Current Topics in Medicinal Chemistry - Volume 8, Issue 6, 2008

Mother nature often provides scientists with new and innovative ideas. One perfect example is enediynes which have now become the core of research in cancer therapeutics. Enediynes are a group of cyclic natural molecules that consist of at least two triple bonds separated by a double bond in conjugation. These products of bacterial origin were first isolated in 1960s and have drawn special attention since mid-eighties following the discovery that they possess powerful antitumor antibiotic activity. The enediyne group is often called a “warhead” because it can easily cyclize to form aromatic ring system via a highly reactive 1,4 benzeniod diradical intermediate. This cyclization process is called the “Bergman cyclo-aromatization reaction” after the name of the discoverer (Bergman, R.G. Accts. Chem. Res., 1973). The diradical intermediate can cause oxidative cleavage to double stranded DNA, giving rise to enediyne's powerful antitumor activity. Depending on the nature of substitution and ring size, the above cyclization can be triggered by temperature, light, pH changes, catalyst, suitable donors, oxidative state, metal coordination/induction as well as transformation from one tautomeric form to the other. Other triggering methods such as release of ring strain, acid base induction and enzyme mediated cleavage of protecting group have also been demonstrated. Recently conditions have been developed for transformation of enediynes to fulvene and indene derivatives. Bergman cyclo-aromatization reaction proceeds more efficiently and under milder condition when enediyne system is present within a constrained cyclic (mostly 9 and 10 membered) system compared to the acyclic one. The size of the ring structure also plays a significant role on the ease of cyclo-aromatisation. Enediynes find very useful applications in the study of biological systems. Thus it is applied to the development of “catalytic antibody or Abzyme” and “novel anticancer agents”. So far four principle effects of the enediynes on mammalian cells have been identified. These are (a) Mutagenicity, (b) Antimitotic activity associated with cell-cycle arrest, (c) Apoptosis induction and (d) Differential induction. Overall enediynes act as antimitotic agents by inducing a temporary delay in the cell lines during division of nucleus and form an important class of antibiotics. So far there are reports of three types of natural enediynes: (i) Type I with 10-membered ring and a 3-ene-1, 5-diyne function eg. calicheamicin/dynemicin), (ii) Type II with 9-membered ring and a 3-ene-1, 5-diyne moiety e.g. kedarcidin and, (iii) Type III with 9-memdered ring containing a dienediyne function eg. neocarzinostatin. Calicheamicin/Dynemicin represent the most potent antitumor agents known. However they are limited by their high toxicity and selectivity. Several pharamaceutical companies including the Bristol-Myers Squibbs made significant contributions in this area. They developed dynemicin class of antibiotics that contain a hydroxyanthraquinone chromophore instead of sugar moiety as found in calicheamicin/esperamicin. Chromoproteins eg. C1027 (discovered in 1988/89) have proteins that wrap around the enediyne, which stabilizes it, and takes it out of the cell. The study of C1027 suggests that all enediynes share a common polyketide biosynthetic pathway. This opens the door to genetic manipulation of these biosynthetic pathways to develop new drug candidates with less toxicity. Of all the enediynes, only neocarzinostatin, which is a chromoprotein, is approved as a drug to treat liver cancer in Japan only. The United States has not approved such a drug. However calicheamicin is now heading most directly to clinical studies, compared to the rest of the enediynes. Efforts to attach calicheamicin to various drug antibody conjugates, peptides or steroids that seem suitable for clinical trials, make calicheamicin an enediyne of interest. Its derivative “Mylotarg” (an antibody conjugate) has now been approved for treatment of Acute Myeloid Leukemia (AML). This drug acts as targeted therapy by delivering the enediyne derivative directly to the cancer cells, thereby not affecting normal cells. Because of these developments enediynes have stimulated considerable synthetic interest, although their clinical use has been limited because of their modest selectivity for cancer cells. The biological mode of action occurs along one of two general pathways, depending on the type of enediyne structure. The majority of enediyne natural products, including calicheamicin undergo Bergman cyclization whereas others such as neocarsinostatin operates via a Myers-Saito pathway. In both cases the end result is cleavage of double stranded DNA leading to apoptosis. Enediynes remain as the major focus of chemists and biologists for future development of more useful and selective antitumor antibiotic drugs which may be useful to degrade harmful DNAs, Proteins, Enzymes and possibly other macromolecules. It is hoped that enediyne based antibiotics may one day be able to kill all types of cancer and bacteria including those of resistant type.

