Exploring an anomaly: The synthesis of 7,7′-diazaindirubin through a 7-azaindoxyl intermediate

Article abstract of DOI:10.1039/d0ra07144g

Two independent methods generating 7-azaindoxyl as an intermediate verify that 7,7′-diazaindirubin is formed exclusively over 7,7′-diazaindigo. This contrasts with long-standing knowledge related to the reactivity of indoxyl, which proceeds via a radical-initiated homodimerization process, leading to indigo. A series of experiments confirms 7-azaindoxyl as an intermediate with results suggesting a condensation pathway followed by oxidation.

Full text of DOI:10.1039/d0ra07144g

RSC Advances
View Article Online
View Journal | View Issue
PAPER
0
Exploring an anomaly: the synthesis of 7,7 -
diazaindirubin through a 7-azaindoxyl
Cite this: RSC Adv., 2020, 10, 36849
intermediate†
James A. Shriver, * Katelyn R. Wang, Andrew C. Patterson, James R. DeYoung
and Richard J. Lipsius
0
Two independent methods generating 7-azaindoxyl as an intermediate verify that 7,7 -diazaindirubin is
Received 19th August 2020
Accepted 24th September 2020
0
formed exclusively over 7,7 -diazaindigo. This contrasts with long-standing knowledge related to the
reactivity of indoxyl, which proceeds via a radical-initiated homodimerization process, leading to indigo.
A series of experiments confirms 7-azaindoxyl as an intermediate with results suggesting a condensation
pathway followed by oxidation.
DOI: 10.1039/d0ra07144g
rsc.li/rsc-advances
converts to indigo in air with selectivity over indirubin. This has
been conrmed through numerous studies of indoxyl-
Introduction
Indirubin (1), a side product from commercial indigo (2) generating compounds under oxidative conditions, and it
production (Fig. 1), is emerging as a promising pharmaceutical proceeds through a free radical-based mechanism. For example,
platform. Indirubin derivatives have been studied for their anti- the action by oxygen under basic conditions suggests a free
1
2
12
cancer properties, potential to treat diabetes, and anti- radical process aer generation of indoxyl.
Moreover,
3
13
inammatory response. Modern understanding began when convincing evidence by Berlin and co-workers supports this
the crystal structures, illustrating the interaction between cyclin observation more broadly with other oxidants and includes the
0
dependent kinase-2 with both indirubin-3 -oxime and possibility of formation under neutral and acidic conditions
4
indirubin-5-sulfonate, were elucidated in 1999. Ultimately, this with the appropriate oxidant.
has led to promising leads inter alia in the treatment of
In contrast, the synthesis of indirubin is thought to occur
gliomas, MLL leukemia cells, and pancreatic cancer for due to formation of small amounts of isatin, 4, by oxidation of
5
6
7
14
indirubin derivatives, and melanoma and non-small cell lung 3. Subsequent condensation occurs between indoxyl and isa-
0
8
cancer cells for 7,7 -diazaindirubin derivatives.
tin as leveraged in the traditional, and still dominant prepara-
Indigo has been used and independently developed as a dye tive method for indirubin introduced by Baeyer in the 1880s and
in many cultures, dated as early as 6000 years ago at an illustrated in Scheme 1. Contemporaneously, there are
archaeological site in Peru. Traditionally, precursors are a limited number of new preparations for indirubin through
derived from natural sources that can vary with geographic classical synthetic methods other than those which employ the
9
15
10
11
location. These compounds give rise to indoxyl (3-hydrox- classical Baeyer approach. In one example, 3-formyl indigo is
yindole, 3, Fig. 1), a reactive intermediate that spontaneously reacted with Oxone® to form indirubin in a 6% yield as
16
a biproduct during the synthesis of isatoic anhydrides.
Though limited in its assessment, 4 was reacted with 2-oxoin-
0 0
17
dole with PCl
promising protocol was accomplished through the reductive
5
to form 5 and 6 -substituted indirubins. A more
18
coupling of isatin using potassium borohydride where yields
Fig. 1 Chemical structures for indirubin (1), indigo (2) and indoxyl (3).
