Both NMS-P937 and the deuterated version, PR00012, were synthesized at ChemPartner (Pudong, Shanghai, China). For cell culture and kinase assays, the compounds were dissolved in DMSO and diluted at a 3-fold or 5-fold gradient starting from a concentration of 10 μM, to achieve the final concentrations for the assays.

Human pancreatic cancer cell lines (PSN1, PANC-1, Panc 04.03, MIA PaCa-2, BxPC-3, and Capan-1) were cultured in their respective culture media. PSN1 cell lines were maintained in RPMI 1640 + 10%FBS. MIA PaCa-2 cells were cultured in DMEM+10%FBS+5% horse serum. Panc 04.03 cells were grown in RPMI 1640 + 15%FBS+10μg/mL Insulin. PANC-1 cells were maintained in DMEM+10%FBS. BxPC-3 cells were cultured in RPMI 1640 + 10%FBS. Capan-1 cells were grown in IMDM+20%FBS. All cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂.

For assays, cells in the logarithmic growth phase were harvested, seeded to different concentrations at 2,000-6,000 cells/well in 96-well plates. Following overnight incubation, test compounds were added at various concentrations and incubated for 72 hours. Cell viability was assessed by the ATP concentration using the CellTiter-Glo reagent (Promega, Madison, WI 53711 USA), and luminescence values were recorded by a Multilabel Reader (PerkinElmer, Waltham, MA, USA).

Test compounds PR000012 and NMS-P937 were dissolved in DMSO and thoroughly mixed by vortexing. Serial dilutions in DMSO were prepared. PLK1 was serially diluted 5-fold from 10 μM, yielding concentrations from 10000 nM to 0.00512 nM. Other targets were serially diluted 3-fold from 10 μM, yielding concentrations from 10000 nM to 0.5 nM. We have used two kinase assays: HTRF assay and ADG-Glo assay.

For HTRF Assay, 2× ATP/substrate solution and 2× kinase solution were prepared in kinase reaction buffer. Using an Echo 655, 50 nL of each diluted compound was transferred to a 384-well detection plate. The plate was centrifuged at 1000 rpm for 1 minute. 2.5 μL of 2× kinase solution was added to each well. The plate was incubated at 25°C for 10 minutes. 2.5 μL of 2× ATP/substrate solution was added to each well. The plate was centrifuged at 1000 rpm for 1 minute. The plate was incubated at 25°C for 40 minutes. 2× XL665 and antibody detection reagent were prepared in detection buffer. 5 μL of the prepared kinase detection reagent was added to each well. The plate was centrifuged at 1000 rpm for 1 minute. The plate was incubated at 25°C for 1 hour. Fluorescence signals at 620 nm (Cryptate) and 665 nm (XL665) were measured using a multifunction plate reader.

For ADP-Glo Assay, 2× ATP/substrate solution and 2× kinase solution were prepared in kinase reaction buffer. Using an Echo 655, 40 nL of each diluted compound was transferred to a 384-well detection plate. The plate was centrifuged at 1000 rpm for 1 minute. 2 μL of 2× kinase solution was added to each well. The plate was incubated at 25°C for 10 minutes. 2 μL of 2× ATP/substrate solution was added to each well. The plate was centrifuged at 1000 rpm for 1 minute. The plate was incubated at 25°C for 60 minutes. 4 μL of ADP-Glo reagent was added to each well. The plate was centrifuged at 1000 rpm for 1 minute. The plate was incubated at 25°C for 40 minutes. 8 μL of detection solution (presumably ADP-Glo detection solution) was added to each well. The plate was centrifuged at 1000 rpm for 1 minute. The plate was incubated at 25°C for 40 minutes. Luminescence signals were measured using a multifunction plate reader.

Approximately 1.0 mg of each compound was weighed into three separate 1.5 mL glass vials. To each vial, 1000 µL of assay buffer was added to achieve a final concentration of 1 mg/mL. A PTFE-encapsulated stainless steel stick stirrer was placed in each vial, which was then sealed with a molded PTDE/SIL silicone plug. The sample plate was shaken at 1100 rpm at 25°C for 24 hours. Post-incubation, the samples were filtered, and 10 µL of the samples was mixed with 10 µL of DMSO. The mixture was then added to 980 µL of methanol, then further diluted 10-fold with methanol:water (1:1) for LC-MS/MS analysis.

