A theoretical and spectroscopic (NMR and IR) study of indirubin in solution and in the solid state

Article abstract of DOI:10.1002/poc.4043

The infrared spectrum of indirubin in the solid state and the ¹H, ¹³C and ¹⁵N NMR spectra in DMSO-d₆ solution as well as the ¹³C and ¹⁵N CPMAS spectra have been recorded. Different types of calculations (B3LYP, GIAO, and GIPAW) have been carried out. Experimental and calculated values were compared yielding commensurate results. E/Z isomerism was explored, as was oxo/hydroxy tautomerism.

Full text of DOI:10.1002/poc.4043

Received: 10 September 2019
DOI: 10.1002/poc.4043
Revised: 28 October 2019
Accepted: 12 November 2019
R E S E A R C H A R T I C L E
A theoretical and spectroscopic (NMR and IR) study of
indirubin in solution and in the solid state
Ibon Alkorta1
Dolores Santa María3
| José Elguero1
| Christophe Dardonville1
| Felipe Reviriego2
|
| Rosa M. Claramunt3 | Marta Marín‐Luna4
1 Instituto de Química Médica, CSIC,
Madrid, Spain
2 Instituto de Ciencia y Tecnología de
Polímeros, CSIC, Madrid, Spain
3 Departamento de Química Orgánica y
Bio‐Orgánica, Facultad de Ciencias,
UNED, Madrid, Spain
4 Departamento de Química Orgánica,
Facultad de Química, Universidad de
Murcia, Regional Campus of International
Excellence “Campus Mare Nostrum”,
Murcia, Spain
Abstract
The infrared spectrum of indirubin in the solid state and the H, 13C and 15N
NMR spectra in DMSO‐d6 solution as well as the 13C and 15N CPMAS spectra
have been recorded. Different types of calculations (B3LYP, GIAO, and
GIPAW) have been carried out. Experimental and calculated values were com-
pared yielding commensurate results. E/Z isomerism was explored, as was
oxo/hydroxy tautomerism.
1
KEYWORDS
GIPAW, indirubin, IR, NMR, tautomerism
Correspondence
Ibon Alkorta, Instituto de Química
Médica, CSIC, Juan de la Cierva, 3, E‐
28006 Madrid, Spain.
Email: ibon@iqm.csic.es
Marta Marín Luna, Departamento de
Química Orgánica, Facultad de Química,
Universidad de Murcia, 30100 Murcia,
Spain.
Email: martamarin@um.es
Funding information
Ministerio de Ciencia e Innovación,
Grant/Award Numbers: PGC2018‐094644‐
B‐C22 and RTI2018‐093940‐B‐I00;
Universidad Nacional de Educacion a
Distancia (UNED); Comunidad de
Madrid, Grant/Award Number: P2018/
EMT‐4329 AIRTEC‐CM; Ministerio de
Ciencia, Innovación y Universidades,
Grant/Award Numbers: RTI2018‐093940‐
B‐I00 and PGC2018‐094644‐B‐C22
1 | INTRODUCTION
angiogenesis, but its anticancer properties are not so
firmly established.[1–4] Besides, some of indirubin
Indirubin, (Z)‐[2,3′‐biindolinylidene]‐2′,3‐dione (1), is a
natural product with several biomedical properties useful
for the treatment of ulcerative colitis, inflammation, and
derivatives (Figure 1), like 3‐monoxime (2)[5] and 5′‐
sulfonate (3),[6] have several applications in diabetes, can-
cer, and inflammation.
Very recently, a group of authors from different coun-
tries have identified indirubin as the active principle
Dedicated to Professor Vicente Arán on the occasion of his retirement.
J Phys Org Chem. 2019;e4043.
wileyonlinelibrary.com/journal/poc
© 2019 John Wiley & Sons, Ltd.
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https://doi.org/10.1002/poc.4043

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ALKORTA ET AL.
Taking account of E/Z isomerism about the 2‐3′ bond
and the oxo/hydroxy tautomerism, there are 26 possible
isomers and tautomers for indirubin (Figure 3). There
are two 3‐oxo, 2′‐oxo tautomers (4, 10), eight 3‐hydroxy‐
2′‐oxo tautomers (5a, 5b, 8a, 8b, 11a, 11b, 14a, 14b), eight
3‐oxo‐2′‐hydroxy tautomers (6a, 6b, 7a, 7b, 12a, 12b, 13a,
13b), and eight 3‐hydroxy‐2′‐hydroxy tautomers (9a, 9b,
9c, 9d, 15a, 15b, 15c, 15d).
FIGURE 1 Indirubin and related compounds
responsible for the anticonvulsant properties of Indigofera
arrecta (a traditional medicinal plant from Congo).
Indirubin itself and 6‐bromoindirubin‐3‐acetoxime, both
potent GSK‐3 inhibitors, are a new therapeutic entry
point for epilepsy.[7,8]
Relative energies of these isomers and tautomers
calculated at the B3LYP/6‐311++G(d,p) level are listed in
Table 1. Those corresponding to rotation of the OH
groups use the same number but different letters. Some
of them are not stable and isomerize during the optimiza-
tion process (see Comments).
