REVIEWS The Chemistry and Biology of Alkannin, Shikonin, and Related Naphthazarin Natural Products** Vassilios P. Papageorgiou,* Andreana N. Assimopoulou, Elias A. Couladouros, David Hepworth, and K. C. Nicolaou* There can be few natural products with histories as rich as those of the enantiomeric naphthoquinones alkannin (1) and shikonin (2). Extracts from the roots of Alkanna tinctoria in Eu-rope and Lithospermum erythrorhizon in the Orient have been used inde-pendently for many centuries as natu-ral red dyes and as crude drugs with the magic property of accelerating wound healing. It was only in 1935 that the major constituents of these extracts, 1 and 2, were correctly identified as enantiomers, and in 1976 that the wound healing properties of alkannin (1) were clinically demonstrated. To-day shikonin (2) has a high value both for its medicinal and coloring proper-ties, especially in Japan. With synthetic chemists unable, until recently, to devise an efficient synthetic route, and sources from the plants unable to meet demands, it was left to biotechnologists to develop the first commercial plant-cell culture manufacturing process. In Europe, alkannin derivatives are the active principles of a pharmaceutical ointment used for the treatment of ulcers and burns. This review is an attempt to tell the ªwhole storyº of 1 and 2, from the 5th century BC and presents up to date aspects of plant biology, pharmacology, and bioorganic, synthetic, and medicinal chemistry. Keywords: bioorganic chemistry ´ natural products ´ phytochemistry ´ quinones ´ wound healing 1. Historical Introduction The story of the enantiomeric naphthoquinone natural products alkannin (1) and shikonin (2; Figure 1) can be traced back many centuries. In fact, the story comprises two sub[*] Prof. V. P. Papageorgiou, A. N. Assimopoulou Organic Chemistry Laboratory, Chemical Engineering Department Aristotle University of Thessaloniki, 54006 Thessaloniki (Greece) Fax: (‡30)31-996252 E-mail:
[email protected] Prof. K. C. Nicolaou, Dr. D. Hepworth Department of Chemistry and The Skaggs Institute for Chemical Biology The Scripps Research Institute 10550 North Torrey Pines Road, La Jolla, California 92037 (USA) Fax: (‡1)619-784-2469 E-mail:
[email protected] and Department of Chemistry and Biochemistry University of California, San Diego 9500 Gilman Drive, La Jolla, California 92093 (USA) Prof. E. A. Couladouros Chemical Laboratories Agricultural University of Athens, Iera Odos 75, 118.55 (Greece) and Organic and Bio-organic Laboratories, NCSR ªDEMOKRITOSº 153.10 Ag. Paraskevi Attikis, POB 60228 (Greece) OH O OH O OH O OH OH O OH 1 Figure 1. Structures of alkannin (1) and shikonin (2). 2 plots that have run remarkably parallel courses in Europe and the Orient, being united only in the 20th century. In Europe, the S enantiomer, alkannin (1, Figures 1 and 2), is found in the roots of the plant Alkanna tinctoria Tausch (AT) (Figure 3a), also known as Anchusa tinctoria, and in common English as alkanet. The compound is a major component of the deep red pigments that are easily extracted from the roots of the plant. It was probably the highly colored nature of alkannin that first captured the attention of botanists. Its use as a dyestuff for fabrics almost certainly dates back centuries BC. Perhaps the first recorded use of AT roots is found in the works of the Greek doctor and philosopher Hippocrates (4th and 5th centuries BC) who described their use for the treatment of ulcers. The botanist and scholar Theophrastus (3rd and 4th centuries BC) also alluded to their application as dyes and medicines. [5] [4] [3] [2] [1] Alexan-der the Great was known to have [6] [**] A list of abbreviations not defined in the main text can be found at the end of the article. Angew. Chem. Int. Ed. 1999, 38, 270 ± 300 employed doctors trained in herbal medicine, seems likely that the medicinal and it thus WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999 1433-7851/99/3803-0271 $ 17.50+.50/0 271 REVIEWS V. P. Papageorgiou, K. C. Nicolaou et al. properties of AT root were used to treat the sick and injured during his campaigns. Dioscorides (Figure 4), who many consider to be the founder of pharmacognosy, described the properties in more detail in his De Materia Medica around [7] 77 AD (Figure 5). While discussing the properties of the related species Anchusa etera, Dioscorides goes on to describe rather more [8] bizarre effects: ª..ye roots red,..having something like blood in them. But ye faculty of it, and ye leaves is to help ye venemous-beast-bitten, especially ye viper-bitten, being eaten or drank or hanged about one. And if one having chewed it, do spitt it out into ye mouth of ye venemous beast, it will kill him.º Despite this blatant detour into fiction, the wound healing properties of AT have been reported by others. Pliny, a Roman herbalist contemporary with Dioscorides, independently made very similar claims for the medicinal properties of [9] AT: ª..It stains the hands the colour of blood; it is used for imparting rich colours to wool. Applied with cerate it heals Figure 2. Computer-generated molecular models of alkannin (1) (left) and shikonin (2) (right). Color code: gray: carbon; white: hydrogen; red: oxygen. V. P. Papageorgiou A. N. Assimopoulou E. A. Couladouros D. Hepworth K. C. Nicolaou Vassilios P. Papageorgiou was born in 1942 in Greece and received his Ph.D. and D.Sc. degree from the Aristotle University of Thessaloniki working on the isolation and structure elucidation of wound-healing agents of plant origin. After postdoctoral work at the Pharmacy School, University of Kentucky, he was elected Professor of Organic Chemistry and then Head of the Department of Chemical Engineering at the University of Thessaloniki. He is currently Dean at the School of Engineering. His research interests include the isolation and study of biologically active molecules of plant origin and the development of novel wound-healing agents. Andreana N. Assimopoulou was born in Greece in 1974. She received her B.Sc. in Chemical Engineering in 1997 from the Aristotle University of Thessaloniki. She is currently a postgraduate student in the Papageorgiou group. Elias A. Couladouros, born in 1956 in Greece, received his B.Sc. from the University of Athens and his Ph. D. from the Agricultural University of Athens under the direction of Professor M. P. Georgiadis. After a postdoctoral stint in the Nicolaou group at the University of Pennsylvania (1988 ± 89), he joined the faculty of the Agricultural University of Athens, where he is currently Associate Professor of Chemistry. He is also a Visiting Professor at the research center ªDemokritosº in Athens. His research interests focus on new synthetic methods and the total synthesis of natural products. David Hepworth, born in 1970 in England, studied Chemistry at the University of Oxford. He completed his D.Phil. under the supervision of Prof. S. G. Davies in 1996, working on asymmetric synthesis with organochromium chemistry and chiral hydroxylamines. Since joining the Nicolaou group in 1997, he has completed a synthesis of alkannin and shikonin, and worked in the epothilone team. K. C. Nicolaou is the Chairman of the Department of Chemistry at The Scripps Research Institute, La Jolla, California where he holds the Darlene Shiley Chair in Chemistry and the Skaggs Professorship of Chemical Biology. He is also Professor of Chemistry at the University of California, San Diego. 272 Angew. Chem. Int. Ed. 1999, 38, 270 ± 300 Int.º Angew.º For centuries Dioscorides De Materia Medica remained one of the standard botanical texts in Europe. English translation of a passage from Dioscorides De Materia Medica.. Earthworms cleaned and purged number 40. stabs or pricking with any pointed weapon. ulcerous sores. and possibly even [10] further afield. and b) Lithospermum erythrorhizon Sieb. Chem. The plants a) Alkanna tinctoria Tausch. then strain out whilst hot and keep it close for use. 270 ± 300 Figure 5... Alkanet Root 3 or 4 ounces. a source of alkannin (1).Alkannin and Shikonin REVIEWS Figure 4. The English ªgentleman student of Physick and astrologyº Nicolas Culpeper. those of the aged in particular: it is employed also for the cure of burns. stated that alkanet helps to cure ªold ulcers. a formula for: ª. 38. Ed. from the 17th century AD. Figure 3. 1710). The founder of pharmacognosy Dioscorides collecting botanical samples (courtesy of Parke Davis).. the medical doctor William Salman described in English Herbs [12] (ca. 273 .deep wounds and punctures of the nerves made with thrusts. a common source of shikonin (2) (courtesy of Medpharm Scientific Publications (a) and Kyoto Pharmaceutical University (b)). and burning by common fireº. Thus. the properties of alkanet became known across Europe. Furthermore. Boil them together. hot inflamma[11] tions. Et Zucc.º The recipe is as follows: ªOlive oil 2lbs. with many other herbals being developed from it. 1999. which he termed shikalkin (1/2). Nicolaou et al. Its application to traditional Chinese medicine may have originated with the great surgeon Hua To (born ca. 136 ± 141 AD). or been forgotten. the use of 1 in Europe. antibacterial. In Europe and North America. and to finally unite the stories of these natural products.4-naphthoquinone. He suggested that this was a consequence of the presence of a quantity of the racemate. P. alkannin (1) is still listed in the Merck [15] Index as an astringent. such compounds have also been shown to exhibit significant antitumor. and hung-tzu ken (reddish-purple root). the R enantiomer. infected crusts. Various preparations that contain 2 and its derivatives are still used today for medicinal purposes in China. proposed structure 3 (Figure 7). Et Zucc. as it is included in the classic compilation of traditional Chinese medicine Pen Ts ao Kang Mu. and anti-inflammatory activities. 2. cines. but incorrect. structural assignment for naphthazarin (64) as 5. Papageorgiou.4naph-thoquinone (4). is set in the Orient. More recently. there has been extensive scientific research concerning these natural products in many areas throughout the disciplines of chemistry and biology. These are notably similar to the medicinal properties claimed independently for AT in many of the ancient herbal texts. 38. Brockmann found that the specific rotations of 1 and 2 were not of identical magnitude. in the extracts of LE. many modern accounts of herbal medicine describe the [12] roots as being. Int. In fact. who is considered to be among the first to have used crude drugs such as antiseptics and [16] anti-inflammatory ointments. These aspects will be dealt with in Section 5. In [19] addition. The development of Chinese medicine is considered to be a process of constant distillation and selection of all that has gone before. Wrongly assigned structures for shikonin (3) and naphthazarin [24] (4). (LE) (Figure 6). Unfortunately. they failed to OH O OH O OH OH OH O O 4 3 Figure 6.3. whereby recent developments are built upon by others. Isolation and Identification of Alkannin and Shikonin Shikonin (2) was first isolated as its acetate (15) from the roots of LE by the Japanese chemists Majima and Kuroda in [24] 1922. which had for so long run in parallel. anal ulcers. but that the values for 1 were generally greater than those for 2. Worldwide. originally in China. known in Chinese by various names including tzu ts ao. and were probably further confused by the widely accepted.6-dihydroxy-1. the medicinal properties of the plant seem either to have drifted into folklore.1 and 5.8[1] dihydroxy-1. 