Ugi Adducts as Novel Anti-austerity Agents against PANC-1 Human Pancreatic Cancer Cell Line: A Rapid Synthetic Approach

Abstract

Pancreatic cancer cells have an inherent tolerance to withstand nutrition starvation, allowing them to survive in hypovascular tumor microenvironments that lack of sufficient nutrients and oxygen. Developing anti-cancer agents that target this tolerance to nutritional starvation is a promising anti-austerity strategy for eradicating pancreatic cancer cells in their microenvironment. In this study, we employed a chemical biology approach using the Ugi reaction to rapidly synthesize new anti-austerity agents and evaluate their structure–activity relationships. Out of seventeen Ugi adducts tested, Ugi adduct 11 exhibited the strongest anti-austerity activity, showing preferential cytotoxicity against PANC-1 pancreatic cancer cells with a PC₅₀ value of 0.5 µM. Further biological investigation of Ugi adduct 11 revealed a dramatic alteration of cellular morphology, leading to PANC-1 cell death within 24 h under nutrient-deprived conditions. Furthermore, the R absolute configuration of 11 was found to significantly contribute to the preferential anti-austerity ability toward PANC-1, with a PC₅₀ value of 0.2 µM. Mechanistically, Ugi adduct (R)-11 was found to inhibit the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway preferentially under nutrition starvation conditions. Consequently, Ugi-adduct (R)-11 could be a promising candidate for drug development targeting pancreatic cancer based on the anti-austerity strategy. Our study also demonstrated that the Ugi reaction-based chemical engineering of natural product extracts can be used as a rapid method for discovering novel anti-austerity agents for combating pancreatic cancer.

INTRODUCTION

Tumor cells require a large amount of energy and nutrients to synthesize biomacromolecules essential for proliferation. Because of the heterogeneous nature of tumor growth, the microenvironment contains niches that exhibit significant gradients of essential nutrients, oxygen, and growth factors.¹⁾ Tumor cells generally depend on angiogenesis to meet their nutrient requirements, and consequently, tumors often possess a large number of blood vessels surrounding them. In contrast to other tumor types, pancreatic tumor cells usually lack an appropriate vasculature system.²⁾ However, without relying on angiogenesis, the tumor cells in their microenvironment can still survive by tolerating nutrition starvation, a distinctive survival mechanism known as ‘austerity’ in cancer biology.³⁾ Eliminating this tolerance to nutrition starvation by tumor cells could selectively kill cancer cells within the tumor microenvironment. The discovery and development of agents that can inhibit the tolerance of cancer cells to nutrition starvation represent a unique anti-austerity strategy in pancreatic cancer therapy. An anti-austerity agent exhibits selective cytotoxicity under nutrient-deprived conditions while not displaying cytotoxicity under normal-nutrient conditions. Following the pioneering discovery of arctigenin (1) as a potent anti-austerity agent against human pancreatic cancer cells PANC-1,³⁾ several anti-austerity agents have been successfully discovered from natural products,^4–16) natural product derivatives,^17–19) and synthetics²⁰⁾ (Fig. 1). Most of these anti-austerity agents are hydrocarbons with some oxygen functional groups. Recently, anti-austerity agents have also been identified from nitrogen-containing compounds such as naturally occurring^21,22) or synthetic amides^23,24) (Fig. 2); these preliminary findings have encouraged us to investigate further structure–activity relationships of the anti-austerity activity of nitrogen-containing compounds.

Fig. 1. Chemical Structures of Arctigenin (1) (a), Recently Discovered Anti-austerity Agents from Natural Products (b), Natural Product-Derived Molecule (c), and Synthetic (d)

Fig. 2. Chemical Structures of Nitrogen-Containing Anti-austerity Agents

The Ugi four-component reaction is a one-step synthetic method of an α-acylamino amide from four different functional group components: an aldehyde or ketone, a primary amine, a carboxylic acid, and an isocyanide.^25,26) This step-economical synthetic method rapidly provides a set of structurally diverse compounds, which can be used to screen for structure–activity relationships and discover anti-austerity agents. During our recent study for the development of the Ugi reaction using natural product extracts as substrates, we have successfully synthesized 17 structurally diverse nitrogen-containing Ugi adducts including natural product-derived molecules in a single operation²⁷⁾ (Chart 1). In this study, the anti-austerity activity of these Ugi adducts against PANC-1 human pancreatic cancer cell lines was examined following the anti-austerity strategy.