Central College, 812 University St., Pella, IA 50219, USA. E-mail: shriverj@central.edu
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/d0ra07144g
Scheme 1 Original Baeyer synthesis of 1.
1
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 36849–36852 | 36849

View Article Online
RSC Advances
Paper
0
Fig. 2 Chemical structures for 7-azaindole (5), 7,7 diazaindirubin (6),
and 7-azaindoxyl (7).
0
Scheme 2 Molybdenum-catalyzed synthesis of 7,7 -diazaindirubin (6).
ranging from 63%–91% were seen for an array of electron-rich
indirubins.
More recently, methods employing biological or enzyme-
promoted pathways have been explored to generate indirubins
and other indigoid species. Insight into this category of reaction
can be gleaned by a look at traditional biological synthesis of
indigo, which is nearly always accompanied by indirubin. An
elaborated example of this is seen through the enzymatic
hydrolysis of indican (indoxyl-b-D-glucoside) isolated from
19
Scheme 3 Trapping of indoxyl intermediates for molybdenum-cata-
lysed procedure.
Polygonum tincturum. A tissue culture where Polygonum tinc-
turum was fed with an array of indoles to produce indigo and
20
indirubin without isolation. Additionally, Maugard identied
isatan C during an early (June) harvest of woad, an indigo-
producing plant, and discerned that it promoted increased
the presence of indoxyl through LCMS during the synthesis of 2.
To independently test for the presence of an indoxyl interme-
diate under these conditions, 4 was employed as a trapping
agent for indoxyl, harkening back to the original process
21
formation of indirubin through an isatin precursor. A novel
approach using Cytochrome P450 enzymes was highlighted in
work by Guengerich which also noted an array of oxidation
15
developed by Baeyer shown in Scheme 1. For indole (8),
indirubin (1) was produced preferentially over 2 in an 8 : 1 ratio.
22
products beyond indoxyl including oxindole and dioxindole.
Further elaboration of this process with Cytochrome P450
mutant enzymes formed an array of synthetic indirubin and
0
For 5, 7 -azaindirubin (9) was formed exclusively under standard
reaction conditions (Scheme 3).
23
indigo compounds. Recently, it was shown that cysteine can
The protocol from Scheme 2 was applied to selected indoles
to help assess the process. Broadly, the results illustrate that
electron-decient indoles are more amenable towards indir-
ubin synthesis than relatively electron rich systems. Notably, 6-
nitroindole, indole-5-carbonitrile and indole-5-carboxylic acid
gave rise to appreciable quantities of the corresponding indir-
ubin, while the parent system and 5-methoxyindole formed only
indigo products. These results are summarized in Table 1,
showing the percent recovery and relative abundance of indir-
ubin and indigo, which have the same molecular weight, as
shi the selectivity of a avin-containing monooxygenase from
indigo to indirubin.
24
Herein, an anomaly to the innate selectivity towards indigo
under indoxyl-generating conditions is explored for 7-azain-
0
dole, 5, which exclusively produces 7,7 -diazaindirubin, 6. In
this instance, the intermediate is specically 7-azaindoxyl, 7
(Fig. 2). This preference was observed for two independent
approaches, both of which show signicant promise as viable
platforms for the low-cost production of a wide array of indir-
ubin derivatives.
1
assessed by H NMR integration.
