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                                                        Supplementary Information



   Indirubin, the active constituent of a Chinese antileukaemia

               medicine, inhibits cyclin-dependent kinases




Ralph HOESSEL, Sophie LECLERC Jane A. ENDICOTT, Martin E. M. NOBLE,
Alison LAWRIE, Paul TUNNAH, Maryse LEOST, Eve DAMIENS,
Dominique MARIE, Doris MARKO, Ellen NIEDERBERGER,
Weici TANG, Gerhard EISENBRAND & Laurent MEIJER



CHEMISTRY
Synthesis of indirubin and 5-substituted indirubin derivatives (general procedure, modified
from Russell and Kaupp1): under argon, isatin or the respective isatin derivative (11.5 mmol)
and sodium carbonate (24.5 mmol) were added to a solution of indoxyl acetate (11.4 mmol) in
methanol (50 ml). The mixture was stirred for half an hour at room temperature and was filtered
after 24 h standing. The residue was washed twice with methanol and several times with cold
water until neutral reaction of the filtrate. Products (dark violet crystals) were dried over
potassium hydroxide in the dessicator. Yields: indirubin, 81.0 %; 5-chloroindirubin, 94.6 %;
                                                                                       1
indirubin-5-sulfonic acid (sodium salt), 75.8 %. Analytical Data: Indirubin:               H-NMR
(DMSO-d6, 25 °C, /ppm): 11.01 (s) and 10.88 (s) (N1H and N1’H), 8.77 (d,3JH,H = 7.7 Hz,
C4H), 7.66 ( d, 3JH,H = 7.4 Hz, C4’H), 7.58 ( m C6’H), 7.42 ( d, 3JH,H = 8.1 Hz, C7’H), 7.26 (m,
C6H), 7.02 (m, C5H and C5’H), 6.91 (d, 3JH,H = 8.1 Hz, C7H). 13C-NMR (DMSO-d6, 90 °C,

/ppm): 188.27 (s, C3’), 171.11 (s, C2), 152.35 (s, C2’), 141.07 (s, C7a’), 138.48 (s, C7a),
136.85 (d, 1JC,H = 161.4 Hz, C6’), 129.15 (d, 1JC,H = 159.8 Hz, C4), 124.62 (d, 1JC,H = 164.6 Hz)
and 124.20 (d, 1JC,H = 163.8 Hz) (C6 and C4’), 121.58 (s, C3a), 121.21 (d, 1JC,H = 162.1 Hz)
and 121.19 (d, 1JC,H = 162.1 Hz) (C5 and C5’), 119.37 (s, C3a’), 113.15 (d, 1JC,H = 168.7 Hz,
C7’), 109.57 (d, 1JC,H = 162.2 Hz, C7), 106.97 (s, C3). MS (70 eV, m/z): 262 (M+, 100%), 234
(43%), 205 (25%), 131 (4%). CHN: found: C, 73.2%; H, 4.0 %; N, 10.6 %, C16H10N2O2
requires: C, 73.3 %; H, 3.8 %; N, 10.7 %. 5-Chloroindirubin: 1H-NMR (DMSO-d6, 25 °C,
/ppm): 11.09 (s) and 10.99 (s) (N1H and N1’H), 8.78 (s, C4H), 7.65 ( d, 3JH,H = 7.6 Hz,
C4’H), 7.58 (m, C6’H), 7.42 (d, 3JH,H = 7.8 Hz, C7’H), 7.27 (d, 3JH,H = 8.3 Hz, C6H), 7.04 (m,
C5’H), 6.89 (d, 3JH,H = 8.3 Hz, C7H). 13C-NMR (DMSO-d6, 25 °C, /ppm): 188.72 (s, C3’),