Chart 1. One-Pot Synthesis of Structurally Diverse Nitrogen-Containing Ugi Adducts

RESULTS AND DISCUSSION

Structure–Activity Relationship of Synthetic Ugi Adducts

First, a set of test compounds, 9–19, were synthesized by the Ugi reaction from petrochemicals²⁷⁾ (Fig. 3). These compounds were then tested for their cytotoxic activity against the PANC-1 cell line in both nutrient-deprived medium (NDM) and nutrient-rich medium (Dulbecco’s Modified Eagle Medium, DMEM) following the anti-austerity strategy. The anti-austerity activity was expressed as the PC₅₀ value, which is the concentration at which 50% of cancer cells were preferentially killed in the NDM without causing apparent toxicity in the DMEM (Table 1). As a result, the Ugi-adduct 9, which was derived from benzaldehyde, benzylamine, acetic acid, and cyclohexyl isocyanide, showed no cytotoxicity under both DMEM and NDM conditions (entry 1). Replacing the methyl group of 9 with a cyclohexyl group displayed an enhanced cytotoxicity with a PC₅₀ value of 9.9 µM preferentially under NDM conditions (entry 2). The Ugi-adduct 11, containing a chloroacetyl group, displayed the most potent preferential cytotoxicity with a PC₅₀ value of 0.5 µM in NDM and a good selectivity index (SI) of 15 between the IC₅₀ in DMEM and PC₅₀ in NDM (entry 3 and Fig. 4). This potency of Ugi-adduct 11 was comparable to arctigenin (1) (PC₅₀ 0.7 µM), a well-established anti-austerity agent (positive control). On the other hand, the PC₅₀ of Ugi-adduct 12, derived from cyclopentanone, was weaker than that of 11, while the IC₅₀ in DMEM was stronger than that of 11, representing the conventional cytotoxicity (entry 4). Similarly, changing the phenyl group of 13 to a cyclopentyl group diminished the activity in NDM and enhanced the activity in DMEM (entries 5 and 6). Aliphatic or partially unsaturated cyclohexyl motifs did not improve the cytotoxic activity in NDM or the SI (entries 7–9). Finally, introducing highly hydrophobic 1-pyrenyl or steroidal groups completely diminished the activity against PANC-1 cell both under DMEM and NDM conditions (entries 10 and 11). Thus, the screening of this small set of diverse nitrogen-containing compounds displayed clear structure–activity relationships and revealed the importance of both the chloroacetyl motif and α-phenylglycine unit for preferential anti-austerity activity in NDM.

Fig. 3. Chemical Structure of the Synthetic Ugi Adducts 9–19

Table 1. Preferential Cytotoxicity of the Synthetic Ugi Adducts 9–19 against the Dulbecco’s Modified Eagle’s Medium (DMEM) and the PANC-1 Human Pancreatic Cancer Cell Line in Nutrient-Deprived Medium (NDM)

Entry	Compound	IC50 (µM) in DMEMa)	PC50 (µM) in NDMb)	SIc)
1	9	>100	>100	—
2	10	>100	9.9	>10
3	11	7.4	0.5	15
4	12	0.9	1.9	0.47d)
5	13	29	1.3	22
6	14	9.7	5.0	1.9
7	15	9.3	7.8	1.2
8	16	4.5	0.7	6.4
9	17	5.9	0.8	7.4
10	18	>100	>100	—
11	19	>100	>100	—
12	Arctigenin (1)e)	>100	0.7	>100

a) Concentration at which 50% of cells were killed in DMEM. b) Concentration at which 50% of cells were killed preferentially in NDM. c) Selectivity index: IC₅₀ in DMEM/PC₅₀ in NDM. d) Compound 12 was more cytotoxic in DMEM than in NDM, and thus represented conventional cytotoxic agent. e) Positive control.