The experimental geometry of indirubin (Figure 2)
compares well with the geometry calculated for
tautomer 4, in particular the following experimental
and (calculated) values: C2═C2′ 1.313 Å (1.368 Å),
N1―C2―C3′―C2 0.78° (0.00°), C4′―H···O3 3.011 Å
(3.057 Å). Note that there are two structures more stable
than isoindirubin (10) (58.9 kJ·mol−1), 7a (57.1
kJ·mol−1), and 11b (56.7 kJ·mol−1). This is due to
IMHBs (see below) and indicates that isoindirubin, an
E‐isomer, should tautomerize into the more stable 11b
structure.
There is a very large number of papers and reviews
dealing with the biological properties of indirubin, but
only few discussing its physical properties and still less
reporting theoretical studies. Amongst the physical prop-
erties, the most discussed is the characteristic red color of
indirubin and the corresponding electronic spectra;
indirubin has characteristic bands at 288.5, 360.6, and
538.6 nm.[9,10] In this sense, aiming to draw an extended
vision of indirubin, we hereby report a combined theoret-
ical and computational study on the tautomerism,
infrared, and nuclear magnetic resonance of this relevant
molecule. The photophysical and electronic aspects will
not be discussed.
A statistical analysis of Erel values considering the dif-
ferent situations present in the molecules of Figure 3 (see
Table S1 in the Supplementary data) was carried out; we
used a multiple regression based on a presence‐absence
or Free‐Wilson matrix.[16–19] The obtained parameters
show that an increase in the stability (lowering Erel) is
due to intramolecular hydrogen bonding, IMHB
(O―H··O═C = −73, O―H···N═C = −49, O―H···O―H
= −31, N―H···O═C = −29, C (Ar)―H···O═C = −31
kJ·mol−1) and that the stability decreases when oxygen
and nitrogen atoms are close (5a, 5b: C═N···O═C = 32
kJ·mol−1). Oxo tautomers are more stable than hydroxy
ones, a general observation in five‐membered ring hetero-
cycles;[20,21] the value depends on the position, 3/2′, and
on the nature of the nitrogen atom, NH/N: oxo‐3 NH =
−96, oxo‐3 N = −74, NH oxo‐2′ = −79 and N oxo‐2′ =
2 | 2. RESULTS AND DISCUSSION
2.1 | Tautomerism
The X‐ray structure (Figure 2) was determined in 1961
although the H atoms were not located.[11–13] Note that
the 1′‐2′ part of 1 is an amide; amides frequently
crystallize forming homodimers.[14,15] In the case of
indirubin, the N―H···O═C hydrogen bonds connect
the molecules into infinite chains running parallel to
the “a” axis.[11]
−30 kJ.mol−1
.
An Atom‐in‐Molecules (AIM) analysis[22–24] showed
the presence of a bond‐critical point between C4′ and
O3 [C (Ar)―H···O═C] besides the traditional
N1―H···O═C2′ in structure 4 (Figure 4). The calculated
electron density (0.017 and 0.026 au, respectively) and
Laplacian (0.061 and 0.097 au) at the bond critical point
are typical of standard hydrogen bonds and related to
the interatomic H···O distance.[19]
The distance between O3 and C4′ (3.011 Å)[11] was
considered by June Sutor as an example of the existence
of C―H···O hydrogen bonds.[25] After a devastating
FIGURE 2 Crystal structure of indirubin[11]

ALKORTA ET AL.
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FIGURE 3 The 26 E/Z isomers and
tautomers of indirubin. Compounds with
a central single bond (atropisomers)
correspond to sZ/sE isomerism
paper by Jerry Donohue that denies the existence of
these HBs,[26] today they are accepted by the scientific
community.[27–29]
For the two more stable tautomers, 4 and 10, we
calculated the chemical shifts using the Gauge Invariant
Atomic Orbital (GIAO) formalism (see Table 3 below).
TD‐DFT benchmark for indigoid dyes.[32] The reason of
these studies is the surprising difference in color between
indirubin (1) and its isomer indigo, (E)‐[2,2′‐
biindolinylidene]‐3,3′‐dione (16)[4] (Figure 5). In the pres-
ent paper, we will focus exclusively on the IR and NMR
data in the solid state.
2.2 | Spectroscopic study
2.2.1 | Infrared
Several theoretical calculations on indirubin have been
reported before, all of them concern the UV‐visible (elec-
tronic) spectra: photophysical and spectroscopic studies
of indigo derivatives,[30] ab initio study of the absorption
spectra of indirubin, isoindigo, and related derivatives,[31]
The IR data of indirubin in solid state have already been
reported.[33] Our first calculations were for the isolated
monomer in the gas phase (Figure 6). Taking into
account the crystal field effects and the hydrogen bonds
present in solid state, the agreement was acceptable.

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ALKORTA ET AL.
TABLE 1 Energetic values, relative energies (kJ·mol−1), dipole moments (Debye), and comments (PT: proton transfer, only possible in the
E isomers)
Isomer
Comp.
