PKC Inhibition and in Vitro Cytotoxicity
Abstract
In this Chapter we revisit the main strategies used for years in synthesizing staurosporine indolocarbazole alkaloid and its analogues, which are promising compounds for treating cancer. In addition to describing the details of the synthesis strategies, including the key challenges that had to be faced, we offer a historical perspective of the development in the field.
Keywords
- Indolocarbazole
- alkaloids
- cancer
- synthesis
- sugar moiety
- glycosylation
1. Introduction
1.1. Aims and significance
Cancer is one of the most serious threats against human health [1], which has motivated extensive research into a plethora of chemotherapeutic agents [2-3]. The need for new anticancer drugs arises not only from the limitations of current drugs, but also from the development of drug resistance [4-6]. Several strategies exist for designing such novel drugs, for which the essential criterion is the selection of a suitable starting point from the vast chemical space [7]. Natural products, in this context, are privileged structures [8] and biologically prevalidated leads, for they contain molecules that probably evolved to exert highly specialized functions. About 74% of anticancer compounds originate from natural products or from natural product-derived products [9]. The variety of structures in products is key for new therapeutics [10].
The indolocarbazole family of natural products (hereafter referred to as ICZ’s) was discovered in 1977 in actinomycetes, bacteria commonly found in soil, and is now investigated by medicinal chemists especially due to its antitumor and neuroprotective properties [11-13]. Figure 1 illustrates that ICZs are a structurally diverse family of natural products. The four types of aglycons include: A) the parent indolo[2,3-a]carbazole nucleus, such as that found in tjipanazole F2 (
We can also further divide ICZs based on the pattern of attachment of aglycon to the sugar moiety into four sub-patterns, viz.: A) ICZs having no sugar moiety, such as
Knolker and Reddy reviewed the synthesis and biological activity of carbazole alkaloids, depicted in Figure 2, where different synthetic strategies for indolocarbazole alkaloids were discussed [15].
1.2. Motivation for the chapter
Potent drugs against cancer normally have to fulfill a number of requirements in terms of its toxicity to tumor cells and solubility for efficient delivery. This requires a full-fledged characterization of drug candidates, including possible synthetic strategies. In this Chapter we concentrate on indolocarbazoles such as staurosporine, the most potent PKC inhibitors isolated to date, which probably act by occupying the ATP binding site and preventing protein phosphorylation. There is hence the need of synthetic routes to prepare indolocarbazole derivatives that are selective toward specific malfunctioning kinases associated with a disease. Furthermore, clinically useful compounds should have enhanced solubility in water, as compared to the poorly soluble ICZs. Since most indolocarbazoles with potent biological activities have substituents on the benzene portion of the core, enhanced solubility has been attempted with at least three approaches. The first is to introduce a hydrophilic group on the imide nitrogen, e.g. the
Well-known examples of pharmaceutically important glycosylated natural products include macrolide antibiotics, aromatic polyketides, glycopeptides, indolocarbazoles, aminoglycosides, and cardiac glycosides. The sugar moieties are often essential for the biological activity in such natural products. Thus, altering the structures and/or substitution patterns of sugar appendages on aglycone moieties, a process known as
1.3. Definition of the problem
The indolocarbazole acceptor is generally a weaker nucleophile than the bis(indoly1)-maleimide or indole acceptor, which limits application of established glycosylation methodologies to the indolocarbazole aglycones.
1.4. History of staurosporine
1.4.1. Isolation
Omura et al reported in 1977 a new alkaloid, isolated from
Structure
This isolation of staurosporine sparked research into related natural and synthetic compounds, particularly for treating cancer with nanomolar inhibition of protein kinases (PKC) [22]. Many staurosporine analogues are in phase III clinical trials to treat cancer and about ten such PKC inhibitors have been approved for use in clinical level.
1.4.2. The importance of protein kinase c inhibitors
Protein kinase C (PKC) is a family comprised of at least eight serine/threonine specific kinases that are approximately 77 kD in size. The importance of PKC in regulating signal transduction pathways and ultimately cellular response has been well-established [59]. Activation of PKC occurs through a series of events that begins with specific binding of an extracellular agonist to a cell surface receptor. This binding results in activation of phospholipase C which then cleaves inositol triphosphate (IP3) from phosphatidylinositol-4-5- biphosphate (PIP2) and leaves behind a molecule of 1,2-diacylglycerol (DAG) in the membrane. Phosphorylation ultimately results in cellular responses by modifying the function of rate-limiting enzymes and regulatory proteins implicated in metabolic pathways.
