A research team has achieved a breakthrough in the production of a well-known drug for the treatment of cancer

Stanford University researchers have discovered a fast and sustainable way to synthetically produce a promising cancer-fighting compound right in the lab. The availability of the compound has been limited because its only known natural source at this time is a single plant species that grows exclusively in a small region of rainforest in northeastern Australia.

The compound, labeled EBC-46 and technically called tigilanol tiglate, works by promoting localized immune response against tumors. The answer is broken down into parts a tumorblood vessels and eventually kills cancer cells. EBC-46 recently joined the lineup human clinical trials after an extremely high success rate in treating a type of cancer in dogs.

However, given its complex structure, EBC-46 proved to be synthetically unavailable, meaning that there seemed to be no plausible way to produce it virtually in the laboratory. However, through a clever process, Stanford researchers have demonstrated for the first time how to chemically convert the abundant plant-derived starting material into EBC-46.

As a bonus, the process could produce EBC-46 “analogs”—compounds that are chemically similar, but which may prove to be even more effective and potentially treat a surprisingly wide range of other serious diseases. All of these diseases, including AIDS, multiple sclerosis and Alzheimer’s disease, share common biological pathways that are affected by EBC-46’s target, a key enzyme called protein kinase C, or PKC.

“We are very excited to report the first scalable synthesis of EBC-46,” said Paul Wender, the Francis W. Bergstrom Professor in the School of Humanities and Sciences, a professor of chemistry and, courtesy of Stanford, of chemical and systems biology. , and the corresponding author of the study describing the findings in the journal Chemistry of nature. “Being able to make EBC-46 in the lab really opens up huge research and clinical opportunities.”

The study was co-authored by Zachary Gentry, David Fanelli, Owen McAteer and Edward Nju, all of whom are Ph.D. students in Wender’s lab, along with former member Quang Luu-Nguyen.

Wender conveyed the immense satisfaction the research team felt at the breakthrough in the synthesis of EBC-46. “If you had visited the lab in the first few weeks after their success,” Wender said, “you would have seen my stellar colleagues grinning from ear to ear. They were able to do what many people thought was impossible.”

From a distant land

Tigilanol tiglate originally emerged from an automated drug candidate screening process conducted by Australian company QBiotics. In nature, this compound is found in the seeds of the pink fruits of the Fontainea picrosperma rose tree. Marsupials, such as muskrat kangaroos, which eat the red fruit, avoid the tigilanol-rich seeds, which cause vomiting and diarrhea when ingested.

Administration of much lower doses of EBC-46 directly into some solid tumors modifies cellular signaling through PKC. Specifically, EBC-46 is thought to activate specific forms of PKC, which in turn affect the activity of various proteins in cancer cells, attracting the host’s immune response. The resulting inflammation makes the tumor’s vasculature (blood vessels) leaky, and this hemorrhage leads to the death of the tumor growth. In the case of external, cutaneous malignancies, the tumors scab and fall off, and routes of delivery of EBC-46 to internal tumors are being investigated.

In 2020, the European Medicines Agency and the Food and Drug Administration in the United States approved an EBC-46-based drug, sold under the brand name Stelfonta, for the treatment of mast cell carcinoma, the most common skin tumor in dogs. The study showed 75% cure after one injection and 88% after the second dose. Clinical trials for skin, head and neck, and soft tissue cancers in humans have since begun.

Based on emerging research and clinical needs, combined with geographic constraints on seed sources, scientists have considered the possibility of establishing special plantations for the roe. But this creates many problems. First, trees require pollination, which means the right pollinator animals must be on hand, and the trees must be planted at the proper density and spacing to promote pollination. In addition, seasonal and climatic changes affect trees along with pathogens. Allocating areas for black forest creates even more problems with land use.

“For sustainable, reliable production of EBC-46 in the quantities we need,” Wender said, “we really need to go the synthetic route.”

Building EBC-46 from scratch

Wender and his colleagues realized that a plant-based compound was a good starting point for creating EBC-46 Farbol. More than 7,000 plant species produce phorbol derivatives worldwide, and phorbol-rich seeds are commercially inexpensive. The researchers chose Croton tiglium, commonly known as cleansing croton, an herb used in traditional Chinese medicine.

The first step in preparing EBC-46, Wender explains, is similar to everyday experience. “You buy a bag of these seeds and it’s like making coffee in the morning,” Wender said. “You grind the seeds and run a hot solvent through them to extract the active ingredient,” in this case an oil rich in phorbol.

After processing the oil to make phorbol, the researchers had to figure out how to overcome the previously insurmountable challenge of lining a part of the molecule called the B ring with carefully spaced oxygen atoms. This is necessary so that EBC-46 can interact with PKC and alter the activity of the enzyme in cells.

To guide their chemical and biological studies, the researchers relied on instruments from the Stanford Neuroscience Microscopy Service, the Stanford Cancer Institute’s proteomics/mass spectrometry shared resources, and the Stanford Sherlock cluster for computer simulations.

With this guidance, the team was able to add additional oxygen atoms to the B ring of the farbol, first using a so-called ene (pronounced “ene”) reaction, which is carried out under flow conditions where the reactants are mixed as they pass through tubes together. The team then introduced other B ring groups in a controlled stepwise manner to obtain the desired spatial arrangement of the atoms. In total, it took only four to six steps to obtain EBC-46 analogs, and a dozen steps to achieve EBC-46 itself.

Wender hopes that the much wider availability of EBC-46 and its cousin compounds that affect PKC through this breakthrough approach will accelerate research into potentially revolutionary new therapies.

“As we learn more and more about how cells function, we learn more about how we can control that functionality,” Wender said. “This control over functionality is particularly important in fighting cells that go haywire in diseases ranging from cancer to Alzheimer’s disease.”

Wender is also a member of Stanford Bio-X and the Stanford Cancer Institute, and a member of Sarafan ChEM-H.