The second method to generate 6 was accomplished through
the synthesis of 7-azaindoxyl acetate (17) and subsequent
solvolysis of the acetate group. Synthesis of 17 was performed by
Results
To initiate indirubin synthesis, a protocol developed by Yama-
moto and co-workers was employed. Their work produced
indigo from indole in up to 82% yield with a molybdenum
Table 1 Relative formation of indirubin : indigo with 2% Mo(CO)
6
catalyst
25
catalyst in the presence of cumene peroxide. Utilizing a scaled-
down variant of this procedure with increased catalyst load, this Indole
Indirubin
Indigo
Indirubin : indigo
Recovery%
method converted 5 to 6 with no evidence of the corresponding
indigo (Scheme 2).‡
The next task was to conrm that the corresponding indoxyl
was produced as an intermediate during the reaction. Germane
a
Indole
NA
6
10
12
14
NA
2
0 : 1
1 : 0
2 : 9
2 : 3
1 : 2
0 : 1
76%
78%
67%
62%
59%
65%
7
6
5
-Aza
NA
11
13
15
16
-NO
-CN
2
to this goal, the original publication by Yamamoto et al. veried 5-CO
2
H
5-OMe
a
25
‡
While initially scaled at
5
mmol, the reaction was performed once
Yield of 82% obtained from Yamamoto et al.
(
non-optimized) at a 100 mmol scale to afford 5.66 g (43% yield) of 6.
36850 | RSC Adv., 2020, 10, 36849–36852
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
0
0
Scheme 4 Acid-catalysed transesterification on 17 leading to 6.
Fig. 3 Structures of 6,6 -dinitroindirubin (10) and 6,6 -dinitroindigo
11).
(
unambiguous switch in selectivity from indigo to indirubin
when utilizing 5 instead of indole as the reactant. It was initially
assumed that this observation was due to the electron decient
character of 5, which was subsequently supported in the eval-
uation shown in Table 1 for other substituted indoles. As this
reaction was already known to proceed through an indoxyl
intermediate from prior research, this strongly suggests that the
ability of the 3-position in indoxyl to behave as an electrophile is
enhanced. To verify the results from Yamamoto for the parent
system and to conrm that an indoxyl intermediate is also
present for 5, a trapping experiment with 4 was run as shown in
Scheme 3. This result helps substantiate that an alternate
mechanism, with initial oxidation at the 2-position, is not in
play.
Given the complexity of the system from Scheme 2, it was
important to independently verify this result, allowing removal
of peroxide and the molybdenum catalyst as variables. Scheme 4
illustrates this alternate approach, which lead to the same result
under acidic (but not basic) conditions. Moreover, the fact that
peroxide was not necessary to promote this transformation
strongly suggests that the reaction is proceeding through
condensation pathway to generate 7 and is followed by oxida-
tion with air to form 6. This was further affirmed by the addition
of a rare earth triate, which promoted the formation of indir-
ubin over indigo for 6-nitroindole. A mechanistic pathway is
illustrated in Scheme 5, which hypothesizes the intermolecular
aldol condensation between the keto and enol forms of 7 fol-
lowed by oxidation with air (or peroxide).
1
Fig. 4 H NMR for the formation of 10 and 11 with and without 2%
Yb(OTf)
3
.
oxidation of 5 with (diacetoxyiodo)benzene (DIB) using a modi-
ed protocol at larger scale (20 mmol), detailed in the ESI.†
While saponication of indoxyl acetate is well known to
produce indigo, mimicking reaction conditions under neutral-
to-acidic conditions is more problematic. Ultimately, acid-
catalysed transesterication§ at 150 C gave rise to 6 as the
sole product as shown in Scheme 3. In contrast, saponication
of 17 leads to the corresponding indigo.
As a condensation pathway was suspected, a Lewis acid was
used to facilitate indirubin formation. It has been established
that rare earth triates as Lewis acids are helpful in facilitating
condensation reactions, including aldol condensations. The
initial screening of indoles shown in Table 1 have generally
produced a mixture of indigo and indirubin derivatives for
electron decient systems. The example of 6-nitroindole
produced a preferred amount of 11 compared to 10 in a 2 : 9
26

ꢀ
8
27
ratio of 10 : 11 (Fig. 3). When 2% Yb(OTf) was added to the
3
reaction, a 3 : 1 ratio of 10 : 11 was observed by NMR (Fig. 4).{ It
is important to note that Yb(OTf) inhibited total recovery of
3
product(s) for all examples explored, likely due to a deleterious
effect on the molybdenum catalyst.