1999. This molecule is the major constituent of the red pigment extracts from the roots of the plant Lithospermum erythrorhizon Sieb. most of which have [23] appeared since the first review in 1980. which confirmed the wound healing properties and antimicrobial properties of AT root extracts (Sections 5. The absolute configurations of 1 and 2 were later [25] determined by degradation to malic acid. and noting its physical and chemical similarities to the known compound naphthazarin (Section 4. More recently. (courtesy of Kyoto Pharmaceutical University). It is thus testimony to the medicinal properties of LE roots that they are still included in compilations of ingredients for traditional Chinese and other oriental medi274 realize that 2 was optically active. they are also used in Japan as cosmetics and [20] dyestuffs. However. tzu-ken (purple root). Its use can be traced back with certainty to the latter days of the Ming dynasty. The treatable indications claimed for LE roots include: burns. the medicinal properties claimed for shikonin (2) have more recently been tested scientifically. The tale of shikonin (2). respectively) and was the first to identify alkannin derivatives as the active components. to correctly assign their structures. Brockmann reinvestigated 2 after the struc-ture of naphthazarin (64) was correctly revised to 5. These workers determined many of the chemical properties of 2. rather than the linear path taken by Western medicine. He was the first to identify alkannin (1) and shikonin (2) as enantiomers. Japan. Chem. the ratio of enantiomers in various sources of alkannin (1) and shikonin (2) have been examined by circular Angew. in 1976 Papageorgiou described the results of experiments. ªrarely used for medicinal purposesº. Figure 7. 21] [13. however. and Korea. Nevertheless. bedsores. which was written in 1596 [17] AD. The interesting findings of these experiments will also be described in Section 5. and what has gone before becomes assumed or forgotten. 270 ± 300 . however. Et Zucc.REVIEWS Since then. This review is an attempt to bring together material from over 400 articles and patents that have been written in this field. K. The use of 2 for dyeing silk probably dates back as far as [18] V. hemorrhoids. 22] pigment for food coloring and cosmetics. and as having ªno medicinal impor[14] tanceº. ªnot [13] widely availableº. 1 is mainly used today as a [13. Roots of the plant Lithospermum erythrorhizon Sieb. Ed. external wounds. As with alkannin (1). C.1). and oozing dermatitis. Further improvements in shikonin production have been made by selection of the most active variant cell lines with improved stability. Chem. and in line with the authors original hypothesis. the entire supply of 2 in Japan had to be imported from China and Korea. 2 can be produced in upwards of 4 gL of liquid culture by this method. they reasoned that the biosynthesis may be triggered by certain environmental factors associated with the production of cork cells. [94] which activate the biosynthesis. together with a wealth of information concerning the biosynthesis of 2 and related molecules. which is consistent with the location of the natural products in the plant roots. 103] consequently. in spite of great efforts over many years by several research groups worldwide. It was suggested that this effect arose from a triggering of the secondary metabolism by a polysaccharide present in the agar. Since the natural product accumulates only in cork layers in the roots of the plant. 45) 275 . hence its rather brief coverage here. Interestingly.2. glutamine has been shown to [97] inhibit pigment formation. 104±107] were met with varying degrees of success. originally derived from germinating LE seeds. which [26. The two key precursors are 4-hydroxybenzoic acid (PHB. 270 ± 300 3. similar to those identified in agar that activate secondary metabolism. At present we have to rely upon nature.4). Int. It was necessary to conduct the whole tissue culture in the dark for successful naphthoquinone pigment formation. together with their occurrence and the biological activities investigated for each. While pigment formation occurred when callus cultures were grown in LS agar medium. upon transfer of cells initially cultured in LS liquid medium to the specially developed production medium M9. complete inhibition of shikonin production was still observed. The generally accepted biosynthetic route is shown in Scheme 1. In this respect. More significantly. been covered in many reviews. The fruits of these studies are a highly effective commercial production method for 2. a process assisted by the brightly colored [99] nature of the natural product. it was found that light disrupts one of the key enzymes in the biosynthetic route (Section 3. the process failed completely when conducted in a LS liquid medium in the absence of agar. The attempted cultivation of LE plants in Japan failed to meet the demand for this valuable natural product. and related compounds have been isolated from many other Boraginaceae. 1 and 2 may be unique among natural products. In fact. while activated charcoal pro[98] motes it.4ÿ6 dichlorophenoxyacetic acid (10 m). However. a much needed viable synthetic route to these enantiomers has remained elusive until very recently (Section 4. 3. have been isolated from these shikonin-producing cells [95] cultured in the M9 medium. and by modification of the [100] culture medium. Production of Alkannin and Shikonin from Cell Tissue Cultures The first successful production of shikonin (2) and a range of ester derivatives from callus cultures was achieved by [91] Tabata and co-workers in 1974. Angew. and remains as one of the few that have been operated on a commercial scale. The possibility that failure of 2 to accumulate in cells illuminated with white light was a result of decomposition of the photosensitive product was ruled out. This process was the world s first commercial application of plant tissue culture for the production of a secondary metabolite. Gibberelin A3 deactivates ÿ7 shikonin biosynthesis even at concentrations as low as 10 m without affecting cell growth. transfer of these callus tissues after three months to a culture medium that contained the auxin 3-indoleacetic acid (IAA) stimulated pigment production.Alkannin and Shikonin dichroism and chiral stationary-phase HPLC. Formerly. However. This [93] hypothesis has been supported by experimentation. could be initiated on a Linsmaier ± [92] ÿ6 Skoog (LS) medium that contained kinetin (10 m) and 2. [99c. the structures of alkannin (1) and shikonin (2) may look misleadingly simple. for commercial sources of shikonin (2). The great success of this method has prompted investigations into shikonin (2) and alkannin (1) production by culturing cells of many other Boraginaceae species. which contains nitrate ions as the source of nitrogen in place of ammonium ions (which have been shown to inhibit shikonin biosynthesis). Production of Alkannin and Shikonin For organic chemists uninitiated in the chemistry of quinones. but these cultures failed to produce any naphthoquinone pigments. The culturing of callus tissues. Remarkably. Plants must grow for five to seven years before the shikonin concentration in their roots reaches one to two percent. [26] [27] REVIEWS When the cells were illuminated only in the period prior to the triggering of pigment formation with IAA. Furthermore. Further experimentation identified additional controlling factors in the biosynthesis of 1 and 2. 1999. Polysaccharides.1. Biosynthesis of Alkannin and Shikonin The extensive research into the production of 1 and 2 from cell cultures has facilitated the elucidation of several stages in [109] the biosynthesis of these and related natural products. 38. This medium also contains Cu ions. 102] ÿ1 ny. it was found that the ratio of 1:2 varies with the species [26] of plant from which it was extracted.2). the cultures started to produce large [94] 2‡ quantities of shikonin. Ed. Table 1 gives an up-to-date compilation of selected natural products in the alkannin and shikonin class. 3. It was suggested that this may be one of the important endogenous regulators of the [96] biosynthesis. 2. These difficulties of supply motivated research into producing 2 from plant cell cultures.3. the master organic chemist. Improvement of the culture process with LE cells is still the subject of intense research and many publications appear each year in [108] this area. It has. 1. These advances have enabled the efficient commercial production of 1 and 2 by the Mitsui Petrochemical Compa[101. In addition to being found in the plants Alkanna tinctoria and Lithospermum erythrorhizon. 2). 1999. [35. [36] [36] Lappula con- Lappula echinata. 276 Angew. antitumor (Section 5. Arnebia hispidissima. hook[42] [42] eri. sichotenze. [35. Int.4). 38. 44] [33] . OH O [a] V. [104b] [26] cell cultures: Alkanna tinctoria. Echium lycopsis. A. Onosma paniculatum.3). hispidissima. 47.1). 32. Macrotomia cephalotes. 39] [41] cephalotes. [36] [36] E. 28±31. O. [104a] [26] [105g] cell cultures: Alkanna tinctoria. antithrombotic (Section 5. 202] [33] [34] Macrotomia 8 O O a-methylbutyrylalkannin 9 b. Macrotomia cephalotes.2). [26] cell cultures: Echium lycopsis. [41] heterophylla. tinctoria. nobilis.3). R OH O Compound 1 R Name alkannin (also arnebin-4) OH Occurrence and biological properties roots: Alkanna tinctoria. Onosma heterophylla. sanguinea. Litho[42] [39] [41] spermum erythrorhizon. 47. Plagiobotrys arizonicus. biological activity: inhibition of topoisomerase-I (Section 5. 39] [38. 159] [2. Onosma heterophylla.3). [30. 39] Macrotomia cephalotes. Chem. nobilis. biological activity: antimicrobial (Section 5.1). 241a] [44] [33] [171] [1. roots: Alkanna tinctoria. biological activity: inhibition of topoisomerase-I and anticancer (Section 5. A. roots: Arnebia densiflora. Echium lycopsis. gutatta. inhibition of topoisomerase-I (Section 5. Arnebia hispidissima.b-dimethylacrylalkannin or arnebin-1 O O roots: Alkanna tinctoria. antithrombotic (Section 5. paniculata. Echium lycopsis.1). hispidissima [26] cell cultures: Echium lycopsis. Onosma echioides. 7 isovalerylalkannin O O roots: Alkanna tinctoria. [36] [36] Eritrichium incanum. O. biological activity: antimicrobial (Section 5. [34] [35] [36] [37] A. antibacterial (Section 5. Papageorgiou. [104a] [26] [105g] cell cultures: Alkanna tinctoria. Macrotomia cephalotes. euchroma. inhibition of topoisomerase-I (Section 5. Selected members of the alkannin/shikonin family of natural products. [85] Onosma 14 O O unnamed roots: Cynoglossum officinale. [105g] cell cultures: Onosma paniculatum. A. biological activity: antimicrobial (Section 5.2). antimicrobial (Section 5.3). Arnebia euchroma. 33] [171] 5 O O acetylalkannin or arnebin-3 6 O O isobutyrylalkannin cell cultures: Echium lycopsis. A. paniculata. Macrotomia cephalotes. A. 202] 12 O OH O b-hydroxyisovalerylalkannin roots: Arnebia euchroma.3).2). P. Mertensia maritima.2). A. biological activity: antimicrobial (Section 5. antithrombotic (Section 5. Onosma paniculatum. tinctoria. C. roots: Alkanna tinctoria.3). 38. Nicolaou et al. Arnebia euchroma. [46] [159] [42±45] [42] [171] 10 O O teracrylalkannin 11 O O angelylalkannin roots: Alkanna tinctoria.REVIEWS Table 1. biological activity: wound healing (Section 5. [38] [35. antiinflammatory (Section 5. [42. Ed.4). antiinflammatory (Section 5. [203] [42±44] Moltkiopsis ciliata. M. A. [35] 13 O OAc O b-acetoxyisovalerylalkannin roots: Alkanna tinctoria. roots: Alkanna tinctoria.3). K.4). nobilis. biological activity: antimicrobial (Section 5. [40] [26] [38. [105g] [26] O. 270 ± 300 . 2. Arnebia euchroma. Eritrichium sichotenze. 99a] cell cultures: Arnebia euchroma. [107f] [26] [91. guttata. M. zerizaminum. (Continued) Compound 2 OH REVIEWS Name shikonin Occurrence and biological properties roots: Arnebia euchroma. stimulatation of peroxidase. 77. Mertensia maritima. antiinflammatory (Section 5. erythrorhizon. 50] Lithospermum arvense.3). Echium lycopsis. [87. Jatro[56] [36] [36] [53] pha glandulifera. [36] [36] Macrotomia euchroma. echinata. erythrorhizon. O. chemopreventive (Section 5. [107e] [26] [91. inhibition of topoisomerase-II [71] [72] (Section 5. hookeri. sericium. E. [26] [91. [107e] [49. paniculatum. L. Echium vulgare. Eritrichium incanum.3). 49] [42. 50] roots: Arnebia decumbens. L. 61.2). L. euchromum. Echium vulgare. erythrorhizon. Echium lycopsis. [36. Onosma paniculatum. echinata. O. Onosma cau[52. biological activity: antimicrobial (Section 5. 99a] [106] cell cultures: Arnebia euchroma. O. 57±61. visianii. biological effects: inhibition of topoisomerase-I (Section 5. biological effects: antitumor (Section 5. erythrorhizon. incanum. Lappula [36] [36] [76] [57. A. guttata. tibetiana. 77] Lappula consanguinea. [44] [74] [42±44. 