Indolocarbazole
Encyclopedia
Indolocarbazoles are a class of compounds that are under current study due to their potential as anti-cancer drugs and the prospective number of derivatives and uses found from the basic backbone alone. First isolated in 1977, a wide range of structures and derivatives have been found or developed throughout the world. Due to the extensive number of structures available, this review will focus on the more important groups here while covering their occurrence, biological activity, biosynthesis, and laboratory synthesis.
(STA) was in 1977 from a culture of Streptomyces staurosporeus found in a soil sample from Iwate Prefecture, Japan. While the structure was initially thought to be as in Figure 3, the proper stereochemistry
was not proven until 1994. Over the course of the next decade, further study of the compound showed some fungi inhibition, hypotensive activity, and most importantly, a broad protein kinase inhibitor. The next landmark discovery came with the detection of rebeccamyacin (REB) in a sample of Lechevalieria aerocolonigenes, again in soil, but this time in a sample from Panama
. REB was found to act against leukemia
and melanoma
in mice, and also against human adenocarcinoma
cells.
Since 1977, ICZs have been discovered all over the world in actinomycetes, bacteria commonly found in soil. Numerous forms have tested positive for anti-tumor activity, such as 7-hydroxy-STA and 7-oxo-STA2. Some of the strains from which ICZ compounds have been found are Actinomadura melliaura in Bristol Cove, California, Streptomyces hygroscopicus in Numazu Prefecture, Japan, Micromonospora sp. L-31-CLO-002 from Fuerteventura Island, Canary Islands, Spain, and Actinomadura sp. Strain 007 from Jiaozhou Bay, China. The wide distribution of the various strains that produce these compounds is not surprising due to the number of properties these compounds can take on with limited functionalization on the specie’s part.
In addition to actinomycetes, ICZs have been found in slime molds (myxomycetes), blue-green algae (cyanobacteria, and marine invertebrates. Like the ones derived from actinomycetes, the ones found in myxomycetes cover an expansive range of derivatives and functionalizations. Two of the more important ones to date have been Arcyriacyanin A, which was found to inhibit a panel of human cancer cells by effecting PKC and protein tyrosine kinase, and lycogallic acid dimethyl ester A (found in Tokushima, Japan from Lycogala epidendrum), which showed strong antiviral activity. A few of the strains of myxomycetes studied are Arcyria ferruginea and Arcyria cinerea, both from Kochi Prefecture, Japan.
Three species of cyanobacteria has been found to produce ICZ compounds. Nostoc sphaericum from Manoa Hawaii, Tolypothrix tjipanasensis from Vero Beach, Florida, and Fischerella ambigua strain 108b from Leggingen, Switzerland. An interesting note on the first two is that many of the ICZ derived from them do not have the annelated pyrrolo[3,4-c] unit.
The final major group in which ICZs are found are various marine invertebrates. Three species of tunicate
, one mollusk
, one flatworm
, and one sponge have been discovered in places ranging from Micronesia to New Zealand. Testing for further invertebrate production is ongoing by both genetic and phylum-based studies.
The general modes of action found in mammalian cells are inhibition of protein kinase
s, inhibition of eukaryotic DNA topoisomerase, and intercalative binding to DNA. The number of protein kinases thought to exist in the human genome exceeds six hundred, making a nanomolar inhibitor such as STA extremely useful for both treatment of various diseases and study of protein kinases in a variety of functions. Since this discovery, a vast effort has been undergone to make highly specific STA and REB derivatives. One of the major lessons learned from initial research on STA was the development of the pharmacophore
model for a protein kinase inhibitor in which a bidentate hydrogen donating system flanked by various hydrophobic groups inserts into the binding site. The information derived from this original pharmacophore has led to the synthesis of highly specific inhibitors against a number of protein kinases, including PKC, cyclin-dependent kinases, G-protein coupled receptor kinases, tyrosine kinase, and cytomegalovirus pUL97 protein.
