Structural Modification of the Propyl Linker of cjoc42 in Combination with Sulfonate Ester and Triazole Replacements for Enhanced Gankyrin Binding and Anti-proliferative Activity

Graphical Abstract

Liver cancer is a complex disease following a chronic liver injury, inflammation, unregulated wound healing, fibrosis, or carcinogenesis.¹ It also typically involves the activation of specific oncogenes and the inactivation of tumor suppressor genes.² The oncoprotein gankyrin was first found to be overexpressed in hepatocellular carcinoma in 2000,^3-7 while more recently its overexpression has also been associated with the proliferation and metastasis of numerous other cancers.^1,4,8-13 The oncogenic nature of gankyrin is driven by its various protein-protein interactions (PPIs) (e.g., 26S proteasome, retinoblastoma protein (Rb),^7,14,15 p53,^7,14-17 cyclin-dependent kinase 4 (CDK4),^14-18 and murine double minute 2 (MDM2)).^14-17 Additionally, gankyrin promotes tumorigenicity by increasing Rb and p53 proteasomal degradation resulting in cell cycle proliferation.^3,8,15,19,20 Interestingly, reduced gankyrin expression has been shown to decrease cell proliferation and colony formation in HepG2, HuH7, and Hep3B cells,^1,18,21-23 while targeting gankyrin has shown promise in inhibiting tumor formation and metastasis.^1,24 Due to its profound impact on the onset and development of various cancers through its numerous PPIs,^11,22 gankyrin is an intriguing target for the treatment of certain cancers.

Despite being an attractive therapeutic target, there have been limited efforts in developing small molecule binders of gankyrin. In 2016, Chattopadhyay et al., discovered the first small molecule gankyrin binder (cjoc42, 1, Figure 1) that disrupts the gankyrin-proteasome interaction.²⁵ Subsequent cell-based assays showed that cjoc42 treatment caused an increase in p53 levels, suggesting that cjoc42 disrupts the proteasomal degradation pathway.^6,25 Cjoc42 alone and in combination with other chemotherapeutic agents has also shown an ability to decrease the proliferation of hepatoblastoma cells. This anti-proliferative effect was attributed to disruption of the gankyrin-26S proteasome interaction, resulting in increased levels of various tumor suppressor proteins (TSPs), such as Rb, p53, C/EBPα, HNF4α and CUGBP1.^6,22,25 While substantial, the limited anti-proliferative activity (IC₅₀ > 50 μM) points towards the need for further optimization of the cjoc42 scaffold to enhance its gankyrin-binding ability and anti-proliferative activity.

Figure 1.

Proposed binding of cjoc42 (Yellow) to Gankyrin (Blue) determined by molecular modeling and visualized using PyMol. PDB: 2DVW.²⁶

Previous work from our group investigated substitutions to the phenyl rings, sulfonate ester, and triazole groups of cjoc42.^12,27,28 Our goal for this project was to modify the propyl linker in combination with previously established alterations to cjoc42 to observe their impact on liver cancer proliferation and gankyrin binding (Figure 2).

Figure 2.

Chemical structures, thermal shift values, and anti-proliferative activity of cjoc42 (1), and proposed cjoc42 derivatives.

It has been previously suggested that cjoc42 binds to gankyrin primarily through interactions with Y15, R41, W46, S49, W74, and K116 (Figure 1).²⁵ The focus of this study was to explore alterations to the three-carbon linker of the cjoc42 scaffold and determine their effect on gankyrin binding and anti-proliferative activity. Molecular modeling studies have suggested that cjoc42 adopts a U-shape conformation when bound to gankyrin. However, the propyl linker does not appear to directly interact with any particular amino acid residue of gankyrin (Figure 1). We hypothesized that adopting this U-shape is important for gankyrin binding to potentially maximize the π-π interactions with W46 and W74. To assess the importance of the flexibility of the propyl linker of cjoc42, the linker was substituted with a series of acyclic linkers. Derivatives were also designed to impart conformational constraint by reducing the distance between the two phenyl rings of cjoc42 by replacing the linker with either a urea or sulfonyl urea group and assessing their impact on anti-proliferative activity.

The synthesis of cjoc42 derivatives 4a-b (Scheme 1) commenced with a copper-catalyzed alkyne-azide cycloaddition (CuAAC) between methyl 4-azidobenzoate (2) and either 3-butyn-1-ol or 5-hexyn-1-ol, to afford triazole intermediates 3a-b. Triazole intermediates 3a-b then underwent nucleophilic substitution reactions with para-toluenesulfonyl chloride in the presence of triethylamine (TEA) and 4-dimethylaminopyridine (DMAP) to generate the desired cjoc42 derivatives 4a-b.

Scheme 1.

Synthesis of compounds 4a and 4b.^a

^a Reagents: (a) 3-Butyn-1-ol or 5-hexyn-1-ol, CuSO₄, sodium ascorbate, THF/^tBuOH/H₂O (53-76%); (b) p-TsCl, DMAP, TEA, CH₂Cl₂, 0 °C (23-51%).

Cjoc42 derivatives 7a-c were then generated with the aim of replacing the sulfonate ester of cjoc42 with a previously optimized amide group¹² in addition to replacing the propyl linker. The synthesis of derivatives 7a-c (Scheme 2) began with an EDCI-mediated amide coupling of para-toluic acid with various alkynyl amines to generate intermediates 6a-c. Amide intermediates 6a-c then underwent a CuAAC with methyl 4-azidobenzoate (2) to afford desired cjoc42 derivatives 7a-c.

Scheme 2.

Synthesis of compounds 7a-c.^a

^a Reagents: (a) Propargylamine, 3-butyn-1-amine, or 5-hexyn-1-amine, EDCI, pyridine, CH₂Cl₂ (47-76%) (b) Methyl 4-azidobenzoate, CuSO₄, sodium ascorbate, THF/_tBuOH/H₂O (13-77%).

To evaluate the impact of these modifications, the anti-proliferative activity of compounds 7a-c was determined against the gankyrin overexpressing cell line, HuH6, as shown in Table 1. Replacing the carbon linker with either a 2 or 4 carbon linker (4a and 4b, respectively) did not improve the anti-proliferative activity as compared to cjoc42. However, compound 7c exhibited a modest improvement in anti-proliferative activity against HuH6 cells (IC₅₀ = 19.5 μM). These findings were in agreement with our previous work where replacing the sulfonate ester of cjoc42 with an amide group improves anti-proliferative activity.²⁹ These results also demonstrated that replacing the propyl linker with an ethyl or butyl linker did not significantly impact the anti-proliferative activity, suggesting that the linker length plays little to no role in influencing the anti-proliferative activity. Additionally, the improvement in anti-proliferative activity observed for 7c suggests that an amide group replacement has more impact on the activity than replacement of propyl linker alone.

Table 1.

Anti-proliferative activity of cjoc42, 4a-b, and 7a-c against HuH6 cells.^a-d

Compound	HuH6 IC50 (μM)
Cjoc42	> 50
4a	> 50
4b	> 50
7a	> 50
7b	> 50
7c	19.5 (± 2.5)

^aHuH6 cells were incubated for 24 h prior to drug addition.

^bHuH6 cells were incubated for 72 h at 37 °C in 5% CO₂ with the respective drug.

^cCell proliferation was determined using an MTT assay.

^dAll experiments were performed in replicates of 6-8.

Previous work from our group also showed that replacing the triazole group of cjoc42 with an amide group improved the scaffold’s anti-proliferative activity.¹² Therefore, derivatives 10a-f were designed and synthesized with an aim of replacing the triazole and sulfonate ester groups of cjoc42 with amide groups while combining them with optimal alkyl linker lengths. The synthesis of derivatives 10a-f (Scheme 3) was performed with an EDCI-mediated amide coupling of either methyl 4-azidobenzoate (8a) or p-toluic acid (8b) with various diamines (9a-c) to generate cjoc42 derivatives 10a-f. Derivatives 13a-h then replaced the triazole and sulfonate ester groups with amide groups. Derivatives 13a-h (Scheme 4) were also synthesized by an EDCI-mediated amide coupling of either p-toluidine (11a) or methyl 4-aminobenzoate (11b) with various dicarboxylic acids (12a-d) to generate cjoc42 derivatives 13a-h.

Scheme 3.

Synthesis of compounds 10a-f.^a

^a Reagents: (a) EDCI, pyridine, CH₂Cl₂ (13-52%).

Scheme 4.

Synthesis of compounds 13a-h.^a

^a Reagents: (a) EDCI, pyridine and CH₂Cl₂ (6-46%).

Cjoc42 derivatives 10a-f and 13a-h were then evaluated for their anti-proliferative activity against HuH6 cells as shown in Table 2. Compounds 10a, 10d, and 13f showed modest improvements in anti-proliferative activity as compared to cjoc42. However, compound 13d substantially improved the anti-proliferative activity against HuH6 cells (IC₅₀ = 3.3 μM). These results again demonstrated that replacement of the propyl linker with other alkyl linkers in combination with replacing the triazole and sulfonate ester groups of cjo42 with amide groups resulted in improved anti-proliferative activity.

Table 2.

Anti-proliferative activity of cjoc42 derivatives 10a-f and 13a-h against HuH6 cells.^a-d

Compound	HuH6 IC50 (μM)
10a	30.2 (± 3.1)
10b	> 50
10c	> 50
10d	19.8 (± 2.5)
10e	> 50
10f	> 50
13a	> 50
13b	> 50
13c	> 50
13d	3.3 (±0.6)
13e	>50
13f	14.1 (± 1.5)
13g	>50
13h	>50

^aHuH6 cells were incubated for 24 h prior to drug addition.

^bHuH6 cells were incubated for 72 h at 37 °C in 5% CO₂ with the respective drug.