Human and mouse liver microsomes in PBS were added to a 96-well incubation plate along with 1 mM NADPH solution and 2 mM UDPGA solution. The reaction was initiated by adding 2 μL of the compound working solution to the plate, carried out at 37°C with shaking at 60 rpm. At 0.5, 15, 30, 45, and 60 minutes, 50 μL samples were transferred to a sample plate containing 200 μL of cold methanol with internal standards (50 nM alprazolam, 50 nM labetalol, and 100 nM ketoprofen). The sample plate was centrifuged at 3,220 g for 40 minutes. Subsequently, 100 μL of the supernatant was transferred to an analysis plate containing water for LC-MS/MS analysis.

Caco-2 cells were cultured in 10% FBS DMEM for 14-21 days before assays. For A-to-B direction, 5 μM compound solution was added to the upper chamber; for B-to-A, to the lower chamber. HBSS was added to the opposite chamber. After 2 hours incubation at 37°C, samples were analyzed by LC-MS/MS. Fluorescein leakage was tested by adding 100 μM fluorescein to the upper chamber and measuring fluorescence after 30 minutes.

Female CD1 mice (6-8 weeks old, 20-30 g) were obtained from SPF Biotechnology Technology Co., Ltd. (Beijing, China). The study was conducted at ICE Bioscience Inc (Beijing, China). Compounds were diluted in a vehicle of 10% DMSO + 15% Solutol HS + 75% (20% SBE-β-CD) for intravenous injection into the mouse tails at a concentration of 10 mg/kg. For oral administration, compounds were diluted in a vehicle of 0.5% MC + 1% Tween-20 with a final dosage of 10 mg/kg. All mice were fasted before dosing. Each route included three animals. Blood samples were collected at 0, 5, 15, 30 minutes; 1, 2, 4, 7, and 24 hours, using EDTA-K2 as the anticoagulant. Samples were centrifuged at 4,000 g, 4°C for 5 minutes, and the plasma were collected and stored at -75°C. The compound concentration in the plasma were analyzed by LC-MS/MS.

For in vivo PK/PD studies, a PSN1 tumor model was established in female M-NSG (NOD.Cg-Prkdc^scidIl2rg^em1Smoc ) mice, which are severely immunodeficient mice lacking the interleukin 2 receptor gamma chain on a NOD.Cg-Prkdc^scid background, at Shanghai Model Organisms (Shanghai, China). Briefly, 5 × 10⁶ PSN1 cells were inoculated subcutaneously into each M-NSG mouse. Once the tumors reached approximately 500 mm³, compounds were administered orally at various dosages. For PK studies, blood samples were collected at 0, 0.25, 0.5, 1, 2, 6, and 24 hours post-dose. The sera from blood were then obtained and stored at -70°C. For PD studies, tumors were harvested 2 hours after dosing and stored at -70°C.The serum and tumor samples were then transported to PRECEDO (Hefei, Anhui, China) for further PK/PD studies.

For PK studies, serum and tumor samples were processed with the internal standard solution. Serum samples were centrifuged and mixed directly, while tumor samples were homogenized first. Supernatants were prepared for LC-MS/MS analysis. Compound concentrations were determined using LC-MS/MS with tolbutamide as the internal standard, employing a reverse-phase column and ESI on a tandem quadrupole mass spectrometer. Quantitative ranges were 2.00-2000 ng/mL for PR00012 and NMS-P937. Data were processed using AnalystTM 1.7.2 and Excel 2010, with pharmacokinetic parameters calculated using WinNonlin 8.2.

For PD study, tumor tissues (~30 mg) were homogenized in 350 µL Lysis Buffer. After centrifugation, protein concentration in supernatants was determined by BCA assay. Samples were prepared for SDS-PAGE and run on 10% gels. Proteins were transferred to PVDF membranes, blocked, and incubated with anti-Phospho-TCTP (Ser46) Antibody (Cell Signaling Technology, Danvers, MA, USA) (1:1,000) overnight at 4°C. After washing, membranes were incubated with HRP-anti-Rabbit IgG (Cell Signaling Technology, Danvers, MA, USA) (1:3,000) for 1 hour, then treated with ECL reagents for imaging.

