New Therapies and Drugs

Bryostatin and the Bryologs

Bryostatin 1, the lead member of the bryostatin family, is a bryozoan-derived macrolide first elucidated in 1982 that exhibits remarkable in vitro and in vivo activities relevant to cancer treatment including restoration of apoptotic function, reversal of multidrug resistance, stimulation of the immune system, and synergism with other oncolytics. Currently in Phase I and II clinical trials for cancer, bryostatin 1 is exceptionally potent with only ~1.2 milligrams required for a full multi-week treatment cycle. Bryostatin 1 has also been shown to facilitate learning and enhance memory in animals, presumably through induction of synaptogenesis, and thus serves as a promising new lead for the treatment of neurodegenerative disorders such as Alzheimer’s disease. Notably, a clinical trial opened in 2008 to test the efficacy of bryostatin 1 against Alzheimer’s disease. Moreover, early reports suggested that bryostatin might induce HIV reservoir clearance, which has been further supported in more recent studies and suggests a first-in-class approach to the eradication of HIV/AIDS.

Function-oriented synthesis approach to potent, simplified bryostatin analogs

That bryostatin 1 has already been tested in human trials increases its appeal as a target and underscores the need for a reliable supply of a more effective agent. In fact, patient accrual in a recent National Cancer Institute clinical trial with bryostatin 1 was terminated given the awareness of the clinical investigators of more potent bryostatin analogs in development. Research on and clinical studies of bryostatin has been impeded by its low natural abundance. Isolated yields from natural sources are low (10^-3 to 10^-8 %) and variable; the good manufacturing practices (GMP) production required 14 tons of the marine bryozoan Bugula neritina to provide just 18 grams of bryostatin 1. While this supply was sufficient to initiate preclinical and clinical research, environmental and economic factors have severely limited further development of the source organism and its aquaculture production. Furthermore, while a superb lead, bryostatin suffers from off-target toxicities that have been revealed in clinical studies.

Convergent late-stage macrocyclization to furnish dioxane and pyran B-ring analogs

In 1988, our group proposed a hypothesis to address both the supply and therapeutic performance problems; this led to the practical syntheses of the first functional analogs that exhibit greater in vitro and in vivo potency than bryostatin and can be tuned for clinical performance. This work is driven by the postulate that the activities of bryostatin could arise from only a subset of its functionality. Using a function-oriented synthesis approach, over 35 analogs with single-digit nanomolar or better potencies have been prepared, some of which outperform the natural product in potency. These analogs can be produced in a practical, convergent fashion via late-stage fragment coupling through esterification then acetal- or Prins-driven macrocyclization. Significantly, these agents can be modified to selectively modulate protein kinase C (PKC) isoform class activity, thereby providing a potential mechanism through which optimized analogs can be applied toward divergent therapeutic applications. Notably, a simplified bryostatin analog has been shown to be well tolerated and efficacious in an in vivo mouse cancer model. More recently, simplified analogs of bryostatin are up to 1000-fold more potent in inducing latent HIV expression than prostratin, the current clinical candidate for latent virus induction. Several of our simplified analogs are currently the most synthetically accessible agents with activity comparable or better than bryostatin and its congeners.

Gnidimacrin

Long an unidentified constituent of traditional (folk) medicines, gnidimacrin (shown below) was only recently found to have selective activity against a variety of cancer cell lines (stomach, non-small cell lung and leukemias) with sub-nanomolar (0.35 nM) IC50 values. Of special therapeutic relevance, gnidimacrin’s anticancer activity has also been shown in animal cancer models, in which significant life extensions and even cures have been reported. Gnidimacrin appears to work through a novel mode of action putatively involving selective modulation of a PKC isozyme. Unfortunately, isolation yields of gnidimacrin are approximately 0.0005%, making its supply from natural sources very limited. The main goal of this project is to achieve the total synthesis of gnidimacrin, and through FOS, compounds with gnidimacrin–like activity which can be used to study its biological activity and develop improved therapeutic agents. Gnidimacrin itself is a formidable synthetic challenge that represents an opportunity to advance the frontiers of synthetic chemistry. Ideally, a synthetic route toward this target would allow for the rapid generation of analogues that can provide information on gnidimacrin's unique mode of action and thus inspire the design of superior anticancer leads.