Erel
0.0
Dipole
Comments
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
E
E
E
E
E
E
E
E
E
E
E
E
E
4
1.31
5.11
5.04
4.65
5.38
2.28
4.43
7.43
5.40
4.26
7.59
2.44
5.32
6.15
0.32
0.54
4.99
8.22
5a
179.8
183.6
75.1
5b
6a
6b
120.0
57.1
7a
7b
138.6
163.1
167.9
230.2
183.9
231.9
190.1
58.9
8a
8b
9a
9b
9c
9d
10
11a
11b
12a
12b
13a
13b
14a
14b
15a
15b
15c
15d
156.6
56.7
107.8
78.6
isomerizes to 11b by O―H···O═C PT
isomerizes to 13a by O―H rotation
205.4
7.56
isomerizes to 12b by O―H···O═C PT
219.5
214.8
179.5
3.68
5.53
1.89
isomerizes to 15c by O―H rotation
Likewise, the simulation of the infrared spectrum of 1 in
solid state by using the GIPAW approach was consistent.
Table 2 lists the frequencies that were extracted from the
experimental and computed infrared spectra. We have
not systematically assigned the bands of Table 2 because
most of them are complex vibrations involving many
atoms; one of the simples are the symmetric and antisym-
metric vibrations of both carbonyl amide groups that
appear at 1701 and 1673 cm−1, respectively, in the gas
phase. Note that a few experimental bands are missed
in the simulated spectra. Nonetheless, we found a linear
correlation between both experimental and computed
values (Equations 1 and 2). In both gas‐phase and solid‐
state calculations, the quality of the linear correlation
was excellent in terms of the correlation factor R2. The
infrared simulation performed on the crystal structure
of 1 being slightly better (0.997 vs 0.990). These results
show the usefulness of the GIPAW approach to reproduce
the experimental infrared spectrum of 1.
FIGURE 4 AIM representation of indirubin 4; green: bond
critical points, red: ring critical points. The values of the electron
density and Laplacian (in parenthesis) at the intramolecular (au)
hydrogen bond critical points are indicated

ALKORTA ET AL.
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FIGURE 5 The colors of indirubin and indigo
FIGURE 6 IR spectrum of: (A) powdered sample of 1,[33] (B) measured in this work (blue), calculated and scaled for the gas phase (gold),
and calculated for the solid state (red) (see experimental section). Spectra shown in b have been processed by the SpectraGryph v1.2.11
[34]
program,
and the peak values could range from the exact values (see Table 2). Some peaks have been omitted for clarity.
2.2.2 | Nuclear magnetic resonance
ν ðExp:Þ ¼ ð20:7 28:0Þ þ ð0:99 0:02Þ*ν ðgas phaseÞ;
n ¼ 21; R2 ¼ 0:991; RMS residual ¼ 23:2 cm−1
;
1
In the literature, we only found data in solution for H
(1)
and 13C NMR of 1 (Table 3) but not for 15N or 17O.
Chemical shifts from Cuong et al[35] were unambiguously
assigned. In a recent paper,[36] a list of signals were
reported but not assigned although they are almost
identical to the previous ones.[35] There are 26 E/Z
isomers and tautomers of indirubin (Figure 3). Tautomer
ν ðExp:Þ ¼ ð−38:8 14:8Þ þ ð1:04 0:01Þ*ν ðsolid stateÞ;
n ¼ 23; R2 ¼ 0:997; RMS residual ¼ 12:7 cm−1
;
(2)

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ALKORTA ET AL.
TABLE 2 Infrared frequencies (cm−1) of experimental (solid‐
state) and calculated, gas phase and solid state, spectra of indirubin 1
Solution studies
Using GIAO/B3LYP/6‐311++G(d,p) calculations and
empirical equations established previously to transform
absolute shielding values (σ, ppm) into chemical shifts
(δ, ppm)[44–54] we calculated the values of Table 3 for
the dioxo isomers 4 and 10.
Experimental Solid
State
Calculated Gas
Phase
Calculated Solid
State
867
869
862
Since the solution values of Table 3 (13C and 1H)
were almost identical, we used those determined by us
891
941
950
983
in this paper that include both 15N signals.
A
961
944
statistical analysis of these data without considering
the NH protons afforded the following linear equations.