84] [53] [36] L. [99a] cell cultures: L. 57. A. A. antipyretic and analgesic (Section 5. roots: Arnebia decumbens.2). echinata. polyphyllum. O. [26] [76] L. 66] [36] echioides. O. 82] [36] Lappula consanguinea. erythrorhizon. O. erythrorhizon. cell cultures: Arnebia euchroma. E.b-dimethylacrylshikonin 22 O O teracrylshikonin roots: Alkanna hirsutissima. O. [36] [78] [42] [42] Mertensia maritima. 57. Echium vulgare. hookeri. Lappula consanguinea. induction and secretion of nerve growth factor. Lithospermum erythro[91. Echium lycopsis. 99a] cell cultures: Arnebia euchroma. O. A. 69] [36] Cynoglossum officinale. 50] [51±53] R 15 O O acetylshikonin 16 O OMe O 1-methoxyacetylshikonin 17 O O propionylshikonin cell cultures: Lithospermum erythrorhizon. Onosma confertum. tauricum. echinata. Mertensia maritima. zerizaminium. 49] [32] [49. 82] guinea.1). Lithospermum arvense. L. 49. L. [73] [19f] protection from uv-radiation. guttata. 65.f] [26] [91. hispidissima. 99a] L. Lithospermum erythrorhizon L. O. antifungal and antibacterial (Section 5. [107e. Lithospermum arvense. Eritrichium [36] [36] [56] [36] incanum. E.3). 99a] [81] [36] [36] [36] [36] [36] 19 O O isovalerylshikonin 20 O O a-methylbutyrylshikonin roots: Cynoglossum officinale. O. paniculatum. euchromum. O. Onosma confertum. [83] [36.[48. Macrotomia [36] euchroma.3). biological activity: antimicrobial (Section 5. [36] [36] [76] [42. officinale. [36] . [107e. Mertensia maritima.Alkannin and Shikonin Table 1. erythrorhi[91. O. [36] [54] [55. livanovii. [36] [36] [36] [36] 21 O O b. Jatropha glandulifera. Macrotomia [63] [52. biological activity: antimicrobial (Section 5. guttata. Other Echium species. erythrorhizon. L. A. erythrorhizon. conferitum. echinata. Lappula consanguinera. ugamensis. [79] 18 O O isobutyrylshikonin roots: Cynoglossum officinale. Chem. Lithospermum erythrorhizon. guttata. cell cultures: Echium lycopsis. sichotenze. Lithospermum erythrorhizon. Echium lycopsis. Macrotomia ugamensis. hookeri. Lithospermum arvense. Lappula consan[36] [36] [76] [36. E.3). echinata. Echium lycopsis. L. erythrorhizon. echioides. O. L. antiamebic (Section 5. Moltkiopsis [85] [78] [84] [84] [53] ciliata. 77] [58±62] L. 1999.f] [26] cell cultures: Arnebia euchroma. 99a] [105] rhizon.2).2). sichotenze. A. M. vulgare. 80] consanguinera. 48. setosum. 49. 42±44. [75] [36. sichotenze. inhibition of microsomal monooxygenase. L. [107e] cell cultures: Arnebia euchroma. 64] [36. 38. wound healing (Section 5. Eritrichium incanum. erythrorhizon. 53. Int. [57. Arnebia euchroma. 84] [49. euchroma. E. Cynoglossum offici[36] [36] Eritrichium [36] [36] [56] nale. 84] [53] 23 O O angelylshikonin roots: Alkanna hirsutissima. L. Angew. euchroma.1). 80. 55] [78] [42] [63] [67] [68] casicum. [49] [49] [86] [86] roots: Arnebia euchroma. L. 88] 24 O OH O b-hydroxyisovalerylshikonin roots: Arnebia euchroma. L. Cynoglossum officinale. 270 ± 300 277 . Mertensia maritima. A. 70] O. 82. zon. O.1). sichotenze. Jatropha glandulifera. tibetiana. L. Echium vulgare. E. Ed. Echium vulgare. roots: Arnebia euchroma. 86] [49] [86] A. inhibition of testosterone-a-reductase. Echium lycopsis. [36] [36] [77. A. L. [63] [63] [69] [52±53. rubrum. [36] [36] [36] [36] Cynoglossum officinale. [26] However. so alleged [26. [85] 27 O OH O unnamed roots: Lithospermum erythrorhizon. 270 ± 300 . [36] [1] [36] [36] [36] [241a] [88] [75] [42±44. Ed. [36. erythrorhizon. biological effects: antidermatophytic and antibacterial (Section 5. [171b] OH 31 O O arnebin-6 roots: Arnebia nobilis. Lappula consanguinea. deoxyshikonin. 25 O OAc O b-acetoxyisovalerylshikonin roots:Macrotomia euchroma. sichotenze. Echium lycopsis. 49. Int. [77] [a] 1) ester hydrolysis may occur during some extraction procedures. 82] [35. Mertensia mar[36] [78] [41] itima. 38. A. guttata. which leads to the isolation of alkannin (1) and shikonin (2) in addition to. the stereochemistry may vary between ester derivatives from the same plant. [107e. [82] OH 35 O O lithospermidin-A roots: Lithospermum erythrorhizon. Eritrichium incanum. 2) in many cases. [61] [36] 34 anhydroalkannin roots: Lithospermum erythrorhizon. Lithospermum erythrorhizon. 278 Angew. [36] Mertensia maritima. Papageorgiou. [89] 26 O O Ph benzoylshikonin roots: Moltkiopsis ciliata. A. [171b] 32 deoxyalkannin. 172] [27] alkannin derivatives may be shikonin derivatives. [77] OH 36 O O lithospermidin-B roots: Lithospermum erythrorhizon. 49] 33 alkannan M. Macrotomia cephalotes. Arnebia decumbens. euchroma.REVIEWS Table 1. while Onosma confertum contains mainly shikonin derivatives. roots: Alkanna tinctoria. O. C. K. Macrotomia euchroma. Eritrichium inca[36] [36] 36] [36] num. A. P. 42. euchroma. [171b] OAc 29 O O acetylarnebin-2 roots: Onosma heterophylla. sichotenze. and even between plants from different locations. paniculatum. heterophylla. their ester derivatives. A. [41] 30 OH arnebin-5 roots: Arnebia nobilis. Echium vulgare. nobilis. 77. E. Nicolaou et al. 166f. echinata. M. euchroma. Cynoglossum officinale. or rather than. hispi[33] [42. Recent circular dichroism (CD) and chiral HPLC experiments have shown that Arnebia euchroma contains mainly alkannin derivatives. A. 99a] [105g] cell cultures: Arnebia euchroma. L. (Continued) Compound R Name Occurrence and biological properties V.2). E. or even racemates.f] [26] [91. antitumor (Section 5. no attempt was made to assign the absolute stereochemistry of each derivative. or arnebin-7 roots: Alkanna tinctoria. hirsutissima. vulgare. Chem. 39] [82] Lithospermum erythrorhizon. 1999.3). O. 50] [90] [36] [36] dissima. Lappula consanguinea. Onosma confertum. [61] OH 28 O O arnebin-2 roots: Arnebia nobilis. L. [36] [36] [36] E. L. [26] Differences may be noted between extracts from plants and those from cell cultures. Cynoglossum officinale. or vice versa. echinata. This question has implications beyond the biosynthesis of shikonin since PHB (45) is a biosynthetic intermediate for many metabolites. cinnamic acid (39). is converted into cinnamic acid (39) by the enzyme phenylalanine ammonia lyase (PAL). 1999. Ed. m-geranylPHB (55). In general. There is some dispute as to whether the biosynthetic conversion of coumaric acid (40) Angew. such as the ubiquinones [113] [114] (62) and vitamin E (63) (Figure 8). Furthermore. and mevalonic acid (50) are all intermediates in the biosynthetic route. far more is known about the early stages of the biosynthetic route to 2 than the later ones. m-geranylPHB (55). A large volume of evidence has been gathered in favor of this biosynthetic route. 54). Feeding experiments have been used to show that shikimic acid (37). while PHB (45) is formed from phenylpropanoid metabolites. GPP (54) is derived from the well-defined isoprenoid pathway that starts from acetyl-CoA (49). and geranyl pyrophosphate (GPP. l-Phenylalanine (38). 270 ± 300 [110] into 45 occurs by the b-oxidative pathway or the retro[112] aldol pathway. Int. PHB (45). PHB (45). and proceeds via mevalonic acid (50). Biosynthesis of shikonin (2). m-geranylhydroquinone (56).Alkannin and Shikonin REVIEWS Scheme 1. with the transformation to coumaric acid (40) then being mediated by cinnamate 4-hydroxylase (C4H). Chem. and m-geranylhydroquinone (56) have been isolated from shikonin-free cell cultures of LE grown in LS liquid medium (in which shikonin biosynthesis is 279 [115] [111] . These components are unified in a reaction mediated by the enzyme PHB geranyl-transferase to yield m-geranylPHB (55). 38. l-phenylalanine (38). originally de-rived from shikimic acid (37). which induces shikonin biosynthesis. Once transferred to the production medium M9. Chemistry of Naphthazarin Naphthazarin (commonly considered as 5. It occurs naturally in the wood [130] bark of Lomatia obliqua and in walnut husks of Juglans [131] mandschurica maxim var. 64b 64c Angew. [128] It has been localized to the Me HO Me Me O 2 63 : vitamin E Figure 8. It can be prepared directly either by heating 1. 280 [125] [115b] cytosol.REVIEWS OH MeO MeO CO2H V. shikonofurans (57). 270 ± 300 . from which it was suggested that its conversion into m-geranylhydroquinone (56) is repressed by ammonium [118] ions. in organic synthesis has [124] been demonstrated.5-dinitronaphthalene [132] with sulfur and fuming sulfuric acid. [126] Me O 62 : ubiquinones (coenzyme Q) PHB glucosyl- 45 : PHB transferase. and the utility of a prenyltransferase. it appears that PHBOG (48) is stored in the vacuoles prior to triggering shikonin [128] biosynthesis. which catalyses the conversion of PHB (45) into PHBOG (48). It has been suggested that all steps subsequent to. Yields for both processes are generally low. naphthazarin (64). Ed. the amount of PHBOG (48) decreased rapidly. GPP synthase. both from plants and cell cultures.8-dihydroxy-1. or by double Friedel ± [133] Crafts acylation of either hydroquinone. which thus implicated PHB (45) or [116] PHBOG (48) as intermediates. inhibited). Papageorgiou. most notably from the laboratories of Mamoru Tabata at Kyoto University. PHB (45) actually accumulates in the vacuoles [116] of shikonin-free cells as its b-d-glucoside PHBOG (48). Structures of naphthazarin (64). Int. 1999. P. Chemistry of Alkannin and Shikonin Before discussing the chemistry of alkannin and shikonin. take place in a special vesicle or biosynthetic site.4-dimethox[134] ybenzene (65). PHB geranyltransferase is the enzyme that is repressed upon [94c] exposure of LE cell cultures to white light. 38. O H n PHB geranyltransferase has been localized to a microsomal fraction of LE cells that contain specific vesicle membranes derived from the endoplasmic reticulum. the cyclic arylether arnebinol (60) is derived from an allylic hydroxylation at the trans terminal methyl group of 59 prior to macroetherifica[122] tion. and the enzyme responsible for GPP (54) [129] production. most likely through disruption of PHB geranyltransferase. An indirect. Sieboldianna Makino. formed by E. OH O O H [135] O O H O OH O O O O O H H 64a Figure 9. This provides the only direct evidence for any of the steps that follow the production of mgeranylhydroquinone (56). It has been isolated from cell-free [123] extracts of LE and its properties were investigated. Publications concerning shikonin biosynthesis continue to appear at a steady rate. and including the coupling of PHB (45) and GPP (54). In support of this. Japan. The other steps from 56 to shikonin (2) are speculative only. a cofactor that may be required for one or more of the oxidation processes that lead to shikonin. possibly to protect it from the glucosidase. Nicolaou et al. three-step procedure with dichloromaleic anhydride is usually employed today to produce larger quantities of naphthazarin for laboratory use (Scheme 2. Upon transfer of the cells to the production medium. but also the transport [126] and secretion of the secondary metabolite. Figure 9) has been used historically as a rather expensive purple dye. Presumably. the FMN photodegradation product lumiflavin was shown to inhibit shikonin biosynthesis. 64a. which is speculated to be a defense mechanism since 45 is [117] known to be toxic to the cells. as has the glucosidase responsible for the reverse transformation. C. has also [127] been partially purified and its characteristics assessed. coli overproduction. and products. It has also been suggested that the biosynthesis may be inhibited by light through the photodegradation of flavin mononucleotide (FMN). this store of PHBOG (48) may be utilized for shikonin biosynthesis. It has been shown more recently that deoxyshikonin (32) is in fact a biosynthetic [115c] precursor to shikonin (2). or 1. it will be instructive to consider first the chemical properties of the parent naphthoquinone. which facilitates not only the orderly biosynthetic reactions. 4-Hydroxybenzoic acid (PHB. with maleic anhydride in fused AlCl3 ± NaCl. Chem. 4. which thus halts shikonin production. K.1. Table 2). Evidence in favor of intermediate 61 comes from the isolation of the furan and dihydrofuran [119] [120] echinofuran (58). Indirect evidence in favor of the biosynthetic route has been gained by the isolation of structurally related secondary metabolites from various species of Boraginaceae. Intermediates up to mgeranylPHB (55) have been detected from these shikoninfree cells. 