The nitrogens are the point. The squares are the frames that carry them. These molecules are called azetidines, and they can be used as building blocks in drug design.

The Rice lab of chemist László Kürti introduced its azetidines in an Angewandte Chemie paper. The lab’s goal is to establish a library of scaffolds for pharmaceutical design through the simple synthesis of a class of molecules that were previously hard to find in nature and very hard to copy.

Azetidines already appear in several drugs and are promising components in the development of treatments for neurological diseases like Parkinson’s disease, Tourette’s syndrome and attention deficit disorder, according to the researchers.

So there’s value in making a fast, inexpensive synthetic route to azetidines with unprotected nitrogen atoms called NH-azetidines — NH for nitrogen and hydrogen — that were first found in several kinds of Pacific sea sponges and have more recently been made in arduous laboratory processes.

Rice graduate students Nicole Behnke and Kaitlyn Lovato, lead authors of the paper, quickly learned why there are so few references to synthetic NH-azetidines in the scientific literature.

It required more than 250 experiments for the students to optimize their process, which takes about 24 hours, including product purification. Azetidine molecules come in many configurations, but they all share the square motif, a four-atom “ring” that contains one nitrogen atom and three carbon atoms. This ring is heterocyclic — that is, it contains at least two different elements.

Kürti noted the square ring is always connected to another ring via one shared carbon atom, a structure called a spiro azetidine. In this way, the two rings are perpendicular to each other, further isolating the highly reactive nitrogen for access by chemists. The nitrogen in the Rice lab’s variations were often, though not always, paired with a hydrogen atom “cap” that still allows the nitrogen to react with outside agents.

“Dr. Kürti was inspired by the mechanism of a synthetic process called the Kulinkovich reaction, which is used to make three-membered all-carbon rings, called cyclopropanes, that have the heteroatom (the nitrogen or oxygen) on the outside,” Lovato said.

“Once we started looking into making four-membered azetidines, we found that most of them didn’t have the NH structures,” she said. “The known synthetic methods predominantly yield azetidines in which the ring nitrogen atom is connected to a carbon outside of the ring, but the NH connectivity was hard to access directly. If there’s a carbon there, the nitrogen is considered protected, but having the hydrogen there leaves it free to engage in further reactions.”

“Once you make this NH heterocycle, you have the flexibility to put whatever you want on the nitrogen,” Kürti said. “Or to leave it as it is.”

A titanium reagent turned out to be a key middleman in the chemical reaction, allowing it to proceed quickly. “This metal complex mediates the overall transformation, and it’s very good because titanium is non-toxic and very abundant,” he said.

“It’s commercially available and cheap,” Behnke said. “If we don’t have the titanium added to the flask, the reaction doesn’t work.”

The Rice team did not patent the process, Kürti said. “The reality is that synthetic organic chemists in academia can contribute a lot to biomedical sciences and pharmaceutical drug discovery when we develop a new mechanism or reaction,” he said.

“Biotech and pharmaceutical companies can use the products of these reactions to build structurally diverse compound libraries and quickly test them for biological activities towards different cancer cell lines, pathogens or other important disease biochemical pathways they have assays for,” Kürti said. “Once they have access to novel core structures like these spiro azetidines, it’s up to medical chemists to decide what diverse functionalities they wish to add on.”

Muhammed Yousufuddin, a lecturer of chemistry at the University of North Texas at Dallas, is co-author of the paper. Kürti is an associate professor of chemistry.

Rice University, the National Institutes of Health, the National Science Foundation (NSF), the Robert A. Welch Foundation, the Amgen Young Investigators Award, the Biotage Young Principal Investigator Award and an NSF Graduate Research Fellowship supported the research.

“Although older adults are capable of empathizing [about] someone’s intentions when making a moral evaluation, they appear less likely to do so than younger individuals when those actions cause harm,” said Janet Geipel, PhD, of the University of Chicago, who presented the research.