981
1001
1017
1096
1143
1177
1192
1206
1259
1293
1302
1320
1355
1380
1460
1479
1595
1610
1618
1659
1702
980
1081
1086
986
988
Exp: ¼ ð0:0 0:4Þ þ ð0:992 0:004Þ Calc:4; n
¼ 26; R2 ¼ 1:000; RMS residual ¼ 2:0 ppm; (3)
1081
1128
1161
1190.1
1190.2
1261
1283.4
1283.6
1304
1335
1377
1438
1462
1567
1592
1595
1614
1648
Exp: ¼ ð1:011 0:006Þ Calc:10; R2 ¼ 0:999; n
1133
1163
1174
1225
1273
1276
1309
1364
1389
1442
1466
1574
1601
1609
1673
1701
¼ 26; RMS residual ¼ 4:0 ppm;
(4)
Only13C; Exp: ¼ ð1:001 0:003Þ Calc:4; R2
¼ 1:000; RMS residual ¼ 1:8; n ¼ 16 (5)
Only13C; Exp: ¼ −ð16 7Þ þ ð1:13 0:05Þ Calc:10; R2
¼ 0:73; RMS residual ¼ 3:8; n ¼ 16 (6)
Only1H; Exp: ¼ ð2:6 0:6Þ
þ ð0:66 0:09Þ Calc:4; R2
¼ 0:91; RMS residual ¼ 0:2; n ¼ 8 (7)
Only1H; Exp: ¼ ð2:6 3:9Þ þ ð0:69 0:55Þ Calc:10; R2
¼ 0:20; RMS residual ¼ 0:6; n ¼ 8 (8)
From Equation (7), the largest residuals were:
Atom H7 Exp:7:41 Calc:4 ¼ 6:76 Fitted exp: ¼ 7:04
4 is identical to indirubin (1), but 1 is representing any
tautomer, while 4 is the 1NH1′NH of Z configuration,
and it corresponds to the structure determined by X‐ray
Atom H4′Exp:8:76 Calc:4 ¼ 9:25 Fitted exp: ¼ 8:71
Table 3 results deserve some comments:
crystallography (Figure 2). The 1NH1′NH of
E
configuration called isoindirubin (10) has been isolated
but without clear characterization (only mass
spectrometry and UV)[37–41] In this paper, NMR data of
1 were measured in solution and solid‐state phases. In
conjunction with this, we simulated the 13C and 15N
NMR by using two theoretical methods: GIAO
• The IMHB present in structure 4 (N1―H···O═C2′)
shifts the 1H of N1H to 11.01 ppm that is well
reproduced by the calculations (10.34 ppm). If the
NH is not involved in an IMHB, N1′H, then it is cal-
culated at 6.41 ppm while experimentally it appears
at 10.87 ppm. This is due to an intermolecular HB
with the DMSO solvent that is absent in the gas‐
phase calculations.
and
GIPAW
(Gauge
Including
Projected
Augmented Wave). The later one is normally used to
reproduce the results obtained by solid‐state NMR of a
crystal whereas the GIAO method is often applied to
simulate NMR in solution phase; nonetheless, it has also
been used successfully in the simulation of solid‐state
NMR.[42,43]
• The most discriminating nucleus between indirubin
(4) and isoindirubin (10) is the 13C.
1
• There are two H NMR signals (H7 and H4′) that
deviate more than usual.

ALKORTA ET AL.
7 of 11
TABLE 3 NMR chemical shifts (ppm)
Experimental DMSO‐d6
CPMAS Calculated
4 (GIAO/gas phase)
1[35]
1
1
10 (GIAO/gas phase)
δC δH
6.37
Solid‐state (GIPAW/INDRUB)
Atom
δC
δH
δC
δH
11.01 ‐‐‐‐
δC
δC
δH
10.34
‐‐‐‐
δC
1
‐‐‐‐
11.01 ‐‐‐‐
‐‐‐‐
‐‐‐‐
2
138.30 ‐‐‐‐
188.58 ‐‐‐‐
118.99 ‐‐‐‐
124.29 7.65
121.21 7.02
137.04 7.57
113.39 7.42
152.46 ‐‐‐‐
138.36 ‐‐‐‐
188.68 ‐‐‐‐
119.03 ‐‐‐‐
124.38 7.64
121.29 7.01
137.14 7.57
113.47 7.41
152.53 ‐‐‐‐
139.1
186.2
120.3
124.0
122.9
136.4
114.3
151.3
139.45
186.80
120.54
126.17
120.42
136.26
109.84
151.76
141.26
178.41
122.58
126.09
121.76
134.71
109.25
150.12
‐‐‐‐
134.35
182.61
117.33
122.16
120.30
133.79
112.59
146.16
3
‐‐‐‐
‐‐‐‐
3a
4
‐‐‐‐
‐‐‐‐
7.79
6.84
7.32
6.74
‐‐‐‐
7.82
6.97
7.37
6.76
‐‐‐‐
5
6
7
7a
1′
‐‐‐‐
10.87 ‐‐‐‐
10.89 ‐‐‐‐
‐‐‐‐
6.41
‐‐‐‐
‐‐‐‐
6.15
‐‐‐‐
2′
170.90 ‐‐‐‐
106.54 ‐‐‐‐
121.42 ‐‐‐‐
124.63 8.77
121.21 7.02
129.22 7.25
109.53 6.90
140.87 ‐‐‐‐
170.94 ‐‐‐‐
106.55 ‐‐‐‐
121.47 ‐‐‐‐
124.69 8.76
121.29 7.01
129.30 7.24
109.59 6.89
140.90 ‐‐‐‐
174.1
104.7
122.9
124.9
122.9
128.5
109.8
140.1
169.83
107.77
123.18
127.85
122.21
129.27
106.80
139.91
160.29
111.07
124.10
121.71
119.91
129.08
107.16
143.27
166.80
103.94
120.21
121.23
120.21
126.44
107.19
135.65
3′
‐‐‐‐
‐‐‐‐
3a′
4′
‐‐‐‐
‐‐‐‐
9.25
7.03
7.15
6.60
‐‐‐‐
7.16
6.84
7.06
6.53
‐‐‐‐
5′
6′
7′
7a′
15N
N1
O3
N1′
O2′
‐‐‐‐
‐‐‐‐
‐‐‐‐
‐‐‐‐
‐‐‐‐
‐‐‐‐
‐‐‐‐
‐‐‐‐
−270.3 ‐‐‐‐
−270.6
‐‐‐‐
−270.5
494.6
‐‐‐‐
‐‐‐‐
‐‐‐‐
‐‐‐‐
−273.8
527.5
‐‐‐‐
‐‐‐‐
‐‐‐‐
‐‐‐‐
−273.8
−243.9
‐‐‐‐
−242.4 ‐‐‐‐
‐‐‐‐ ‐‐‐‐
‐‐‐‐
−242.3
‐‐‐‐
−250.5
302.0
−250.1
331.5
• The calculated 1H and 13C values for isoindirubin (10)
CPMAS ¼ ð2:4 1:4Þ þ ð0:993 0:009Þ Calc:10; n
¼ 18; R2 ¼ 0:999; RMS residual
¼ 4:8 ppm
can be used for its identification.