4.4naphthoquinone. Similar enzymes have been identified and characterized in ubiqui[114] none biosynthesis. Thus. [121] dihydroechinofuran (59). 45) as a versatile biosynthetic intermediate. Studies in this area are presumably on-going and we wait with anticipation for more detailed information on the later stages of the biosynthetic route. Several of the enzymes involved in shikonin biosynthesis have been identified and their location within the cell defined. the quinone is extracted into the aqueous phase as its deep ªcornflower Angew. since it has not been possible to split the aromatic and quinonid signals below a coalescence temperature.5.4. Upon treatment of these solutions with strong alkali. NH4I (89%) [152] (94%)[152] The symmetry of naphthazarin (64) has been a matter of [136] some debate. 72 OH AcOH (40%) O OR OR Reduction of naphthazarin (64) may be effected with a number of reagents.3. This feature has allowed the more efficient largescale preparation of naphthazarin (64) by a three-step sequence.4-addition prod[153] ucts. 38.13. which was formerly used in printing. [142] O OH O OH O OR OR 75 73 71 Scheme 2. In fact. Naphthazarin (64) has been described in the crystalline state as deep brown ± red crystals with a green ± golden luster [139] and as deep green crystals with a metallic reflex. this property has allowed the use of naphthazarin (64) as a pH indicator. which exhibits only a single signal at dH ˆ 7. During unsuccessful attempts to synthesize shikonin (2). Scheme 12). [142] Tin(ii) chloride reduction of 2. Chemistry of naphthazarin (64). AcOH or Na2S2O4 67 SnCl2. Friedel ± Crafts acylation of 1. 1999.4-quinoid structure (64a). Reduction of halogenated naphthazarins by this method occurs with concomitant dehalogenation and formation of 68. midway between the signals expected for the aromatic (ca. In fact. presumably due to the stability of the naphthazarin dianion. Me2SO4 . HCl (93%) 66 65 air OH OH 69 OR A O recovered. The adjacent keto and phenolic oxygen atoms make naphthazarins ideal substrates for the formation of metal [136. HCl (70%) O OH O O H OH O OR O Pb(OAc)4 . Zn MTMSTFA. phenyliodonium bis(tri[140] [141] Highly reactive diquinone (124) has been 68 74 + H OH O 64 O BF ·OEt 3 2 70 B C used for the synthesis of cage compounds. [140] fluoroacetate). alkaline solutions of 69 are rapidly oxidized to the naphthazarin [135] dianion upon exposure to atmospheric oxygen. Ed. Pd-C or OH O OH OH blueº dianion. Reduction of 64 with tin(ii) chloride in hydrochloric acid affords the more air-stable crystalline diketo tautomer of leuconaphthazarin [142±143] (68). followed by air oxidation of an alkaline solution of leuconaphthazarin (68). acidification of the Cl Cl OH O AlCl 3-NaCl (97%) OMe [139] Upon immediate blue solution.5. as exemplified [145] by the synthesis of cycloshikonin (73) (Scheme 2). air (93%) SnCl2. DMF Ac TMS TBS Ac2O. Figure 9) than [138] the more commonly depicted 1. DMF 2) NaH. R Me A MeOTs. NaOAc MTMSTFA [151a] (100%) MTBSTFA [133] (72%)[150] Ac2O. an efficient procedure 281 . dH ˆ 6. Complex mixtures generally result. Reaction conditions for pathways A ± C in Scheme 2. all give rise to the highly air-sensitive [139] 1. Furthermore. dH ˆ 7. the interconversion of naphthazarin structures may be more accurately described as a tunneling process. Satisfactory yields of alkylated products are obtained upon reaction with oxonium ions derived from cyclic enolethers. which leads to a structure that is essentially centrosymmetric as 1 evidenced from its H NMR spectrum. K2CO3 (44%)[147±148] B Na2S2O4 . which included alkene derivatives such as 75 [1] (Scheme 2). Int. It is readily soluble in organic solvents such as benzene and dichloromethane to form blood-red solutions. Chem. These have been studied in some detail with regards to a number of specific properties such as magnetic [136.4-naphthoqui[132] none). while prolonged exposure to atmospheric oxygen prior to acidification causes oxidation of the aromatic nucleus to give naphthopurpurin (2.5-quinoid structures (64b. c.3.8-trihydroxy-1. naphthazarin can be Cl Zn. and in a synthesis of cycloshikonin (Section 4. Brockmann demonstrated that condensation reactions of aldehydes with 64 under acidic conditions provided complex mixtures of products. affords naphthazarin (64) in very good [135] overall yield. sodium dithionite. NaOH MeI (44%) [149] C Na2S2O4 . NaOAc.3-dichloronaphthazarin (67). Methods for the bis-protection.4-dimethoxybenzene (66) and dichloromaleic anhydride (65) proceeds. See also Section 5. One of the only useful compounds in this respect is the bisulfite addition compound Alizarin Black S. and reductive tetra-protection of naphthazarin (64) are summarized in Scheme 2. NaOAc Zn[133] ± ± (100%)[151b] MTBSTFA. The scope of these reactions is probably limited by the tendency of 64 to bind Lewis acids. or zinc. including both 1. 146] exchange interactions between paramagnetic ions.Alkannin and Shikonin REVIEWS OMe O + O O Cl H2. In particular. in very high yield. NaOH MeI (48%) [149] or 1) H2 Pd-C.8-tetrahydroxynaphthalene (69).2. in contrast [144] to the reaction with maleic anhydride. Naphthazarin (64) reacts with only a limited range of electrophiles.and 1. appear to be more consistent with a 1. Etherification of the phenolic groups is rather difficult.95). Table 2. Controlled nucleophilic addition reactions with naphthazarin (64) have not yet been achieved.25) and quinoid protons [137] (ca. Use of catalytic hydrogenation. the spectroscopic properties of naphthazarin (64).2. or more effectively. NH4I [133] Ac2O. The most important aspect from the synthetic viewpoint is that rapid ªtautomerizationº occurs. including its intense color in solution. 270 ± 300 Recently. 146] chelates. none Naphthazarin (64) may be oxidized to the naphthodiqui(124) (see Scheme 12) upon treatment with [133] OH O OH NaOH. The acid/base color chemistry of 1 and 2 (Figure 10) resembles that of naphthazarin (64). The quinone may be selectively reduced [139] with Zn or Na2S2O4 to provide a highly air-sensitive tetrahydroxynaphthalene deriv-ative. and their derivatives. and even a photoacoustic technique for [178] transdermal ad-sorption measurements. 159] [157] OH H2. 167] 13C NMR. 166c. DMAP OH O 79 NaOH NaOH (RCO)2O or RCOCl. All other protection-deprotection chemistry resembles that of naphthazarin (64). 166f. barely visible). and its derivatives were undertaken by Shukla and co-workers. 155. gel permeation. 167. 2. py or RCOOH.[27. 81 Compounds 1 and 2 have also been shown to undergo [1] racemization under strongly acidic conditions. Diels ± Alder reactions with 64 have found extensive use in the construction of anthracyclinone antibiotics. Quinones 1 and 2 are also susceptible to photooxidation by exposure to air and light. The aliphatic hydroxyl group in shikonin may be selectively acylated in the presence of the phenolic hydroxyl [160] groups by standard esterification methods. Cycloaddition reactions are generally the most useful and V. 50. 44. py ROCO ROCO O [153] successful reactions of naphthazarin (64). [175] [176] raphy. who performed a double Mannich-type addition to afford 83. while use of catalytic PtO 2 allows for selective alkene reduction to dihydroshikonin 82 [156a] (Scheme 4). 38. 170. 164] Chromatographic techniques used include TLC. 40. and light. 1999. Papageorgiou. occurs readily by the action of acids. [38. (Scheme 5). 2. [87. and a reaction with diazomethane. 173] and indirect atomic absorption. 171a] mass circular dichroism. 44. DCC. and HPLC. Angew. P. 172. The only examples of ring functionalization of 1. 156] occurs readily. differential pulse [177] voltammetry.2. to provide 85 282 Figure 10. which has been [162] trapped as the pentaacetyl derivative 80 (Scheme 4). OH O O2 OH O –H2O OH O OH OOH OH OOH OH OH OOH O 2 76 O [157] 77 Scheme 3. PtO2 O OH OAc OAc OAc 2 EtOAc (69%) OH O 80 H 2. 53.[33.4-addition has been reported via the leuco form of the respective bromo derivative. 52. 270 ± 300 [165] [159] . Solutions of alkannin (1) in CH2Cl2 (red) and in 1n aqueous NaOH (cornflower blue) as contrasted with a solution of pentaacetate derivative 80 in CH2Cl2 (colorless. 43. 50. The major tautomeric form of the quinone nucleus substituted by an electron-donating group is that depicted in [137] 1 and 2. and their derivatives far more sensitive than 64. have been utilized for qualitative and quantitative determinations of 1. 166] The properties studied include UV/Vis. bases. 77. C. heat. 42. Ac2O AcONa OAc 4. cyclization of the side chain to the tetrahydrofuran isomer 73 (cyclo-shikonin) [1. Chem. In particular. OH (RCO)2O or O OCOR OOCOR 78 RCOCl. Nicolaou et al. Hydrogenation of 2 with Pd catalysis causes reduction of both the quinone and the alkene groups to afford 81. Polymerization of 1 and 2 [158. 89. 2. A large number of physical techniques have been applied for the analysis and study of 1. 2. 50. K. Reactivity of Alkannin and Shikonin In general. 52. This aspect [154] has been dealt with extensively elsewhere and will not be discussed here. and their derivatives. Ed. acylshikonin derivatives 78 can be produced through the selective hydrol-ysis of triacylated shikonin derivatives 79 [161] (Scheme 4). 77. Photooxidation of shikonin (2). Selected reactions of shikonin (2). In acidic conditions (either Brùnsted or Lewis). py OAc Zn. [174] Polarog[26a. Pd-C OH O AcOH (30%) OH O OH OH O OH 82 Scheme 4. OH O NaOH (RCO)2O or RCOCl. An interesting rearrangement to the cyclobutane derivative 84 has been observed during the isolation [87] of certain alkannin esters. 53. the chemistry of alkannin (1) and shikonin (2) has many similarities to the chemistry of naphthazarin (64). 77. 168] [44.h. 167] 1 [33. spectrometry. which proceeds by a sequence of cycloaddition followed by N-methylation. The major photolytic product 77 is thought to arise through the mechanism indicated in Scheme 3. Int. 49. 2. The hydroxyl-containing side-chain introduces some interest-ing properties that render 1. 166c. Conversely. H NMR. 170] IR. voltammetry.[169a.REVIEWS for formal 1. densi- [163] tometry. Br2. since polymeric materials are formed. such as the strong affinity of naphthazarins for both silica and alumina. a two-step procedure was developed (Scheme 7. is the development of an effective protecting system for the naphthoquinone core. K2CO3. EtOH (95%) 2. POCl3. the selectivity being dependent upon the nature of the substituent R. CH2Cl2 (88%) 88 Scheme 5. which makes chromatographic purification difficult. CuI. which avoids the use of this costly material. While 88 can be prepared directly from naphthazarin (64) by reductive methylation (Scheme 2). the cheap starting material 1. CCl4 (58%) OH OMe Br BrOMe 86 MeONa.5-dihydroxynaphthalene (86. [87. MeOH.5.8-tetramethoxynaphthalene (88. The sensitivity of the natural product to a variety of reagents and conditions besides acids. successfully converts tetramethoxynaphthalene (88) into naphthazarin (64) in 68% yield after air oxidation of the originally produced tetrahydroxynaphthalene. Scheme 6) was O-methylated before bromination. This meth-od is. An alternative approach to 89 has recently been described by the Coula-douros group (Scheme 6). Subsequent O-methylation and DIBAL reduction provided 89. Angew. with reducing agents and nucleophiles) severely limits the options available for the final deprotection steps of the synthesis. however. 1999. Methods for the release of naphthazarins from tetramethoxynaphthalene derivatives have been thoroughly investigated by Tanoue and Terada. As mentioned above. Int. 163] OMe OMe OH LDA. DIBAL. an ideal synthesis of 1 and 2 would be one in which protection of the naphthazarin system was not required. 270 ± 300 283 . Me2SO4. Perhaps the most challenging aspect of this synthetic problem. Table 3). one of the standard reagents for aromatic ether cleavage. unsuitable for the preparation of 2-substituted derivatives of 64. There are further practical difficulties. 90 [149.Alkannin and Shikonin REVIEWS OH 1. 