170.52 (s, C2), 152.39 (s, C2’), 139.31 (s) and 139.10 (s) (C7a and C7a’), 137.21 (d, 1JC,H =
159.8 Hz, C6’), 128.16 (d, 1JC,H = 167.0 Hz, C4), 125.00 (s, C5), 124.41 (d, 1JC,H = 163.8 Hz)
and 123.76 (d, 1JC,H = 170.2 Hz) (C6 and C4’), 122.85 (s, C3a), 121.54 (d, 1JC,H = 165.4 Hz,
C5’), 118.85 (s, C3a’), 113.50 (d, 1JC,H = 168.6 Hz, C7’), 110.61 (d, 1JC,H = 164.6 Hz, C7),
104.95 (s, C3). MS (70 eV, m/z): 298 (M+, 33%) 296 (M+, 100%), 268 (39%), 233 (35 %), 205
(50 %). CHN: found: C, 64.5%; H, 3.0 %; N, 9.5 %, C16H9ClN2O2 requires: C, 64.8 %; H, 3.1
%; N, 9.4 %. Indirubin-5-sulfonic acid (sodium salt) : 1H-NMR (DMSO-d6, 25 °C, /ppm):
11.05 (s) and 10.99 (s) (N1H and N1’H), 9.13 (s, C4H), 7.67 (d, 3JH,H = 7.4 Hz, C6H), 7.57 (m,
C4’H and C6’H), 7.43 (d, 3JH,H = 8.0 Hz, C7’H), 7.04 (m, C5’H), 6.84 (d, 3JH,H = 8.0 Hz, C7H).
13
     C-NMR (DMSO-d6, 25 °C, /ppm): 188.33 (s, C3’), 171.14 (s, C2), 152.39 (s, C2’), 141.64

(s) and 140.89 (s) (C7a and C7a’), 138.45 (s, C5), 137.02 (d, 1JC,H = 161.0 Hz, C6’), 126.83 (d,
1
    JC,H = 163.3 Hz, C4), 124.27 (d, 1JC,H = 165.6 Hz, C4’), 122.57 (d, 1JC,H = 169.4 Hz, C6),
121.28 (d, 1JC,H = 157.9 Hz, C5’), 120.50 (s) and 118.98 (s) (C3a and C3a’), 113.39 (d, 1JC,H =
168.6 Hz, C7’), 108.20 (d, 1JC,H = 163.3 Hz, C7), 106.25 (s, C3). CHN: found: C, 49.2%; H,
3.1 %; N, 7.2 %, C16H9N2NaO5S (1.5 H2O) requires: C, 49.1 %; H, 3.1 %; N, 7.2 %.
Indirubin-3’-oxime2: indirubin (1.91 mmol) was refluxed together with hydroxylamine
hydrochloride (5.04 mmol) in pyridine (15 ml). After 1.5 h the reaction mixture was poured into
1 N hydrochloric acid (100 ml). The precipitate was filtered off, redissolved in 1 N sodium
hydroxide (50 ml) and reprecipitated in 1 N hydrochloric acid. The precipitate was filtered off
and washed with water. Recrystallization from ethanol/water (7:2) gave deep red crystals.
Yield: 78.9 %. 1H-NMR (DMSO-d6, 25 °C, /ppm): 13.48 (s, NOH), 11.73 (s) and 10.72 (s)
(N1H and N1’H), 8.65 (d, 3JH,H = 7.2 Hz, C4H), 8.24 (d, 3JH,H = 7.6 Hz, C4’H), 7.41 (m, C6’H
and C7’H), 7.13 (m, C6H), 7.03 (m, C5’H), 6.95 (m, C5H), 6.90 (d, 3JH,H = 7.6 Hz, C7H). 13C-
NMR (DMSO-d6, 25 °C, /ppm): 170.95 (s, C2), 151.22 (s, C3’), 145.32 (s, C2’), 144.83 (s,

C7a’), 138.34 (s, C7a), 132.02 (d, 1JC,H = 159.5 Hz, C6’), 127.95 (d, 1JC,H = 164.4 Hz, C4’),
125.92 (d, 1JC,H = 154.0 Hz, C4), 123.09 (d, 1JC,H = 165.1 Hz, C6), 122.66 (s, C3a), 121.49 (d,
1
    JC,H = 161.6 Hz, C5), 120.43 (d, 160.2 Hz, C5’), 116.54 (s, C3a’), 111.52 (d, 1JC,H = 165.8 Hz,
C7’), 108.91 (d, 1JC,H = 162.3 Hz, C7), 98.88 (s, C3). MS (70 eV, m/z): 277 (M+, 100 %), 260
(M+, 87 %), 247 (24 %), 220 (14 %), 205 (11 %), 130 (13 %), 103 (16 %), 76 (12 %). CHN:
found: C, 68.4 %; H, 4.1 %; N, 14.8 %, C16H11N3O2 (0.25 H2O) requires: C, 68.2%; H 4.1 %;
N, 14.9 %. Isoindigo 3: Oxindole (10.14 mmol) and isatin (10.19 mmol) were stirred (95 °C) in
a mixture of glacial acetic acid (30 ml) and concentrated hydrochloric acid (0.5 ml). After 3 h,
the resulting precipitate (dark brown crystals) was filtered off and washed with methanol and
diethyl ether. Yield: 83.9 %. 1H-NMR (DMSO-d6, 25 °C, /ppm): 10.90 (s) (N1H and N1’H),
9.07 (d, 3JH,H = 8.0 Hz, C4H and C4’H), 7.34 (m, C6H and C6’H), 6.97 (m, C5H and C5’H),
6.85 (d, 3JH,H = 7.7 Hz, C7H and C7’H).       13
                                                   C-NMR (DMSO-d6, 25 °C, /ppm): 168.88 (s, C2