•
17O chemical shifts have not been determined; the cal-
culated 17O chemical shifts are rather different for Z
and E isomers.
(11)
Equation (9) shows that the solid‐state effects are very
small (with chemical shifts about 1% higher in solid
state than in solution). Although the values are very sim-
ilar, structure 4 fits better than 10 (compare Equations 10
and 11), which is consistent with the X‐ray determination
(Figure 2).[11] We can conclude that the structure of
indirubin in solution is also 4.
Solid‐state studies
The CPMAS (13C, 15N) chemical shifts reported in Table 3
were compared with the solution chemical shifts and
with the gas‐phase calculations for indirubin 4 (a Z‐
isomer) and with isoindirubin 10 (an E‐isomer).
Finally, chemical shifts for crystal structure INDRUB
were simulated by using the GIPAW approach. Com-
puted absolute shielding values were transformed into
chemical shifts, according to linear equations established
previously for the azole‐family compounds in solid
state,[55] and reported in Table 3 as solid state (SS, see
below). The comparison of experimental CPMAS values
CPMAS ¼ ð1:001 0:002Þ DMSO − d6; n ¼ 18; R2
¼ 1:000; RMS residual ¼ 1:4 ppm
(9)
CPMAS ¼ ð1:4 0:8Þ þ ð0:991 0:005Þ Calc:4; n ¼ 18;
R2 ¼ 1:000; RMS residual ¼ 2:7 ppm
(10)

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ALKORTA ET AL.
15N signals. Our empirical equations (Equations 13‐15,
see Experimental section) relate the calculations in the
gas phase to the experimental data in solution,[44–54]
and the new Equation (9) relates the solution to the
CPMAS values; therefore, for molecules where the crystal
structure is not very different from that of the monomer,
the much less expensive B3LYP/GIAO calculations could
be used instead of the GIPAW ones.
4 | EXPERIMENTAL SECTION
4.1 | Synthesis
Indirubin was synthesized in 93% yield following the pro-
tocol of Cheng et al.[56]
FIGURE 7 The HBs in indirubin crystal (INDRUB [12,13]). The
NH protons (black dots) were added manually
4.1.1 | General
with these calculated ones provided a linear equation
with an R2 of 1.000 (Equation 12). These results indicate
that both GIAO and GIPAW approaches are comparable
enough to reproduce the experimental NMR parameters
of indirubin 1 in solid state.
Anhydrous methanol (Macron Fine Chemicals) was
degassed by sonication before use. LC‐MS spectrum was
recorded on a WATERS apparatus integrated with a
HPLC separation module (2695), PDA detector (2996),
and Micromass ZQ spectrometer using electrospray ioni-
zation (ES+). Analytical HPLC was performed with a
SunFire C18‐3.5 μm column (4.6 mm × 50 mm). Mobile
phase A: CH3CN + 0.08% formic acid and B: H2O +
0.05% formic acid. UV detection was carried over 190 to
440 nm.
CPMAS ¼ ð2:8 0:4Þ þ ð1:004 0:003Þ Calc:SS; n
¼ 18; R2 ¼ 1:000; RMS residual
¼ 1:5 ppm
(12)
3 | CONCLUSIONS
There is a fair agreement between experimental data
(solution and solid state) and calculated ones (gas phase
and solid‐state phase), but surprisingly there is a near
identity between solution and solid‐state NMR chemical
shifts. The isolated molecule of indirubin in DMSO
should have one IMHB and one N―H···DMSO HB. In
solid state (Figure 7), there are one IMHB
(N1―H1···O2′═C2′) and one intermolecular hydrogen
bond between pairs of molecules: N1′―H1′···O3═C3.
Although both GIPAW and GIAO methods succeed in
simulating the IR and NMR spectra of molecules in solid
state, they present some disadvantages. For instance, in
case of the GIPAW approach, it is generally necessary to
count with the X‐ray crystal structure of the molecule
under study to reproduce the desired spectroscopic infor-
mation successfully and in an assumable computational
time. On the other hand, GIAO computes the properties
in an isolated molecule without taking into account the
periodicity of the system, what in some cases could lead
to errors. However, in the case of indirubin, apparently
both situations are similar in what concerns 13C and
4.1.2 | Synthesis of indirubin
A suspension of 3‐indoxyl acetate (200 mg, 1.1 mmol),
isatin (185 mg, 1.2 mmol), and anhydrous potassium car-
bonate (117 mg, 1.1 mmol) in degassed anhydrous meth-
anol (25 mL) was stirred at room temperature under
argon atmosphere for 5 hours. The precipitate was col-
lected by filtration, washed successively with MeOH (10
mL) and water, and dried under vacuum to yield
indirubin as reddish solid (268 mg, 93%). HPLC (UV) >
95%, m.p. > 330°C, lit., m.p. 348 to 353°C.[57] LRMS
(ESI+) m/z 263 [(M + H)+, 100%].
