Construction and Biological Evaluation of Small Libraries Based on the Intermediates within the Total Synthesis of Uvaretin.

Discovering therapeutic agents: New bioactive agents, either as sole or combinational agents, have been constructed through the synthetic manipulation of the intermediates within the total synthesis of the uvaretin class of natural products. It was found that increasing the hydrophobic character of the phenolic core correlates to a decrease in sole agent cytotoxicity. The synthesis of new, small chemical screening libraries (CSL) constructed from the intermediates of our total synthesis route of the uvaretin class of natural products is demonstrated herein. Numerous chalcone-based CSLs with various substitution on the phenolic groups within the chalcone core were assembled. Through cytotoxicity investigations, it was found that the level of hydrophobicity of the phenolic core of the chalcones gives biases: less cytotoxicity with more hydrophobic cores. In addition, it was observed that the potentiation, evaluated with 6-thiopurine in the pancreatic cancer cell line MIA PaCa-2, is tunable by the inclusion of less-hydrophobic character on the phenolic core. The role of the o-hydroxybenzyl group, present within the uvaretin family, was revealed to be cytotoxic in character. Merging all of the structure-activity relationship studies performed on the CSLs constructed in this effort led to the construction of a new chalcone hybrid possessing both a cytotoxic enone group and a small-molecule-potentiating, reduced enone group.

The winding road of the uvaretin class of natural products: from total synthesis to bioactive agent discovery.

Herein, we disclose the development of a synthetic route to gain access to the uvaretin class of chalcone natural products. In this, the construction of a small library was achieved, and the collection was evaluated for cytotoxicity and other biological properties. Uvaretin (1) was accessed via a seven-step route in an overall yield of 15.1%. Within this route, the unsaturated enone variant of uvaretin (2), also a natural product, was accessed in a 16.7% yield over six steps. This route provides a nearly three-fold increase in yields of 1 and 2 in comparison to the previous synthetic route accessing them in 5.8% and 3.0% overall yields, respectively. Evaluation of 1 and 2 revealed IC50 values between 2.0 and 5.1 µM in the cancerous cell lines HeLa, U937, A549, and MIA PaCa-2. Screening of the whole chalcone library set led to the discovery of over 30 compounds, within six cancerous cell lines, possessing single digit µM IC50 activity as sole agents. Furthermore, multiple library members were found to possess promising potentiating properties with known chemotherapeutic agents.

New methods to assess 6-thiopurine toxicity and expanding its therapeutic application to pancreatic cancer via small molecule potentiators.

6-Thiopurine (6TP) is a potent cytotoxic agent that is a clinically prescribed anti-metabolite employed in the treatment of numerous blood cancers since 1952. However, its reported severe toxicities limit its general usage in the clinic. We previously have undertaken investigations into identifying the mode of toxicity for 6TP, and have found that the oxidative metabolites of 6TP, specifically 6-thiouric acid (6TU), is responsible for the in vitro inhibition of UDP-glucose dehydrogenase (UDPGDH) in a UV-vis method. In this method, inhibition was quantified through the quantification of NADH production, however, purines absorb at the same wavelength and thereby can interfere with the NADH detection. Herein, we report a HPLC method that allows for direct quantification of UDP-glucuronic acid, product from UDPGDH, for the assessment of inhibition towards UDPGDH with no interference from purines. In this method it was revealed that 6TP possesses a greater inhibitory properties than previously observed; 111 vs. 288 µM. Building upon the data collected from a previously performed rat hepatocyte study, which correlated our in vitro to in vivo inhibition theories about UDPGDH, we have developed a bio-mimic in vitro assay to aid in the inhibitory assessment of 6TP and analogs. In our efforts to expand the use of 6TP, and analogs constructed, our laboratory has undertaken a screening campaign to identify small molecule potentiators that work in synergy with 6TP in other types of cancers. Three chalcone-based compounds have been discovered through our total synthesis campaign of uvaretin, and it has been found that 11c has strong synergism with 6TP in the pancreatic cancer cell line MIA PaCa-2. Through the work presented herein, we reveal new methods to assess toxicity of 6TP and future analogs and new small molecules that work in synergy to expand the therapeutic applications of this neglected cytotoxic agent.

Efforts in redesigning the antileukemic drug 6-thiopurine: decreasing toxic side effects while maintaining efficacy.