179] lation reaction of the phthalide anion derived from 90 with [180] acrylonitrile to afford 91. This intermediate is common to most of the syntheses of 1 and 2 reported thus far. NaOH. light. CHCl3 (99%) H OMe OMe O OMe OMe OMe OMe 89 1. this has not been realized. a more economical approach. Most of the successful syntheses of 1 and 2 have been achieved through the use of 1. [179] [149] OH O O OH 95 96 [181] This method employs an annu- Scheme 7. They found that boron tribromide. HNO3 Method 1 OH O R O 94 AlCl 3 Method 2 OH R subsequent copper(i)-assisted methoxylation afforded 88. however.1. This method is notable for the high overall yield obtained and the possibility that hydroquinone 91 may be used to produce a naphthalene intermediate with orthogonal protection of the two aromatic rings. Total Syntheses of Alkannin and Shikonin 4. Initial oxidation with ceric ammonium nitrate (CAN) affords two regioisomeric products. light. For [149] [181] OMe OMe R CAN OMe OMe OMe O R + OMe O O OMe R O OMe 92 93 AgO. Me2SO4 (71%. Instead. This may be of assistance for improving the efficiency of its subsequent demasking to a naphthazarin (see below). So far. and oxygen. Ed. THF CN SO2Ph O O + CN 4.8-Tetramethoxynaphthalene (88) by the Vilsmeir method produces 89 (Scheme 6) in almost quantitative yield. Scheme 6) and its derivatives. Introduction There are several problems that must be overcome in order to accomplish an efficient synthesis of alkannin (1) and/or shikonin (2). has been devised by Terada and co-workers. 38. Naturally.3. Chemistry of alkannin ester 79.4. Thus. and [149] [149] OMe OH OMe 91 Scheme 6. the Friedel ± Crafts acylation of hydroquinones with maleic anhydride derivatives (Section 4. DMF (84%) 87 OMe OMe DMF. 2 steps) 2.5. Clearly. as a protected form of the naphthazarin core. Two syntheses of key intermediate 89.4. the natural product is sensitive to Brùnsted and Lewis acids.3. and oxygen (for example. Formylation of 1. Chem. Deprotection of tetramethoxynaphthalene derivatives.1) is not applicable here. the most general approach to substituted naphthazarins. although the AgO/ HNO3 demethylation. to the aldehyde product 112. and a double Grignard addition to form the key intermediates 109. 28%). as described previously. THF (86%) OMe O CAN (70%) OH OMe O 98 OMe OMe OH OMe OMe OH OH electron-donating groups such as hydroxyalkyl (Table 3. HNO3 (27%) 99 OH O OAc O OH AcOH py (88%) OH OAc O OAc OH O OH 101 102 SOCl2. TsOH. [183] [182] opened by treatment with the organolithium reagent 105 to afford 106. until recently. and not to 1/2. a cyclopropanation. entries b ± g). and 96 the major isomer when [137] R is electron withdrawing. Torii and co-workers have reported a synthesis of 1/2 also starting from formyl derivative 89 (Scheme 10). More recently. although generally in low yield. py (34%) OH O 1) NaOH 2) CH3CO2H (37%) OAc O OH O OH OAc O OAc 1/2 workers. Treatment of 101 with acetic anhydride in pyridine selectively produced triacetate 102. tetramethoxynaphthalene derivatives were. favor isomer 94 as the major product. as ever. regioselectivity is usually modest. This intermediate was common to their alternative approach. 1999. the low yield being a consequence. Ketal hydrolysis and subsequent addition of methylmagnesium iodide afforded diol 99. K. The initial step is a McMurry-type coupling between 89 and the unsaturated aldehyde 113 mediated by a low-valent vanadium species. Closure of diols 114 to the cyclic carbonates 115 (presumably a mixture of diaster-eoisomers) set the stage for a palladium-catalyzed allylic reduction.2. 38. MeMgI. the initial CAN oxidation proceeded with reasonably high selectivity in favor of the desired isomer 100. which employed an anodic oxidation. 284 [184] The epoxide was . and a rather elaborate reaction sequence was required before acetate 116 could be obtained in a pure form. and required the action of AlCl3 . OO BrMg OMe OMe OO OMe OMe 97 H THF (96%) OMe OMe O OH 89 1. while electron-withdrawing groups such as acetyl and formyl.[149. The synthesis was completed by alkaline hydrol-ysis of the acetate groups to yield shikalkin (1/2). The first synthesis of shikalkin (1/2) by Terada and co- 4. Unfortunately. the only protected form of naphthazarins suitable for the synthesis of 1 and 2. In the former case.3. into 1/2 was effected by the usual two-step sequence. Nicolaou et al. Yields for the reactions shown in Scheme 7. This allowed subsequent elimination with thionyl chloride and pyridine to afford 103. CH3COCH3 (87%) 2. the regioselectivity of the initial CAN oxidation was negligible. In this case. 100 AgO. rather curiously. 270 ± 300 [185] [186] [187] Researchers from Russia have reported two approaches to 1/2 from the formyl derivative 89 (Scheme 9). Angew. Methoxyquinones of form 93 can be further demethylated by treatment with AgO/ HNO3 .REVIEWS Table 3. in part. These latter conditions would be unsuitable in a synthesis of 1 and 2. Syntheses of Shikalkin (1/2) After Terada and co-workers had developed a method for the conversion of tetramethoxynaphthalenes into naphtha-zarins they were able to complete the first synthesis of shikalkin 1/2 in 1983 (Scheme 8). of the production of significant amounts of the terminal alkene regioisomer. R a b c d e f g h i H CH2OH CH(OH)CH3 CH(OH)(CH2)3CH3 CH(OH)(CH2)3CH(CH3)2 CH(OH)(CH2)3C(CH3)2OH CH(OH)(CH2)3COCH3 COCH3 CHO CAN oxidation 93 94 [%] [%] 70 75 61 42 52 70 33 3 0 ± 13 13 16 17 15 33 77 85 Demethylation method yield [%] ± 1 1 1 1 1 1 2 2 ± 53 52 22 22 28 27 71 43 OMe OMe V. Papageorgiou. Int. C. which utilized a Wittig olefination. The regioisomeric quinones 94 failed to react under these conditions. and once again the second demethylation reaction was low-yielding. Conversion of 106. P. 182] 103 Scheme 8. Conversion of aldehyde 89 into epoxide 104 was achieved in good yield with standard sulfur ylid methodology. Attempts to convert the undesired regioisomer 111 into 1/2 by AgO/HNO3 treatment led. this reaction afforded a significant amount of a regioisomeric alkene (ca. Ed. Chem. obtained from both routes. At this stage. the substituted ring is preferentially oxidized. and in moderate yield. with 95 being favored when R is electron donating. The transformation into 106 was accomplished by an intriguing acid-catalyzed rearrangement. Tautomerization between 95 and 96 occurs readily. This research group also used this method for the preparation of isomers of 1. these researchers opted to release the naphthoquinone from its protected form by the twostep CAN/AgO/HNO3 sequence. Addition of the Grignard reagent 97 to the formyl derivative 89 produced intermediate 98 in good yield. In this case. gave only a poor yield of the naphthoquinone 101. Despite being far from ideal. These workers developed an alternative method for the deprotection of the tetramethoxy-naphthalene core. Pd(OAc)2. DMAP OMe OMe OAc Li 105 (57%) OMe OMe CO2Et 116 3. In fact. whereupon selective oxidation of the methoxy-lated ring could be achieved by a second anodic oxidation. Ac2O. 3 steps) electrooxidation 115 108 MeMgI (66%) OMe OMe HClO4 (58%) OMe OMe OMe O O OMe + OMe OMe OH OMe OMe OH 106 CAN OMe O O 109 OMe O M e O O Ac O 117 41% OMe OAc 118 31% Zn. the anodic oxidation method is complementary to the previously [181] described two-step demethylation procedure. Further syntheses of shikalkin (1/2) from the formyl derivative 89. [188] Thus. the desired isomer 118 (which would be the unwanted one for a AgO/HNO3 demethylation) was the minor product of this reaction. PPh3. Alkaline hydrolysis of 103 completed the synthesis. although its transformation into acetylshikonin (15). and then to 1/2. The approach of Torii and co-workers for a total synthesis of shikalkin (1/2). Et3N OMe OMe O O (67%.Alkannin and Shikonin OMe OMe OMe OMe REVIEWS OMe OMe VCl3. 4. toluene (82%) OMe OMe O OMe OMe 104 107 N2CHCO2Et (71%) OMe OMe OMe OMe 1. THF HCO2H. py. Et3N OMe OMe 2. THF (73%) + _ OMe OMe O H Me2S CH2 (71%) _ Ph3P CH 2 (65%) + H O OMe OMe O H OMe OMe OH 89 OMe OMe OMe OMe 89 113 114 CDI. In order to achieve the second demethylation.3. a reductive acetylation to provide 119 was required. should be possible by AgO/HNO3 treatment. they were unable to improve the regioselectivity of the initial oxidative demethylation. Thus. Approaches via Cycloshikonin Terada and co-workers have also shown that cycloshikonin (73) may be converted into shikalkin (1/2) itself (Scheme . THF HCO2H. Ac2O. PPh3. 11). however.[184. a synthesis of 73 constitutes a formal Despite significant experimentation. These researchers apparently made no attempt to demethylate their unwanted regioisomer 117. Et3N (81%) + OMe O OAc OMe O OH OMe OH 110 AgO/HNO3 47% 111 (19%) O OMe 27% AgO/HNO3 (39%) OH O O A c OMe OAc 119 electrooxidation (43%) OH O OAc O 1) NaOH OH O OH O OMe O 1/2 112 OH O OH OAc 2) CH3CO2H (72%) Scheme 9. Zn. 185] O OAc 1/2 : shikalkin [186] 103 Scheme 10. Pd(OAc)2.3. Angew. after hydrogenolysis. Conversion into cycloshikonin (73) was then achieved in a rather inefficient 12-step linear sequence. 38. Ed.3] sigmatropic rearrangements to produce. this route may be of some utility for the synthesis of analogues. leuconaphthazarin (129) in 80% overall yield. The key step is the opening of the tetrahydrofuran ring to 123 with p-toluenesulfonic acid in the presence of acetic anhydride. in which the key steps are addition of the Grignard reagent [188] 120. Although impractical for the synthesis of shikonin (1) and cycloshikonin (73). followed by acid-catalyzed ring closure. Chem. although rather long. The initial approach to cyclo-shikonin (73) developed by this group (Scheme 11). 270 ± 300 An interesting. which was an intermediate in their [149. starting either from 73 itself or more efficiently from the diacetate 122. Int. The g-addition of silyl keteneacetal 125 to the highly reactive naphthodiquinone 124 afforded 126. 182] earlier synthesis of shikalkin. has been super-seded by the report of the direct synthesis of 73 from [145] naphthazarin (64) (Scheme 2). 285 [141] . route to cycloshikonin (73) has recently been reported by Aso and Kanematsu (Scheme 12). which underwent two consecutive reductive [3. Alkaline hydrolysis of 123 provided 101.synthesis of 1/2. 1999. The 286 Scheme 14.2. py (96%) O MOMO OMe OAc O OH O O OH O MOMO OOTBS MOMO OH OTBS OAc O 134 + H . Unfortunately.and (S)-(2-hydroxy[193] 1. [189] OH OOH OAc OOAc 101 3 steps (11% combined yield) 123 1/2 : shikalkin Scheme 11. by using their asymmetric acetate enolate equivalents derived from (R). Thermolysis with enantiomerically pure alkyne 132 provided the protected naphthazarin 133. PhH (51%) 2. [192] Angew. that the published synthesis of alkyne 132 involves a fivestep linear sequence from commercially available starting materials and uses a Sharpless asymmetric epoxidation to [191] secure the required stereochemistry. Chem. A stereoselective synthesis of shikonin (2) by the Dötz annulation reaction. CAN (70%) 3. K. Ed. [189] and involves an elegant application of the Dötz [190] annulation reaction (Scheme 13). the authors failed to quote yields for the synthesis.3. which was then converted into shikonin (2) by acid hydrolysis. THF 2 : shikonin 133 122 Ac2O. Thus. [Cr(CO)6] + Me3O BF4 – OMe Cr(CO)5 120 H OMe OMe O THF (95%) OMe OMe OH MOMO MOMO 89 1. H2O 126 CO2tBu phenolic group in 133 allowed selective oxidation of the more functionalized ring of the naphthalene to produce quinone 134. Int. after ortho lithiation of 130. by the standard method. however. TsOH (26%) OAc O OAc Scheme 13. Asymmetric Approaches The first asymmetric synthesis of 2 was disclosed in a patent. P. 38. It should be noted. 1999. While these reagents generally give very high stereocontrol. The asymmetric approach to 1 and 2 taken by Braun and Bauer was to perform an asymmetric aldol addition onto the [192] formyl derivative 89. Braun s synthesis of shikonin (2) by an asymmetric aldol reaction. 