Fig. 4. Preferential Cytotoxicity of 11 against PANC-1 Cell Lines in NDM (Red Circle) and DMEM (Blue Circle)

Structure–Activity Relationship of Natural Product-Derived Ugi Adducts

Another set of test compounds, comprising natural product-derived Ugi-adducts 20–24 and a natural product hybrid 25, were synthesized by the Ugi reaction using natural product extracts as substrates²⁷⁾ (Fig. 5). Among them, Ugi adducts 23 and 24 had a good cytotoxicity in NDM, with PC₅₀ values of 27.8 and 16.9 µM, respectively (Table 2, entries 4, 5). However, these adducts also showed cytotoxicity in DMEM, with IC₅₀ values of 9.3 and 0.7 µM, respectively. The natural product hybrid 25 had a potent PC₅₀ value of 6.8 µM against PANC-1 cells under NDM conditions and no cytotoxicity was observed at the applied concentrations under DMEM conditions, exhibiting a good selectivity index (SI) of >15 between the IC₅₀ in DMEM and PC₅₀ in NDM (entry 6). Interestingly, all the starting natural products, such as (+)-ricinoleic acid (26) and curcumenone (27), were almost inactive against PANC-1 cells under both DMEM and NDM conditions (entries 7, 8). Therefore, the Ugi reaction-based chemical engineering of natural product extracts allowed a rapid access to novel anti-austerity agents.

Fig. 5. Chemical Structure of Natural Product-Derived Ugi Adducts 20–24, Natural Product Hybrid 25, and Natural Products 26 and 27

The structural motifs shown in colors are derived from natural products.

Table 2. Anti-austerity Activity of Natural Product-Derived Ugi Adducts 20–24, Natural Product Hybrid 25, and Natural Products 26 and 27

Entry	Compound	IC50 (µM) in DMEMa)	PC50 (µM) in NDMb)	SIc)
1	20	>100	>100	—
2	21	>100	>100	—
3	22	>100	54	>1.9
4	23	9.3	27.8	0.33d)
5	24	0.7	16.9	0.041d)
6	25	>100	6.8	>15
7	(+)-Ricinoleic acid (26)	>100	48	>2.1
8	Curcumenone (27)	>100	59	>1.7

a) Concentration at which 50% of cells were killed in DMEM. b) Concentration at which 50% of cells were killed preferentially in NDM. c) Selectivity index: IC₅₀ in DMEM/PC₅₀ in NDM. d) Compounds 23 and 24 were more cytotoxic in DMEM than in NDM, and thus represented conventional cytotoxic agent.

Live Cell Imaging

The mode of action of Ugi-adduct 11, which displayed the most potent cytotoxicity (PC₅₀, 0.5 µM) and good preferential anti-austerity activity (SI, 15), was then investigated. To evaluate the real-time effect of 11 on PANC-1 cell morphology, a live imaging study was conducted. PANC-1 cells were treated with either 0.5 and 1 µM of compound 11, and then placed in a CO₂ incubator equipped with a CytoSmart real-time microscopy system. Images were automatically taken every 15 min for a period of 24 h (Supplementary Fig. S1). The results showed that the untreated PANC-1 cells retained their intact cell morphology during the entire experiment. In contrast, the cells exposed to compound 11 exhibited cellular shrinkage and membrane blebbing within 4 h and total cell death within 8 h (Fig. 6, Supplementary video).

Fig. 6. Captures of the Live Imaging of the Effect of Compounds 11 (0.5 and 1 µM) on PANC-1 Cells at Different Intervals of Time in NDM

Ethidium Bromide–Acridine Orange (EB/AO) Staining

To investigate the effect of 11 on the alternation of morphology and PANC-1 cell death under nutrient-deprived conditions, EB-AO double-staining fluorescence assay was employed. In this assay, a cell-membrane permeable dye, AO emits green fluorescence in the live cells. On the other hand, dead cells are stained with EB, which is only permeable when the membrane integrity is lost during the cell death process, giving red fluorescence. PANC-1 cells were treated with 0.5 and 1 µM of 11 and incubated for 24 h. The untreated PANC-1 human pancreatic cancer cells showed intact cell membranes and displayed green fluorescence, indicative of live cells. Treatment of PANC-1 cells with 1 µM of 11 induced a rounded cellular morphology and emitted exclusive red fluorescence, suggestive of total cell death (Fig. 7).

Fig. 7. Morphological Changes of PANC-1 Cells Treated with 11 in Comparison to Untreated Cells (i.e., the Control) in NDM

PANC-1 tumor cells were treated with 11 at the indicated concentrations (0.5 and 1 µM) and incubated for 24 h in NDM. Cells were stained with ethidium bromide (EB) and acridine orange (AO) and photographed under the fluorescence (red and green) and phase contrast modes using an EVOS FL digital microscope.

