).
(2Z)-(1,2-Dihydro-7-methoxy-4-methyl-2-oxo-3H-indol-3-ylidene)(sulfanyl)ethanoic Acid (6i). 4-
Methyl-7-methoxyisatin (6.36 g, 0.33 mol) was dissolved together with rhodanin (4.5 g, 0.34 mol) in

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50 ml of AcOH. AcONa (4 g) was melted to dryness and added. The mixture was refluxed for 8 h. A dark
solid formed, which was isolated by suction and washed with dist. H2O. Yield 6.42 g (64%). Hydrolysis
with 12 g of NaOH in 75 ml of dist. H2O at 608 for 4 h, stirring overnight at r.t., and neutralizing with
conc. HCl to pH 1 yielded a solid, which was stirred for some hours, whereby a red cristalline powder
formed. Yield 5.6 g (68%).
(2Z)-2-(1,2-Dihydro-7-methoxy-4-methyl-2-oxo-3H-indol-3-ylidene)-2-(methylsulfanyl)ethanoic
Acid. Yellow to green needles. 1H-NMR ((D6)DMSO): 6.72 (d, J ¼ 8.3, 1 H); 6.58 (d, J ¼ 8.3, 1 H); 3.73
(s, 3 H); 2.4 (s, 3 H).
The Klinge-Stiftung of the late G. Klinge for funding, Prof. Dr. H. Langhals, Ludwig-Maximilians
Universitꢄt Mꢀnchen, for contributing the fluorescence spectra, and U. Ammari, R. Dumitrescu, and G.
Krutsch, TU Mꢀnchen, for supporting numerous analytical data, are greatfully acknowledged.
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Received January 30, 2012