and C2’), 143.98 (s, C7a and C7a’), 133.24 (s, C3 and C3’), 132.52 (d, 1JC,H = 159.9 Hz, C4
and C4’), 129.21 (d, 1JC,H = 166.5 Hz, C6 and C6’), 121.58 (s, C3a and C3a’), 121.04 (d, 1JC,H =
161.2 Hz, C5 and C5’), 109.42 (d, 1JC,H = 162.5 Hz, C7 and C7’). MS (70 eV, m/z): 262 (M+,
100 %), 234 (85 %), 205 (18 %), 132 (17 %). CHN: found: C, 73.0 %; H, 3.8 %; N, 10.9 %,
C16H10N2O2 requires: C, 73.3 %; H 3.8 %; N, 10.7 %.
References
1           Russell G. A. and Kaupp G. Oxidation of Carbanions. IV. Oxidation of Indoxyl to
            Indigo in Basic Solution. J. Am. Chem. Soc., 91, 3851-9 (1969).
2           Farbwerke vorm. Meister Lucius & Brüning in Hoechst a.M. Verfahren zur
            Herstellung von Derivaten der Indirubine. DRP 283726 (1913).
3           Wahl A., Bayard P. Sur un nouvel isomère de l’indigo. Comptes Rendues
            Hebdomadaires des Séances de l’Académie des Sciences, 148, 716-719 (1909).

CRYSTAL STRUCTURE SOLUTION, REFINEMENT AND REPRESENTATION
The sulfonate indirubin derivative structure was built in SYBYL1 with reference to the small
molecule crystal structure of indirubin2. The indirubin-3'-monoxime structure was taken from
the Cambridge Structural Database. Refinement of the model was then pursued with alternating
cycles of interactive model building3 and maximum likelihood refinement using the program
REFMAC4. Towards the end of the refinement, water molecules were added using ARP5.
References
1           Tripos Associates Inc. Sybyl Molecular Modelling Software, St. Louis, MO (1992)
2           Pandraud, H. Structure cristalline de l'Indirubine. Acta Cryst. 14, 901-908 (1961).
3        Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. Improved method for building
         models in electron density maps and the location of errors in these models. Acta Cryst.
         A47, 110-119 (1991).
4        Murshudov, G.N., Vagen, A.A. & Dodson, E.J. Refinement of macromolecular
         structures by the maximum -likelihood method. Acta Cryst. D53, 240-255 (1997).
5        Lamzin, V.S. & Wilson, K.S. Automated refinement of protein models. Acta Cryst.
         D49, 129-147 (1993).
6        Connolly, M. Molecular surface triangulation. J. Appl. Cryst. 18, 499-505 (1985).
7        Goodford, P. Multivariate characterisation of molecules for QSAR. J. Chemometrics
         10, 107-117 (1996).
8        Nicholls, A. & Honig, B. A rapid finite difference algorythm, utilising successive over
         relaxation to solve the Poison Boltzmann equation. J. Comp. Chem. 12, 435-445
         (1991).