Influence of Absolute Configuration of 11 on Anti-austerity Activity

With a potential anti-austerity agent 11 in hands, we then investigated which enantiomers of 11 contributed to the preferential cytotoxicity against PANC-1 in NDM. Both enantiomers were synthesized as described in Chart 2. The synthesis began with the condensation of tert-butoxycarbonyl (Boc) L- or D-phenylglycine (Boc-Phg-OH) with cyclohexyl amine using condensing agents. Although the chiral α-carbon stereocenter of Phg is known to be susceptible to racemization, the use of (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU) as a condensing agent at 0 °C was found to afford Boc L- or D-Phg-NHC₆H₁₁ (28) in a perfect enantiomer ratio (approx. 99 : 1). Subsequently, the N-Boc deprotection of 28 followed by reductive amination using benzaldehyde gave Bn-Phg-NHC₆H₁₁ (29). Finally, the N-chloroacetylation of 29 successfully provided both (S)-11 and (R)-11 in an excellent enantiomer ratio (approx. 99 : 1). In sharp contrast to this 4-step synthetic process, the Ugi reaction (Chart 1), although racemic, can be used to rapidly synthesize and evaluate initial screening compounds^28,29) for identifying a potential anti-austerity agent that should be further investigated. Then, the cytotoxic activity of (S)-11 and (R)-11 were tested against the PANC-1 cell line in both NDM and DMEM; both the R and S enantiomers demonstrated concentration-dependent anti-austerity activity against PANC-1 cell lines (Fig. 8), with the R enantiomer showing a PC₅₀ value of 0.2 µM, which was a 7-fold superior to the S enantiomer (PC₅₀, 1.5 µM) (Table 3). The average PC₅₀ value for the 1 : 1 enantiomeric mixture was calculated to be 0.87 µM. This value closely approximated the observed preferential cytotoxic concentration of the racemic 11 showing PC₅₀ value 0.5 µM (Table 1, entry 3). In contrast, when tested in DMEM, there was no significant difference in the IC₅₀ values between the enantiomers. Thus, the R configuration of Phg was significantly responsible for the preferential cytotoxicity against PANC-1 cell line, identifying a novel anti-austerity agent (R)-11 with potent activity.

Chart 2. Synthesis of (S)-11 and (R)-11

Fig. 8. Preferential Cytotoxicity of (S)-11 (a) and (R)-11 (b) against PANC-1 Cell Lines in NDM (Red Circle) and DMEM (Blue Circle)

Table 3. Anti-austerity Activity of (S)-11 and (R)-11

Entry	Compound	IC50 (µM) in DMEMa)	PC50 (µM) in NDMb)	SIc)
1	(S)-11	11.5	1.5	7.7
2	(R)-11	9.2	0.2	46

a) Concentration at which 50% of cells were killed in DMEM. b) Concentration at which 50% of cells were killed preferentially in NDM. c) Selectivity index: IC₅₀ in DMEM/PC₅₀ in NDM.

Western Blot Analysis

The cancer cell survival, progression, angiogenesis, and metastasis are linked with the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and autophagy signaling pathways.^30–32) These pathways are closely associated with the resistance of human pancreatic cancer cells to nutrient starvation and their survival in the austere tumor microenvironment.²⁾ In randomized controlled trials, therapies containing AKT/mTOR pathway inhibitors have demonstrated significant improvements in progression-free survival.³³⁾ Given this context, we investigated whether the most potent Ugi adduct (R)-11 is involved in suppressing the Akt/mTOR pathway. For this purpose, we performed Western blot analysis on PANC-1 cells exposed to various concentrations of Ugi-adduct (R)-11 for a short period of 6 h, under both normoxic and nutrient-deprived conditions (NDM and DMEM). The effects of (R)-11 on key proteins in the PI3K/Akt/mTOR and autophagy signaling pathways were then analyzed. As depicted in Fig. 9, exposure to (R)-11 for 6 h under NDM conditions resulted in a concentration-dependent and significant downregulation of PI3K, p-Akt, Akt, and p-mTOR, with complete inhibition of these proteins observed at 2.5 µM. Similarly, in DMEM at 25 µM, (R)-11 showed significantly near-complete inhibition of PI3K and complete inhibition of p-Akt, Akt, and p-mTOR. These findings strongly indicate that (R)-11 adduct specifically targets the PI3K/Akt/mTOR signaling pathway, leading to cytotoxicity against PANC-1 human pancreatic cancer cells in both NDM and DMEM conditions. Notably, the concentration of (R)-11 adduct required to induce the inhibition of these proteins under NDM conditions was significantly lower compared to DMEM. These results suggest that (R)-11 adduct holds promising therapeutic potential for pancreatic cancer treatment, especially under conditions that mimic nutrient-deprived tumor microenvironments, where targeting the PI3K/Akt/mTOR pathway could be particularly effective.