4.2 | IR
A Perkin Elmer Spectrum Two, fitted with a diamond
single‐bounce ATR, was used to collect spectrum of
indirubin at 4 cm−1 spectral resolution with four co‐adds.
Indirubin was pressed on the diamond crystal and mea-
sured directly without any further sample preparation.

ALKORTA ET AL.
9 of 11
½52ꢀ
δ1H ¼ 31:0 − 0:97*σ1H; ðreference TMS; 0:00 ppmÞ
4.3 | Solution NMR
(13)
Solution NMR spectra were recorded on a Bruker DRX 400
(9.4 Tesla, 400.13 MHz for H, 100.62 MHz for 13C and
1
½53ꢀ
40.54 MHz for 15N) spectrometer with a 5‐mm BBFO direct
detection probe 1H‐19F/31P‐15N equipped with a z gradient
coil, at room temperature. Chemical shifts (δ in ppm) are
δ13C ¼ 175:7 − 0:963*σ13C; ðreference TMS; 0:00 ppmÞ
(14)
1
given from internal solvent, DMSO‐d6 2.49 for H and
δ15N ¼ –152:0 − 0:946*σ15N;
ðreference CH3NO2; 0:00 ppmÞ
39.5 for 13C. For 15N NMR, nitromethane (0.00) was used
as external standard. 2D (1H‐1H) gs‐COSY and inverse pro-
ton detected heteronuclear shift correlation spectra,
(1H‐13C) gs‐HMQC, (1H‐13C) gs‐HMBC, and (1H‐15N) gs‐
HMQC, were carried out with the standard pulse
sequences[58] to assign the 1H, 13C, and 15N signals.
(15)
½53ꢀ
The geometry of indirubin crystal, which has been
obtained from the CSD crystallographic database
(INDRUB), has been optimized using the GGA (PBE)
exchange correlation functional with the Grimme dis-
persion correction, and employing the optimization
method based on Broyden‐Fletcher‐Goldfarb‐Shanno
(BFGS) algorithm and both norm‐conserving and ultra-
soft pseudopotentials. A plane wave kinetic cutoff
energy of 900 eV, k‐point spacing of 0.05 Å−1, and ultra-
fine convergence criteria have been applied. Cell param-
eters have been remained fixed during the geometry
optimization process. These settings have been also used
for the GIPAW calculation of IR and NMR parameters.
The phonon spectrum was calculated at the gamma
point. Acoustic sum rule correction <40.154634 cm−1
was applied. CASTEP package has been used to run
all calculations.
4.4 | Solid‐state NMR
Solid‐state 13C (100.73 MHz) and 15N (40.60 MHz)
CPMAS NMR spectra have been obtained on a Bruker
AV WB 400 spectrometer at 300 K using a 4‐mm triple
channel probehead. Samples were carefully packed in a
4‐mm diameter cylindrical zirconia rotor with Kel‐F
end‐caps. 13C spectra were originally referenced to a
adamantane sample, and then the chemical shifts were
recalculated to the Me4Si [for the CH2 atom
δ
(adamantane)
=
29.5 ppm] and 15N spectra to
15NH415NO3 and then converted to nitromethane scale
using the relationship: δ15N(nitromethane) = δ15N
(ammonium nitrate) −358 ppm. Typical acquisition
parameters for 13C CPMAS were: 2.75 μs 90° H pulses
1
NOTES
and decoupling field strength of 90.9 kHz by TPPM
sequence spectral width, 35 kHz; recycle delay, 4 s; acqui-
sition time, 50 ms; contact time, 3 ms; and spin rate, 10
kHz. Typical acquisition parameters for 15N CPMAS
were: 2.5 μs 1H 90° pulses (decoupling field 100 kHz
tppm15) spectral width, 50 kHz; recycle delay, 6 s; acqui-
sition time, 20.5 ms; contact time, 7.5 ms; and spin rate, 7
kHz.
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
This work was carried out with financial support from
the Ministerio de Ciencia, Innovación y Universidades
(PGC2018‐094644‐B‐C22,
RTI2018‐093940‐B‐I00),
Comunidad de Madrid (P2018/EMT‐4329 AIRTEC‐CM),
and Universidad Nacional de Educacion a Distancia
(UNED). Thanks are also given to the CTI (CSIC) for
their continued computational support.
4.5 | Computational details
Gas‐phase monomer calculations of the optimized geom-
etries were carried out with the B3LYP/6‐311++G(d,p)
method[49,50] using the facilities of the Gaussian 16 suite
of programs.[51] Frequency calculations of the structures
of Figure 6 proved that they were minima (no imaginary
frequencies); they were used to simulate the infrared
spectra of Figure 4. A scaling factor of 0.9679 was applied
for correcting the frequency values.[59] Chemical shifts
were obtained from absolute shielding values calculated
within the GIAO approximation[44,45] using empirical
equations previously obtained:
ORCID
Ibon Alkorta https://orcid.org/0000-0001-6876-6211
José Elguero https://orcid.org/0000-0002-9213-6858
Christophe Dardonville
https://orcid.org/0000-0001-5395-
1932
Felipe Reviriego https://orcid.org/0000-0002-4126-7305
Dolores Santa María
https://orcid.org/0000-0001-5125-
2206

10 of 11
ALKORTA ET AL.