Gnidimacrin: A Selective Anti-Cancer Lead

Daphnetoxins

The daphnane diterpene orthoesters (DDOs) constitute a structurally fascinating, synthetically challenging, and biologically intriguing family of natural products. Plants containing DDOs have been used medicinally for over 2000 years, and more than 160 DDOs have been identified to date. Many are exceptional leads for the treatment of cancer, diabetes, neurodegenerative diseases, neuropathic pain, and other illnesses. A major goal of our research program is the development of a flexible and efficient synthetic route that would allow access to a large number of these compounds. Because of the extreme scarcity of nearly all known DDOs, this would be an invaluable step towards elucidating the biological mode of action for not only the natural products, but also currently unknown structural variants.

Yuanhuapin Functional Analogs

The daphnetoxins and 12-hydroxydaphnetoxins, two subsets of DDOs, are currently a primary focus for our general synthetic strategy. Yuanhuapin, a 12-hydroxydaphnetoxin, has been the inspiration for some of our most recent work. It possesses anti-cancer activity due in part through inhibition of DNA topoisomerase I. Pursuing this target has provided a platform to gain valuable information regarding synthetic transformations on these challenging scaffolds and has allowed us to access biologically active analogues. We expect the knowledge that we have gained working towards this natural product to be widely applicable to the broader family of daphnetoxins. In addition to pursuing natural products, we are also designing and synthesizing function-oriented analogues based on a model of the pharmacophoric elements of this family of molecules. These analogues are more accessible than the natural products and share many of the key functionalities found in the parent natural products thought to be responsible for potency; however, their syntheses still present challenges.

Kirkinine: A Potent Lead for Neurotrophic Activity

Another DDO of interest is kirkinine, a 12-hydroxydaphnetoxin isolated from Synaptolepis kirkii (an African medicinal plant), which displays remarkable activity. Like other members of this class, kirkinine has a very complex molecular architecture featuring a highly oxygenated tricyclic ring system, an orthoester side chain, an epoxide, and 11 stereogenic centers. More importantly, it is kirkinine's remarkable neurotrophic activity that makes it a very attractive synthetic target. It effectively promotes neuronal survival against serum deprivation in nanomolar concentrations. As a result, kirkinine and its analogs present very promising therapeutic opportunities against neurodegenerative diseases such as Alzheimer’s.

Prostratin

Over the last two decades impressive progress has been made in the treatment of HIV/AIDS. Many antiviral therapies can halt progression of the disease. However, these medications must be used chronically, elicit resistance, require disciplined self medication, and are costly, especially in countries where rates of infection are high. While antiviral therapies show efficacy against active virus they do not affect the latent viral reservoirs. As such, if one stops anti-viral therapy, disease rebound occurs as the active virus is re-supplied by latent virus reservoirs. Induced activation of the latent virus would purge these reservoirs which in combination with antiretroviral therapy would provide a first strategy to eradicate HIV/AIDS, a potentially transformative approach to this disease.

Prostratin is a naturally occurring tigliane and represents the active constituent of a traditional tonic used by Samoan healers to treat a local type of hepatitis. After its identification through an NIH effort to discover novel medicinal leads, prostratin was shown to stimulate the proliferation of HIV in latently infected cells. Unfortunately, the current isolation process is low yielding (0.005%) and at the moment does not appear to provide the required quantities for therapeutic advancement of prostratin. More importantly, we could in principle design more effective agents.

We have established a program that addresses the prostratin supply problem, has produced the first synthetic source of prostratin and is directed at the optimization of the therapeutic properties of prostratin through function oriented analog design and synthesis. We have shown that prostratin can be generated through a concise synthesis starting with phorbol (5 steps, ~16% overall yield), which can be obtained in quantity either from commercial sources or directly through isolation from croton oil. More importantly, we have now produced agents that are significantly more potent than prostratin.

Five Step Synthesis of Prostratin

Our program draws on synthesis and design to make such analogs and study their biological activity using potency and functional assays. These research efforts are central to the advancement of prostratin as a viable therapeutic agent. Our work is conducted in collaboration with several other research groups including universities, companies and clinical groups.