^cCell proliferation was determined using an MTT assay.

^dAll experiments were performed in replicates of 6-8.

Previous work from our lab demonstrated that a 4-phenyl substitution on the phenyl ring connected to the sulfonate ester of cjoc42 improved gankyrin binding and anti-proliferative activity. Additionally, a 4-trifluoromethyl or 4-methoxy substitution on the phenyl ring connected to the triazole of cjoc42 enhanced gankyrin binding and modestly improved its anti-proliferative activity against certain gankyrin overexpressing cancer cell lines.^12,27,28 As previously discussed, compounds 10d, 13d, and 13f improved anti-proliferative activity against HuH6 cells, as compared to cjoc42. Therefore, cjoc42 derivatives 17a-j and 21a-b were designed to combine the optimized features of 10d, 13d, and 13f with the previously optimized aryl sulfonate ester and aryl triazole substitutions. The synthesis of derivatives 17a-j (Scheme 5) then began with an EDCI-mediated amide coupling between various p-substituted benzoic acid derivatives and specific mono-Boc-protected diamines 14a-c to generate intermediates 15a-e. These intermediates then underwent an acid-catalyzed decarboxylation to provide intermediates 16a-e. Lastly, intermediates 16a-e then underwent another EDCI-mediated amide coupling with specific p-substituted benzoic acids to generate desired derivatives 17a-j.

Scheme 5.

Synthesis of compounds 17a-j.^a

^a Reagents: (a) p-toluic acid, monomethyl terephthalate, [1,1′-biphenyl]-4-carboxylic acid, or 4-methoxybenzoic acid, EDCI, pyridine, and CH₂Cl₂ (47-74%); (b) 6 M HCl and EtOH, 0 °C (75-98%); (c) p-toluic acid, monomethyl terephthalate, 4-(trifluoromethyl) benzoic acid, [1,1′-biphenyl]-4-carboxylic acid, or 4-methoxybenzoic acid, EDCI, pyridine, and CH₂Cl₂ (13-48%).

The synthesis of compounds 21a-b (Scheme 6) began with an EDCI-mediated amide coupling of p-toluidine or 4-aminobiphenyl with monoesters 18a or 18b to generate intermediates 19a-b. Intermediates 19a-b then underwent ester hydrolysis to afford intermediates 20a-b followed by a second EDCI-mediated amide coupling with p-toluidine or methyl 4-aminobenzoate to generate desired cjoc42 derivatives 21a-b.

Scheme 6.

Synthesis of compounds 21a-b.^a

^a Reagents: (a) p-toluidine or 4-aminobiphenyl, EDCI, pyridine, CH₂Cl₂ (75-80%); (b) 10 M NaOH and MeOH (90-98%); (c) p-toluidine or methyl 4-aminobenzoate, EDCI, pyridine, CH₂Cl₂ (15-37%).

Derivatives 17a-j and 21a-b were then evaluated for their anti-proliferative activity against HuH6 cells as shown in Table 3. Compounds 17a and 17c showed modest improvements in anti-proliferative activity while compound 17e (IC₅₀ = 2.4 μM) showed a substantial improvement in activity against HuH6 cells, as compared to cjoc42. This further demonstrated that a 4-phenyl substitution or 4-methoxy substitution improves the anti-proliferative activity against HuH6 cells. However, changing the propyl linker length resulted in little to no impact on the anti-proliferative activity.

Table 3.

Anti-proliferative activity of cjoc42 derivatives 17a-j and 21a-b against HuH6 cells.^a-d

Compound	HuH6 IC50 (μM)
17a	31.0 (± 5.0)
17b	> 50
17c	20.3 (± 1.4)
17d	> 50
17e	2.4 (± 0.2)
17f	> 50
17g	> 50
17h	> 50
17i	> 50
17j	> 50
21a	> 50
21b	> 50

^aHuH6 cells were incubated for 24 h prior to drug addition.

^bHuH6 cells were incubated for 72 h at 37 °C in 5% CO₂ with the respective drug.

^cCell proliferation was determined using an MTT assay.

^dAll experiments were performed in replicates of 6-8.

Compounds 23a-b, 24a-c, 26a-b, and 27a-c were then designed and synthesized to incorporate a constrained linker (urea and sulfonyl urea: Schemes 7 and 8) in place of the propyl linker to assess its impact on anti-proliferative activity. The syntheses of compounds 23a-e and 25a-e were performed with a nucleophilic addition between either p-tolyl isocyanate or p-toluenesulfonyl isocyanate with various anilines or benzylamines to generate desired compounds 23a-e and 25a-e.

Scheme 7.

Synthesis of compounds 23a-e.^a

^a Reagents: p-toluidine, methyl 4-aminobenzoate, 4-methoxybenzylamine, or (aminomethyl) benzoate, DIPEA, CH₂Cl₂ (97-99%).

Scheme 8.

Synthesis of compounds 25a-e.^a

^a Reagents: p-toluidine, methyl 4-aminobenzoate, 4-methoxybenzylamine, or methyl 4-(aminomethyl) benzoate, DIPEA, CH₂Cl₂ (95-100%).

Compounds 23a-e and 25a-e were then evaluated for their anti-proliferative activity against HuH6 cells, as shown in Table 4. Interestingly, only modest to no improvement in anti-proliferative activity was observed, suggesting that reducing the distance and flexibility of the linker between the phenyl rings of the cjoc42 scaffold has a detrimental effect on anti-proliferative activity.

Table 4.

Anti-proliferative activity of cjoc42 derivatives 23a-e and 25a-e against HuH6 cells.^a-d

Compound	HuH6 (μM)
23a	> 50
23b	38.8 (± 1.9)
23c	> 50
23d	> 50
23e	> 50
25a	> 50
25b	> 50
25c	> 50
25d	> 50
25e	> 50

^aHuH6 cells were incubated for 24 h prior to drug addition.

^bHuH6 cells were incubated for 72 h at 37 °C in 5% CO₂ with the respective drug.

^cCell proliferation was determined using an MTT assay.

^dAll experiments were performed in replicates of 6-8.

Anti-proliferative activity against non-cancerous cells

To assess the relative selectivity of these next-generation cjoc42 derivatives, the most potent compounds were also evaluated against the non-cancerous HEK-293 cell line (Table 5). Compounds 10d and 13d exhibited IC₅₀ values > 50 μM against HEK-293, demonstrating they preferentially target cancer cells and potentially target gankyrin which is overexpressed in HuH6 cells but not HEK-293 cells. Interestingly, compound 17e (IC₅₀ =3.3 μM) showed similar potency against both HEK-293 cells and HuH6 cells, suggesting that this compound exhibits little to no selectivity for cancer cells and therefore was not pursued further. Compound 13d was then chosen for subsequent biological evaluation due to its potent anti-proliferative activity and high degree of selectivity for cancer cells.

Table 5.

Anti-proliferative activity of compounds 10d, 13d, and 17e potent derivatives against HEK-293.^a-d

Compound	Structure	HuH6 IC50 (μM)	HEK-293 IC50(μM)
10d		19.8 (± 2.5)	> 50
13d		3.3 (± 0.6)	> 50
17e		2.4 (± 0.2)	3.3 (± 0.3)

^aHuH6 and HEK293 cells were incubated for 24 h prior to drug addition.

^bHuH6 and HEK-293 cells were incubated for 72 h at 37 °C in 5% CO₂ with the respective drug.

^cCell proliferation was determined using an MTT assay.

^dAll experiments were performed in replicates of 6-8.

Cellular Thermal Shift Assay (CETSA)

To determine if compound 13d effectively binds gankyrin in HuH6 cells, it was evaluated in a cellular thermal shift assay (CETSA, Figure 3). When HuH6 cells were treated with compound 13d, it demonstrated the ability to bind and thermally stabilize gankyrin in HuH6 cells at its respective anti-proliferative IC₅₀ value (Figure 3A). Interestingly, this is the first gankyrin-binding small-molecule to demonstrate a stabilizing effect on gankyrin by CETSA and these results suggest that compound 13d effectively binds gankyrin in HuH6 cells.

Figure 3.

CETSA to assess the binding of 13d to gankyrin. (A) Representative blot of gankyrin stability in HuH6 cells treated with compound 13d for 5 h. (B) CETSA melt curve for gankyrin from HuH6 cells in the presence of compound 13d. Data represents the mean ± SD (n = 3).

Western Blot Analysis

Gankyrin facilitates the proteasomal degradation of essential TSPs, such as p53 and Rb, and previous work with cjoc42 and subsequent derivatives has demonstrated an ability to bind gankyrin and increase the levels of p53 and/or Rb in various cancers.^6,25 Western blot analysis of compound 13d in HuH6 cells was then performed (Figure 4B). Our data shows that 13d caused an increase in both Rb and p53 levels, while gankyrin levels were relatively constant, which is in agreement with previous data for cjoc42 and some of its derivatives.^12,22 This suggests that compound 13d exhibits a similar mechanism of action to cjoc42 and its derivatives by disrupting the proteasomal degradation pathway resulting in reduced cancer cell proliferation.

Figure 4.

Western blot analysis of HuH6 cells treated with compound 13d. (A) Chemical structure and IC₅₀ value of 13d in HuH6 cells. (B) Western blot analysis of TSP levels from HuH6 lysate upon treatment with 13d. All experiments were conducted in duplicate, and a representative result is shown.