Of the commonly recognized structural elements within nucleic acids, bulges are among the least developed as targets for small molecules. Bulges in DNA and RNA have been linked to biomolecular processes involved in numerous diseases, thus probes with affinity for these targets would be of considerable utility to chemical biologists and medicinal chemists. Despite such opportunity, there is a dearth of small molecules available with affinity for bulges, which has hampered exploitation of these key targets. We have used guided chemical synthesis to prepare small molecules capable of binding to DNA and RNA bulges. Our design is based on a template which mimics a metabolite of the enediyne neocarzinostatin. The key spirocylic building block was formed through an intramolecular aldol process and the parent template shows pronounced affinity for 2 base bulges. Functionalization with specific aminosugar moieties confers nanomolar binding affinity for selected bulged DNA targets, and installation of reactive functional groups allows covalent modification of bulges. These rationally designed agents can now be used to study the stereochemistry and architecture of bulge-drug complexes and investigate the molecular biology of bulge induced processes. Members of this class have been shown to induce slipped synthesis of DNA, suggesting the agents, in addition to recognizing and binding to pre-formed bulges, can also induce bulge formation on demand.

The enediyne polyketides are secondary metabolites isolated from a variety of Actinomycetes. All members share very potent anticancer and antibiotic activity, and prospects for the clinical application of the enediynes has been validated with the recent marketing of two enediyne derivatives as anticancer agents. The biosynthesis of these compounds is of interest because of the numerous structural features that are unique to the enediyne family. The gene cluster for five enediynes has now been cloned and sequenced, providing the foundation to understand natures' means to biosynthesize such complex, exotic molecules. Presented here is a review of the current progress in delineating the biosynthesis of the enediynes with an emphasis on the model enediyne, C-1027.

The concept of photoswitchable enediynes, which are stable in the dark but undergo efficient cycloaromatization reaction to produce p-benzyne diradical after irradiation with light of an appropriate wavelength, is discussed. Two novel methods for the generation of reactive enediyne compounds from thermally stable precursors have been developed. In the first approach, one of the triple bonds of cyclodeca-3-ene-1,5-diynes is replaced with cyclopropenone group. Cyclopropenone-containing enediyne precursors are unable to undergo cycloaromatization because the enediyne fragment is incomplete. Photolysis of cyclopropenones results in the efficient decarbonylation and the regeneration of a triple bond thus completing the enediyne π-system. The second method employs photo-Wolff reaction to achieve ring contraction of stable eleven-membered ring precursor enediynes. Benzannulated cyclic enediynes produced by the photodecomposition of enediyne precursor containing 2-diazo-1,3-diketones possess enolized β-ketoester fragment and undergo remarkably facile τ36o = 5 min - 3 h) Bergman cyclization. The generation of reactive enediyne was also achieved with NIR light by non resonant two-photon excitation.

It has been shown that the enediyne anticancer antibiotics e.g., calicheamicin, neocarzinostatin and others cleave DNA via the putative intermediate 1,4-diradical formed in the Bergmann cyclization and are thus useful for cancer chemotherapy. The pharmacological activity of these drugs is based, in general, on the activation of the pharmacophore and the subsequent cyclization leading to the formation of a radical, the rate of which is, in part, based on the terminal distance of the 1,5-diyne functionality, also known as the critical distance. But the critical distance alone cannot govern the rate of cyclization of the enediynes. A theoretical model has been developed to predict the rate of cyclization, and the thus the pharmacological activity, of these antibiotics based on the critical distance and the energy of activation.

Enediynes continue to fascinate scientists working in various domains because of their structural complexity and fascinating biological mode of action. They represent a masterpiece of nature's ingenuity. Besides the warhead which is the enediyne moiety, these molecules are equipped with a locking device, a delivery system and a chemical trigger for activation. Upon triggering, the molecules become active and undergo a thermal rearrangement that was disclosed in the early '70 by Masamune and Bergman and commonly known as Bergman cyclization. The reaction is believed to precede through a diradical benzenoid species (a p-benzyne). This review describes the various strategies employed for the synthesis of highly strained enediynes and dienediynes, both naturally occurring and the designed ones.