4. AgO. [188] O O OTMS OtBu O OH 125 CH2Cl2 O O O O 124 Na2S2O4 Et2O.4. MOM-protected hydroquinone 130 was converted into the chromium carbene intermediate 131. Terada s approach to shikalkin (1/2) via cycloshikonin (73). the selectivity OH O CO2tBu O OH OH O O O 128 H2. H2O. TsOH. C. 270 ± 300 .REVIEWS OMe OMe BrMg OMe OMe MOMO V. Nicolaou et al. TsOH (47%) OH O OH NaOH (78%) 73 Ac2O. Papageorgiou. MOMO nBuLi. so it is not possible to comment on the efficiency of their route. Pd-C (80% from 120) OH O CO2tBu 12 steps 127 OH O CO tBu 2 O OH O OH O 129 73 [141] Scheme 12. Kanematsu s synthesis of cycloshikonin (73). HNO3 (30%) 130 TBSO 131 121 132 MOMO O DDQ Ac2O.2-triphenylethyl) acetate (Scheme 14). by an asymmetric allylation reaction with allyldiisopinocampheylborane [194] (Scheme 15). The enantiomeric excess (ee) of shikonin (2) produced by this route was determined to be approximately 65% by CD measurements. Nicolaou s stereocontrolled total synthesis of alkannin (1) OMe OMe OMe OMe Ph3 P=C(CH 3)2 (74%) OMe OMe OTBS and shikonin (2). [195] the two naphthazarin isomers D and E. but spontaneous elimination of formaldehyde should afford Angew. The Couladouros group has recently achieved a synthesis [179] of alkannin intermediate 138. and 3) to devise a novel protecting system for the naphthazarin core. Improved approach to 138 by asymmetric allylation A B –CH2O OH O C –CH2O O OH Recently. K3[Fe(CN)6] 3. Construction of the side-chain was completed after hydroxyl protection by a high-yielding three-step sequence. REVIEWS OMe OMe Ipc2Ballyl (78%) H OMe OMe O OMe OMe OMe OMe OH 89 1. and lead to the poor selectivity. the Nicolaou group reported highly efficient total syntheses of both alkannin (1) and shikonin (2) (Scheme [195] 16). It was reasoned that ketone 143 should be an ideal intermediate to access both enantiomeric series.3-dichloronaphthazarin (67) as the readily available starting material (Sec-tion 4. alcohol 139 was produced in 82% ee (based on an 80% ee of the chiral reagent). and preferential formation of naphthazarin isomer D (Nicolaou and Hepworth). [179] 140 R OO oxidation + R HOOO OOOH R Scheme 15. DMF (97%) 2. These workers considered it prudent to protect the aliphatic hydroxyl group in an attempt to improve the efficiency of the final deprotection steps. Chem. and subsequent bromination with NBS. Halogen ± metal exchange with tert-butyllithium followed by the addition of Weinreb amide 142 afforded ketone 143. 270 ± 300 [196] [144] tautomerization R OH O O OH R D E Figure 11. NaIO4 (98%. but the electron-rich nature of aldehyde 89 may disfavor this scenario. bromine-substituted methyleneacetal 141 b (Scheme 16) was prepared from 2. [197] [142] [137] O-alkylation with bro- mochloromethane. TBSCl. This strategy was designed with three considerations in mind: 1) to use a commercially viable source of the naphthazarin core. 2 steps) 139 Scheme 16. In this case. no improvement in yield for the demethylation sequence was observed. which could be cleaved at the end in one step. 38.3-dichloronaphthazarin (67) by tin(ii) chloride reduction. An early transition state is usually invoked to explain the asymmetric induction obtained with these reagents. Separate reduction of 143 with each enantiomer of DIP-Cl [198] 287 . Thus. [195] O O O HO O O O O OH OMe OMe OTBS 138 (Couladouros and co-workers).Alkannin and Shikonin in this case was rather modest (ca. since arylalkyl ketones of this type are generally considered to be excellent substrates for many asymmetric reducing agents. 1999. The plans came to fruition in a concise and highly efficient synthesis. 2) to produce a late-stage intermediate from which it would be possible to form both alkannin (1) and shikonin (2) in high ee. 5:1 after purification). Furthermore. Ed. it was anticipated that bis(methyleneacetal) derivatives such as A (Figure 11) would provide a solution to the deprotection problem. K2[OsO2(OH)4]. The first aim was achieved by using 2. Thus. These isomers should rapidly tautomerize in favor of the desired form D by virtue of the stabilizing effect of the electron-donating alkyl group. Conversion of the initial aldol adduct 136 into 138 was achieved by standard methods.1). However. oxidative treatment may still produce two regioisomeric intermediates B and C. Projected deprotection of bis(methyleneacetal) derivatives. imidazole. Int. and under mild conditions. and acute anal fissures. ªKoushikonº (both of which are LE roots that contain mainly shikonin (2). A power supply that forced a constant potential difference across the cell of 3 V. [203] [159] [2] Several in vivo animal studies have been undertaken in Japan to assess the efficacy of shikonin (2) and its derivatives on various aspects of wound healing. and pigments. MDS-004 Angew. Figure 13). Each increased proliferation of granuloma tissue induced by a subcutaneous cotton pellet implant. Figure 12. being applied topically each day around the edge of the ulcer. C. The results were dramatic (Figure 12). Int. but equally active to the pentaacetyl derivative MDS-004 (80. with no skin inflamma-tion being observed at any stage of the treatment. This preparation was then tested clinically on patients suffering from indolent ulcers of the lower leg. Shikonin. The yields of shikonin and alkannin were high (about 80%. 2. and seemed to be independent of both the side-chain stereochemistry and the aliphatic ester group. Cleavage of the methyleneacetal protecting groups was achieved by anodic oxidation. In 1977 Hayashi reported the results of extensive tests on rats with 2. and within five to six weeks. 1999. [208] [209] decubitus. These extracts were tested by topical application on skin ulcers induced in laboratory animals (for example. and that the active components are 1. This sequence may provide a commercially viable synthetic route to 1 and 2. cats. antitumor. fluorescent compounds. and ªNanshikonº (the root of Macrotomia euchroma Pauls which contains mainly alkannin esters). Each of the major biological effects. Furthermore. free alkannin (1) was [204] not detected during the isolation. and antithrombotic properties will be considered in turn. was observed. These preparations. angelate (11). rats. After three to four weeks of treatment. when applied topically. 5. in contrast to 2 and 15. and Their Derivatives Biological investigations over the last 25 years have shown that many of the medicinal properties claimed for AT and LE in the historical texts do indeed have a sound scientific basis. based on recovered starting alcohol) provided the reaction was taken no further than 50% conversion. References for other biological proper-ties not discussed are given in Table 1. It has also been applied successfully to the treatment of leprous ulcers. acetylshikonin (15). He concluded that these drugs were effective for the treatment of cutaneous injuries. 288 [206] [47. and uv radiation. Wound Healing and Anti-Inflammatory Effects As described in the introduction. Papageorgiou. was this medicinal property confirmed experimentally. isovalerate (7). the initial hexane extract was [201] divided into: waxes. Biological Activity of Alkannin. and its esters). which had resisted all previous attempts at treat-ment. and promoted wound healing. A separate comparison of the antiinflammatory activities of alkannin (1) and shikonin (2) also led to the conclusion that the absolute configuration has little influence on biological activity. however. 38. natural polymers. the oxidation did not require any elaborate electrochemical equipment. and the active components identified [200] by Papageorgiou. The efficacy of this ointment has been further tested for the treatment of burns. including anti-inflammatory effects. P. Furthermore.REVIEWS afforded the enantiomeric alcohols 144 and 145 in high yield and excellent ee. The success rate was 80%. [210] and crude ether extracts of ªshikonº (LE root).1. Ed. there was proliferous growth of granulation tissue. dogs). and the roots extracted. Not until 1976. The patient was a 62 year old woman and the ulcer on her right leg was previously unresponsive to treatment for ten years. After further extraction and chromatographic separation. heat. Ozaki and co-workers compared Japanese ªShikonº. also showed anti-inflammatory effects against acute edema induced by several methods. K. An indolent ulcer before (right) and after (left) four weeks of treatment with HISTOPLASTIN RED. Nicolaou et al. The pigment-contain-ing fraction showed excellent healing effects. An ointment containing the alkannin esters extracted from AT root (HISTOPLASTIN RED and afterwards HELI-DERM) was formulated and patented following these suc-cessful animal trials. Mild antipyretic and analgesic effects were also observed. in which it was shown to be significantly [207] superior to the drugs Betadin and Fucidin. namely: wound healing. Chem. 270 ± 300 Interestingly. Shikonin (2) was shown to be more active than acetylshikonin (15) with respect to acceleration of granuloma formation. AT and LE roots have been used for the treatment of wounds since ancient times. They found that the proliferation of granulation tissue induced by the ether extracts from these roots correlated well with the total naphthoquinone pigment content. and the novel bacetoxyisovalerate (13).b-dimethylacrylate [159] [202] [202] (9). V. complete healing. Chemical analysis of the pigments identified the following esters of alkannin: b. 5. Seto and co-workers studied the effects of shikonin and several derivatives as pure compounds on various aspects of wound healing. antimicrobial. Many samples of AT taken from different locations were [159] obtained. including histamine. serotonin. 205] [211] [212] [162] . or at least considerable reduction in ulcer size. with the other fractions being completely inactive. and low-cost graphite-rod [199] electrodes were used. the Japanese preparation Shiunko. and their derivatives. traumatic ulcers. 220] mice. MDS-004 (80) was shown to have improved pharmacological properties relative to shikonin (2). have been studied and have shown [162. Walker carcinosarcoma 256 (W256). In recent years. Chemopreventive effects have also been demonstrated in vivo. While various types of Anchusa have been used for cancer treat[218] ment. AT appears not to have been used in this regard. and 4-nitroquinoline-1-oxide on the bacterium Salmonella typhimurium TA98 was inhibited in vitro by extracts from LE root. 1999. Sarcoma 180. all exhibited in vitro [219] cytotoxicity against KB cells. Int.2. alkannin (1). it now being second only to cardiovascular disease as a cause of death.b-dimethylacrylate (9). which is a highly complex and delicate balance of [216] degradative and regenerative processes. in Japan. significant advances have been made in understanding the biochemistry of wound healing and tissue repair. although significant cytotoxicity was also noted at the concentrations tested. and b. catalyzed by enzymes such as NADPH-cytochrome P-450 reductase. but was inactive at lower doses. Platinum complexes of naphthazarins. Antitumor Activity In addition to the wound healing properties described above. but with lower nephrotoxicity. 1. none of the derivatives tested showed any healing ability for incised and open wounds. More recently. The chemopreventive properties of alkannin (1) and shikonin (2) derivatives have been examined by several other groups. For example. and its b. Nevertheless. and exhibited a tendency to heal gastric ulcers induced by acetic acid. Interestingly. it has been shown that while alkannin (1) and shikonin (2) both exhibit cytotoxic activity at high ÿ1 ÿ1 concentrations (100 mg mL ± 10 ngmL ). and their derivatives have been investigated as potential drug candidates for various aspects of cancer treatment within the last 25 years. 210. however. 212] similar results to those previously described. which the authors suggested may play a major role in the [215] mechanism of its anti-inflammatory effects. its isobutyrate (6). Structures MDS-004 (80). There are a number of ways in which [228] quinones can disrupt cellular processes. 270 ± 300 . dating back as far as the 12th century AD. [156] Similar results were obtained by Sankawa et al. 38. Copper complexes of shikonin (2) have been patented as DNA cleaving [227] agents. The increasing problem of cancer in the developed world. Shikonin (2) and a range of simple derivatives completely inhibited tumor growth in mice at a dose of 5 ± ÿ1 ÿ1 10 mgkg day . rather than a brightly colored pigment. 2. their precise mode of action remains undetermined. benzo[a]pyrene. in these model studies. This radical is thought to initiate a redox cycle since it can readily autooxidize in the presence of dioxygen to regenerate the quinone and form the 289 5. Of over 1500 quinones screened in one campaign in 1974. and their derivatives produce their cytotoxic activity. 