Cell Cycle Analysis

Gankyrin’s critical role in regulating p53 and Rb levels in turn results in cell cycle regulation.^4,8 As previously discussed, compound 13d significantly improved the anti-proliferative activity against HuH6 cells while also increasing levels of Rb and p53, therefore it was selected for cell cycle analysis in HuH6 cells (Figure 5). Rb and p53 are essential TSPs for cell cycle regulation, where p53 upregulation is known to arrest the cell cycle in the G₁ phase,^30,31 and Rb is a checkpoint for G₁ while restricting S-phase entry.^31,32 Treatment of HuH6 cells with 3.3 μM of compound 13d caused a significant increase in the number of cells in the G₀-G₁ phase and a significant decrease in the number of cells in the S phase (Figure 5). This suggests that compound 13d halts cell cycle progression in the G₀-G₁ phase, which is further supported by our western blot results (Figure 4). This data also suggests that the possible mode of action of 13d is gankyrin binding resulting in disruption of the proteasomal degradation pathway and ultimately inhibition of cell cycle progression.

Figure 5.

Cell cycle analysis of compound 13d in HuH6 cells. (A) Cell cycle analysis by flow cytometry of HuH6 cells treated with DMSO. (B) Cell cycle analysis by flow cytometry of HuH6 cells treated with compound 13d. (C) Representative histogram data of the cell cycle analysis of HuH6 cells treated with DMSO and compound 13d. Data represents the mean ± SD (n = 3). Note: *0.01 < p < 0.05.

Anti-proliferative activity against other liver cancer cell lines

Since compound 13d significantly improved anti-proliferative activity against HuH6 cells, it was also evaluated for its anti-proliferative activity against three other gankyrin-overexpressing liver cancer cell lines: HuH7, HepG2, and Hep3B (Table 6). Compound 13d then showed a modest improvement in anti-proliferative activity against HepG2 cells with an IC₅₀ of 24.0 μM. However, no improvement in activity was observed against Hep3B and HuH7 cells (IC₅₀ > 50 μM). The lack of potency against Hep3B and HuH7 cells could be due to their p53 status, as Hep3B is p53-null³³ and HuH7 has a mutant p53 status,³⁴ while HuH6 and HepG2 possess a wild-type p53 status.³⁵ Additionally, HuH6 and HepG2 cells are a type of hepatoblastoma (HBL),³⁵ while HuH7 and Hep3B are hepatocellular carcinoma cells (HCC). ^34,35 These results suggest that both p53 status and liver cancer type (HBL vs. HCC) play important roles in the anti-proliferative activity of cjoc42 derivatives.

Table 6.

Anti-proliferative activity of 13d against HuH7, HepG2, and Hep3B cells.^a-d

Cell line	IC50 (μM)
HepG2	24.0 (± 2.6)
Hep3B	>50
HuH7	> 50

^aHuH7, HepG2 and Hep3B cells were incubated for 24 h prior to drug addition. HuH7, HepG2 and Hep3B cells were incubated for 72 h at 37 °C in 5% CO₂ with the respective drug.

^cCell proliferation was determined using an MTT assay.

^dAll experiments were performed in replicates of 6-8.

Compound effects on gankyrin conformation

Circular Dichroism (CD) was performed to assess the effect of compounds lOd and 13d on gankyrin’s global 3D structural fold. CD is a robust biophysical method that can be used to monitor the α-helical content of gankyrin over a gradient of increasing compound concentration. To assess the specificity of compounds 10d and 13d towards gankyrin, we also conducted parallel analyses on ANKRA2, another ankyrin-repeating containing protein with a similar 3D fold to gankyrin.

The CD spectra for gankyrin and ANKRA2 alone revealed characteristic spectra minima at 208 nm and 222 nm confirming α-helical content of the native state. Subsequent examination of gankyrin spectra in the presence of each small molecule revealed a significant loss of gankyrin structural integrity, indicating that compounds 10d and 13d destabilize gankyrin’s 3D fold. Conversely, the spectra of ANKRA2 in the presence of both small molecules exhibits no significant global structure change. These findings confirm that compounds 10d and 13d are specific for targeting gankyrin and disrupt its structural integrity, with no effect on ANKRA2.

Conclusion

To assess the importance of the flexibility of the cjoc42 propyl linker on its gankyrin binding ability and anti-proliferative activity, various acyclic linkers were evaluated in combination with triazole and sulfonate ester replacements. Compound 13d was found to significantly improve anti-proliferative activity against HuH6 cells, while CETSA experiments showed that compound 13d effectively binds gankyrin in HuH6 cells. Our western blot analyses also showed that compound 13d could effectively increase levels of p53 and Rb in HuH6 cells, demonstrating its ability to bind gankyrin and inhibit the proteasomal degradation pathway. This is also supported by our CD spectra that shows compound 13d disrupts the global fold of gankyrin which is a core component of the 19S regulatory cap of the 26S proteasome. Compound 13d was then able to arrest the cell cycle of HuH6 cells in the G₀-G₁ phase, which was expected due to the observed increases in Rb and p53 levels. Our cumulative data suggests that replacing the propyl linker with alkyl linkers of various lengths did not have a major impact on the potency of the derivatives. However, replacing the propyl linker of cjoc42 along with appropriate triazole and sulfonate ester isosteres improved gankyrin specific binding and anti-proliferative activity. Compound 13d also modestly improved anti-proliferative against another gankyrin overexpressing cell line, HepG2. Interestingly, no improvement in activity was observed for Hep3B and HuH7, which do over express gankyrin but are either have p53 null or mutated p53 status in addition to being HCC cell lines.

In summary, these results indicate that compound 13d is only effective against cell lines with wild-type p53 status (HuH6 and HepG2) and are HBL cell lines. This finding indicates a potentially unique mechanism by which these cjoc42 derivatives inhibit the gankyrin-proteasome interaction. Although further evaluation is required to fully understand the exact mechanism of these derivatives, they can be used to further clarify gankyrin’s role in HBL development.

Materials and Methods

All commercially available reagents were purchased and used without further purification. Commercially available solvents (> 99.0% purity) were used for column chromatography without further purification. Proton (Ή) and carbon (¹³C) NMR spectra were recorded on a 400 MHz spectrometer. Proton chemical shifts are reported in ppm (δ) relative to residual Acetone-d₆ (2.05 ppm), DMSO-d₆ (2.49 ppm), CDCl₃ (7.26 ppm), or Methanol-d₄ (4.78 ppm, 3.31) ppm. Data are reported as follows: chemical shift (multiplicity [singlet (s), doublet (d), doublet of doublets (dd), doublet of doublets of doublets (ddd), triplet (t), quartet (q), quintet (p), sextet (h), multiplet (m)], coupling constants [Hz], integration). Carbon chemical shifts are reported in ppm with the respective solvent resonance as the internal standard: Acetone-d₆ (206.7 ppm and 29.9 ppm), DMSO-d₆ (39.5 ppm), CDCl₃ (77.2 ppm), or Methanol-d₄ (49.15 ppm). Unless otherwise noted, all NMR spectra were acquired at ambient temperature. Analytical thin-layer chromatography (TLC) was performed using Silica Gel 60 Å F254 precoated plates (0.25 mm thickness). UV absorption was used as the primary visualization method for monitoring reactions and column chromatography. UPLC/HPLC was performed using reverse-phase C18 column with UV detector. Accurate mass measurements/high resolution mass spectra (HRMS) were obtained from the Columbia University Chemistry Department Mass Spectrometry Facility on a Waters Xevo G2-XS QToF mass spectrometer equipped with an H-Class UPLC inlet and a LockSpray ESI source.

Purity analysis

Purity analysis of compounds 4a-b, 7a-c, 10a-f, 13a-h, 17a-j, 21a-b, 23a-e, and 25a-e was carried out using an Agilent 1260 infinity series HPLC system (Agilent, Santa Clara, CA). Purity analysis was performed using an Agilent Eclipse plus C18, 3.5 μm, 4.6 mm × 100 mm column and the runs were monitored at 254 nm. Methanol and water mixtures were used as the mobile phase. An isocratic run was performed with 90% methanol in water over 5 min, and the flow rate was 0.5 mL/min. Purity analysis of compounds 4a-b, 7a-c, 10a-f, 13a-h, 17a-j, 21a-b, 23a-e, and 25a-e was carried out using an Agilent 1100 infinity series HPLC system (Agilent, Santa Clara, CA). Purity analysis was performed using an Agilent Eclipse plus C18, 3.5 μm, 4.6 mm × 100 mm column and the runs were monitored at 254 nm. Methanol and water mixtures were used as the mobile phase. An isocratic run was performed with 90% methanol in water over 12 min with a flow rate of 0.5 mL/min.

All target compounds were analyzed to be > 95% pure by the methods described above unless otherwise noted.

General procedure 1 - Amide coupling.

To a stirred solution of carboxylic acid (1 equiv) in dichloromethane was added EDCI (3 equiv) and amine (1 equiv), followed by the addition of pyridine (5 equiv). The reaction mixture was then stirred overnight at room temperature. The reaction mixture was then diluted with dichloromethane and washed 3 times with water. The organic layer was then dried over sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography, unless otherwise stated.

General procedure 2 - Copper-catalyzed azide-alkyne cycloaddition.

To a stirred solution of azide (1 equiv) in a THF/^tBuOH/H₂O (2:3:5) mixture was added alkyne (1 equiv), copper sulfate (0.3 M, 0.3 equiv), and sodium ascorbate (0.1 M, 0.01 equiv). The reaction mixture was then stirred overnight at room temperature. Upon completion, the reaction mixture was partitioned between ethyl acetate and water, and then washed 3 times with water. The organic layer was then dried over sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography, unless otherwise stated.

General procedure 3 – Urea and sulfonylurea formation.

To a stirred solution of either isocyanate or sulfonyl isocyanate (1 equiv) in dichloromethane was added amine (1 equiv) and DIPEA (3 equiv). The reaction mixture was then stirred overnight at room temperature. Upon completion, the reaction mixture was partitioned between ethyl acetate and water, and then washed 3 times with water. The organic layer was then dried over sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography, unless otherwise stated.