The pituitary is the preeminent gland of the body, synthesizing and releasing key hormones including growth hormone, thyroid-stimulating hormone, prolactin, luteinizing hormone, follicle-stimulating hormone and adrenocorticotropic hormone. The secretion of these hormones is regulated by the hypothalamus via the hypothalamus-hypophyseal portal veins. The target organ of adrenocorticotropic hormone is the adrenal gland, which regulates serum levels of cortisol. The trio of the hypothalamus, pituitary, and adrenal glands, termed the HPA axis, is responsible for coordinating an organism's response to exogenous stressors. A number of hormone-receptor systems within the HPA axis provide likely targets for pharmacological manipulation, three of which are reviewed in this issue. For some time, the importance of the hormone corticotropin releasing factor (CRF) as a regulator of stress states in mammals has been recognized. It is a key controller of the neuroendocrine system and is the dominant hormone responsible for governing stress response. However, despite the fact that peptide CRF receptor antagonists have been known for twenty years and small molecule antagonists have been known for more than a decade, no phase two, placebo-controlled proof of concept study showing efficacy has been disclosed. In their review, Tellew and Luo discuss recent advances in the field of CRF receptor antagonists and speculate on the characteristics of the ideal molecule. In contrast to CRF receptor antagonists, which have not been used medicinally, modulators of the glucocorticoid (GC) receptor have been used to treat inflammatory and immune conditions for more than half of a century. These drugs, however, come with significant side effects, which limit their use as first line therapies. One innovative solution to the unfavorable side effect profile of current glucocorticoids is to find organ (tissue) selective agonists. Takahashi, Razavi and Thomson discuss progress towards this goal and also speculate on the issues that may arise from the discovery and development of GC receptor antagonists for the treatment of diseases resulting from excess circulating cortisol. Calcitonin gene-related peptide (CGRP) and its receptor are best known for their role in vasodilation and their potential application to migraine. However, CGRP has wide expression throughout the central nervous system and has been shown to interact with the HPA. Rudolf, Arndt, Muller and Doods review recent reports in the field of CGRP receptor antagonists and speculate on the potential of a CNS-penetrating GCRP receptor antagonist. These reviews present an update on several drug targets related to the HPA axis. It is hoped they will not only provide the background and state-of-the-art for these molecular targets, but will also help to spur further research in this area.

Antagonists of the corticotropin releasing factor (CRF or CRH) receptor have shown promise for the treatment of anxiety, depression, and irritable bowel syndrome. In the present article, medicinal chemistry developments surrounding small molecule CRF receptor antagonists are reviewed, focusing on publications and patents from mid-2004 through the first quarter of 2006. While the CRF type 2 receptor remains an intractable target, incremental progress has been made in the search for drug-like antagonists of the CRF type 1 receptor. Most recent work has not ventured far from previously-established pharmacophoric topologies. A common theme in recent patent disclosures is the addition of novel polar substituents to known heterocyclic core structures to reduce overall lipophilicity. New disclosures of pharmacokinetic (PK) data for several series of antagonists reveal that achieving appropriate PK remains a challenge for the field. The recent publication of selection patents and patents relating to salt and crystal forms of particular compounds suggests that several second generation compounds are nearing or have entered clinical development.

Glucocorticoids have been used in modern clinical practice for over fifty years. Although they have demonstrated potent anti-inflammatory and immunosuppressive activities, their association with debilitating and lifethreatening side effects has been a major drawback. Recent insights into glucocorticoid biology have lent support to the hypothesis that the glucocorticoid anti-inflammatory activities could be dissociated from their adverse side effects. Inspired by these biological findings, the search for dissociated glucocorticoid receptor agonists has intensified. Antagonists of the glucocorticoid receptor that offer therapeutic benefits for the treatment of diseases such as diabetes have also been pursued. These efforts have been partly focused on the development of tissue, especially liver, selective glucocorticoid receptor antagonists, which are thought to have improved safety profiles. This review offers a summary of the research and development activities in this field and covers journal and patent publications from 2003 to March 2006.