Discussion
The collective results supply insight into the likely mechanism
for this reaction. The reaction in Scheme 2 illustrated an
§
The reaction was performed for 24 h with either trichloroacetic acid (14%) or
Yb(OTf)
3
(34%) to yield solely 6 without further optimization.
1
{
Due to low solubility, the H NMR was taken with 2000 scans at 300 MHz on the
crude precipitate aer ltration.
Scheme 5 Proposed mechanism for the synthesis of 6 from 7.
RSC Adv., 2020, 10, 36849–36852 | 36851
This journal is © The Royal Society of Chemistry 2020

View Article Online
RSC Advances
Paper
7
B. Marchand, I. Tremblay, S. Cagnol and M. J. Boucher,
Carcinogenesis, 2012, 33, 529.
Conclusions
In summary, the anomalous behaviour where certain indoxyl
compounds form indirubin, illustrated in detail for 7, is likely
due to a previously unidentied condensation pathway shown
in Scheme 4. This is a striking result given the ubiquitous
nature of indigo and the extent of research around it. Moreover,
8 X. Cheng, K.-H. Merz, S. Vatter, J. Christ and S. W ¨o l, Bioorg.
Med. Chem., 2014, 22, 247.
9 Indirubin, the Red Shade of Indigo, ed. L. Meijer, N. Guyard, L.
A. Skaltsounis and G. Eisenbrand, Life in Progress, Station
Biologique, Roscoff, France, 2006.
there is signicant opportunity for the scope to include, at the 10 J. C. Splitstoser, T. D. Dillehay, J. Wouters and A. Claro, Sci.
least, an array of electron decient indoles. This is particularly
Adv., 2016, 2, 1.
true given the indirubin-enhancing effect seen for Yb(OTf) . In 11 K. G. Gilbert, D. J. Hill, C. Crespo, A. Mas, M. Lewis,
3
addition to possible methodology development, future studies
B. Rudolph and D. T. Cooke, Phytochem. Anal., 2000, 11, 18.
will explore the extent of keto–enol tautomerization and its role 12 G. A. Russell and G. Kaupp, J. Am. Chem. Soc., 1969, 91, 3851.
in promoting this anomalous pathway.
13 A. N. Zelentskii, G. V. Fomin, N. V. Kutana and A. A. Berlin,
Russ. Chem. Bull., 1970, 19, 1105.
1
4 For a review: A. Medvedev, O. Buneeva and V. Glover,
Biologics, 2007, 1, 151.
Conflicts of interest
There are no conicts to declare.
15 (a) A. Baeyer, Ber. Dtsch. Chem. Ges., 1882, 15, 50; (b)
F. S ´a nchez-Viesca and R. G ´o mez, Am. J. Chem., 2018, 8, 85.
1
6 A. C. Nelson, E. S. Kalinowski, T. L. Jacobson and P. Grundt,
Tetrahedron Lett., 2013, 54, 6804.
Acknowledgements
The authors would like the thank Central College and the 17 H. Saito, K. Tabata, S. Hanada, Y. Kanda, T. Suzuki and
Chemistry Department for support of this research. In addition,
we would like to thank the following for summer funding for
undergraduates: The Moore Family Foundation (Andrew Pat-
terson), the Arthur J. Bosch Endowment for Student Research
S. Miyairi, Bioorg. Med. Chem. Lett., 2011, 21, 5370.
8 C. Wang, J. Yan, M. Du, J. A. Burlison, C. Li, Y. Sun, D. Zhao
and J. Liu, Tetrahedron, 2017, 73, 2780.
9 T. Maugard, E. Enaud, A. d. L. Sayette, P. Choisy and
M. D. LeGoy, Biotechnol. Prog., 2002, 18, 1104.
1
1
(Katelyn Wang) and the John Wesselink Endowment (Katelyn
Wang). Katelyn Wang will continue this research during the 20 J.-Y. Shim, Y.-J. Chang and S.-U. Kim, Biotechnol. Lett., 1998,
2020–21 academic year as a recipient of a Goldwater Fellowship.