This study was conducted at Shanghai Model Organisms (Shanghai, China). Female M-NSG mice, averaging 25 g in body weight, were randomly divided into 3 groups, each receiving one of the following treatments: 60 mg/kg for NMS-P937, 60 mg/kg and 120 mg/kg for PR00012. Oral gavage was chosen to match the clinical route, with dosing once daily for 6 days. Mice were monitored for an additional 18 days.

Rat toxicity was studied at JOINN Laboratories (Suzhou, Jiangsu, China). Sprague-Dawley rats were randomly divided into 2 groups, each receiving 15 mg/kg at 1.5 mg/mL, with 3 female animals per group. Group 1 received PR00012, and Group 2 received NMS-P937. Oral gavage was chosen to match the clinical route, with dosing once daily for two weeks at 15 mg/kg. Using 2 mL sterile syringes and gavage tubes, animals were dosed based on their most recent body weight, with volumes rounded to one decimal place. Test compounds were stirred continuously for at least 15 minutes before and during administration. The rats were dosed once a day for 14 days.

These studies were performed at Yikon Medical (Beijing, China). Cancer cell line HCT 116 or BxPC-3 were cultured in 10% FBS RPMI-1640 medium plus penicillin, streptomycin, and glutamine at 37°C and 5% CO2 until reaching confluency. Cells were then prepared for inoculation. 5×10⁷ cells/mL cells in 100 µL were inoculated subcutaneously into the right flank of each female BALB/c nude mouse for HCT 116 cell line or female NOD SCID mouse for BxPC-3 cell line. When tumors reached 100-150 mm³, mice were divided into 4-5 groups (6-7 mice/group). Different groups were dosed with Vehicle, PR00012 or NMS-P937 at various concentrations. Mice were dosed at least 10 days and were monitored for 30 days.

TGI was calculated as follows. TGI(%)=100×(1−ΔT/ΔC), ΔT represents the volume change for treated tumors and ΔC represents the volume change for control tumors.

All statistical analyses were conducted using Prism 10.1.2 (GraphPad). Extra sum-of-squares F test was used. Data was considered statistically significant at p < 0.05.

The molecule of PR00012 was generated by replacing all the hydrogen atoms with deuterium on piperazine of the molecule NMS-P937 ( Figure 1 ).

We compared the inhibitory function of PR00012 and its non-deuterated form, NMS-P937, against six human pancreatic cancer cell lines. Cell lines were plated in 96-well plates and cultured overnight. Test compounds were then added at various concentrations in triplicates and incubated for 72 hours. Cell viability was finally assessed. The data demonstrated the dose-dependent inhibitory effects of the compounds. However, there was no significant difference between PR00012 and NMS-P937 ( Figure 2 ). Also summarized in Table 1 , PR00012 displayed similar or slightly higher IC₅₀ value compared to NMS-P937, with no significant differences.

Next, we investigated the kinase inhibition ability of PR00012 and NMS-P937 against 416 kinases at the concentration of 0.1 μM. As shown in the kinase trees, the deuterated PR00012 and NMS-P937 exhibited similar kinase activities ( Figures 3A, B ). The correlation in the inhibitory percentage between PR00012 and its non-deuterated counterpart, NMS-P937, was substantial ( Figure 3C ), confirming their similarities in receptor binding affinity. As depicted in Figure 3C , the correlation between these two compounds was robust, with an R² value of 0.6392, underscoring their highly similar pharmacological profiles. There are only one notable outlier, which is tyrosine-phosphorylation-regulated kinase 2 (DYRK2) ( Figure 3C ).

We further performed a dose-dependent assay for those kinases inhibited for more than 40% by PR00012 at 0.1 μM to determine their IC₅₀ value ( Table 2 ). For the 15 kinases tested, PR00012 had a similar IC₅₀ value to NMS-P937 among 14 kinases. However, the kinase activity of DYRK2 was significantly inhibited by PR00012, but not NMS-P937. The IC₅₀ of NMS-P937 for DYRK2 was about 14-fold higher than PR00012. The function of DYRK2 in tumorigenesis is not clear, it may function either as a promoter or inhibitor of cancer, depending on the organ context (20). However, DYRK2 Inhibitor has been developed for the treatment of prostate cancer (21). Furthermore, DYRK2 was targeted by a small molecule LDN192960 for the treatment of for triple-negative breast cancer and multiple myeloma (22). In addition, high expression of DYRK2 led to lower survival probability in pancreatic cancer, breast cancer, and renal cancer, among others (proteinatlas.org).