General procedure 4 - Tosylation.

To a stirred solution of an alcohol intermediate in dichloromethane at 0 °C was added triethylamine (1 equiv) and 4-dimethylaminopyridine (0.1 equiv). p-Toluene sulfonyl chloride (1.5 equiv) was then added, and the reaction mixture was stirred for an additional 15 min at 0 °C. The reaction mixture was then stirred overnight at room temperature. Upon completion, the reaction mixture was partitioned between dichloromethane and water. The organic layer was then washed 3 times with water, dried over sodium sulfate, filtered, and the solvent was removed under reduced pressure. The compounds were then purified using column chromatography.

General procedure 5 – Boc deprotection.

To a stirred solution of Boc-protected intermediates 16a, 16b, 16c, 16d or 16e in ethanol at 0 °C was added 6M HCl, and the reaction mixture was stirred for an additional 1 h at 0 °C. The reaction mixture was then stirred overnight at room temperature. Upon completion, the reaction mixture was concentrated to obtain the desired compounds which were then used in the next reaction without further purification.

General procedure 6 – Ester hydrolysis.

Ester intermediates 19a or 19b were dissolved in MeOH, followed by addition of 10M NaOH, and the reaction mixture was then stirred overnight at room temperature. Upon completion, the reaction mixture was concentrated to remove MeOH. The crude mixture was then partitioned between dichloromethane and water, and the organic layer was then washed 3 times with water, dried over sodium sulfate, filtered, and the solvent was removed under reduced pressure to obtain the desired carboxylic acid intermediate which was used in the next reaction without further purification.

Methyl 4-(4-(2-hydroxyethyl)-1H-1,2,3-triazol-1-yl) benzoate (3a)

General procedure 2, (150 mg, 53%). ¹HNMR (400 MHz, DMSO-d₆) δ 8.70 (s, 1H), 8.19 – 8.09 (m, 2H), 8.10 – 7.97 (m, 2H), 4.82 (s, 1H), 3.88 (s, 3H), 3.72 (d, J = 3.1 Hz, 2H), 2.87 (t, J = 6.8 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 165.81, 146.56, 140.38, 131.42 (2C), 129.43, 121.30, 119.95 (2C), 60.57, 52.82, 29.59.

Methyl 4-(4-(4-hydroxybutyl)-1H-1,2,3-triazol-1-yl) benzoate (3b)

General procedure 2, (248 mg, 76%). ¹H NMR (400 MHz, CDCl₃) δ 8.20 (d, J = 8.8 Hz, 2H), 7.85 (d, J = 8.8 Hz, 2H), 7.85 (s, 1H), 3.98 (d, J = 6.3 Hz, 3H), 3.73 (t, J = 6.4 Hz, 2H), 2.87 (t, J = 7.5 Hz, 2H), 1.87 (dt, J = 12.6, 7.4 Hz, 3H), 1.72 (dd, J = 14.9, 6.5 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 165.98, 149.22, 140.18, 131.30, 129.87, 119.66 (3C), 118.88, 62.22, 32.06, 25.49 (2C), 25.23.

Methyl 4-(4-(2-(tosyloxy)ethyl)-1H-1,2,3-triazol-1-yl)benzoate (4a)

General procedure 4, (75 mg, 23%). ¹H NMR (400 MHz, CDCl₃) δ 8.30 – 8.17 (m, 2H), 7.91 (s, 1H), 7.90 – 7.72 (m, 4H), 7.32 (d, J = 8.0 Hz, 2H), 4.38 (t, J = 6.3 Hz, 2H), 3.99 (s, 3H), 3.21 (t, J = 6.2 Hz, 2H), 2.43 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 165.90, 148.44, 144.84, 140.18, 132.98, 131.27 (2C), 129.89 (2C), 127.81 (2C), 119.64 (2C), 118.98 (2C), 70.23, 52.38, 28.15, 25.11, 24.79, 21.60. HRMS (ESI+): m/z calculated for C₁₉H₁₉N₃O₅S⁺ [M+H]⁺ 402.1045, found 402.1124.

Methyl 4-(4-(4-(tosyloxy)butyl)-1H-1,2,3-triazol-1-yl)benzoate (4b)

General procedure 4, (198 mg, 51%). ¹H NMR (400 MHz, CDCl₃) δ 8.13 (d, J = 8.7 Hz, 2H), 7.76 (d, J = 9.5 Hz, 3H), 7.71 (d, J = 8.3 Hz, 2H), 7.27 (d, J = 8.2 Hz, 2H), 4.00 (t, J = 5.8 Hz, 2H), 3.89 (s, 3H), 2.72 (t, J = 7.0 Hz, 2H), 2.37 (s, 3H), 1.77 – 1.67 (m, 4H). ¹³C NMR (100 MHz, CDCl₃) δ 165.90, 148.44, 144.84, 140.18, 132.98, 131.27 (2C), 129.89 (2C), 127.81 (2C), 119.64 (2C), 118.98 (2C), 70.23, 52.38, 28.22, 25.11, 24.79, 21.60. HRMS (ESI+): m/z calculated for C₂₁H₂₃N₃O₅S⁺ [M+H]⁺ 430.1358, found 430.1452.

4-methyl-N-(prop-2-yn-1-yl)benzamide (6a)

General procedure 1, (160 mg, 47%). ¹H NMR (400 MHz, CDCl₃) δ 7.71 (d, J = 8.2 Hz, 2H), 7.25 (d, J = 7.9 Hz, 2H), 6.39 (s, 1H, NH), 4.26 (dd, J = 5.2, 2.6 Hz, 2H), 2.41 (s, 3H), 2.29 (t, J = 2.5 Hz, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 167.15, 142.25, 130.91, 129.26 (2C), 127.09 (2C), 79.69, 71.73, 29.71, 21.47.

N-(but-3-yn-1-yl)-4-methylbenzamide (6b)

General procedure 1, (330 mg, 56%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.58 (s, 1H), 7.74 (d, J = 8.0 Hz, 2H), 7.27 (d, J = 7.9 Hz, 2H), 2.85 (s, 1H, NH), 2.51 (s, 2H), 2.41 (d, J = 2.4 Hz, 2H), 2.35 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.56, 141.53, 131.96, 129.27 (2C), 127.62 (2C), 82.73, 72.57, 38.80, 21.40, 19.17.

N-(hex-5-yn-1-yl)-4-methylbenzamide (6c)

General procedure 1, (110 mg, 76%). ¹H NMR (400 MHz, CDCl₃) δ 7.68 (d, J = 8.1 Hz, 2H), 7.15 (d, J = 7.9 Hz, 2H), 6.94 (s, 1H, NH), 3.39 (dd, J = 13.0, 6.7 Hz, 2H), 2.35 (s, 3H), 2.17 (td, J = 6.9, 2.4 Hz, 2H), 1.95 (t, J = 2.5 Hz, 1H), 1.80 – 1.59 (m, 2H), 1.61 – 1.47 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 167.74, 141.59, 131.84, 129.07 (2C), 127.02 (2C), 84.12, 68.76, 39.45, 28.70, 25.80, 21.39, 18.10.

Methyl 4-(4-((4-methylbenzamido)methyl)-1H-1,2,3-triazol-1-yl)benzoate (7a)

General procedure 2, (80 mg, 30%). ¹H NMR (400 MHz, CDCl₃) δ 8.22 – 8.06 (m, 3H), 7.76 (d, J = 8.5 Hz, 2H), 7.65 (d, J = 8.0 Hz, 2H), 7.21 – 7.13 (m, 2H), 7.11 (s, 1H, NH), 4.72 (d, J = 5.7 Hz, 2H), 3.88 (s, 3H),2.31 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 167.58, 165.89, 146.05, 142.29, 140.01, 131.34 (2C), 130.97, 130.29, 129.29 (2C), 127.05 (2C), 120.87, 119.92 (2C), 52.47, 35.30, 21.47. HRMS (ESI+): m/z calculated for C₁₉H₁₈N₄O₃⁺ [M+H]⁺ 351.1379 found 351.1476.

Methyl 4-(4-(2-(4-methylbenzamido)ethyl)-1H-1,2,3-triazol-1-yl)benzoate (7b)

General procedure 2, purified by recrystallization, (25 mg, 12.85%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.80 (s, 1H), 8.58 (s, 1H, NH), 8.16 (d, J = 8.2 Hz, 2H), 8.07 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 7.7 Hz, 2H), 7.26 (d, J = 7.8 Hz, 2H), 3.90 (s, 3H), 3.59 (d, J = 6.0 Hz, 2H), 2.99 (t, J = 7.0 Hz, 2H), 2.34 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.65, 165.84, 146.59, 141.44, 140.40, 132.17, 131.49 (2C), 129.55, 129.25 (2C), 127.65 (2C), 121.31, 120.07 (2C), 52.87, 26.01, 21.40 (2C). HRMS (ESI+): m/z calculated for C₂₀H₂₀N₄O₃⁺ [M+H]⁺ 365.1535, found 365.1617.

Methyl 4-(4-(4-(4-methylbenzamido)butyl)-1H-1,2,3-triazol-1-yl)benzoate (7c)

General procedure 2, (140 mg, 77%). ¹H NMR (400 MHz, CDCl₃) δ 8.19 (d, J = 8.8 Hz, 2H), 7.89 (s, 1H), 7.84 (d, J = 8.8 Hz, 2H), 7.69 (d, J = 8.1 Hz, 2H), 7.22 (d, J = 7.9 Hz, 2H), 6.52 (s, 1H, NH), 3.97 (s, 3H), 3.51 (dd, J = 12.9, 6.8 Hz, 2H), 2.87 (t, J = 7.3 Hz, 2H), 2.39 (s, 3H), 1.93 – 1.81 (m, 2H), 1.81 – 1.67 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 167.60, 165.97, 148.82, 141.79, 140.20, 131.73, 131.30 (2C), 129.93, 129.20 (2C), 126.88 (2C), 119.70 (2C), 119.02, 52.43, 39.64, 28.94, 26.50, 25.00, 21.42. HRMS (ESI+): m/z calculated for C₂₂H₂₄N₄O₃⁺ [M+H]⁺ 393.1848, found 393.1927.