ORIGINS OF INDIGO (AND INDIRUBIN) FOR USE AS A TEXTILE DYE
The use of indigo as a textile dye dates back to the Bronze age (-7000). This dye was produced
from the Brassicaceae plant woad (Isatis tinctoria) all over Europe. It should be stressed that
cultivation of this plant and production of the dye were essential features of the Renaissance
agriculture and commerce . (for reviews on historical aspects of indigo and indirubin see refs
12, 13 of our paper). The main production and trade centres were Albi/Toulouse (‘Pays de
Cocagne’) in France, Somerset in Great Britain, Thüringen in Germany and Florence in Italy.
This widespread and important economy collapsed in a few decades in the sixteenth century
with the introduction of indigo blue from a very different plant, Indigofera tinctoria, a Fabaceae
cultivated in Asia (India, Bangladesh) and later from other Indigofera species in the Caribbean
and the American colonies. Indigofera had a higher indigo content than its European
competitor. Furthermore, the presence of a small and variable percentage of indirubin in indigo
blue from Indigofera sp. (up to 15 % of the total indigoïds content in Indigofera arrecta from
Java) also provided a richer palette of hues to this new source of indigo dye. Nevertheless this
new trade also collapsed a few centuries later, following the total chemical synthesis of indigo
(at a much reduced prize) from aniline (itself derived from coal tar) by Adolf von Baeyer in
18781-6, who received a Nobel Prize of Chemistry for his work in 1905. Twenty years later, all
indigo was definitively produced by the chemical industry (review in ref. 7). Indigo, the oldest
dye of mankind, is still widely used all over the world (blue jeans — ‘bleu de Gênes’, blue
Denim — ‘bleu de Nîmes’, etc.).
       According to Julius Caesar and Pliny, the inhabitants of Britain used woad indigo to
colour themselves to generate a fearsome appearance (Picti, Scoti, Britanni mean ‘painted
men’; Bryth in gaellic means ‘paint’, and gave the word ‘brython’). Indigo is also the origin of
the colour of the well-known blue-skinned Tuaregs from the Sahara. The famous, tropical rain
forest, resistant, maya blue paint is a mixture of palygorskite clay and indigo obtained from a
plant called xiuquilit (Indigofera sp.)8.
       Another very different natural source of indigoids should be mentioned, the gastropod
molluscs of the Muricidae and Thaididae families (review in ref. 9). These marine invertebrates
have been used in numerous parts of the world as the source of a vivid purplish red dye, known
as ‘Tyrian purple’ around the Mediterranean sea. The hypobranchial gland of these gastropods
indeed contains colourless precursors, indoxyl-sulphate, its mercaptan derivative and their
bromo derivatives, which release various indoxyls upon maceration (in fact hydrolysis by
purpurase). When exposed to light and oxygen, these indoxyls dimerize in a mixture of indigo
and indirubin, and, mainly, their 6,6'-dibromo-derivatives. The composition of the famous
‘Tekhelet’ described in the Bible was recently identified as these four indigoids specifically
obtained from Hexaplex trunculus10, 11 . The highly prized beautiful Tyrian purple obtained at
great expense was the object of an important trade in Crete (fifth century BC), Phoenicia, Greece
and later in Rome. The colour was considered a privilege of the emperors Neron and Caligula.
In Britain and Ireland the ‘Dog-Welk’ (Nucella lapillus) had also been used for ages as a source
of purple dye.

1        Adolf von Baeyer. Synthese des Isoatins und Indigblaus. Chem. Ber. 11, 1228-1229
         (1878).
2        Adolf von Baeyer. Synthese des Indigblaus. Chem. Ber. 11, 1296-1297 (1878).
3        Adolf von Baeyer. Synthese des Oxindols. Chem. Ber. 11, 582-584 (1878).
4        Heumann K. Neue Synthese des Indigos und verwandter Farbstoffe 1. Chem. Ber. 23,
         3043-3045 (1890)
5        Heumann K. Neue Synthese des Indigos und verwandter Farbstoffe 2. Chem. Ber. 23,
         3431-3435 (1890)
6        Friedländer P. Fortschr., Teerforbenfabr. Verw. Industriezweige 1900-1902, 6, 567;
         DRP 137955 (1901)
7       Fairbanks, V.F. Blue gods, blue oil, and blue people. Mayo Clin. Proc. 69, 889-892
        (1994).
8       José-Yacamàn, M., Rendon, L., Arenas, J., Serra Puche, M. C. Maya blue paint: an
        ancient nanostructured material. Science 273, 223-225 (1996).
9       Cardon, D. & du Chatenet, G. Guide des Teintures Naturelles. Delachaux et Niestlé,
        400 pp. (1990).
10      Friedländer, P. Uber den Farbstoff des antiken Purpurs aus Murex brandaris. Angew.
        Chem. 22, 2492-2494 (1909).
11      Fouquet, H. & Bielig, H.J. Biological precursors and genesis of Tyrian-Purple. Angew.
        Chem. Internat. Edit. 10, 816-817 (1971).


INDIGO AND INDIRUBIN IN THE URINE
Interestingly indican, indigo and indirubin appear in the urine of patients with various
                                   1, 2
pathologies including leukaemias          . The ‘purple urine bag’ and ‘blue diaper’ syndromes
derive from the metabolism of tryptophan into indole which is absorbed and further oxidized in
the liver to indoxyl. This indoxyl is then excreted in urine as a sulphate conjugate and it is
decomposed to indigo and indirubin by bacteria.