Topoisomerase
I and II
cleave and relegate one and two sides of a DNA strand, respectively, and are consequently vital parts of cell reproduction. Studies have found that in REB-like structures, the imide function of the pyrrole segment acts to interact with Topoisomerase I, the main carbon backbone acts as an intercalative inhibitor, and the sugar moiety undergoes DNA groove binding. The latter two actually act in unison due to the three dimensional structure of a glycosylated REB molecule. The Top1 inhibitor section binds to cleavable DNA-Top1 complexes so as to prevent the relegation step. Because of this, sensitivity is based on quantity of Top1 present, making cells undergoing constant reproduction and growth (namely tumor cells) most vulnerable.
At this point, bacterial inhibition of Top1 has not been founding using ICZs. Because of this, it is thought that most of the anti-cell growth function of ICZs comes from inhibition of various protein kinase groups and intercalative DNA binding. Studies on Streptomyces griseus
with in vitro protein labelling have led to inhibition of a wide range of cellular functions. This led to the theory that there were several eukaryotic protein kinases present required for secondary metabolism.
to 7-chloro-L-tryptophan. This is done by catalysis using RebH in vitro halogenation and RebF (a flavin reductase) to provide FADH2 for the halogenase. RebO (a tryptophan oxidase) then deaminates, after which it is further reacted with another one of itself and RebD (a heme containing oxidase). This forms the majority of the carbon backbone, which then undergoes decarboxylative ring closure using RebC and RebP. A glycosylation
occurs using RebG and NDP-D-Glucose, which finally goes through methylation by RebM.
Information for this pathway, along with those of K252a and STA, was derived from information on known genes, enzymes, and intermediates. The two types of studies done on these pathways are in vivo studies of gene disruption of L. aerocolonigenes or recombinant strains of S. albus. The second type of experiment consisted of in vitro experiments done on cell extracts.
based catalysts, which have been found to be excellent activators for use in formation of carbon-carbon bonds.
Occurrence
The first isolated ICZ, dubbed staurosporineStaurosporine
Staurosporine is a natural product originally isolated in 1977 from the bacterium Streptomyces staurosporeus.It was the first of over 50 alkaloids to be isolated with this type of bis-indole chemical structure...
(STA) was in 1977 from a culture of Streptomyces staurosporeus found in a soil sample from Iwate Prefecture, Japan. While the structure was initially thought to be as in Figure 3, the proper stereochemistry
Stereochemistry
Stereochemistry, a subdiscipline of chemistry, involves the study of the relative spatial arrangement of atoms within molecules. An important branch of stereochemistry is the study of chiral molecules....
was not proven until 1994. Over the course of the next decade, further study of the compound showed some fungi inhibition, hypotensive activity, and most importantly, a broad protein kinase inhibitor. The next landmark discovery came with the detection of rebeccamyacin (REB) in a sample of Lechevalieria aerocolonigenes, again in soil, but this time in a sample from Panama
Panama
Panama , officially the Republic of Panama , is the southernmost country of Central America. Situated on the isthmus connecting North and South America, it is bordered by Costa Rica to the northwest, Colombia to the southeast, the Caribbean Sea to the north and the Pacific Ocean to the south. The...
. REB was found to act against leukemia
Leukemia
Leukemia or leukaemia is a type of cancer of the blood or bone marrow characterized by an abnormal increase of immature white blood cells called "blasts". Leukemia is a broad term covering a spectrum of diseases...
and melanoma
Melanoma
Melanoma is a malignant tumor of melanocytes. Melanocytes are cells that produce the dark pigment, melanin, which is responsible for the color of skin. They predominantly occur in skin, but are also found in other parts of the body, including the bowel and the eye...
in mice, and also against human adenocarcinoma
Adenocarcinoma
Adenocarcinoma is a cancer of an epithelium that originates in glandular tissue. Epithelial tissue includes, but is not limited to, the surface layer of skin, glands and a variety of other tissue that lines the cavities and organs of the body. Epithelium can be derived embryologically from...
cells.
Since 1977, ICZs have been discovered all over the world in actinomycetes, bacteria commonly found in soil. Numerous forms have tested positive for anti-tumor activity, such as 7-hydroxy-STA and 7-oxo-STA2. Some of the strains from which ICZ compounds have been found are Actinomadura melliaura in Bristol Cove, California, Streptomyces hygroscopicus in Numazu Prefecture, Japan, Micromonospora sp. L-31-CLO-002 from Fuerteventura Island, Canary Islands, Spain, and Actinomadura sp. Strain 007 from Jiaozhou Bay, China. The wide distribution of the various strains that produce these compounds is not surprising due to the number of properties these compounds can take on with limited functionalization on the specie’s part.