6-Thiopurine (6TP) is a currently prescribed drug in the treatment of diseases ranging from Crohn's disease to acute lymphocytic leukemia. While its potent mode of action is through incorporation into DNA as a thiol mimic of deoxyguanosine, severe toxicities are associated with its administration which hinder the potential therapeutic application. We have previously reported in vitro that the oxidative metabolites of 6TP, specifically 6-thiouric acid (6TU, K i 7 µM), are potent inhibitors of UDP-glucose dehydrogenase (UDPGDH), an enzyme that is responsible for the formation of UDP-glucuronic acid (UDPGA), an essential substrate that is used in detoxification processes in the liver. An in vivo investigation was undertaken to probe if 6TU inhibits UDPGDH in rat hepatocytes, and it was observed that 6TU does greatly suppress the conjugation of bilirubin with UDPGA. The failed excretion of bilirubin is linked to a majority of the reported toxicities associated with 6TP administration. Efforts were undertaken for the construction of 6TP analogs, substituted at the C8 position, to reduce inhibition of UDPGDH while retaining therapeutic efficacy. Three new 6TP analogs bearing a halogen (Br, Cl, and F) at the C8 position have been achieved over five-synthetic steps in overall yields of 16 to 32%. Each of these analogs were shown to have reduced inhibition towards UDPGDH, with K i values of 192, 163, 215 µM, respectively. In addition, the bromine, chlorine, and fluorine analogs were shown to possess cytotoxicity towards the REH cell line (acute lymphocytic leukemia) having IC50 values of 9.54 µM (±0.97), 3.95 µM (±1.94), and 4.71 µM (±1.40), respectively. These three new 6TP analogs represent the first steps in the redesign of this potent anticancer agent into a better drug that possesses reduced toxic side effects while retaining therapeutic potency.

Inhibition of UDP-glucose dehydrogenase by 6-thiopurine and its oxidative metabolites: Possible mechanism for its interaction within the bilirubin excretion pathway and 6TP associated liver toxicity.

6-Thiopurine (6TP) is an actively prescribed drug in the treatment of various diseases ranging from Crohn's disease and other inflammatory diseases to acute lymphocytic leukemia and non-Hodgkin's leukemia. While 6TP has beneficial therapeutic uses, severe toxicities are also reported with its use, such as jaundice and liver toxicity. While numerous investigations into the mode in which toxicity originates has been undertaken. None have investigated the effects of inhibition towards UDP-Glucose Dehydrogenase (UDPGDH), an oxidative enzyme responsible for UDP-glucuronic acid (UDPGA) formation or UDP-Glucuronosyl transferase (UGT1A1), which is responsible for the conjugation of bilirubin with UDPGA for excretion. Failure to excrete bilirubin leads to jaundice and liver toxicity. We proposed that either 6TP or its primary oxidative excretion metabolites inhibit one or both of these enzymes, resulting in the observed toxicity from 6TP administration. Inhibition analysis of these purines revealed that 6-thiopurine has weak to no inhibition towards UDPGDH with a Ki of 288 µM with regard to varying UDP-glucose, but 6-thiouric (primary end metabolite, fully oxidized at carbon 2 and 8, and highly retained by the body) has a near six-fold increased inhibition towards UDPGDH with a Ki of 7 µM. Inhibition was also observed by 6-thioxanthine (oxidized at carbon 2) and 8-OH-6TP with Ki values of 54 and 14 µM, respectively. Neither 6-thiopurine or its excretion metabolites were shown to inhibit UGT1A1. Our results show that the C2 and C8 positions of 6TP are pivotal in said inhibition towards UDPGDH and have no effect upon UGT1A1, and that blocking C8 could lead to new analogs with reduced, if not eliminated jaundice and liver toxicities.

Total Synthesis of Reniochalistatin E.

Reniochalistatin E (1) is one of the five related cyclic peptides isolated from the marine sponge Reniochalina stalagmitis. The discovery of these compounds resulted from a screening program directed toward the identification of proline-rich bioactive compounds. Reniochalistatin E is the only member of the family to possess a tryptophan amino acid residue. Given the cytotoxicity observed for 1, efforts were directed toward developing a synthetic route to 1. The first total synthesis of 1 has been accomplished in a 15-step route in an overall 5.0% yield. The synthetic sample of reniochalistatin E was shown to have similar activity toward HeLa and RPMI-8226 cell lines compared to the natural sample, with IC50 values of 16.9 vs 17.3 µM and 4.5 vs 4.9 µM, respectively. Interestingly, both of the fully deprotected octapeptides constructed toward the synthesis of reniochalistatin E were shown to have cytotoxicity. The route provides a means to probe the structure-activity relationship of 1 and further biological investigations.