As already mentioned, indolocarbazoles such as K252a and staurosporine are the most powerful PKC inhibitors isolated to date. This mode of PKC binding, illustrated in Figure 5, unfortunately results in a relatively non-selective inhibition of several kinases. The preparation of indolocarbazole derivatives possessing selectivity toward specific malfunctioning kinases associated with a disease state would be a solution; thus, an efficient and general synthetic route to the indolocarbazoles is desirable.
1.4.3. Pharmacology of staurosporine and its analogues
The recent literature on staurosporine analogues has provided valuable inputs into their biochemical pharmacology and generated discussion on the suitability of protein kinase C as potential target for anticancer drugs. The following conclusions are particularly pertinent with respect to pharmacological mechanisms [23]:
staurosporine analogues such as UCN-01 and CGP 41251 are inhibitors not only of PKC, but of a ‘cocktail’ of kinases;
the composition of this cocktail and expression of its constituent kinases in a given neoplasm determine the nature and extent of pharmacological efficacy; and
slight alterations in molecular structure dramatically alter individual components of this cocktail.
Indolocarbazoles are all biologically active and display such properties as antimicrobial, antifungal, and antitumor activity, in addition to acting as hypotensive or platelet aggregation agents [24-27]. Three representative examples of this class are staurosporine (
2. Synthesis of staurosporine and its analogues
2.1. Introduction
Staurosporine can be divided into two distinct parts: the "northern" indolocarbazole aglycon and the ‘‘southern’’ carbohydrate portion of the molecule, as shown in Figure 6. One can envision that by so dissecting the molecule, a convergent synthetic approach would be possible in which a lactam-protected derivative of aglycon could be coupled with a bis-glycal derivative (no commitment is made as to the functional nature of R1 or R2).
From Figure 7 one may infer that aglycon
2.2. Biosynthetic pathway of staurosporine
2.2.1. Biogenesis of the indolocarbazole nucleus
Cordell and Pearce independently reported the first indolocarbazole biosynthesis in 1988 [33-35], both identifying aglycon units of ICZs (
2.2.2. Biosynthesis of indolocarbazole carbohydrates
The carbohydrate precursor to staurosporine has been shown to be D-glucose and the
2.2.3. About this pathway
The first enzyme identified in staurosporine biosynthesis was the one catalyzing the very last step (3'-
The next step is glycosylation, which is catalyzed by two enzymes. K252c
2.3. First total synthesis of staurosporine and ent-staurosporine (Danishefsky et al., 1995)
It was not until 1995 that the first total synthesis of staurosporine (
Triisopropylsilyl-L-glucal
Compound
A methodology was developed to convert the 7-oxo compound
2.4. Staurosporine and ent-staurosporine: The first total syntheses, prospects for a regioselective approach, and activity profiles (Danishefsky et al., 1996)
The total syntheses of staurosporine and
The authors dealt with the problem of indole glycosylation, functional group management in the pyranose ring, and regiochemical harmonization in the course of the first total synthesis of staurosporine (
Danishefsky et al. assumed the upcoming C3´ methoxy and C4´ methylamino vestiges would be existing in an oxazolidinone ring. Protecting the nitrogen with a benzyloxymethyl group, C1´-
Consistent with the discussion above, they formulated the donor to be a glucal of the type
Oxazolidinone glycal
Alteration of the functional group was essential to construct the second glycosidic bond. It was performed by deoxygenating the newly created alcohol at C5´, deprotecting the indole moiety, establishing 2,2´ indolic bond, and finally formation of exo-glycal (Scheme 7).
Early screening of the reaction of indolocarbazole glycoside
To complete the total synthesis of
Upon successfully completing the chemistry in the
Danishefsky et al. evaluated
2.5. Wood and Stolz’s synthesis of staurosporine
A total synthesis of the natural product (+)-staurosporine has been achieved [46] along with other ICZs. The synthetic strategy involved steroselective ring expansion of a furanosylated indolocarbazole [(+)-79] to a pyranosylated congener [(+)-80] that serves a common intermediate in the production of 1 and other desired ICZs.