N,N’-(ethane-1,2-diyl)bis(4-methylbenzamide) (10a)

General procedure 1, purified by trituration, (244 mg, 22%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.53 (s, 2H), 7.76 (d, J = 8.0 Hz, 4H), 7.26 (d, J = 7.9 Hz, 4H), 3.49 – 3.38 (m, 4H), 2.35 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.96 (2C), 141.41 (2C), 132.21 (2C), 129.49 (4C), 127.67 (4C), 40.21 (2C), 21.40 (2C). HRMS (ESI+): m/z calculated for C₁₈H₂₀N₂NaO₂⁺ [M+Na]⁺ 319.1422 found 319.1437. This matches the previously reported spectra.³⁶

Dimethyl 4,4'-((ethane-1,2-diylbis(azanediyl))bis(carbonyl))dibenzoate (10b)

General procedure 1, purified by trituration, (120 mg, 9%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.83 (s, 2H), 8.03 (s, 4H), 7.98 (s, 4H), 3.88 (s, 6H), 3.47 (d, J = 2.6 Hz, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.21 (2C) δ 166.15 (2C), 139.13 (2C), 132.17 (2C), 129.58 (4C), 128.10 (4C), 52.84 (2C), 40.78 (2C). HRMS (ESI+): m/z calculated for C₂₀H₂₁N₂O₆⁺ [M+H]⁺ 385.1321, found 385.1423. This matches the previously reported spectra.³⁶.

N,N'-(propane-1,3-diyl)bis(4-methylbenzamide) (10c)

General procedure 1, (490 mg, 52%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.44 (t, J = 5.4 Hz, 2H), 7.75 (d, J = 8.1 Hz, 4H), 7.27 (d, J = 7.9 Hz, 4H), 3.33 – 3.23 (m, 4H), 2.35 (s, 6H), 1.84 – 1.67 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 165.78 (2C), 146.97, 140.00, 131.63 (2C), 131.07, 130.97 (2C), 128.89 (2C), 122.09, 120.49 (2C), 52.95 (2C), 39.51 (2C), 21.69. HRMS (ESI+): m/z calculated for C₁₉H₂₂N₂NaO₂⁺ [M+Na]⁺ 333.1579, found 333.1580.

Dimethyl 4,4'-((propane-1,3-diylbis(azanediyl))bis(carbonyl))dibenzoate (10d)

General procedure 1, (450 mg, 38%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.72 (t, J = 5.4 Hz, 2H), 8.04 (d, J = 8.4 Hz, 4H), 7.96 (d, J = 8.4 Hz, 4H), 3.88 (s, 6H), 3.33 (s, 4H), 1.88 – 1.74 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 167.29, 166.31, 138.08 (2C), 132.85 (2C), 129.94 (4C), 127.07 (4C), 77.22 (2C), 52.42, 36.33, 29.80. HRMS (ESI+): m/z calculated for C₂₁H₂₃N₂O₆⁺ [M+H]⁺ 399.1556, found 399.1562. This matches the previously reported spectra.³⁶

N,N'-(butane-1,4-diyl)bis(4-methylbenzamide) (10e)

General procedure 1, purified by trituration, (130 mg, 16%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.40 (t, J = 5.2 Hz, 2H), 7.74 (d, J = 7.9 Hz, 4H), 7.25 (d, J = 7.9 Hz, 4H), 3.27 (d, J = 5.1 Hz, 4H), 2.34 (s, 6H), 1.55 (s, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.52 (2C), 141.43 (2C), 132.41 (2C), 129.20 (4C), 127.61 (4C), 40.19 (2C), 27.26 (2C), 21.44 (2C). HRMS (ESI+): m/z calculated for C₂₀H₂₄N₂NaO₂⁺ [M+Na]⁺ 347.1735, found 347.1825.

Dimethyl 4,4'-((butane-1,4-diylbis(azanediyl))bis(carbonyl))dibenzoate (10f)

General procedure 1, purified by trituration, (120 mg, 13%), ¹H NMR (400 MHz, DMSO-d₆) δ 8.69 (t, J = 5.1 Hz, 2H), 8.03 (d, J = 8.2 Hz, 4H), 7.95 (d, J = 8.2 Hz, 4H), 3.88 (s, 6H), 3.31 (d, J = 5.7 Hz, 4H), 1.59 (s, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.22 (2C), 165.77 (2C), 139.23 (2C), 132.09 (2C), 129.57 (4C), 128.01 (4C), 52.82 (2C), 39.35 (2C), 27.04 (2C). HRMS (ESI+): m/z calculated for C₂₂H₂₅N₂O₆⁺ [M+H]⁺ 413.1713, found 413.1713. This matches the previously reported spectra.³⁶

N¹,N³-di-p-tolylmalonamule (13a)

General procedure 1, (277 mg, 30%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.05 (s, 2H, NH), 7.48 (d, J = 8.4 Hz, 4H), 7.11 (d, J = 8.2 Hz, 4H), 3.42 (s, 2H), 2.25 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 165.69 (2C), 136.94 (2C), 132.75 (2C), 129.60 (4C), 119.56 (4C), 46.27, 20.91 (2C). HRMS (ESI+): m/z calculated for C₁₇H₁₈N₂NaO₂⁺ [M+Na]⁺ 305.1266, found 305.1282. This matches the previously reported spectra.²⁷

Dimethyl 4,4'-(malonylbis(azanediyl)) dibenzoate (13b)

General procedure 1, (120 mg, 31%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.56 (s, 2H, NH), 7.94 (d, J = 8.7 Hz, 4H), 7.75 (d, J = 8.7 Hz, 4H), 3.83 (s, 6H), 3.58 (s, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.39 (4C), 143.64 (2C), 130.82 (4C), 124.64 (2C), 118.98 (4C), 52.37 (2C), 46.70. HRMS (ESI+): m/z calculated for C₁₉H₁₉N₂O₆⁺ [M+H]⁺ 371.1243, found 371.1258.

N¹,N⁴-di-p-tolylsuccinamide (13c)

General procedure 1, purified by recrystallization, (110 mg, 13%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.87 (s, 2H), 7.46 (d, J = 8.2 Hz, 4H), 7.08 (d, J = 8.1 Hz, 4H), 2.62 (s, 4H), 2.23 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 170.59 (2C), 137.42 (2C), 132.21(2C), 129.49 (4C), 119.68 (4C), 31.68 (2C), 20.88 (2C). HRMS (ESI+): m/z calculated for C₁₈H₂₀N₂NaO₂⁺ [M+Na]⁺ 319.1422, found 319.1434. This matches the previously reported spectra.³⁷

Dimethyl 4,4'-(succinylbis(azanediyl))dibenzoate (13d)

Purified by recrystallization. General procedure 1, (50 mg, 7%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.38 (s, 2H), 7.91 (d, J = 8.7 Hz, 4H), 7.73 (d, J = 8.7 Hz, 4H), 3.82 (s, 6H), 2.74 (s, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 171.61 (2C), 166.53 (2C), 144.14 (2C), 130.57 (4C), 124.05 (2C), 118.62 (4C), 52.48 (2C), 31.74 (2C). HRMS (ESI+): m/z calculated for C₂₀H₁₉N₂NaO₆⁺ [M+Na]⁺ 407.1219, found 407.1222.

N¹,N⁵-di-p-tolylglutaramide (13e)

General procedure 1, (121 mg, 46%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.82 (s, 2H), 7.48 (d, J = 8.2 Hz, 4H), 7.09 (d, J = 7.8 Hz, 4H), 2.35 (t, J = 7.3 Hz, 4H), 2.24 (s, 6H), 1.96 – 1.81 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 171.02 (2C), 137.30 (2C), 132.25 (2C), 129.47 (4C), 119.43 (4C), 36.00 (2C), 21.44, 20.89 (2C). HRMS (ESI+): m/z calculated for C₁₉H₂₂N₂Nao₂⁺ [M+Na]⁺ 333.1579, found 333.1587.

Dimethyl 4,4'-(glutaroylbis(azanediyl))dibenzoate (13f)

General procedure 1, (60 mg, 10%). ¹H NMR (400 MHz, DMSO) δ 10.31 (s, 2H), 7.91 (d, J = 7.5 Hz, 4H), 7.75 (d, J = 7.7 Hz, 4H), 3.82 (s, 6H), 2.44 (t, J = 7.2 Hz, 4H), 2.25 (t, J = 7.7 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 167.60, 165.97, 148.82, 141.79, 140.20, 131.73, 131.30 (2C), 129.93, 129.20 (2C), 126.88 (2C), 119.70 (2C), 119.02, 52.43, 39.64, 28.94, 26.50, 25.00, 21.42. HRMS (ESI+): m/z calculated for C₂₁H₂₂N₂O₆⁺ [M+H]⁺ 399.1566, found 399.1566.