[26] J. Donohue, in Struct. Chem. Mol. Biol., (Eds: A. Rich, N.
Davidson), W. H. Freeman, San Francisco 1968.
Rosa M. Claramunt
2677
https://orcid.org/0000-0001-6634-
[27] G. Desiraju, T. Steiner, The Weak Hydrogen Bond, Oxford
University Press 2001.
Marta Marín‐Luna https://orcid.org/0000-0003-3531-6622
[28] C. H. Schwalbe, Crystallogr. Rev. 2012, 18, 191.
REFERENCES
[29] A. M. Vibhute, U. Deva Priyakumar, A. Ravi, K. M. Sureshan,
[1] M. T. Hamann, Bioactive Compounds from Natural Sources:
Isolation, Characterisation and Biological Properties, Publishing,
Taylor & Francis, London 2001.
Chem. Commun. 2018, 54, 4629.
[30] J. Seixas de Melo, A. P. Moura, M. J. Melo, J. Phys. Chem. A
2004, 108, 6975.
[2] L. Meijer, N. Guyard, L. Skaltsounis (Eds), Indirubin, the Red
Shade of Indigo, Life In Progress Editions, Roscoff 2006.
[31] E. A. Perpète, J. Preat, J.‐M. André, D. Jacquemin, J. Phys.
Chem. A 2006, 110, 5629.
[3] M. J. Moon, S. K. Lee, J.‐W. Lee, W. K. Song, S. W. Kim, J. Il
Kim, C. Cho, S. J. Choi, Y.‐C. Kim, Bioorg. Med. Chem. 2006,
14, 237.
[32] E. A. Perpète, D. Jacquemin, J. Mol. Struct. THEOCHEM 2009,
914, 100.
[33] M. Pan, W. Pei, Y. Yao, L. Dong, J. Chen, Molecules 2018, 23,
[4] R. Christie, Biotech. Histochem. 2007, 82, 51.
2743.
[5] A. Martinez, A. Castro, I. Dorronsoro, M. Alonso, Med. Res.
Rev. 2002, 22, 373.
[34] F. Menges, “SpectraGryph v1.2.11,” can be found under
https://www.effemm2.de/spectragryph/, 2019.
[6] T. Blažević, E. H. Heiss, A. G. Atanasov, J. M. Breuss, V. M.
Dirsch, P. Uhrin, Evidence‐Based Complement. Altern. Med.
2015, 2015, 1.
[35] N. M. Cuong, B. H. Tai, D. H. Hoan, Nat. Prod. Res. 2010, 24,
99.
[36] C. Wang, J. Yan, M. Du, J. A. Burlison, C. Li, Y. Sun, D. Zhao,
[7] N. Aourz, A.‐S. K. Serruys, J. N. Chabwine, P. B. Balegamire, T.
Afrikanova, R. Edrada‐Ebel, A. I. Grey, A. R. Kamuhabwa, L.
Walrave, C. V. Esguerra, et al., ACS Chem. Nerosci. 2019, 10,
1992.
J. Liu, Tetrahedron 2017, 73, 2780.
[37] T. Maugard, E. Enaud, P. Choisy, M. D. Legoy, Phytochemistry
2001, 58, 897.
[38] M. Puchalska, K. Polec‐Pawlak, I. Zadrozna, H. Hryszko, M.
Jarosz, J. Mass Spectrom. 2004, 39, 1441.
[8] C. Drahl, Chem. Eng. News 2008, 86, 10.
[9] N. A. Al‐Dhabi, C. Balachandran, M. K. Raj, V. Duraipandiyan,
C. Muthukumar, S. Ignacimuthu, I. A. Khan, V. S. Rajput,
BMC Complement. Altern. Med. 2012, 12, 242.
[39] P. Aobchey, S. Sinchaikul, S. Phutrakul, S.‐T. Chen, Chiang
Mai J. Sci. 2007, 34, 329.
[40] J. Zhou, F. Qu, Afr. J. Tradit. Complement. Altern. Med. 2011, 8.
[10] R. M. Christie, Colour Chemistry, Royal Society Of Chemistry,
Cambridge 2007.
https://doi.org/10.4314/ajtcam.v8i5S.13
[41] L. Warjeet, S. D. Wangkheirakpam, Int. J. Plant Physiol.
Biochem. 2011, 3, 108.
[11] H. Pandraud, Acta Crystallogr. 1961, 14, 901.
[12] C. R. Groom, F. H. Allen, Angew. Chem. Int. Ed. 2014, 53, 662.
[13] F. H. Allen, O. Kennard, Chem. Des. Autom. News 1993, 8, 31.
[14] C. H. Langley, N. L. Allinger, J. Phys. Chem. A 2003, 107, 5208.
[42] M. M. Quesada‐Moreno, J. R. Avilés‐Moreno, J. J. López‐
González, K. Jacob, L. Vendier, M. Etienne, I. Alkorta, J.
Elguero, R. M. Claramunt, Phys. Chem. Chem. Phys. 2017, 19,
1632.
[15] D. Tzeli, G. Theodorakopoulos, I. D. Petsalakis, D. Ajami, J.