Fig. 9. A) Effect of Treatment of PANC-1 Human Pancreatic Cancer Cells with Different Concentrations of (R)-11 for 6 h on Key Proteins Involved in the Akt/mTOR Signaling Pathway, and B) Quantitative Estimation of GAPDH Normalized Western Blot Data of Three Independent Experimental Results

The statistical significance was calculated using one-way ANOVA (for NDM) and unpaired t-test (for DMEM) of three independent experimental results using GraphPad Prism 9. * p < 0.05; ** p < 0.01; *** p < 0.001 indicates a significant difference from the control.

CONCLUSION

The present study has demonstrated the efficacy of the Ugi reaction as a practical and step-economical strategy for generating structurally diverse anti-austerity agents. The results have established a clear structure–activity relationship, with the chloroacetic motif and α-phenylglycine unit being essential for the anti-austerity activity. Among the synthesized Ugi-adducts, compound (R)-11 exhibited the most promising anti-austerity activity, with a PC₅₀ value of 0.2 µM. Mechanistically, Ugi adduct 11 was found to inhibit Akt/mTOR activation. In addition, natural product hybrid 25 also displayed a strong anti-austerity activity with a PC₅₀ value of 6.8 µM. The findings suggest that Ugi reaction-based chemical engineering provides a means to rapidly synthesize and identify potential drug candidates against pancreatic cancer based on an anti-austerity strategy.

MATERIALS AND METHODS

General Experimental Information

Analytical TLC was performed using TLC Silica gel 60 F254 (Merck) and visualized by UV light at 254 and 345 nm and stained with an acidic solution of p-anisaldehyde (concentrated H₂SO₄ in EtOH). Silica gel column chromatography was performed using Silica gel 60 (spherical) 40–50 µm (Kanto, Tokyo, Japan). Medium pressure liquid chromatography (MPLC) was performed using a Yamazen EPCLC AI-580S (Yamazen, Osaka, Japan). Optical rotations were measured with an Anton Paar polarimeter MCP300 (Anton Paar, Graz, Austria) using a cell with a pathlength of 10 cm. Concentrations (c) are given in grams per 100 mL. Enantiomeric ratios (er) were determined by HPLC analysis using SHIMADZU HPLC Prominence (SHIMADZU, Kyoto, Japan) equipped with a column packed with an appropriate optically active material, as described below. NMR spectra were recorded at room temperature on a JEOL ECA 600 instrument with tetramethylsilane (TMS) as an internal standard. ¹H-NMR data are presented as follows. Chemical shift (δ in ppm), integration, multiplicity, and coupling constant J (in Hz and rounded to 0.1 Hz). Splitting patterns are abbreviated as follows: singlet (s), doublet (d), triplet (t), quintet (quint), multiplet (m), and broad (br), or a combination of them. ¹³C-NMR data are reported in terms of chemical shift (δ in ppm and rounded to 0.1 Hz). IR spectra were recorded using a JASCO FT/IR 4700 with substance as a pellet in a mixture with KBr, and described as wave numbers (cm⁻¹). Time-of-Flight (TOF) MS spectra were obtained from a Bruker Daltonics micrOTOF-KSIfocus spectrometer (Bruker, Billerica, MA, U.S.A.). Dimethyl sulfoxide (DMSO) for spectrophotometry was purchased from TCI (Tokyo, Japan) and used as received. Milli-Q water was used throughout. Other reagents and solvents were purchased from TCI, Nacalai (Kyoto, Japan), FUJIFILM Wako (Osaka, Japan), Sigma-Aldrich (St. Louis, MO, U.S.A.), and Kanto, and used as received. The Ugi adducts 9–24, except for (S)-11, (R)-11, and 17, were synthesized in pure form according to the previous procedure.²⁷⁾

Synthesis of 17 by the Ugi Reaction

To a solution of safranal (0.5 mmol, 1.0 equivalent (equiv.)) in methanol (0.5 M), benzylamine (0.5 mmol, 1.0 equiv.) was added at room temperature and the resulting mixture was stirred at room temperature for 1 h. Then, chloroacetic acid (0.5 mmol, 1.0 equiv.) was added and the resulting solution was cooled to 0 °C. Then, cyclohexyl isocyanide (0.5 mmol, 1.0 equiv.) was added at 0 °C and the reaction mixture was stirred at 0 °C for 1 h and then at room temperature for 7 d. After the resulting solution was concentrated in vacuo, the crude mixture was purified by silica gel column chromatography (hexane/AcOEt = 5 : 1) to afford 17 (73 mg, 0.164 mmol) as a colorless amorphous solid in 33% yield.

