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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%; indirubin-5-sulfonic acid (sodium salt), 75.8%. Analytical Data: Indirubin: 1H-NMR (DMSO-d6, 25 °C, /ppm): 11.01 (s) and 10.88 (s) (N1H and N1’H), 8.77 (d,3JH,H = 7.7Hz, C4H), 7.66 ( d, 3JH,H = 7.4Hz, C4’H), 7.58 ( m C6’H), 7.42 ( d, 3JH,H = 8.1Hz, C7’H), 7.26 (m, C6H), 7.02 (m, C5H and C5’H), 6.91 (d, 3JH,H = 8.1Hz, 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 = 161.4Hz, C6’), 129.15 (d, 1JC,H = 159.8Hz, C4), 124.62 (d, 1JC,H = 164.6Hz) and 124.20 (d, 1JC,H = 163.8Hz) (C6 and C4’), 121.58 (s, C3a), 121.21 (d, 1JC,H = 162.1Hz) and 121.19 (d, 1JC,H = 162.1Hz) (C5 and C5’), 119.37 (s, C3a’), 113.15 (d, 1JC,H = 168.7Hz, C7’), 109.57 (d, 1JC,H = 162.2Hz, 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 = 7.6Hz, C4’H), 7.58 (m, C6’H), 7.42 (d, 3JH,H = 7.8Hz, C7’H), 7.27 (d, 3JH,H = 8.3 Hz, C6H), 7.04 (m, C5’H), 6.89 (d, 3JH,H = 8.3Hz, 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 = 159.8Hz, C6’), 128.16 (d, 1JC,H = 167.0Hz, C4), 125.00 (s, C5), 124.41 (d, 1JC,H = 163.8Hz) and 123.76 (d, 1JC,H = 170.2 Hz) (C6 and C4’), 122.85 (s, C3a), 121.54 (d, 1JC,H = 165.4Hz, C5’), 118.85 (s, C3a’), 113.50 (d, 1JC,H = 168.6Hz, C7’), 110.61 (d, 1JC,H = 164.6Hz, 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 = 7.4Hz, 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 = 8.0Hz, C7H). 13C-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 = 161.0Hz, C6’), 126.83 (d, 1JC,H = 163.3 Hz, C4), 124.27 (d, 1JC,H = 165.6Hz, 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 = 168.6Hz, C7’), 108.20 (d, 1JC,H = 163.3Hz, 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, 1JC,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 %. Isoindigo3: 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). 13C-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

1Russell 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).

2Farbwerke vorm. Meister Lucius & Brüning in Hoechst a.M. Verfahren zur Herstellung von Derivaten der Indirubine. DRP283726 (1913).

3Wahl 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

1Tripos Associates Inc. Sybyl Molecular Modelling Software, St. Louis, MO (1992)

2Pandraud, H. Structure cristalline de l'Indirubine. Acta Cryst. 14, 901-908 (1961).

3Jones, 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).

4Murshudov, G.N., Vagen, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum -likelihood method. Acta Cryst. D53, 240-255 (1997).

5Lamzin, V.S. & Wilson, K.S. Automated refinement of protein models. Acta Cryst. D49, 129-147 (1993).

6Connolly, M. Molecular surface triangulation. J. Appl. Cryst.18, 499-505 (1985).

7Goodford, P. Multivariate characterisation of molecules for QSAR. J. Chemometrics10, 107-117 (1996).

8Nicholls, 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.

3Adolf von Baeyer. Synthese des Isoatins und Indigblaus. Chem. Ber. 11, 1228-1229 (1878).

4Adolf von Baeyer. Synthese des Indigblaus. Chem. Ber. 11, 1296-1297 (1878).

5Adolf von Baeyer. Synthese des Oxindols. Chem. Ber. 11, 582-584 (1878).

6Heumann K. Neue Synthese des Indigos und verwandter Farbstoffe 1. Chem. Ber.23, 3043-3045 (1890)

7Heumann K. Neue Synthese des Indigos und verwandter Farbstoffe 2. Chem. Ber.23, 3431-3435 (1890)

8Friedländer P. Fortschr., Teerforbenfabr. Verw. Industriezweige 1900-1902, 6, 567; DRP 137955 (1901)

9Fairbanks, V.F. Blue gods, blue oil, and blue people. Mayo Clin. Proc.69, 889-892 (1994).

10José-Yacamàn, M., Rendon, L., Arenas, J., Serra Puche, M. C. Maya blue paint: an ancient nanostructured material. Science273, 223-225 (1996).

11Cardon, D. & du Chatenet, G. Guide des Teintures Naturelles. Delachaux et Niestlé, 400 pp. (1990).

12Friedländer, P. Uber den Farbstoff des antiken Purpurs aus Murex brandaris. Angew. Chem.22, 2492-2494 (1909).

13Fouquet, 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 pathologies including leukaemias 1, 2. 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.

1Blanz, 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).

2Arnold, W.N. King George III’s urine and indigo blue. The Lancet34, 1811-1813 (1996).

Figure 1. Chemical structure of the natural precursors of indigo, indirubin and isoindigo and a selection of synthetic analogues.