In addition to actinomycetes, ICZs have been found in slime molds (myxomycetes), blue-green algae (cyanobacteria, and marine invertebrates. Like the ones derived from actinomycetes, the ones found in myxomycetes cover an expansive range of derivatives and functionalizations. Two of the more important ones to date have been Arcyriacyanin A, which was found to inhibit a panel of human cancer cells by effecting PKC and protein tyrosine kinase, and lycogallic acid dimethyl ester A (found in Tokushima, Japan from Lycogala epidendrum), which showed strong antiviral activity. A few of the strains of myxomycetes studied are Arcyria ferruginea and Arcyria cinerea, both from Kochi Prefecture, Japan.
Three species of cyanobacteria has been found to produce ICZ compounds. Nostoc sphaericum from Manoa Hawaii, Tolypothrix tjipanasensis from Vero Beach, Florida, and Fischerella ambigua strain 108b from Leggingen, Switzerland. An interesting note on the first two is that many of the ICZ derived from them do not have the annelated pyrrolo[3,4-c] unit.
The final major group in which ICZs are found are various marine invertebrates. Three species of tunicate
Tunicate
Tunicates, also known as urochordates, are members of the subphylum Tunicata, previously known as Urochordata, a group of underwater saclike filter feeders with incurrent and excurrent siphons that is classified within the phylum Chordata. While most tunicates live on the ocean floor, others such...
, one mollusk
Mollusca
The Mollusca , common name molluscs or mollusksSpelled mollusks in the USA, see reasons given in Rosenberg's ; for the spelling mollusc see the reasons given by , is a large phylum of invertebrate animals. There are around 85,000 recognized extant species of molluscs. Mollusca is the largest...
, one flatworm
Flatworm
The flatworms, known in scientific literature as Platyhelminthes or Plathelminthes are a phylum of relatively simple bilaterian, unsegmented, soft-bodied invertebrate animals...
, and one sponge have been discovered in places ranging from Micronesia to New Zealand. Testing for further invertebrate production is ongoing by both genetic and phylum-based studies.
Biological activity
Indolocarbazoles have been found to exhibit a wide range of activities, which makes their range of presence in nature unsurprising. Because of this variety, the following section will examine their modes of action in bacterial and mammalian cells independently, with special attention paid to cancer cell effects.The general modes of action found in mammalian cells are inhibition of protein kinase
Protein kinase
A protein kinase is a kinase enzyme that modifies other proteins by chemically adding phosphate groups to them . Phosphorylation usually results in a functional change of the target protein by changing enzyme activity, cellular location, or association with other proteins...
s, inhibition of eukaryotic DNA topoisomerase, and intercalative binding to DNA. The number of protein kinases thought to exist in the human genome exceeds six hundred, making a nanomolar inhibitor such as STA extremely useful for both treatment of various diseases and study of protein kinases in a variety of functions. Since this discovery, a vast effort has been undergone to make highly specific STA and REB derivatives. One of the major lessons learned from initial research on STA was the development of the pharmacophore
Pharmacophore
thumb|right|300px|An example of a pharmacophore model.A pharmacophore is an abstract description of molecular features which are necessary for molecular recognition of a ligand by a biological macromolecule....
model for a protein kinase inhibitor in which a bidentate hydrogen donating system flanked by various hydrophobic groups inserts into the binding site. The information derived from this original pharmacophore has led to the synthesis of highly specific inhibitors against a number of protein kinases, including PKC, cyclin-dependent kinases, G-protein coupled receptor kinases, tyrosine kinase, and cytomegalovirus pUL97 protein.
Topoisomerase
Type I topoisomerase
Type I topoisomerases cut one strand of double-stranded DNA, relax the strand, and reanneal the strands. They are further subdivided into two structurally and mechanistically distinct topoisomerases: type IA and type IB....