17: N-Benzyl-2-chloro-N-(2-(cyclohexylamino)-2-oxo-1-(2,6,6-trimethylcyclohexa-1,3-dien-1-yl)ethyl)acetamide

¹H-NMR (600 MHz, CDCl₃) δ: 1.03 (3H, s), 1.03–1.20 (3H, m), 1.09 (3H, s), 1.32–1.39 (2H, m), 1.57–1.68 (3H, m), 1.59 (3H, s), 1.86–1.93 (2H, m), 1.99 (1H, dd, J = 5.7, 16.8 Hz), 2.08 (1H, d, J = 16.8 Hz), 3.76–3.82 (1H, m), 4.00 (2H, s), 4.56 (1H, d, J = 18.0 Hz), 4.77 (1H, d, J = 18.0 Hz), 5.26 (1H, br d, J = 6.0 Hz), 5.69–5.71 (2H, m), 5.81–5.84 (1H, m), 7.25 (1H, t, J = 7.2 Hz), 7.35 (2H, t, J = 7.2 Hz), 7.54 (2H, d, J = 7.2 Hz); ¹³C-NMR (151 MHz, CDCl₃) δ: 20.6 (CH₃), 24.7 (CH₂), 25.6 (CH₂), 26.4 (CH₃), 33.1 (CH₂), 35.2 (C), 39.1 (CH₂), 42.3 (CH₂), 48.5 (CH), 48.8 (CH₂), 58.7 (CH), 126.7 (CH), 127.5 (CH), 128.9 (CH), 132.1 (C), 135.1 (C), 137.8 (C), 168.4 (C), 169.6 (C); IR (KBr) cm⁻¹: 3422, 2932, 2853, 1679, 1650, 1515, 1452, 1410, 735, 700; high resolution (HR)MS (electrospray ionization (ESI)-TOF): m/z Calcd for C₂₆H₃₅ClN₂NaO₂ ([M + Na]⁺): 465.2279. Found 465.2278.

Synthesis of (S)-11 and (R)-11

Condensation

To a solution of Boc-L-Phg-OH or Boc-D-Phg-OH (125 mg, 0.5 mmol) in N,N-dimethylformamide (DMF) (0.2 M), COMU (214 mg, 0.5 mmol), ⁱPr₂NEt (85.6 µL, 0.5 mmol), and cyclohexylamine (54.4 µL, 0.5 mmol) were added successively at 0 °C and the resulting mixture was stirred at 0 °C for 1 h. The reaction mixture was poured into 1 M HCl at 0 °C. After adding AcOEt, the organic layer was extracted with 1 M HCl (two times), sat. NaHCO₃ aq. (two times), and brine, dried over MgSO₄, filtered, and concentrated. The crude mixture was purified by MPLC (hexane/AcOEt = 43 : 7 to 13 : 7) to afford (S)-28 (139 mg, 0.418 mmol, 84%) or (R)-28 (134 mg, 0.403 mmol, 81%) as colorless solids.