Discovery of mAChR subtype selective M4 positive allosteric modulators. There are two types of cholinergic receptors through which the neurotransmitter acetylcholine (ACh) acts: nicotinic acetylcholine receptors (nAChRs), ligand gated ion channels, and muscarinic acetylcholine receptors (mAChRs), class A G protein-coupled receptors. To date, five mAChR subtypes have been identified (M1- M5) and are thought to mediate the majority of the actions of ACh in the peripheral and central nervous systems. Of these, M1 and M4 are the most heavily expressed in the CNS and represent attractive therapeutic targets for cognition, Alzheimer's disease, schizophrenia and movement disorders such as dystonia and Parkinson's disease. In contrast, the adverse effects of cholinergic agents are due to activation of peripheral M2 and M3 mAChRs [1-3]. Phase III clinical trials with Xanomeline an M1/M4 preferring orthosteric agonist, demonstrated efficacy as both a cognition enhancing agent, are more interestingly, as an antipsychotic agent [4]. In follow-up studies in rats, xanomeline displayed an antipsychotic phenotype comparable to clozapine [5]. However, was the antipsychotic phenotype mediated by activation of M1, M4 or a combination of M1/M4? Data from mAChR knock-out mice suggest that a selective M1 agonist would be beneficial for cognition whereas an M4 agonist would provide antipsychotic effects for the treatment of schizophrenia [6]. This proposal is further supported by recent studies demonstrating that M4 receptors modulate the dynamics of cholinergic and dopaminergic neurotransmission and that loss of M4 function results in a state of dopamine hyperfunction - a hallmark of schizophrenia [7]. These data, coupled with new information that schizophrenic patients possess altered hippocampal M4, but not M1, receptor expression argue well for the need for selective activators of M4 as a novel treatment for schizophrenia [8]. Due to the high sequence homology and conservation of the orthosteric ACh binding site among the mAChRs, development of chemical agents that are selective for a single subtype have been largely unsuccessful - until now [1-7]. In a recent paper from the Conn laboratory at Vanderbilt (Shirey et.al. Nat. Chem. Bio. 2008, 4 (1), 42-50), a novel, mAChR subtype-selective M4 positive allosteric modulator, VU100010, was disclosed [9]. Employing a cheminformatics approach, coupled with medicinal chemistry, Conn identified a new series of ligands that interact with an allosteric site on the M4 receptor (does not displace [3H]-NMS) which not only activates the receptor, but also confers complete selectivity versus M1, M2, M3 and M5 - a ‘Holy Grail’ for molecular pharmacology as this tool will enable researchers to probe the pharmacological role of selective M4 activation and evaluate the potential of M4 as a novel therapeutic target for multiple CNS disorders. VU100010 possesses an EC50 for potentiation of ∼400 nM and potentiates the ACh response curve 47-fold to the left. Mechanistic studies indicate that VU100010 exerts its allosteric activation of M4 by increasing the affinity of ACh and coupling to G proteins. In electrophysiology studies, VU100010 was shown to modulate hippocampal synaptic transmission. Specifically, the M4 positive allosteric modulator increased carbacholinduced depression of transmission at excitatory, but not inhibitory synapses in the hippocampus. Importantly, this effect was absent when inactive analogues of VU100010 were employed or in M4 knockout mice [9]. No in vivo data was disclosed for VU100010, but the in vitro profile of this novel and highly selective M4 positive allosteric modulator represents a major advance in the muscarinic field and will allow many important studies to be performed to dissect the contribution of M4 to the efficacy of xanomleine and ultimately, to the potential of selective M4 activation as a therapeutic mechanism for CNS disorders. REFERENCES [1] Felder, C.C.; Bymaster, F.P.; Ward, J.; DeLapp, N. ‘Therapeutic opportunities for muscarinic receptors in the central nervous system’ J. Med. Chem. 2000, 43, 4333-4353. 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[7] Tzavara, E.T.; Bymaster, F.P.; Davis, R.J.; Wade, M.R.; Perry, K.W.; Wess, J.; McKinzie, D.L.; Felder, C.; Nomikos, G.G. ‘M4 muscarinic receptors regulate the dynamics of cholinergic and dopaminergic neurotransmission: relevance to the pathophysiology and treatment of cnetral nervous system disorders’ FASEB J. 2004, 18, 1410-1412. [8] Scarr, E.; Sundram, S.; Keriakous, D.; Dean, B. ‘Altered hippocampal muscarinic M4, but not M1, receptor expression from subjects with schizophrenia’ Biol. Psych. 2007, 61, 1161-1170. [9] Shirey, J.K.; Xiang, Z.; Orton, D.; Brady, A.E.; Johnson, K.A.; Williams, R.; Ayala, J.E.; Rodriguez, A.L.; Wess, J.; Weaver, D.; Niswender, C.M.; Conn, P.J. ‘An allosteric potentiator of M4 mAChR modulates hippocampal synaptic transmission’ Nat. Chem. Bio. 2008, 4, 42-50.