Geipel and her colleagues conducted a series of experiments examining how younger adults (ages 21 to 39) and older adults (ages 63 to 90) would morally evaluate accidentally harmful and accidentally helpful actions.

The first experiment involved 60 participants equally split into younger and older adults. Each participant was presented with eight hypothetical scenarios in which a person’s actions resulted in either a positive or negative outcome. In each case, the scenario was described in such a way that the participant could infer whether the act was intended to cause the outcome that it did, as opposed to simply being an accident.

After each scenario with a negative outcome, participants were asked to judge the immorality of the described action and how much it should be punished. In the case of a positive outcome, participants were asked to judge the goodness of the action and how much it should be rewarded. Participants answered all questions on a scale of zero to ten.

For instance, in one scenario, a character named Joanna and one of her friends are in a boat in a part of the sea with lots of poisonous jellyfish. Her friend asks if it is OK to go swimming, and Joanna (knowing the water is not safe) tells her to go ahead. The friend goes swimming, gets stung and goes into shock. In another version of the scenario, Joanna has read (incorrectly) that the local jellyfish were harmless and unknowingly puts her friend at risk.

The researchers found that older adults were more likely to condemn accidentally harmful acts and recommend that the person be punished, even when it appeared that the harmful action was unintentional. Interestingly, they did not find any age difference in how accidentally helpful actions were evaluated.

A second experiment involved 82 participants and was similar to the first experiment. Participants were presented with four different scenarios: One in which accidental harm was caused by negligence (e.g., Chloe sold a sick dog that was infected with rabies because she did not check the animal carefully), one in which the agent acted with due care (e.g., Chloe sold a dog with rabies after a careful assessment of the dog made her believe it was healthy), one with a neutral outcome (e.g., Chloe intended to sell a healthy dog and did so) and one in which the agent acted with negative intentions (e.g., Chloe knew the dog had rabies and sold it anyway).

“We found that while younger adults condemned more severely negligent than non-negligent actions, older participants condemned both equally,” said Geipel.

In a second part of the same experiment, participants were presented with the accidental harm scenarios from the first experiment and asked to what extent they thought the person was negligent and whether his or her actions should be condemned.

“We found that older adults condemned the accidental transgressors more than did younger adults and were more likely to attribute negligence to the actions,” said Geipel. “Further analysis showed that perceived negligence mediated the relationship between age group and the judgment of moral wrongness.”

Geipel believes this phenomenon may have something to do with the fact that people experience cognitive decline as they age. Making moral judgments based on intent requires more cognitive effort than simply condemning outcomes. Since older adults may find considering intent more mentally taxing than younger adults, they would be more likely to condemn even unintentional harm.

These findings may have important implications, especially for the legal system, said Geipel. For example, a jury member who has to evaluate whether someone is guilty needs to consider intent.

“The present results suggest that older adults may attend less to the intentions of the accused and more to the negative outcomes that the accused produced,” said Geipel. “Put simply, the present findings imply that older adults may be more likely to convict.

The study published today in Science Immunology suggests that giving the two therapies as short course, first-line treatment might work better than the current practice of providing one treatment early and the other treatment only if tumors relapse.

“This study reveals the importance of the proper combination and timing of tyrosine kinase inhibitors and immunotherapy such as the programmed death-ligand 1 (PD-L1) inhibitor, also known as immune checkpoint blockade. If borne out by future research, these findings might open new treatment avenues for many cancer patients,” said corresponding author Dr. Yang-Xin Fu, Professor of Pathology, Immunology, and Radiation Oncology and a member of the Harold C. Simmons Comprehensive Cancer Center.

“Many cancers have high and abnormal tyrosine kinase activities. Tyrosine kinase inhibitors, or TKIs — which target specific cancers — are a common first-line treatment for rapidly shrinking tumors such as those associated with kinase-driven breast cancer and lung cancer. But tumor relapse or resistance often occurs. The standard of care is to use TKIs first and then use immunotherapy such as PD-L1 inhibitors after relapse occurs,” Dr. Fu explained. “Our study showed that immunotherapy should be used together with the TKIs as the first-line — not second-line or third-line — treatment. We also demonstrated that the dose and timing of the TKI is important.”