2. quinones can undergo a one-electron reduction. and in vitro cytotoxicity against KB cells in culture. acetylshikonin 80 (15). [221] OH O OH OH O OAc OAc OAc OAc 2 Figure 13. 5. The anti-inflammatory effects of extracts from Arnebia [213] euchroma (alkannin esters). Among the active quinones tested. its acetate (5).b-dimethylacrylalkan[214] nin (9) in particular. and [210] in contrast to the study by Hayashi. Chemoprevention is based on the idea that noncarcinogenic. Although the efficacy of alkannin and shikonin derivatives have been demonstrated in vivo. exhibit similar antitumor activities to [226] cisplatin. shikonin (2) has been shown to inhibit the biosynthesis of leukotriene B4 and 5-hydroxyeicosatetranoic acid. More recently. Adenocarcinoma 755. they displayed immunostimulating activities at extremely low concentrations ÿ1 ÿ1 (10 ngmL ± 10 fgmL ) in vitro on human granulocytes and lymphocytes. MDS-004 (80) has the significant marketing advantage of being a colorless compound. including 1. Thus. and 28 (Table 1) isolated from the roots of Arnebia nobilis were previously shown to be active against W256 in rats and P388 lymphoid leukemia in [163. The synthetic derivatives 83 and 85 (Scheme 5) were shown to have comparable activities. has motivated the extensive growth of cancer research in recent years. is missing from several current [18] pharmacopoeias of Chinese medicine. Alkannin derivatives 1. Again. and may be less common than the applications already described. They found that 2 caused acute toxicity at higher doses (see Section 5. Ed. MDS-004 was also orally active against carrageenaninduced hind paw edema. however.5). more than 10% exhibited significant in vivo (mice or rats) activity against at least one of the following cancer cells: L1210 leukemia. Chem.Alkannin and Shikonin OH O OH O OAc OAc REVIEWS the USA have identified the quinone moiety as a pharmaco[219] phore that commonly affords cytotoxic activity. 2. No reports of clinical trials for either 80 or 2 have yet been published. of 15 shikonin (2). 9. and their derivatives. Its use for this purpose. LE root extracts have been used in Chinese traditional [217] medicine for many years as a cancer treatment. to [228] form a semiquinone radical. Mass screening programs of natural products and synthetic compounds by the National Cancer Institute of Angew. synthetic or natural products can inhibit the process of carcinogenesis. in contrast to 2 and 15. which arises from the capacity of these compounds to enter redox cycles. Several research groups have investigated the biological mechanism through which 1. Shikonin (2) has been shown to inhibit the early antigen activation by 12-O-tetradecanoylphorbol-13-acetate (TPA) of the Epstein ± Barr virus in Raji [224] cells. and (80) showed strong inhibition when applied topically against delayed-type allergies (ear edema) induced by oxazolone and dinitrofluorobenzene. with shikonin (2) reducing the incidence of azoxymethane-induced intestinal tumors in [225] rats. The mutagenic effects of picrolonic [222] [223] acid. Lewis lung carcinoma. One of the most commonly proposed mechanisms of quinone cytotoxicity is through ªoxidative stressº. These racemic analogues [149. by a chain mechanism may also cause cellular damage. or the semiquinone.REVIEWS superoxide radical anion. A range of shikonin esters have been [160] tested for their inhibitory effects on topoisomerase-I. In a protection study. It has been proposed that the reaction of quinones as electrophiles with cellular nucleophiles may be. OH O [161] [228] OH OOCOR Compound 15 146 147 R CH3 CH2ˆCHCH2 CH3(CH2)2 IC50 [mm)] 45 3 40 3 144 10 148 149 shikonin (2) camptothecin CH3(CH2)4 CH3(CH2)6 ± ± 207 15 > 625 208 10 127 9 intercalate with the DNA. The process. Ahn and co-workers have undertaken more indepth investigations to elucidate the mechanism of cytotoxicity for 1. The selective inhibition of topoisomerase-I with no activity against topoisomserase-II (from Hella cells) was reported in this [233] study. 2. the shorter chain esters (C2 ± C6) were more potent than longer chain derivatives. and recombination. perhaps most famously for the [230] antitumor natural product mitomycin C. the general order was: 6-substituted DMNQs > 2-substituted DMNQs > naphthazarins. Papageorgiou. 2. Nicolaou et al. is well established. responsible for their cytotoxic behavior. gave a protective effect of 50% at 1 mm (Table 6). 38. These studies suggest that the cytotoxic activity of 1. K. A plausible bioreductive alkylation mechanism of alkannin (1) and shikonin (2) (Moore). these authors were also able to establish that 2 did not 290 . 231] DNA topoisomerases. at least in part.8-dimethoxy-1. From these experiments the authors deduced that redox cycling plays a prominent role in the cytotoxicity of 2-substituted naphthazarins. Inhibition of topoisomerase-I by shikonin esters. Recently. such as glutathione or DNA. In this mechanism reduction of the molecule in vivo promotes the elimination of a suitably positioned leaving group to produce a highly potent alkylating agent. 153] were synthesized by a ªmodified Terada methodº. a general inhibitor of endonu[235] cleases. Through the application of a DNA unwinding assay. at least partly. and DMNQ derivatives 150d ± f against L1210 cells were compared. OH O a) OH OH V. As predicted. Table 4. Moore was the first to suggest that 1. However. to their interactions with these important enzymes. A selection of typical examples is given in Table 4. at least from a chemists viewpoint. Perhaps the most appealing of the cytotoxicity mechanisms for quinones. and their derivatives could function in this manner. To test whether electrophilic arylation was an important mechanism for these compounds. reduced the cytotoxicity of shikonin (2) by 25%. In general. P. Furthermore. Chem. 2. the latter were found to be three.4-naphthoquinone (DMNQ) deriva- [234] OH O OR OH H O OR –HOR OH OH + H OH OH OH OH Nu OH O Nu – Figure 14. and their derivatives may be due. shown in Figure 14 for 1. C. transcription. Their ability to capture cellular nucleophiles was assessed by incubation of the quinones with glutathione. 270 ± 300 a) Reduction. is that of bioreductive [229] alkylation. a separate study showed shikonin (2) can induce DNA cleavage mediated by topoisomerase-II (from calf thymus). these researchers have prepared a series of analogues in an attempt to identify structure ± activity relationships and subsequently to produce more active derivatives. which may eventually lead to cell death.to tenfold less active (Table 5). 2. These enzymes control the topological state of DNA by concerted breaking and rejoining [232] of DNA strands. Nu ˆ nucleophile. through the formation of a cleaveable com[231] plex. Topoisomerases have been identified as targets for chemotherapy since they are involved in many important processes such as DNA replication. Ed. They reasoned that this structural modification should increase the electrophilicity of the quinone moiety (as it prevents the rapid ªtautome[137] rizationº present in the naphthazarins). tocopherol. Int. 1999. This electrophile may serve to capture cellular nucleophiles. When the cytotoxicity of 2-substituted naphthazarins 150a ± c. while aurintricarboxylic acid (ATA). Acetylation of the 2-substituted DMNQ derivatives 150d ± f Angew. The generation of free radicals from this. and their derivatives. and their derivatives. but decrease the susceptibility of the naphthoquinone to one-electron reduction (since the semiquinone produced on reduction of the latter should achieve greater delocalization than the corresponding radical from a DMNQ derivative). Ahn and coworkers prepared 5. In this regard it has been demonstrated in vitro that shikonin (2) and its derivatives can interact strongly with [160. which could contribute to their cytotoxicity. Inhibition of enzymes vital for cell metabolism or replication may also be a process through which quinones are toxic to cells. Further trends in cytotoxicity can be deduced from Table 5. A combination of ATA and tocopherol produced a protective effect of 80%. which also shows that several of these derivatives are more potent in vitro inhibitors than camptothecin. The mechanism of action of the well-known anticancer agent camptothecin involves inhibition of this enzyme. although no evidence was presented to support [229] this hypothesis. however. [229] tives and compared the biological data with those of the corresponding naphthazarin derivatives. These results suggest that at least two different mechanisms of cytotoxic action may be operating with these agents. an antioxidant which has been used to scavenge radicals derived from redox cycling. 2. correlated to their anti-cell [238] adhesive properties. [235] ÿ1 Table 6. Shikonin concentration was 60 nm throughout. however. leads to compounds 150g ± i. Although initial reports indicated that 1.41 0. This was not sufficient. Nevertheless. and their derivatives were found to be active against Gram-positive bacteria such as Staphylococcus aureus. Protection from shikonin cytotoxicity. 240a. [b] Life-span increase. and Bacillus subtilis with minimum inhibitory concentrations (MIC) of between 0. 243] mL . 240a. Enterococcus faecium. The 4-(N. the ana-logues tested.09 0. 2. such as topoisomerase-I. Recent reports from the same group have concentrated along these lines in the study of the antitumor activity of [237. and K562 cancer cells. was one of the most active. were therefore undertaken to show that this is.05 0. Extracts from pigment-producing callus cultures of LE [242] showed antibacterial activity similar to the plant roots.25 mg ÿ1 [231.15 0. it was suggested that shikonin (2) may be a useful therapeutic agent against MRSA. such as Escherichia coli and Pseu-domonas [231. 1. . The results (Table 5) can be compared with the cytotoxicity values from in vitro screens.23 50 0. 241b] aeruginosa. 241] [171] LE. 241a. indeed the case. the question of stereochemistry should no longer be ignored.06 0. In order to achieve this. This same study showed that such derivatives are selectively toxic to methicillin-resistant and methicillin-susceptible Staphylococcus aureus (MRSA and MSSA. 1 3 OR OOR 2 R 2 R 1 OR O OR 3 1 OR O [234±238] REVIEWS improved antitumor properties over shikonin (2).3. By variation of R and R a range of 6-substituted DMNQ analogues (151d ± j) were investigated. In general. a recent kinetic study has demonstrated that the action is. Plant extracts containing 1.3mm 1 mm not given [a] Protection [%] 25 0.03 0. Cytotoxicities for 17 analogues were determined in vitro against A549. relative to a control. A dose of 10 mgkg day was administered and the increase in life-span relative to a control was recorded. L1210.03 0. 2. and systematic studies for each of the common sources of 1 and 2. 240] [164. and their derivatives have also been shown to act against various [244] species of lactic acid bacteria. which showed an approximately threefold increase in activity. 270 ± 300 .04 0.37 0. which have recently been patented. None of these were significantly more potent in vitro than 2 or any of the other analogues examined. which was synthesized with the aim of improving the water solubility. They were inactive against Gramnegative bacteria. Cytotoxicity and in vivo activity of shikonin analogues. showing a T/C value of 192%. 