References

Bryostain and Bryolog Lead References:

DeChristopher, B.A.; Loy, B.A.; Marsden, M.D.; Schrier, A.J.; Zack, J.A.; Wender, P.A. “Designed, synthetically accessibility bryostatin analogues potently induce activation of latent HIV reservoirs in vitro.” Nature Chem., 2012, 4, 705-710.
Wender, P.A.; Billingsley, K.L. “Lead Diversification through a Prins-Driven Macrocyclization Strategy: Application to C13-Diversified Bryostatin Analogues.” Synthesis, 2013, 45 (13), 1815-1824.
Ogawa, Y.; Painter, P.P.; Tantillo, D.J.; Wender, P.A. "Mechanistic and Computational Studies of Exocyclic Stereocontrol in the Synthesis of Bryostatin-like Cis-2, 6-Disubstituted 4-Alkylidenetetrahydropyrans by Prins Cyclization." J. Org. Chem., 2013, 78(1), 104-115.
DeChristopher, B. A.; Fan, A. C.; Felsher, D. W.; Wender, P. A. “ ‘Picolog,’ a synthetically-accessible bryostatin analog, inhibits growth of MYC-induced lymphoma in vivo.” Oncotarget, 2012, 3, 58-66.
Wender, P. A.; Baryza, J. L.; Brenner, S. E.; DeChristopher, B. A.; Loy, B. A.; Schrier, A. J. “Design, synthesis and evaluation of potent bryostatin analogs that modulate PKC translocation selectivity.” Proc. Natl. Acad. Sci. USA, 2011, 108, 6721-6726.
Wender, P. A.; Schrier, A. J. “Total Synthesis of Bryostatin 9.” J. Am. Chem. Soc., 2011, 133, 9228-9231.
Wender, P. A.; Loy, B. A.; Schrier, A. J. “Translating nature’s library: the bryostatins and function-oriented synthesis.” Isr. J. Chem., 2011, 51, 453-472.
Wender, P. A.; DeChristopher, B. A.; Schrier, A. J. “Efficient Synthetic Access to a New Family of Highly Potent Bryostatin Analogues via a Prins-Driven Macrocyclization Strategy,” J. Am. Chem. Soc., 2008, 6658.
Wender, P. A.; Baryza, J.; Bennett, C.; Bi, C.; Brenner, S. E.; Clarke, M.; Horan, J.; Kan, C.; Lacote, E.; Lippa, Nell, P.; Turner, T.“The Practical Synthesis of a Novel and Highly Potent Analog of Bryostatin” J. Am. Chem. Soc., 2002, 124, 13648.

Gnidimacrin and Daphnetoxins Lead References:

Wender, P.A.; Buschmann, N.; Cardin, N.B.; Jones, L.R.; Kan, C.; Kee, J-M.; Kowalski, J.A.; Longcore, K.E. “Gateway Synthesis of daphnane congeners and their protein kinase C affinities and cell-growth activities” Nature Chem. 2011, 3, 615.
Wender, P. A.; D'Angelo, N.; Elitzin, V. I.; Ernst, M.; Jackson-Ugueto, E. E.; Kowalski, J. A.; McKendry, S.; Rehfeuter, M.; Sun, R.; Voigtlaender, D. “Function-Oriented Synthesis: Studies Aimed at the Synthesis and Mode of Action of 1-Alkyldaphnane Analogues ” Org. Lett. 2007, 1829.
Wender, Bi, Buschmann, Gosselin, Kan, Kee, Ohmura, "Studies on Oxidopyrylium [5+2] Cycloadditions: Toward a General Synthetic Route to the C12-Hydroxy Daphnetoxins" Org. Lett. 2006, 8, 5373.
Wender, Jesudason, Nakahira, Tamura, Tebbe, Ueno "The First Synthesis of a Daphnane Diterpene: The Enantiocontrolled Total Synthesis of (+)-Resiniferatoxin" J. Am. Chem. Soc. 1997, 119, 12976.
Wender, Rice, Schnute, "The First Formal Asymmetric Synthesis of Phorbol" J. Am. Chem. Soc. 1997, 7611-7612.

Prostratin Lead Reference:

Beans, E.J.; Fournogerakis, D.; Gauntlett, C.; Heumann, L.V.; Kramer, R.; Marsden, M.D.; Murray, D.; Chun, T.-W.; Zack, J.A.; Wender, P.A. “Highly potent, synthetically accessible prostratin analogs induce latent HIV expression in vitro and ex vivo.” Proc. Natl. Acad. Sci., USA, 2013, 110 (29), 11698-11703.
Wender, P.A.; Kee, J.-M.; Warrington, J.M. “Practical Synthesis of Prostratin, DPP, and Their Analogs, Adjuvant Leads Against Latent HIV.” Science, 2008, 320 (5876), 649-652.