The team, which included first authors and postdoctoral researchers Drs. Zhida Liu and Chuanhui Han, also found that hypofractionated TKI (HypoTKI) — giving a high dose over a short time — was more effective in mouse models of breast cancer and lung cancer than standard hyperfractionated TKI (HyperTKI) — lower doses over a longer time. Compared with standard therapy, the high-dose/short time TKI approach appeared to significantly reduce tumor burden and limit relapse with fewer side effects.

Earlier studies that found increased side effects when TKI and PD-L1 were given together had used HyperTKI rather than the HypoTKI provided in this investigation, said Dr. Fu, a physician-scientist who holds the Mary Nell and Ralph B. Rogers Professorship in Immunology.

The researchers found that TKIs work in part by activating both innate and adaptive immunity — that is, both of the body’s immune systems. The innate immune system is activated as soon as a threat is identified. Adaptive immunity is the body’s response to threats it has learned to recognize, Dr. Fu explained.

PD-L1 inhibitors are thought to enhance adaptive immunity to overcome the ability of some tumor cells to develop resistance to TKIs, he added.

Compared with standard HyperTKI therapy, the HypoTKI approach triggered greater innate immune sensing and a more potent release of type I interferon and other cytokines through an innate immunity signaling pathway to enhance tumor-specific T cell infiltration and reactivation. The researchers also found that HypoTKI was more potent than HyperTKI in limiting tumor relapse in a host immune response-dependent manner. More importantly, they observed that PD-L1 blockade could further enhance the anti-tumor effectiveness of HypoTKI treatment in advanced large tumors and limit the relapse. The timing of the dose of the drugs was important, he said.

The study is among the first to find that this class of TKIs can trigger the innate immune response, Dr. Fu said. “We think there might be a threshold effect with HypoTKI in which the treatment more effectively alerts the innate immune system. It appears that higher-dose/shorter duration TKI treatment may spark a more potent anti-tumor response by better triggering the innate immunity system.”

In one series of experiments, the researchers found that tumors were completely resistant to anti-PD-L1 therapy alone, so-called monotherapy. When HypoTKI was given by itself, it markedly reduced tumor burden initially, but all tumors relapsed eventually.

When the two treatments were given concurrently and early, it resulted in the best tumor regression and most mice showed no relapse over more than two months of follow-up. Tumor relapse in mice usually occurs within two to three weeks.

“The maximum synergistic effect of combination therapy depends on the timing of the anti-PD-L1 treatment. When it was started within three days of TKI administration, there was total tumor regression and only two of eight tumors relapsed,” Dr. Fu said.

Almost no synergistic effect was seen if PD-L1 inhibitors were provided a week after TKI therapy.

“Our study demonstrates that HypoTKI and PD-L1 blockade can work in combination to effectively control advanced large tumors, increase overall survival, and reduce tumor relapse. These data suggest that proper timing — giving the two agents together at the start of treatment — provides maximum synergistic anti-tumor effects in these tumors and should be studied further,” Dr. Fu said.

UTSW co-authors include Research Associate Dr. Chunbo Dong, Drs. Zhenhua Ren, Changzheng Lu, Longchao Liu, Anli Zhang, all postdoctoral researchers in the Fu laboratory; Eric Hsu and Casey Timmerman, both students in the Medical Scientist Training Program that grants dual M.D./Ph.D. degrees; Dr. Mingyi Chen, Associate Professor of Pathology; and Dr. Jian Qiao, Assistant Professor of Pathology. Former laboratory manager Yang Wang and former postdoctoral researchers Aijun Shen, and Yang Pu also participated.

The study received support from the National Institutes of Health and the Cancer Prevention and Research Institute of Texas (CPRIT).