1       Blanz, J., Ehninger, G. & Zeller, K.P. The isolation and identification of indigo and
        indirubin from urine of a patient with leukemia. Res. Comm. Clin. Pathol. Pharmacol.
        64, 145-156 (1989).
2       Arnold, W.N. King George III’s urine and indigo blue. The Lancet 34, 1811-1813
        (1996).
                                                                                                                                                     OR2
Figure 1. Chemical structure of the natural precursors of indigo, indirubin and isoindigo
                                                                                                                                                     3
                                                                                                                                                         2
and a selection of synthetic analogues.                                                                                                                       R1
                                                                                                           6

                                                                                              R3                                                 N
                                                                                                                                                 H


              [1] INDICAN                                                 [2] ISATAN B                                                       [3] INDOXYL SULFATE                    TYRINDOXYL SULFATE AND ANALOGUES
 1H-indole-3-yl-ß-D-glucopyranoside                        (R)-1H-indol-3-yl-D-xylo-5-hexulo-                                                potassium 1H-indol-3-yl sulfate        [4] potassium 6-bromo-2-(methylthio)-   R1 = SCH3
                                                           furanosonic acid ester                                                                                                       1H-indol-3-yl sulfate               R2 = SO3- K+
                  (plant indican)                                                                                                                    (urinary indican)
                                                                                                                                                                                                                            R3 = Br
         R1 = H                                                 R1 = H                                                                                   R1 = H                                    (tyrindoxyl sulfate)
                            CH2 OH                                                                                                                       R2 = SO3- K+
                                                                                                               O
                                                                           HOH2C                                                                         R3 = H                                                             R1 = SCH3
              2                     O                               2                                      O                                                                              potassium 2-(methylthio)-
         R =                                                    R =
                            OH                                                                              HO                                                                            1H-indol-3-yl sulfate             R2 = SO3- K+
                                                                                                                                                                                                                            R3 = H
                    HO                                                              HO
                                                                                                                                                                                                                             1
                                    OH                                                        HO                                                                                          potassium 6-bromo-1H-indol-3-yl   R =H
                                                                                                                                                                                                                             2     - +
         R =H
              3
                                                                R =H
                                                                    3                                                                                                                     sulfate                           R = SO3 K
                                                                                                                                                                                                                             3
                                                                                                                                                                                                                            R = Br
                                                                                                                                                                                                                             1
                                                                                                                                                                                          potassium 6-bromo-2-(methyl-      R = SO2CH3
                                                                                                                                                                                                                             2     - +
                                                                                                                                                                                          sulfonyl)-1H-indol-3-yl sulfate   R = SO3 K
                                                                                                                                                                                                                             3
                                                                                                                                                                                                                            R = Br



                                                                                                      OH                                                          O


                                                                                                                                                                      O
                                                                R
                                                                    3                             N                                      3                    N
                                                                                                  H                                 R                         H
                                                                         [5] INDOXYL                                                             [6] ISATIN
                                                                                    R3 = H                                                           R3 = H


                                                                                                                                                                                                       H1        7
                                                                                                                                                                                                       N             6
                                                                                                                                                                                              2
                                                                                                                                                 5
                                                                                                                                                                                         O
                                                                                                                                                         6                                                           5
                             O                                                                                              O            4                                                         3
         4'                             H1        7                                                   4'                                                                            4'                           4
                       3'                                                                                              3'                                                                     3'
    5'                                  N                                                5'                                                              7                     5'
                                                            6
                               2'   2                                                                                          2'    3                                                                  2'
                                                                                                                                                                                                             O
                                                                                                                                                     1
    6'                                                      5                            6'                                                      NH                            6'
                         N1'             3
                                                                                                                        N 1'                 2
                                                                                                                                                                                               N 1'
         7'              H                        4                                                   7'                H                                                           7'         H
                                    O                                                                                                O
                         [7] INDIGO                                                                            [8] INDIRUBIN                                                             [9] ISOINDIGO




                                                                                                  R
                                                                                                                            [10] INDIRUBIN-3'-MONOXIME
                                                                                                  5
                                                                                                                   6                R=H
                                                                          X              4
                                                                                                                                    X = NOH
                                                      4'
                                                                    3'
                                             5'                                                                    7
                                                                               2'    3
                                                                                                                            [11] INDIRUBIN-5-SULFONIC ACID
                                                                                                           1
                                                                                                                                    R = SO3H
                                             6'                                                       NH                            X= O
                                                                        N 1'                  2
                                                      7'                H
                                                                                      O                                     [12] 5-CHLORO INDIRUBIN
                                                                                                                                    R = Cl
                                                                                                                                    X= O

				
Jun Wang Jun Wang Dr
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