N¹,N⁶-di-p-tolyladipamide (13g)

General procedure 1, (97 mg, 10%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.79 (s, 2H), 7.46 (d, J = 8.2 Hz, 4H), 7.08 (d, J = 8.2 Hz, 4H), 2.30 (s, 4H), 2.23 (s, 6H), 1.61 (s, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 171.32 (2C), 137.26 (2C), 132.28 (2C), 129.47 (4C), 119.55 (4C), 36.70 (2C), 25.40 (2C), 20.88 (2C). HRMS (ESI+): m/z calculated for C₂₀H₂₅N₂O₂⁺ [M+H]⁺ 325.1916, found 325.1919. This matches the previously reported spectra.³⁸

Dimethyl 4,4’-(adipoylbis(azanediyl))dibenzoate (13h)

General procedure 1, (36 mg, 6%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.26 (s, 2H), 7.90 (d, J = 7.8 Hz, 4H), 7.73 7.73 (d, J = 7.8 Hz, 4H), 3.84 (d, J = 16.8 Hz, 6H), 2.39 (s, 4H), 1.65 (s, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 172.18 (2C), 166.30 (2C), 144.16 (2C), 130.73 (4C), 124.13 (2C), 118.82 (4C), 52.32 (2C), 36.81 (2C), 25.12 (2C). HRMS (ESI+): m/z calculated for C₂₂H₂₅N₂O₆⁺ [M+H]⁺ 413.1713, found 413.1715.^39,40

tert-butyl (2-(4-methylbenzamido)ethyl)carbamate (15a)

General procedure 1, (255 mg, 47%). ¹H NMR (400 MHz, CDCl₃) δ 7.71 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 8.1 Hz, 2H), 7.16 (s, 1H), 5.09 (s, 1H), 3.54 (dd, J = 10.8, 5.2 Hz, 2H), 3.40 (d, J = 5.1 Hz, 2H), 2.38 (s, 3H), 1.43 (s, 9H). ¹³C NMR (100 MHz, CDCl₃) δ 167.85, 157.46, 141.79, 131.32, 129.13 (2C), 127.01 (2C), 79.90, 41.90, 40.03, 28.34 (3C), 21.43.

tert-butyl (3-(4-methylbenzamido)propyl)carbamate (15b)

General procedure 1, (300 mg, 59%). ¹H NMR (400 MHz, CDCl₃) δ 7.71 (d, J = 7.6 Hz, 2H), 7.60 (s, 1H), 7.15 (d, J = 7.7 Hz, 2H), 5.43 (s, 1H), 3.44 (dd, J = 11.8, 5.9 Hz, 2H), 3.16 (d, J = 4.8 Hz, 2H), 2.33 (s, 3H), 1.64 (s, 2H), 1.40 (s, 9H). ¹³C NMR (100 MHz, CDCl₃) δ 167.78, 156.90, 141.58, 131.67, 129.06 (2C), 127.05 (2C), 79.17, 37.08, 36.21, 30.06, 28.39 (3C), 21.37.

tert-butyl (3-([1,1'-biphenyl]-4-carboxamido)propyl)carbamate (15c)

General procedure 1, (410 mg, 73%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.51 (t, J = 5.4 Hz, 1H), 7.94 (d, J = 8.3 Hz, 2H), 7.75 (dd, J = 15.7, 7.9 Hz, 4H), 7.50 (t, J = 7.5 Hz, 2H), 7.42 (d, J = 7.3 Hz, 1H), 6.85 (s, 1H), 3.28 (dd, J = 12.8, 6.6 Hz, 2H), 2.99 (dd, J = 12.7, 6.4 Hz, 2H), 1.74 – 1.59 (m, 2H), 1.38 (s, 9H). ¹³C NMR (100 MHz, CDCl₃) δ 167.21, 157.03, 144.10, 140.16, 133.24, 128.89 (2C), 127.90, 127.53, 127.21 (2C), 79.60, 36.04, 30.29, 28.41 (3C).

tert-butyl (4-(4-methylbenzamido)butyl)carbamate (15d)

General procedure 1, (350 mg, 74%). ¹H NMR (400 MHz, CDCl₃) δ 7.69 (d, J = 7.9 Hz, 2H), 7.20 (d, J = 7.9 Hz, 2H), 6.63 (s, 1H), 4.74 (s, 1H), 3.46 (q, J = 6.5 Hz, 2H), 3.15 (d, J = 6.2 Hz, 2H), 2.38 (s, 3H), 1.60 (ddd, J = 21.4, 10.7, 5.1 Hz, 4H), 1.44 (s, 9H). ¹³C NMR (100 MHz, CDCl₃) δ 167.57, 156.21, 141.70, 131.76, 129.13 (2C), 126.96 (2C), 79.24, 40.38, 39.58, 28.42 (3C), 27.69, 26.69, 21.42.

tert-butyl (4-([1,1'-biphenyl]-4-carboxamido)butyl)carbamate (15e)

General procedure 1, (250 mg, 64%). ¹H NMR (400 MHz, CDCl₃) δ 7.81 (d, J = 7.9 Hz, 2H), 7.56 (dd, J = 15.2, 7.8 Hz, 4H), 7.39 (t, J = 7.5 Hz, 2H), 7.32 (t, J = 7.3 Hz, 1H), 6.49 (s, 1H), 4.59 (s, 1H), 3.45 (dd, J = 12.5, 6.4 Hz, 2H), 3.11 (d, J = 5.9 Hz, 2H), 1.62 – 1.51 (m, 4H), 1.38 (s, 9H). ¹³C NMR (100 MHz, CDCl₃) δ 167.28, 156.23, 144.14, 140.09, 133.31, 128.90, 127.94, 127.50, 127.19 (2C), 127.16, 79.28, 39.67, 28.42 (3C), 27.78, 26.48.

N-(2-aminoethyl)-4-methylbenzamide (16a)

General procedure 5, (250 mg, 98%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.82 (t, J = 4.7 Hz, 1H), 8.30 (s, 2H), 7.87 (d, J = 7.9 Hz, 2H), 7.29 (t, J = 10.5 Hz, 2H), 3.56 (s, 2H), 3.01 (d, J = 5.4 Hz, 2H), 2.35 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.09, 141.70, 131.62, 129.20 (2C), 127.93 (2C), 39.04, 37.53, 21.42.

N-(3-aminopropyl)-4-methylbenzamide (16b)

General procedure 5, (255 mg, 85%). ¹H NMR (400 MHz, DMSO) δ 8.74 (d, J = 4.9 Hz, 1H), 8.15 (s, 2H), 7.81 (d, J = 7.8 Hz, 2H), 7.27 (d, J = 7.8 Hz, 2H), 3.33 (dd, J = 11.8, 5.9 Hz, 2H), 2.82 (d, J = 5.7 Hz, 2H), 2.32 (d, J = 21.7 Hz, 3H), 1.91 – 1.74 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.75, 141.51, 131.92, 129.25 (2C), 127.71 (2C), 37.10, 36.53, 27.70, 21.41.

N-(3-aminopropyl)-[1,1'-biphenyl/-4-carboxamide (16c)

General procedure 5, (645 mg, 81%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.86 (s, 1H), 8.09 (s, 2H), 8.00 (d, J = 8.2 Hz, 2H), 7.76 (dd, J = 17.6, 7.8 Hz, 4H), 7.50 (t, J = 7.6 Hz, 2H), 7.42 (d, J = 7.2 Hz, 1H), 3.37 (dd, J = 12.3, 6.2 Hz, 2H), 2.85 (dd, J = 12.7, 6.4 Hz, 2H), 1.89 – 1.81 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.54, 143.22, 139.62, 133.51, 129.51 (2C), 128.53, 128.39 (2C), 127.33 (2C), 126.96 (2C), 37.16, 36.66, 27.74.

N-(4-aminobutyl)-4-methylbenzamide (16d)

General procedure 5, (200 mg, 90%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.55 (t, J = 5.1 Hz, 1H), 8.07 (s, 2H), 7.78 (d, J = 7.9 Hz, 2H), 7.26 (d, J = 7.9 Hz, 2H), 3.34 – 3.17 (m, 2H), 2.79 (d, J = 5.5 Hz, 2H), 2.35 (s, 3H), 1.58 (s, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.51, 141.32, 132.23, 129.20 (2C), 127.67 (2C), 38.95, 38.84, 26.63, 24.95, 21.40.

N-(4-aminobutyl)-[1,1'-biphenyl]-4-carboxamide (16e)

General procedure 5, (188 mg, 75%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.64 (t, J = 5.5 Hz, 1H), 7.96 (d, J = 8.3 Hz, 2H), 7.75 (dd, J = 16.5, 7.8 Hz, 4H), 7.56 – 7.36 (m, 3H), 3.31 (d, J = 5.5 Hz, 2H), 2.81 (d, J = 5.5 Hz, 2H), 1.60 (s, 4H). ³C NMR (100 MHz, DMSO-d₆) δ 166.29, 143.10, 139.65, 133.81, 129.50, 128.51, 128.32, 127.32 (2C), 126.93, 39.05, 38.92, 26.63, 25.02.

Methyl 4-((2-(4-methylbenzamido)ethyl)carbamoyl)benzoate (17a)

General procedure 1, (240 mg, 46%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.82 (s, 1H), 8.55 (s, 1H), 8.04 (d, J = 8.3 Hz, 2H), 7.97 (d, J = 8.3 Hz, 2H), 7.76 (d, J = 8.0 Hz, 2H), 7.26 (d, J = 8.0 Hz, 2H), 3.88 (s, 3H), 3.45 (s, 4H), 2.35 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.87, 166.21, 166.13, 141.42, 139.14, 132.20, 132.17, 129.58 (2C), 129.23 (2C), 128.09 (2C), 127.68 (2C), 52.83 (2C), 21.40 (2C). HRMS (ESI+): m/z calculated for C₁₉H₂₁N₂O₄⁺ [M+H]⁺ 341.1501, found 341.1517.