I and II
Type II topoisomerase
Type II topoisomerases cut both strands of the DNA helix simultaneously in order to manage DNA tangles and supercoils. They use the hydrolysis of ATP, unlike type I topoisomerase. In this process, these enzymes change the linking number of circular DNA by +/-2....
cleave and relegate one and two sides of a DNA strand, respectively, and are consequently vital parts of cell reproduction. Studies have found that in REB-like structures, the imide function of the pyrrole segment acts to interact with Topoisomerase I, the main carbon backbone acts as an intercalative inhibitor, and the sugar moiety undergoes DNA groove binding. The latter two actually act in unison due to the three dimensional structure of a glycosylated REB molecule. The Top1 inhibitor section binds to cleavable DNA-Top1 complexes so as to prevent the relegation step. Because of this, sensitivity is based on quantity of Top1 present, making cells undergoing constant reproduction and growth (namely tumor cells) most vulnerable.
At this point, bacterial inhibition of Top1 has not been founding using ICZs. Because of this, it is thought that most of the anti-cell growth function of ICZs comes from inhibition of various protein kinase groups and intercalative DNA binding. Studies on Streptomyces griseus
Streptomyces griseus
Streptomyces griseus is a member bacterial species of the genus Streptomyces and are commonly found in soil. A few strains have been also reported from deep sea sediments. These are Gram positive bacterium with high GC content. Along with most other streptomycetes S...
with in vitro protein labelling have led to inhibition of a wide range of cellular functions. This led to the theory that there were several eukaryotic protein kinases present required for secondary metabolism.
Biosynthesis
Unfortunately, only biosynthesis of REB, STA, and K252a have been studied in depth. This section will emphasize the REB pathway due to how well studied it is. The pathway begins with the modification of L-tryptophanTryptophan
Tryptophan is one of the 20 standard amino acids, as well as an essential amino acid in the human diet. It is encoded in the standard genetic code as the codon UGG...
to 7-chloro-L-tryptophan. This is done by catalysis using RebH in vitro halogenation and RebF (a flavin reductase) to provide FADH2 for the halogenase. RebO (a tryptophan oxidase) then deaminates, after which it is further reacted with another one of itself and RebD (a heme containing oxidase). This forms the majority of the carbon backbone, which then undergoes decarboxylative ring closure using RebC and RebP. A glycosylation
Glycosylation
Glycosylation is the reaction in which a carbohydrate, i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule . In biology glycosylation refers to the enzymatic process that attaches glycans to proteins, lipids, or other organic molecules...
occurs using RebG and NDP-D-Glucose, which finally goes through methylation by RebM.
Information for this pathway, along with those of K252a and STA, was derived from information on known genes, enzymes, and intermediates. The two types of studies done on these pathways are in vivo studies of gene disruption of L. aerocolonigenes or recombinant strains of S. albus. The second type of experiment consisted of in vitro experiments done on cell extracts.
Synthesis
Laboratory synthesis of ICZs has been a topic of great interest since their discovery. Unfortunately, due to the somewhat complex nature of the molecule and the high level of reactivity of carbons on indole molecules, a facile high yield synthesis has yet to be found. Despite this, there have been many ways found to produce this compound in its various forms. Of special interest is one of the better REB syntheses, found in 1999. The process begins by producing 7-chloroindole-3-acetamide by treating 7-chloroindole with a series of reagents, shown farther down. This molecule is then glycosylated and reacted with methyl 7-chloroindole-3-glyoxylate to produce an intermediate that goes on to stabilize into the final product. While this process is one of the better ones to date, it is still work and time intensive, going through 12 total steps and only yielding 12%.Further developments
Ever since the birth of ICZ research in the late seventies, the field has been burgeoning with continued advances in both technology and organic chemistry techniques. While only a handful of ICZ based compounds have made it past stage II clinical trials, the sheer variety that these molecules can take on leaves much still unexplored territory. Of particular recent interest in synthesis techniques is the use of palladiumPalladium
Palladium is a chemical element with the chemical symbol Pd and an atomic number of 46. It is a rare and lustrous silvery-white metal discovered in 1803 by William Hyde Wollaston. He named it after the asteroid Pallas, which was itself named after the epithet of the Greek goddess Athena, acquired...
based catalysts, which have been found to be excellent activators for use in formation of carbon-carbon bonds.