2.5.1. Retrosynthetic analysis: The development of a ring expansion approach to the pyranosylated indolocarbazoles
Wood and Stolz began by considering approaches that involved ring expansion of a furanosylated intermediate. Noting the striking structural homology of
The inspiration for developing this approach derived from Wood’s recognition that ketone
2.5.2. Completion of staurosporine
Next, Wood and Stolz [46] treated (+)-
3. The synthesis of carbohydrates for indolocarbazole synthesis
Only a few methodologies have been developed for synthesizing complex carbohydrate intermediates for use in the total synthesis of indolocarbazole alkaloids such as staurosporine (
3.1. Synthesis of staurosporine monosaccharide (Weinreb et al.)
Weinreb published the synthesis of aminohexose fragment of staurosporine
3.2. Staurosporine glycal precursor (Danishefsky et al).
Danishefsky exploited glycal epoxide
3.3. Methods describing the combination of carbohydrate and indolocarbazole
3.3.1. The Danishefsky synthesis of (+)- and (-)-staurosporine
Danishefsky formulated a strategy to staurosporine [41], in which epoxidation of glycal (-)-
3.3.2. Syntheses, biochemical and biological evaluation of staurosporine analogues from the microbial metabolite rebeccamycin
To synthesize staurosporine analogues from rebeccamycin, different structural variations were exploited by Prudhomme et al., including coupling of the sugar moiety to the second indole nitrogen, dechlorination and then reduction of imide to amide [49].
The synthesized compounds
3.3.3. Synthetic studies on indolocarbazoles: Total synthesis of staurosporine aglycon
Mohankrishnan et al synthesized staurosporine aglycon and its analogues with 28-36% overall yield, using 2-methylindole (
Triphenylphospite-mediated nitrene insertion of 2-nitroarylcarbazole was performed at a moderate temperature using anhydrous ZnBr2 as catalyst. In addition, an alternative synthetic protocol for preparing ICZs involving concurrent electrocyclization followed by nitrene insertion was adopted as in Scheme 17 by Mohankrishna et al. [50].
3.3.4. Synthesis of pyrrolidin-2-ones and staurosporine aglycon (K-252c) by intermolecular Michael reaction
3,4-Disubstituted pyrrolidin-2-ones, a group of compounds with interesting biological properties, are related to staurosporinone. The most important property is inhibition of protein kinase C (PKC), so that this antiproliferative agent can interfere with the cell cycle. The synthetic strategy permits preparation of said compounds using an intermolecular Michael addition, starting from nitroethene derivatives and substituted acetate Michael donors [51].
Enantioselective syntheses can also be carried out using chiral auxiliaries in this strategy. Reduction of the nitro group using raney nickel and subsequent lactamization, the desired lactam precursor of staurosporine, which is essential for the biological activity, is obtained according to Scheme 18. The easiest and shortest (in contrast to the published routes of staurosporinone) synthetic strategy of staurosporinone within three steps with good to moderate yields is obtained.
3.4. Syntheses of the indolo[2,3-a]carbazole nucleus
Synthetic strategies for preparing the indolo[2,3-a]carbazole nucleus have been already summarized in Figure 2 based on the key bond formations, type of structure synthesized (aglycon), and research group. In the following section some of the methodologies are described briefly.
3.4.1. Winterfeld’s strategy to synthesis of staurosporinone
In 1983, Winterfeld published the first synthesis of K252c as shown in Scheme 19 [52-53]. The synthesis of lactam
3.4.2. Magnus’ approach
Magnus published a synthetic methodology to selectively protect staurosporinones, just after Winterfeld’s report [54]. Intramolecular Diels-Alder cycloaddition of indole-2,3-quinidomethane
3.4.3. The Weinreb approach
Weinreb exploited a synthetic strategy for the synthesis of bis indolyl maleimides to furnish maleimide
3.4.4. Raphael’s approach
Raphael staurosporinone synthesis based on intermolecular Diels-Alder methodology and nitrene insertion chemistry is depicted in Scheme 22 [55-56]. Reaction of numerous dienophiles with diene
4. Conclusion
In this book chapter, a brief introduction to biologically active indolocarbazole alkaloids was presented, with emphasis on the isolation and synthetic pathways of powerful protein kinase inhibitors such as Staurosporine indolocarbazole alkaloid and its analogues. Glycosylation on indolic moiety and concerns were discussed apart from the synthesis of staurosporinone aglycon and sugar portion. We do hope that this book chapter will be a valuable addition to the chemists dealing with indolocarbazole alkaloids from pharmaceutical industry and synthetic organic point of view.
Acknowledgments
Dr. Ravi Varala heartfully thanks Prof. Sirasani Satyanarayana, Vice-Chancellor (RGUKT-Basar) and Prof. Appala Naidu, RGUKT-AP, for their kind cooperation and support. And also, specially thank FAPESP-Brazil for the award of ‘Visiting Researcher’ grant (2014/25784-7). Profound thanks to Prof. S. J. Danishefsky, Prof. B. M. Stolz and Prof. J. L. Wood et al. for their valuable contributions to the field.
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