The metabolic stability was assessed by assays using human and mouse liver microsomes. PR00012 exhibits a slightly longer T_1/2 (half-life) and a slightly slower clearance rate in both human and mouse liver microsomes than NMS-P937 ( Table 3 ). In human liver microsomes, the T_1/2 was 90 minutes and 86 minutes for PR00012 and NMS-P937, respectively ( Table 3 ). The T_1/2 in mouse liver microsomes was 76 minutes and 66 minutes for PR00012 and NMS-P937, respectively ( Table 3 ). The intrinsic clearance (CLint) in human liver microsomes was 15 and 16 μL/min/mg protein for PR00012 and NMS-P937, respectively ( Table 3 ). In mouse liver microsomes, the CLint was 18 and 21 μL/min/mg protein for PR00012 and NMS-P937, respectively ( Table 3 ).

​The Caco-2 permeability assay is critical for evaluating the intestinal absorption of compounds as well as the compound permeability in tissues. AS shown in Table 4 , by comparing Propranolol, Digoxin, and Prazosin to our test compounds, PR00012 and NMS-P937 are characterized as candidate drugs with medium permeability. Their efflux ratios are similar, and the recoveries showed no significant difference.

As previously described, the lower energy state of the carbon-deuterium (C-D) bond hinders the ability of drug transporters and enzymes to metabolize the compound efficiently, thereby reducing its rate of metabolism (3). Deuterated drugs, which incorporate deuterium in place of hydrogen, take advantage of this kinetic isotope effect. Deuterium, being twice as heavy as hydrogen, forms stronger C-D bonds compared to C-H bonds. This subtle change can significantly impact the pharmacokinetics of the drug, including its absorption, distribution, metabolism, and excretion (ADME) properties (7).

Since our compound is intended to treat cancer patients such as those with pancreatic cancer, it is important to evaluate the PK and PD properties of PR00012 and NMS-P937 in tumor-bearing mice.

The PSN-1 cell line, derived from a human pancreatic adenocarcinoma, is characterized by specific genetic mutations that are significant for cancer research. A critical mutation present in the PSN-1 cell line is a point mutation in the KRAS gene. This mutation involves a substitution from GGT to CGT at codon 12 of the gene, which is presented at KRAS G12R, leading to a 3 to 6-fold amplification of KRASmRNA ( 23). In addition, PSN-1 cell lines were previously found to have 50-fold amplification of c-Myc mRNA (23), while the aberrant high expression of c-Myc has been associated with more aggressive cancer phenotypes.

Here we established PSN-1 CDX model in M-NSG mice. M-NSG mice are a modified version of the NOD scid gamma (NSG) mouse strain. These mice lack mature T cells, B cells, and natural killer (NK) cells. M-NSG mice further enhance this immunodeficiency, enabling even higher efficiency in the engraftment of human cells. The PNS-1 tumor-bearing mice were dosed with PR00012 and NMS-P937 at 60 mg/kg, 30 mg/kg or 15 mg/kg, and the blood samples were collected at seven time points. The compound concentration in the blood were determined and theT_1/2 was calculated. As shown in Figure 4 , the T_1/2 of PR00012 was consistently higher across all three concentrations than NMS-P937. When mice were dosed at 60 mg/kg, the T_1/2 were 5.5 h and 2.4 h for PR00012 and NMS-P937, respectively ( Figure 4 ). Similar data were observed after mice were dosed at 30 mg/kg and 15 mg/kg.

Thus, PR00012, a deuterated NMS-P937 compound, demonstrates slightly high T_1/2 in both in vitro liver microsomes and in vivo tumor-bearing mice.

To thoroughly evaluate pharmacodynamic effect of PLK1 inhibition in vivo, tumors from the PSN-1 xenograft tumor model in M-NSG mice were collected at 2 and 6 hours following a single oral dose of the compounds at varying concentrations (60, 30, and 15 mg/kg). Western blot analysis was performed to assess the levels of p-TCTP, a direct substrate of PLK1. Phosphorylation of TCTP has been demonstrated as a marker for PLK1 activity in vivo (15, 24).

After 2-hour in vivo treatment, both PR00012 and NMS-P937 exhibited reduced p-TCTP signal, indicating a decrease in PLK1 activity ( Figure 5A ). Notably, PR00012 treatment significantly reduced TCTP phosphorylation compared to NMS-P937 treated group, particularly at the lower doses. This suggests that PR00012 has a stronger PLK1 inhibition capability. Six hours after administration of the PLK1 inhibitors, p-TCTP levels from both drugs began to return to baseline ( Figure 5B ), demonstrating a time-dependent PLK1 inhibition effect.