38. AT. the emergence of a clinically useful anticancer agent remains to be seen.03 0. In vivo tests for many of the analogues were undertaken on mice with S-180 cells. They suggest that making structural modifications that increase selective binding to endonucleases while decreasing the nonselective binding to cellular nucleophiles and participation in redox cycling may be the best approach for future developments. however. OR 1 O 150 Compound 2 150a 150b 150c 150d 150e 150 f 150g 150h 150i 150j 150k 151a 151b 151c 151d 151e 151 f 151g 151h 151i 151j 151 R1 H H H H Me Me Me Me Me Me H H Me Me Me Me Me Me Me Me Me Me R2 3-methyl but-2-enyl Bu pentyl i-pentyl Bu pentyl i-pentyl Bu pentyl i-pentyl pentyl nonyl Bu pentyl i-pentyl Me Bu i-pentyl i-pentyl i-pentyl i-pentyl i-pentyl R3 H H H H H H H Ac Ac Ac Ac Ac H H H Ac Ac COPr Ac COPr COpentyl COheptyl ED50 0. The analogues were found to be much less active against the solid tumor cell line A549 than the leukemic cells L1210 and K562. The cytotoxicity of these analogues against A549 cells was.10 0. to implicate 1.03 0. Because of its low toxicity.b. The investigators concluded that binding to endonucleases.05 0. and their derivatives as the active components of the plant roots.b.3 and 6. [236] [156a] ÿ1 ÿ1 1 3 showed Angew. Crude preparations derived from plant roots containing these substances were initially shown to exhibit [239] antibacterial and antifungal properties. Ed. The 6-substituted derivatives of DMNQ 151a ± c showed cytotoxicities similar to those of the corresponding naphthazarin derivatives (150a ± c) (Table 5). 291 [246] [245] [a] Cytotoxicity was measured in vitro against L1210 cancer cells. and those for shikonin (2) obtained from a previous study with an identical dosage. While further research in the field is certain to continue. namely [82. Int.06 1.81 80 [a] Concentration of protective agent.05 ± ± 0.03 0. which they suggest may be their mode of action. 1999. bactericidal.N-dimethylamino)phenylacetyl derivative of shikonin (2). in fact. 238] arylacetyl esters of shikonin.02 0. Chem.5 ± ± ± ± ± ± ± ± ± 255 197 141 110 ± ± ± ± 148 198 217 205 5.Alkannin and Shikonin Table 5. Antimicrobial Activity Over the past 30 years the antimicrobial activity of 1. may be the most plausible mode of in vivo antitumor action of these compounds. 2. and Arnebia nobilis. There was no correlation between in vitro cytotoxicity and life-span increase.07 0. respectively).10 [mgmL ] ÿ1 [a] T/C [b] (%) 92.02 0. and their derivatives has been investigated by a number of groups. Protective agent tocopherol ATA tocopherol and ATA Concentration 0. 2.b. and their derivatives exhibit a bacteriostatic effect. for mice bearing S-180 cells (dose 10 mgkg ÿ1 day ). 5 4. The action was shown to be fungistatic rather than fungicidal. (1) and esters. Autopsies showed the animals had no Heinz bodies and their vital organs were of unchanged morphology. and Other Pharmacological Aspects In vitro and in vivo potencies are not the only factors to be considered if 1. cerebral. Compound shikonin (2) acetylshikonin ( esters to mice by intraperitonal ÿ1 LD50 [mgkg ] [210b] ) 15 b. 2.7 ÿ1 Control 89. Alkannin was excreted in the urine and not deposited in abdominal fat. [42. Chem.3 b. sulfureum. C.b-dimethylacrylshikonin (21) 20 5 41 48 5 [210b] 10 .b-dimethylacrylshikonin (21) 21. Tonsulans var. Pharmacokinetics. while other researchers have shown them to be active. No evidence of toxicity was observed when 1 was fed to mice for 15 weeks at 1% of their diet (average total intake 3. acetylshikonin (15). metabolism. mentagrophytes. and their [231. 241a] The crude ethanol extract from Arnebia nobilis has been tested in vivo on guinea pigs against [250] experimentally induced Candida albicans infection. Int. and/or their derivatives are to become therapeutically useful drugs. and 4) since 1.b-dimethylacrylshikonin (21). 2 and their derivatives in mice and rats. and Epidermophyton fluccosum (MIC < ÿ1 [247] 25 mgmL ). 5. Inhibition of collagen-induced platelet aggregation by alkannin Platlet aggregation [%] [a] IC50 [mgmL ] ± 4. T.8 2. the infection was cleared within six days. Results of in vitro tests are presented in Table 7. 2) alkylation of the phenolic groups leads to complete loss of activity. Aspects such as toxicity. Papageorgiou. 44] note 2).75 [213] [214] 5. but the therapeutic effect was weak when administered orally to rats [251] with experimental intestinal amebiasis. Platelet aggregation is considered to be a major pathogenic mechanism that leads to arterial thrombosis. It was thus suggested that screening a variety of ester derivatives may lead to products with greater potency. toxicity has been observed.9 ÿ1 [a] Measured with a substrate concentration of 20 mgmL . 1999. Antithrombotic Activity Thrombus formation in humans can restrict blood flow to vital tissues or organs and cause peripheral.1 10. four compounds were isolated from crude extracts and shown to inhibit platelet aggregation induced by collagen.4. and their derivatives. the aliphatic side chain may act as a delivery system for the naphthazarin. extracted from the cell cultures of LE. Shikonin 2 and its derivatives are rather more toxic to mice by intraperitonal administration. Toxic doses of 1. and reached the following conclusions: 1) the antibacterial effect of the hexane extract arises from the naphthoquinone pigment. tives (1. Embolism of the thrombosis can be catastrophic. 2. and their derivatives for antitumor effects.0 0. 10) were isolated from this source (see Table 1. It was suggested that 15 acts through [253b] suppression of phosphoinositide breakdown. Recently. Trichophyton rubrum. By topical application twice daily of 0. which is a goal that remains to be reached. Compound shikonin (2) shikonin (2) alkannan (33) alkannan leucoacetate cycloalkannin diacetate b. 9. Microsporum gypseum.[171a. and formulation are also vitally important. they remain under suspicion. 243.b-dimethylacrylalkannin (9) a-methoxyphenylacetyl shikonin Toxic dose 10 5 mgkg ÿ1 ÿ1 day ÿ1[156a] 30 mgkg day 30 mgkgÿ1 dayÿ1[156b] 30 mgkgÿ1 dayÿ1[156b] 30 mgkgÿ1 dayÿ1[156b] 10 mgkgÿ1 dayÿ1[220] ÿ1[156a] 7. during in vivo testing of 1. 22.5. 270 ± 300 . 3) polymerization results in complete loss of activity. 239. Deoxyshikonin (32) was generally more active. 2. including Candida [247] albicans.38 g). Alkannin (1) was shown to have low toxicity when [254] administered orally in a feeding study. Similar results were [210] demonstrated for shikonin (2) and acetylshikonin (15). 2. Arnebia euchroma is one of the so-called ªvasoactiveantithromboticº herbs of Chinese medicine. Papageorgiou and co-workers studied structure ± activity relationships for the antimicrobial properties of the constit[241b] uents of AT. Honda and co-workers have shown that shikonin (2) and deoxyshikonin (32). Table 9 indicates the derivatives for which this was the case (dose administered intraperitoneally). K. Results are summarized in Table 8. 2. since alkannin deriva292 [253] In addition. and teracrylshikonin (22). Table 7. An ointment for the control of athlete s foot with this [248] as the active component has been patented. or coronary ischemia. Several groups have also shown that 1. Its activity against [252] Aedes aegypti larvae has also been demonstrated. Shikonin (2) has been shown to exhibit a significant antiamebic effect in vitro on Entamoeba histolytica. 38. with the naphthazarin system being necessary for activity. Shikonin (2) and deoxyshikonin (32) were shown to be inactive (MIC > ÿ1 200 mgmL ) against several other fungi.75 mgkg ÿ1 day ÿ1[237] Angew. Acetylshikonin 15 also inhibits arachidonic acid induced platelet aggregation. Table 8.1 mL of a 5% solution of the arnebins in polyethylene glycol. Toxicity. 247] derivatives were inactive against Candida albicans. Other antimicrobial preparations containing shikonin as the active [249] principle have also been patented.REVIEWS There is generally less agreement in the literature concerning the antifungal properties of 1.0 18. Nicolaou et al. 5.2 2. and their esters are all active. exhibited significant antidermatophytic properties against five fungi tested: Saccharomyces sake. Ed. Compound [253] V. Although their structures were assigned as shikonin (2). Alkannin (1) [255] and Table 9.1 teracrylshikonin (22) acetylshikonin (15) shikonin (2) 0 0 67. and is a common cause of death. Toxicity of shikonin and administration. b. The LD50 was ÿ1 ÿ1 3 gkg in mice and less than 1 gkg in rats. P. T. is still in development. E. the total radioactivity excreted was 40. Orfanos. shikonin (2). great Angew. These investigations resulted in the identification of alkannin (1). 2. To provide literature coverage up to November 30. Other results are presented in Table 10.7 Addendum Since the initial submission of this review a number of papers concerning various aspects of the chemistry and biology of alkannin and shikonin have appeared in the literature. Table 10. Int. Ed. shikonin (2). cell cultures and biosynthesis. including anhydroalkannin (34). Chem. This study was undertaken at the Freie Universität of Berlin under the supervision of Prof. 1998 these are included here under the following [260] categories: medicinal properties. 38. with radioactivity detectable in plasma after about one minute.4-benzoquinone diisobutylaluminum hydride B-chlorodiisopinocampheylborane 4-dimethylaminopyridine N. The pioneering work of Terada. Kadota and co-workers have incubated 2 with Bacteroides fragilis [258] subsp. at least in part. has been gained from this work.6-dicyano-1. From this study the authors concluded that "Shikonin mixture is safe and effective for later-stage cancer". and related naphthazarins. C. and related derivatives in other therapeutic areas. biology and medicine of alkannin (1). Summary and Outlook The ancient medicinal properties claimed for AT and LE have. cycloshikonin (73). the wound-healing ability of HELIXDERM has been evaluated in further clinical trials. Furthermore. In addition. OH OH O OH OH OH O O OH 152 Cheng and co-workers were able to dramatically improve the pharmaceutical properties of 1 and 2 through the formation of a 1:1 inclusion complex with hydroxypropyl-b[259] cyclodextrin.156 h . The value of 2 in Japan motivated biotechnologists to develop the world s first manufacturing process utilizing plant cell cultures. particularly in cancer chemotherapy. Significant developments are to be expected in this area in the future.3 64. Administration oral intramuscular Peak level in plasma [min] 5. These developments should allow for many future adventures in the chemistry. 3 A pharmacokinetic study has been described using [ H]shi[257] konin in mice.Alkannin and Shikonin alkannin esters (5 ± 13) are nonmutagenic according to the [256] Ames test. 1999. Pharmacokinetic data for shikonin (2) in mice. thetaotus from human feces.78 7. these claims are certainly necessary. The absorption was rapid after both oral and intramuscular administration. been confirmed by scientific experimentation within the last 25 years. In addition. it has come to our attention that further research concerning the anticancer properties of "shikonin mixture" has been conducted in China. The research in this area has provided a wealth of knowledge to the field of biotechnology. Perhaps more significantly.3-dichloro-5.5% in the urine and 40. [264] and miscellane-ous biological properties. The clinical application of preparations that contain ester derivatives of 1 extracted from AT for the treatment of burns and ulcers is. [266] [265] As a predictive model to mimic the transformation of shikonin (2) in the human gastrointestinal tract.N'-carbonyldiimadazole dicyclohexylcarbodiimide 2. with 3. 270 ± 300 CDI DCC DDQ DIBAL DIP-Cl DMAP DMF hn Ipc LDA MOM MTBSTFA MTMSTFA NBS py TBAF N. The challenge of synthesizing these deceptively simplelooking compounds has kept a number of synthetic chemists occupied worldwide for many man-years.6 and 7. Ten metabolites were detect-ed: five monomers. clinical trials have been conducted using shikonin mixture for 19 patients with later stage lung cancer. Water solubility was improved 200-fold and photochemical decomposition rate constants decreased ÿ1 from 0. deoxy-shikonin (32).3% in feces. and five dimeric structures such as shikometabolin A (152). The use of 1. and showed that HELIXDERM gave very good results in terms of granulation and epithelization. and others in the field. the release rate of 1 and 2 was enhanced dramatically at one tenth of the control preparation Shiunko ointment.7%. laid the foundations upon which recent advances in the chemical synthesis of these substances relied. and related derivatives as the active components. Further studies to test Appendix: List of Abbreviations Used 6. Bioavailability [%] 34. Within 96 hours of intravenous injection. 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