Rebek, J. Am. Chem. Soc. 2011, 133, 16977.
[43] C. I. Nieto, P. Cabildo, R. M. Claramunt, P. Cornago, D. Sanz,
M. C. Torralba, M. R. Torres, M. B. Ferraro, I. Alkorta, M.
Marín‐Luna, et al., Struct. Chem. 2016, 27, 705.
[16] C. Hansch, A. Leo, Exploring QSAR: Fundamentals and
Applications in Chemistry and Biology, American Chemical
Society, Washingthon, DC 1995.
[44] F. London, J. Phys. le Radium 1937, 8, 397.
[45] R. Ditchfield, Mol. Phys. 1974, 27, 789.
[17] W. Holzer, L. Castoldi, V. Kyselova, D. Sanz, R. M. Claramunt,
M. C. Torralba, I. Alkorta, J. Elguero, Struct. Chem. 2019, 30,
1729.
[46] A. D. Becke, J. Chem. Phys. 1993, 98, 5648.
[47] A. D. Becke, Phys. Rev. A 1988, 38, 3098.
[48] C. Lee, W. Yang, R. G. Parr, Phys. Rev. B 1988, 37, 785.
[18] I. Alkorta, J. Elguero, Chem. Phys. Lett. 2018, 691, 33.
[19] I. Alkorta, L. Barrios, I. Rozas, J. Elguero, J. Mol. Struct.
THEOCHEM 2000, 496, 131.
[49] R. Ditchfield, W. J. Hehre, J. A. Pople, J. Chem. Phys. 1971, 54,
[20] J. Elguero, C. Marzin, A. R. Katritzky, P. Linda, The Tautomer-
ism of Heterocycles, Academic Press, New York 1976.
724.
[50] M. J. Frisch, J. A. Pople, J. S. Binkley, J. Chem. Phys. 1984, 80,
[21] V. I. Minkin, A. D. Garnovskii, J. Elguero, A. R. Katritzky, O.
3265.
V. Denisko, Adv. Heterocycl. Chem., New York 2000 157.
[51] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A.
Robb, J. R. Cheeseman, G. Scalmani, V. Barone, G. A.
Petersson, H. Nakatsuji, et al., 2016.
[22] R. F. W. Bader, Chem. Rev. 1991, 91, 893.
[23] R. F. W. Bader, Atoms in Molecules, A Quantum Theory, Oxford
University Press, Oxford 1990.
[52] A. M. S. Silva, R. M. S. Sousa, M. L. Jimeno, F. Blanco, I.
Alkorta, J. Elguero, Magn. Reson. Chem. 2008, 46, 859.
[24] P. L. A. Popelier, Atoms In Molecules. An Introduction, Prentice
Hall, Harlow 2000.
[53] F. Blanco, I. Alkorta, J. Elguero, Magn. Reson. Chem. 2007, 45,
[25] D. J. Sutor, J. Chem. Soc. 1963, 1105.
797.

ALKORTA ET AL.
11 of 11
[54] D. Sanz, R. M. Claramunt, I. Alkorta, G. Sánchez‐Sanz, J.
Elguero, Magn. Reson. Chem. 2012, 50, 246.
[66] S. J. Clark, M. D. Segall, C. J. Pickard, P. J. Hasnip, M. I. J.
Probert, K. Refson, M. C. Payne, Zeitschrift für Krist. ‐ Cryst.
Mater. 2005, 220. https://doi.org/10.1524/zkri.220.5.567.65075
[55] M. Marín‐Luna, I. Alkorta, J. Elguero, Magn. Reson. Chem.
2018, 56, 164.
[56] X. Cheng, K.‐H. Merz, S. Vatter, J. Zeller, S. Muehlbeyer, A.
Thommet, J. Christ, S. Wölfl, G. Eisenbrand, J. Med. Chem.
2017, 60, 4949.
SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of the
article.
[57] G.‐H. Du, Natural Small Molecule Drugs from Plants, Springer
Singapore, Singapore 2018.
[58] S. Berger, S. Braun, 200 and More Basic NMR Experiments,
Wiley‐VCH Verlag, New York 2004.
[59] M. P. Andersson, P. Uvdal, J. Phys. Chem. A 2005, 109, 2937.
[60] J. P. Perdew, K. Burke, M. Ernzerhof, Phys. Rev. Lett. 1996, 77,
How to cite this article: Alkorta I, Elguero J,
Dardonville C, et al. A theoretical and spectroscopic
(NMR and IR) study of indirubin in solution and in
the solid state. J Phys Org Chem. 2019;e4043.
https://doi.org/10.1002/poc.4043
3865.
[61] J. P. Perdew, K. Burke, Y. Wang, Phys. Rev. B 1996, 54, 16533.
[62] S. Grimme, J. Antony, S. Ehrlich, H. Krieg, J. Chem. Phys. 2010,
132, 154104.
[63] S. Grimme, J. Comput. Chem. 2004, 25, 1463.
[64] S. Grimme, J. Comput. Chem. 2006, 27, 1787.
[65] M. D. Segall, P. J. D. Lindan, M. J. Probert, C. J. Pickard, P. J.
Hasnip, S. J. Clark, M. C. Payne, J. Phys. Condens. Matter
2002, 14, 2717.