In summary, both PR00012 and NMS-P937 effectively inhibit PLK1 activity in vivo, with PR00012 demonstrating a stronger inhibition of PLK1 in PSN-1 xenograft tumors. These findings suggest that PR00012 may offer enhanced therapeutic efficacy in targeting PLK1-related pathways for tumor growth suppression.

We initially aimed to investigate the in vivo toxicity and efficacy using M-NSG mice. Mice were received oral administration of both compounds for the first six days, followed by an observation period until the 18th day. In the group treated with NMS-P937 at a dose of 60 mg/kg, all three mice significantly lost body weight, and eventually died before day 9 ( Figure 6 ). Conversely, in the group treated with PR00012 at a double dosage of NMS-P937 (120 mg/kg), only two out of three mice died before day 11. Interestingly, when PR00012 was administered at 60 mg/kg, the same dosage of NMS-P937, all mice survived throughout the observation period without significant loss of body weight.

In addition, in our CDX efficacy models, we observed higher mortality rates in NMS-P937-treated mice compared to those treated with deuterated PR00012, regardless of whether the mice were from BALB/c nude or NOD SCID background ( Figures 7A, B ). In the HCT 116 CDX studies, there were three mice died in the NMS-P937 (60 mg/kg)-treated group while only one mouse died in the PR00012 (60 mg/kg)-treated group. No mice died in the PR00012 (30 mg/kg) group, whereas one mouse died when dosed with NMS-P937 at 30 mg/kg ( Figure 7A ). In the BxPC-3 CDX studies, two mice died in the NMS-P937 dosed group while only one mouse died in PR00012 dosed group ( Figure 7B ). Furthermore, when mice dosed at 60 mg/kg, the body weights were significantly lower in the NMS-P937-dosed group ( Figures 7A, B ).

We extended our toxicity studies to include a rat model. Initially, we evaluated the response of different dosages of PR00012 in Sprague-Dawley rats. At 10 mg/kg, the rats were still tolerable (data not shown).

Subsequently, we conducted a 14-day repeated administration toxicity study. Rats were orally dosed with PR00012 or NMS-P937 at 15 mg/kg once a day. After 9-day dosing, all three rats treated with PR00012 survived, while 1 out of 3 rats treated with NMS-P937 died ( Figure 8 ). The rat that died exhibited severe symptoms, including arching back, watery diarrhea, decreased spontaneous activity, pale ears, perianal soiling, ruffled fur, yellowing of fur around the groin, urethra, and vaginal area, and weight loss (data not shown). The body weight of NMS-P937-treated rats were also lower than PR00012-treated group ( Figure 8 ). Upon completion of the 14-day dosing, all PR00012-treated rats remained alive.

These findings suggest that PR00012 possesses a more favorable safety profile compared to NMS-P937, particularly when administered at the same dosage level. The improved survival rates and minimal weight impact highlight PR00012’s potential as a safer therapeutic option.

CDX models are essential in preclinical oncology research. They allow for the evaluation of anti-cancer therapies through the implantation of in vitro cultured human cell lines into immunocompromised mice, creating a tumor-bearing system.​ These models facilitate the assessment of drug efficacy, pharmacokinetics, and the underlying mechanisms of action in an in vivo environment. CDX models are valued for their reproducibility and ability to mimic human tumor characteristics, providing a controlled environment to test various therapeutic agents and analyze tumor growth kinetics. This makes them a practical choice for preclinical evaluation.

We first established CDX model in BALB/c nude mice using HCT116 cells, a human colon cancer cell line. Our compounds were tested after the models were established. Both PR00012 and NMS-P937 inhibited the growth of tumors in a dose-dependent manner. The TGI of PR00012 (60 mg/kg)-treated group was slightly better than that of NMS-P937-treated group ( Figure 9A ). In addition, more mice died in the NMS-P937-treated groups, and there was significant loss of body weight in the NMS-P937 (60 mg/kg)-treated group ( Figure 7A ).

For pancreatic cancer cell lines, we have established two CDX models: one with deletion of the SMAD4 gene and the other from a KRAS mutation cell line.