28: tert-Butyl (2-(Cyclohexylamino)-2-oxo-1-phenylethyl)carbamate

¹H-NMR (600 MHz, CDCl₃) δ: 0.95–1.00 (1H, m), 1.09–1.15 (2H, m), 1.26–1.38 (3H, m), 1.41 (9H, s), 1.58–1.61 (1H, m), 1.66–1.69 (1H, m), 1.74–1.77 (1H, m), 1.89–1.92 (1H, m), 3.72–3.78 (1H, m), 5.06 (1H, br s), 5.45 (1H, br s), 5.81 (1H, br s), 7.30–7.36 (5H, m); ¹³C-NMR (151 MHz, CDCl₃) δ: 24.7 (CH₂), 24.8 (CH₂), 25.4 (CH₂), 28.3 (CH₃), 32.6 (CH₂), 32.8 (CH₂), 48.5 (CH), 58.3 (CH), 79.8 (C), 127.1 (CH), 128.0 (CH), 128.7 (CH), 138.7 (C), 155.3 (C), 169.3 (C). IR (KBr): cm⁻¹ 3325, 3277, 2935, 2855, 1691, 1673, 1649, 1555, 1519, 1367, 1249, 1171, 702; HRMS (ESI-TOF): m/z Calcd for C₁₉H₂₈N₂NaO₃ ([M + Na]⁺): 355.1992. Found 355.1988; [α]_D²⁰ +87.53 (c 0.39, CHCl₃, er 99 : 1) for (S)-28; [α]_D²⁰ −87.03 (c 0.42, CHCl₃, er 99 : 1) for (R)-28; HPLC: Daicel CHIRALPAK ID, hexane/2-propanol = 1 : 1, flow = 1 mL/min, λ = 210 nm, t_R(R) = 6.2 min, t_R(S) = 14.2 min.

Boc-Deprotection and Reductive Amination

A solution of (S)-28 (80.1 mg, 0.241 mmol) or (R)-28 (80.9 mg, 0.243 mmol) in 4 M HCl/dioxane (2 mL) was stirred at 0 °C for 2.5 h and the volatiles were concentrated in vacuo to give H-Phg-NHC₆H₁₁ as a HCl salt, which was then dissolved in dichloromethane (DCM) and ⁱPr₂NEt (1.0 equiv.) was added at 4 °C and the resulting mixture was stirred at 0 °C for 20 min. Then, benzaldehyde (1.0 equiv.) was added at 0 °C and the mixture was stirred at 0 °C for 2 h. Finally, NaBH(OAc)₃ (1.0 equiv.) was added at 0 °C and the reaction mixture was stirred at 0 °C for 2 h. The resulting mixture was diluted with DCM and water and the aqueous layer was extracted with DCM (three times), dried over MgSO₄, filtered, and concentrated. The crude mixture was purified by MPLC (hexane/AcOEt = 77 : 23 to 14 : 11) to afford (S)-29 (48.0 mg, 0.149 mmol, 62% in 2 steps) or (R)-29 (45.7 mg, 0.142 mmol, 58% in 2 steps) as colorless solids.

29: 2-(Benzylamino)-N-cyclohexyl-2-phenylacetamide

¹H-NMR (600 MHz, CDCl₃) δ: 1.13–1.22 (3H, m), 1.32–1.41 (2H, m), 1.57–1.72 (4H, m), 1.84–1.92 (2H, m), 3.75–3.82 (1H, m), 3.78 (2H, s), 4.20 (1H, s), 7.14 (1H, br d, J = 7.2 Hz), 7.27–7.37 (10H, m); ¹³C-NMR (151 MHz, CDCl₃) δ: 24.8 (CH₂), 24.9 (CH₂), 25.6 (CH₂), 33.2 (CH₂), 33.2 (CH₂), 47.7 (CH), 52.7 (CH₂), 67.2 (CH), 127.4 (CH), 127.5 (CH), 128.2 (CH), 128.3 (CH), 128.7 (CH), 128.9 (CH), 139.4 (C), 139.6 (C), 171.0 (C); IR (KBr) cm⁻¹: 3357, 3286, 2931, 2852, 1622, 1551, 1453, 743, 731, 697; HRMS (ESI-TOF): m/z Calcd for C₂₁H₂₇N₂O ([M + H]⁺): 323.2118. Found 323.2120; [α]_D²⁰ +34.59 (c 0.71, CHCl₃, er 99 : 1) for (S)-29; [α]_D²⁰ −33.17 (c 0.50, CHCl₃, er 99 : 1) for (R)-29; HPLC: Daicel CHIRALPAK IM, hexane/2-propanol = 7 : 3, flow = 0.6 mL/min, λ = 210 nm, t_R(S) = 9.6 min, t_R(R) = 11.1 min.