Methyl 4-((3-(4-methylbenzamido)propyl)carbamoyl)benzoate (17b)

General procedure 1, (200 mg, 48%). ¹H NMR (400 MHz, CDCl₃) δ 8.11 (d, J = 8.0 Hz, 2H), 7.98 (d, J = 8.1 Hz, 2H), 7.76 (d, J = 7.9 Hz, 2H), 7.72 (d, J = 5.8 Hz, 1H), 7.27 (t, J = 7.5 Hz, 2H), 7.08 (s, 1H), 3.96 (s, 3H), 3.56 (dd, J = 12.8, 6.4 Hz, 4H), 2.42 (s, 3H), 1.84 (d, J = 4.9 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 168.49, 166.99, 166.41, 142.20, 138.29, 132.64, 131.20, 129.84 (2C), 129.34 (2C), 127.15 (2C), 126.99 (2C), 52.37 (2C), 36.32, 29.86, 21.46. HRMS (ESI+): m/z calculated for C₂₀H₂₃N₂O₄⁺ [M+H]⁺ 355.1658, found 355.1686.

N-(3-(4-methylbenzamido)propyl)-[1,1'-biphenyl]-4-carboxamide (17c)

General procedure 1, purified by trituration, (200 mg, 16%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.70 (t, J = 5.5 Hz, 1H), 8.57 (t, J = 5.5 Hz, 1H), 8.01 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 8.2 Hz, 4H), 7.81 – 7.76 (m, 2H), 7.55 (t, J = 7.5 Hz, 2H), 7.46 (t, J = 7.3 Hz, 1H), 7.32 (d, J = 8.0 Hz, 2H), 3.41 – 3.36 (m, 4H), 2.40 (s, 3H), 1.85 (p, J = 6.8 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.59, 166.34, 143.08, 141.34, 139.68, 133.87, 132.28, 129.49 (2C), 129.25 (2C), 128.48, 128.29 (2C), 127.61 (2C), 127.32 (2C), 126.96 (2C), 37.44, 29.78, 21.40 (2C). HRMS (ESI+): m/z calculated for C₂₄H₂₅N₂O₂⁺ [M+H]⁺ 373.1916, found 373.1932.

Methyl 4-((3-([1,1'-biphenyl]-4-carboxamido)propyl)carbamoyl)benzoate (17d)

General procedure 1, (45 mg, 13%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.77 (s, 1H), 8.61 (s, 1H), 8.13 – 7.85 (m, 6H), 7.75 (dd, J = 14.4, 7.8 Hz, 4H), 7.47 (dd, J = 24.9, 17.4 Hz, 3H), 3.88 (s, 3H), 3.30 – 3.23 (m, 2H), 1.88 – 1.76 (m, 2H), 1.23 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.35, 166.21, 165.88, 143.10, 139.68, 139.18, 133.85, 132.15, 129.61 (2C), 129.49 (2C), 128.49, 128.27 (2C), 128.01 (2C), 127.33 (2C), 126.96 (2C), 52.83, 37.75, 37.60, 29.62. HRMS (ESI+): m/z calculated for C₂₅H₂₅N₂0₄⁺ [M+H]⁺ 417.1814, found 417.1840.

N-(3-(4-methoxybenzamido)propyl)-[1,1'-biphenyl]-4-carboxamide (17e)

General procedure 1, purified by trituration, (72 mg, 15%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.58 (s, 1H), 8.39 (s, 1H), 7.94 (d, J = 8.2 Hz, 2H), 7.83 (d, J = 8.6 Hz, 2H), 7.75 (dd, J = 16.5, 7.8 Hz, 4H), 7.50 (t, J = 7.5 Hz, 2H), 7.42 (d, J = 7.2 Hz, 1H), 7.00 (d, J = 8.6 Hz, 2H), 3.80 (s, 3H), 3.31 (s, 4H), 1.86 – 1.70 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.34, 166.22, 161.93, 143.09, 139.67, 133.86, 129.49 (2C), 129.39 (2C), 128.49, 128.27 (2C), 127.33 (2C), 127.28, 126.97 (2C), 113.93 (2C), 55.79, 37.57, 37.45, 29.88. HRMS (ESI+): m/z calculated for C₂₄H₂₅N₂NaO₃⁺ [M+Na]⁺ 411.1685, found 411.1698.

N-(3-(4-(trifluoromethyl)benzamido)propyl)-[1,1'-biphenyl]-4-carboxamide (17f)

General procedure 1, purified by trituration, (69 mg, 20%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.77 (d, J = 5.2 Hz, 1H), 8.59 (t, J = 5.2 Hz, 1H), 8.05 (d, J = 8.0 Hz, 2H), 7.95 (d, J = 8.1 Hz, 2H), 7.86 (d, J = 8.1 Hz, 2H), 7.75 (dd, J = 16.2, 7.9 Hz, 4H), 7.50 (t, J = 7.5 Hz, 2H), 7.42 (d, J = 7.2 Hz, 1H), 3.35 – 3.29 (m, 2H), 2.51 (s, 2H), 1.93 – 1.73 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.37, 165.54, 143.11, 139.67, 138.82, 133.84, 131.65, 131.34, 129.50 (2C), 128.52 (2C), 128.28 (2C), 127.33 (2C), 126.97 (2C), 125.75, 123.09, 40.90, 37.77, 37.58, 29.61. HRMS (ESI+): m/z calculated for C₂₄H₂₂F₃N₂O₂⁺ [M+H]⁺ 427.1633, found 427.1638.

Methyl 4-((4-(4-methylbenzamido)butyl)carbamoyl)benzoate (17g)

General procedure 1, (150 mg, 27%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.68 (s, 1H), 8.39 (s, 1H), 8.03 (d, J = 8.3 Hz, 2H), 7.96 (d, J = 7.8 Hz, 2H), 7.74 (d, J = 7.7 Hz, 2H), 7.25 (d, J = 7.9 Hz, 2H), 3.88 (s, 3H), 3.30 (dd, J = 9.4, 4.3 Hz, 4H), 2.35 (s, 3H), 1.57 (s, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.44, 166.21, 165.74, 141.26, 139.25, 132.34, 132.08, 129.57 (2C), 129.20 (2C), 128.02 (2C), 127.61 (2C), 52.82, 27.23 (2C), 27.08 (2C), 21.39. HRMS (ESI+): m/z calculated for C₂₁H₂₅N₂O₄⁺ [M+H]⁺ 369.1814, found 369.1833.

Methyl 4-((4-([1,1'-biphenyl]-4-carboxamido)butyl)carbamoyl)benzoate (17h)

General procedure 1, purified by trituration, (150 mg, 30%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.70 (s, 1H), 8.54 (s, 1H), 8.10 – 8.01 (m, 4H), 7.99 – 7.91 (m, 3H), 7.74 (dd, J = 12.9, 7.9 Hz, 3H), 7.48 (d, J = 7.6 Hz, 2H), 7.41 (t, J = 7.1 Hz, 1H), 3.88 (d, J = 4.6 Hz, 4H), 3.32 – 3.28 (m, 3H), 1.59 (s, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.22, 165.76, 143.02, 139.69, 139.26, 133.93, 132.08, 129.57 (2C), 129.49 (2C), 128.47, 128.29 (2C), 128.02 (2C), 127.32 (2C), 126.92 (2C), 52.82, 27.19 (2C), 27.08 (2C). HRMS (ESI+): m/z calculated for C₂₆H₂₆N₂NaO₄⁺ [M+Na]⁺ 453.1790, found 453.1805.

N-(4-(4-methylbenzamido)butyl)-[1,1'-biphenyl]-4-carboxamide (17i)

General procedure 1, (203 mg, 45%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.54 (t, J = 5.4 Hz, 1H), 8.40 (t, J = 5.3 Hz, 1H), 7.94 (d, J = 8.3 Hz, 2H), 7.82 – 7.67 (m, 6H), 7.49 (s, 2H), 7.41 (t, J = 7.3 Hz, 1H), 7.25 (d, J = 7.9 Hz, 2H), 3.30 (dd, J = 10.5, 5.5 Hz, 4H), 2.34 (s, 3H), 1.58 (s, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.45, 166.21, 143.02, 141.26, 139.69, 133.94, 132.35, 129.49 (2C), 129.20 (2C), 128.47, 128.29 (2C), 127.61 (2C), 127.32 (2C), 126.92 (2C), 39.47, 27.23 (2C), 21.39 (2C). HRMS (ESI+): m/z calculated for C₂₅H₂₇N₂O₂⁺ [M+H]⁺ 387.2073, found 387.2090.

N-(4-(4-(trifluoromethyl)benzamido)butyl)-[1,1'-biphenyl]-4-carboxamide (17j)

General procedure 1, purified by trituration, (120 mg, 23%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.74 (t, J = 5.3 Hz, 1H), 8.55 (t, J = 5.4 Hz, 1H), 8.05 (d, J = 8.1 Hz, 2H), 7.95 (d, J = 8.3 Hz, 2H), 7.85 (d, J = 8.2 Hz, 2H), 7.75 (dd, J = 14.0, 7.8 Hz, 4H), 7.50 (t, J = 7.5 Hz, 2H), 7.41 (t, J = 7.3 Hz, 1H), 3.33 (d, J = 3.4 Hz, 4H), 1.61 (s, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.23, 165.43, 143.03, 139.69, 138.89, 133.93, 131.53, 129.48 (2C), 128.53 (2C), 128.47, 128.29 (2C), 127.31 (2C), 126.92 (2C), 125.77, 125.73, 27.19 (2C), 27.05 (2C). HRMS (ESI+): m/z calculated for C₂₅H₂₃F₃N₂NaO₂⁺ [M+Na]⁺ 463.1609, found 463.1619.