20, 1139.
2
2
2
1 E. E. Maugard, P. Choisy and M. D. Legoy, Phytochem, 2001,
58, 897.
2 E. M. J. Gillam, L. M. Notley, H. Cai, J. J. DeVoss and
F. Guengerich, Biochem, 2000, 39, 13817.
3 (a) F. P. Guengerich, J. L. Sorrells, S. Schmitt, J. A. KRauser,
P. Aryal and L. Meijer, J. Med. Chem., 2004, 47, 3236; (b)
Z.-L. Wu, P. Aryal, O. Lozach, L. Meijer and
F. P. Guengerich, Chem. Biodiversity, 2005, 2, 51.
4 J. Kim, J. Lee, P.-G. Lee, E.-J. Lee, W. Kroutil and B.-g. Kim,
ACS Catal., 2019, 9, 9539.
Notes and references
1
(a) Y. Wang, P. M. Hoi, J. Yuet-Wa Chan and M.-Y. Simon,
Anti-Cancer Agents Med. Chem., 2014, 9, 1213; (b) G. Y. Wu,
J. Z. Liu, F. D. Fang and J. Zuo, Sci. Sin., Ser. B, 1982, 10, 1071.
(a) S. Sharma and R. Taliyan, Biochem. Biophys. Res.
Commun., 2014, 452, 1009; (b) T. Konno, K. Sasaki,
K. Kobayashi and T. Murata, Mol. Med. Rep., 2000, 21, 1552.
W. Gao, Y. Guo, C. Wang, Y. Lin, L. Yu, T. Sheng, Z. Wu and
Y. Gong, Acta Histochem., 2016, 118, 606.
(a) R. Hoessel, S. LeClerc, J. A. Endicott, M. E. Noble,
A. Lawrie, P. Tunnah, M. Leost, E. Damiens, D. Marie,
D. Marko, E. Niederberger, W. Tang, G. Eisenbrand and
L. Meijer, Nat. Cell Biol., 1999, 1, 60; (b) T. G. Davies,
P. Tunnah, L. Meijer, D. Marko, G. Eisenbrand,
J. A. Endicott and M. E. M. Noble, Structure, 2001, 9, 389.
S. Kotliarova, S. Pastorino, L. C. Kovell, Y. Kotliarov, H. Song,
W. Zhang, R. Bailey, D. Maric, J. C. Zenklusen, J. Lee and
H. A. Fine, Cancer Res., 2008, 68, 6643.
2
2
2
3
4
5 (a) Y. Inoue, Y. Yamamoto, H. Suzuki and U. Takaki, New
Dev. in Selective Oxidation II, Proceedings of the Second
World Congress and Fourth European Workshop Meeting
Studies in Surface Science and Catalysis, 1994, p. 615; (b)
Y. Yamamoto, Y. Inoue, U. Takaki and H. Suzuki, Bull.
Chem. Soc. Jpn., 2011, 84, 82.
2
2
6 K. Lie, P. Wen, J. Lie and G. Huang, Synthesis, 2010, 21, 3623.
7 (a) S. Kobayashi, M. Sugiura, H. Kitagawa and W. W.-L. Lam,
Chem. Rev., 2002, 102, 2227; (b) T. Mukaiyama, K. Naraska
and T. Banno, Chem. Lett., 1973, 2, 1011; (c) A. Kumar,
I. Ahmad and M. S. Rao, Can. J. Chem., 2008, 86, 899.
5
6
Z. Wang, K. S. Smith, M. Murphy, O. Piloto, T. C. Somervialle
and M. L. Cleary, Nature, 2008, 455, 1205.
36852 | RSC Adv., 2020, 10, 36849–36852
This journal is © The Royal Society of Chemistry 2020