Acylation

To a solution of (S)-29 (46.8 mg, 0.145 mmol) or (R)-29 (44.5 mg, 0.138 mmol) and ⁱPr₂NEt (1.2 equiv.) in DCM (1 mL), chloroacetyl chloride (1.0 equiv.) was added dropwise at 0 °C and the reaction mixture was stirred at 0 °C for 2 h. The reaction mixture was poured into sat. NaHCO₃ aq. at 0 °C and the aqueous layer was extracted with CHCl₃ (three times) and the combined organic layers were dried over MgSO₄, filtered, and concentrated. The crude mixture was purified by MPLC (hexane/AcOEt = 87 : 13 to 4 : 1) to afford (S)-11 (36.5 mg, 0.0915 mmol, 63%) or (R)-11 (33.9 mg, 0.0849 mmol, 62%) as colorless amorphous solids. All spectral data of (S)-11 and (R)-11 were in good agreement with our previously reported values.²⁷⁾

11: N-Benzyl-2-chloro-N-(2-(cyclohexylamino)-2-oxo-1-phenylethyl)acetamide

[α]_D²⁰ +43.70 (c 0.95, CHCl₃, er 99 : 1) for (S)-11; [α]_D²⁰ −43.99 (c 0.62, CHCl₃, er approx. 99 : 1) for (R)-11; HPLC: Daicel CHIRALPAK IM, hexane/2-propanol = 9 : 1, flow 0.6 mL/min, λ = 210 nm, t_R(R) = 14.9 min, t_R(S) = 20.4 min.

Cell Line

The PANC-1 human pancreatic cancer cell line (RBRC-RCB2095) was procured from the Riken BRC cell bank and cultured in standard Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotic-antimycotic solution. The cells were maintained at 37 °C in a humidified atmosphere containing 5% CO₂.

Preferential Cytotoxicity Assay

Preferential cytotoxicity assay was conducted to evaluate the toxicity of Ugi adducts against PANC-1 human pancreatic cancer cells.³⁾ Cells (2 × 10⁴ cells/100 µL/well) were seeded into 96-well plates at a density of 2 × 10⁴ cells/100 µL/well and incubated for 24 h to allow cell attachment. Following this, the cells were washed with Dulbecco’s phosphate buffered saline (PBS) and treated with serially diluted compounds in nutrient-deprived medium (NDM) and nutrient-rich DMEM medium. The samples were treated in three replications for both NDM and DMEM, and placed in a CO₂ incubator for 24 h. Then, the medium was replaced with 100 µL of DMEM containing 10% WST-8 cell counting kit solution and incubated for 3 h before measuring the absorbance at 450 nm. The cell viability was calculated using the mean values of three wells according to the following equation.

  

Morphological Analysis

The assessment of cell morphology was carried out according to an established protocol. PANC-1 cells were exposed to NDM with or without Ugi adduct 11 for 24 h, and then stained with a dual fluorescent reagent consisting of acridine orange and ethidium bromide for 15 min. The stained cells were then visualized by an EVOS FL cell imaging system.

Live Cell Imaging

1 × 10⁵ PANC-1 cells were cultured in a 60 mm dish and allowed to adhere overnight. The media were then replaced with NDM for the control, or NDM containing Ugi adduct 11 at concentrations of 0.5 and 1 µM, and the dish was then placed in a live cell imaging device within a CO₂ incubator. Time-lapse images were taken in parallel, with an interval of 15 min, over a period of 24 h. The image data were further analyzed using ImageJ software.

Western Blot Analysis

Western blotting was performed on PANC-1 cells treated with Ugi adduct 11 in NDM (0, 0.5, 1, 2.5 µM) and DMEM (0, 25 µM) for 6 h. Proteins were separated on a 0.1% sodium dodecyl sulfate (SDS)-containing polyacrylamide gel and then transferred to polyvinylidene fluoride membranes. The membranes were blocked with Block Ace (DS Pharma Medical, Tokyo, Japan) and washed with PBS containing 0.1% polyoxyethylenesorbitan monolaurate (FUJIFILM Wako). Subsequently, the membranes were incubated overnight with primary antibodies diluted in Can Get Signal (Toyobo, Osaka, Japan). Subsequently, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-goat immunoglobulins for 45 min at room temperature as the secondary antibody. Finally, the bands were revealed with enhanced chemiluminescence solution from Bio-Rad.

Acknowledgments

This research was supported by JSPS KAKENHI (JP21K05290) and TOBE MAKI Scholarship Foundation to KT, and JSPS KAKENHI (JP23H02104) and Kobayashi International Scholarship to SA.

Author Contributions

KT and SA conceived and supervised this research. KT, JM, NO, RF, MJK, and SS performed the experiments and analyzed the data. All authors reviewed and approved the manuscript.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

Live cell imaging (photograph and video), spectra data, and quantitative estimation of protein expression in Western blot.

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