Methyl 4-([1,1'-biphenyl]-4-ylamino)-4-oxobutanoate (19a)

General procedure 1, (1975 mg, 75%). ¹H NMR (400 MHz, CDCl₃) δ 7.92 (s, 1H), 7.54 (d, J = 13.9 Hz, 6H), 7.40 (s, 2H), 7.35 – 7.15 (m, 1H), 3.71 (s, 3H), 2.73 (d, J = 25.4 Hz, 4H). ¹³C NMR (100 MHz, CDCl₃) δ 173.76, 169.89, 140.50, 137.23, 137.07, 128.79 (2C), 127.56 (2C), 127.09, 126.83 (2C), 120.16 (2C), 52.05, 32.05, 29.26.

Methyl 6-oxo-6-(p-tolylamino)hexanoate (19b)

General procedure 1, (286 mg, 80%). ¹H NMR (400 MHz, CDCl₃) δ 7.73 (s, 1H), 7.42 (d, J = 8.0 Hz, 2H), 7.11 (d, J = 8.0 Hz, 2H), 3.68 (s, 3H), 2.36 (t, J = 6.8 Hz, 4H), 2.31 (s, 3H), 1.81 – 1.63 (m, 4H). ¹³C NMR (100 MHz, CDCl₃) δ 174.08, 170.91, 135.46, 133.77, 129.40 (2C), 120.03 (2C), 51.61, 37.06, 33.68, 25.00, 24.33, 20.85.

4-([1,1'-biphenyl]-4-ylamino)-4-oxobutanoic acid (20a)

General procedure 6, (1530 mg, 90%). ¹H NMR (400 MHz, DMSO-d₆) δ 11.80 (s, 1H), 7.69 – 7.51 (m, 6H), 7.43 (t, J = 7.3 Hz, 2H), 7.31 (t, J = 7.3 Hz, 1H), 2.45 – 2.33 (m, 2H), 2.23 (t, J = 6.4 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 176.24, 175.14, 172.95, 140.33, 139.92, 134.47, 129.32 (2C), 127.25 (2C), 126.61 (2C), 119.53 (2C), 35.30, 34.59.

6-oxo-6-(p-tolylamino)hexanoic acid (20b)

General procedure 6, (263 mg, 92%). ¹H NMR (400 MHz, DMSO-d₆) δ 12.11 (s, 1H), 9.85 (s, 1H), 7.53 (d, J = 8.2 Hz, 2H), 7.15 (d, J = 8.2 Hz, 2H), 2.43 – 2.30 (m, 4H), 2.28 (s, 3H), 1.76 – 1.52 (m, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 174.85, 171.23, 137.28, 132.25, 129.48 (2C), 119.51 (2C), 36.52, 33.89, 25.15, 24.62, 20.89.

Methyl 4-(4-([1,1'-biphenyl]-4-ylamino)-4-oxobutanamido)benzoate (21a)

General procedure 1, purified by trituration, (40 mg, 13%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.09 (s, 1H), 9.90 (s, 1H), 7.70 (s, 1H), 7.62 (t, J = 9.0 Hz, 3H), 7.54 – 7.40 (m, 4H), 7.32 (t, J = 7.3 Hz, 1H), 7.09 (d, J = 8.2 Hz, 2H), 2.50 (s, 4H), 2.24 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 170.95, 170.54, 140.20, 139.29, 137.31, 135.02, 132.20, 129.51 (2C), 129.49, 129.35 (2C), 127.42, 127.34 (2C), 126.66 (2C), 119.74, 119.45, 119.39, 31.78, 31.64, 20.89. HRMS (ESI+): m/z calculated for C₂₃H₂₂N₂NaO₂⁺ [M+Na]⁺ 381.1579 found 381.1578.

Methyl 4-(6-oxo-6-(p-tolylamino)hexanamido)benzoate (21b)

General procedure 1, (130 mg, 37%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.26 (s, 1H), 9.80 (s, 1H), 7.97 – 7.88 (m, 2H), 7.74 (t, J = 9.8 Hz, 2H), 7.52 – 7.38 (m, 2H), 7.08 (d, J = 8.1 Hz, 2H), 3.82 (s, 3H), 2.35 (d, J = 27.5 Hz, 4H), 2.24 (s, 3H), 1.63 (s, 4H). ¹³C NMR (100 MHz, DMSO-d₆) δ 172.25, 171.30, 166.32, 144.14, 137.24, 132.30, 130.72 (2C), 129.48 (2C), 124.14, 119.55 (2C), 118.84 (2C), 52.33, 36.84, 36.66, 25.33, 25.19, 20.88. HRMS (ESI+): m/z calculated for C₂₁H₂₄N₂NaO₄⁺ [M+Na]⁺ 391.1634, found 391.1649.

1,3-di-p-tolylurea (23a)

General procedure 3, purified by trituration, (320 mg, 89%). ¹H NMR (400 MHz, DMSO-d₆) δ 8.50 (s, 2H), 7.33 (d, J = 8.3 Hz, 4H), 7.08 (d, J = 8.2 Hz, 4H), 2.24 (s, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 153.01, 137.59 (2C), 131.02 (2C), 129.62 (4C), 118.67 (4C), 20.80 (2C). HRMS (ESI+): m/z calculated for C₁₅H₁₇N₂O⁺ [M+H]⁺ 241.1341, found 241.1361. This matches the previously reported spectra.^41-43.

Methyl 4-(3-(p-tolyl)ureido)benzoate (23b)

General procedure 3, purified by trituration, (100 mg, 24%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.06 (s, 1H), 8.70 (s, 1H), 7.89 (d, J = 8.7 Hz, 2H), 7.58 (d, J = 8.6 Hz, 2H), 7.35 (d, J = 8.3 Hz, 2H), 7.10 (d, J = 8.2 Hz, 2H), 3.82 (s, 3H), 2.25 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.17, 152.74, 144.97, 137.17, 131.56, 130.88 (2C), 129.70 (2C), 122.76, 118.98 (2C), 117.69 (2C), 52.24, 20.82. HRMS (ESI+): m/z calculated for C₁₆H₁₇N₂O₃⁺ [M+H]⁺ 285.1239, found 285.1252.

1-(4-methylbenzyl)-3-(p-tolyl)urea (23c)

General procedure 3, purified by trituration, (450 mg, 71%) ¹H NMR (400 MHz, DMSO-d₆) δ 8.39 (s, 1H), 7.27 (d, J = 8.3 Hz, 2H), 7.15 (dd, J = 19.5, 7.9 Hz, 4H), 7.02 (d, J = 8.2 Hz, 2H), 6.48 (t, J = 5.8 Hz, 1H), 4.23 (d, J = 5.8 Hz, 2H), 2.27 (s, 3H), 2.21 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 155.73, 138.38, 137.80, 136.19, 130.23, 129.50 (2C), 129.30 (2C), 127.60 (2C), 118.24 (2C), 42.95, 21.13, 20.76. HRMS (ESI+): m/z calculated for C₁₆H₁₉N₂O⁺ [M+H]⁺ 255.1497, found 255.1521.

Methyl 4-((3-(p-tolyl)ureido)methyl)benzoate(23d)

General procedure 3, purified by trituration, (180 mg, 36%). ¹H NMR (400 MHz, DMSO-d₆)) δ 8.52 (s, 1H), 7.93 (d, J = 8.1 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 7.28 (d, J = 8.3 Hz, 2H), 7.03 (d, J = 8.2 Hz, 2H), 6.66 (s, 1H), 4.36 (d, J = 5.9 Hz, 2H), 3.84 (s, 3H), 2.21 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆ δ 166.61, 155.79, 146.84, 138.28, 130.36, 129.71 (2C), 129.51 (2C), 128.48, 127.65 (2C), 118.34 (2C), 52.51, 42.96, 20.76. HRMS (ESI+): m/z calculated for C₁₇H₁₉N₂O₃⁺ [M+H]⁺ 299.1396, found 299.1412.

1-(4-methoxybenz.yl)-3-(p-tolyl)urea (23e)

General procedure 3, purified by trituration, (145 mg, 36%). ¹H NMR (400 MHz, DMSO-d₆)) δ 8.38 (s, 1H), 7.27 (d, J = 8.2 Hz, 2H), 7.22 (d, J = 8.3 Hz, 2H), 7.02 (d, J = 8.2 Hz, 2H), 6.89 (ds, J = 8.3 Hz, 2H), 6.46 (d, J = 5.4 Hz, 1H), 4.20 (d, J = 5.8 Hz, 2H), 3.73 (s, 3H), 2.21 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 158.64, 155.69, 138.38, 132.76, 130.20, 129.50 (2C), 128.97 (2C), 118.22 (2C), 114.17 (3C), 55.52, 42.67, 20.76. HRMS (ESI+): m/z calculated for C₁₆H₁₈N₂NaO₂⁺ [M+Na]⁺ 293.1266, found 293.1284.

4-methyl-N-(p-tolylcarbamoyl)benzenesulfonamide (25a)

General procedure 3, purified by recrystallization, (205 mg, 67%). ¹H NMR (400 MHz, CDCl₃) δ 9.95 (s, 1H), 8.05 (s, 1H), 7.90 (d, J = 8.2 Hz, 2H), 7.28 (d, J = 8.1 Hz, 2H), 7.21 (d, J = 8.3 Hz, 2H), 7.03 (d, J = 8.2 Hz, 2H), 2.39 (s, 3H), 2.26 (s, J = 8.4 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 149.45, 144.37, 136.95, 134.87, 133.35, 129.57 (2C), 129.39 (2C), 127.68 (2C), 119.65 (2C), 21.59, 20.76. HRMS (ESI+): m/z calculated for C₁₅H₁₇N₂O₃S⁺ [M+H]⁺ 305.0960 found 305.1461. This matches the previously reported spectra.^44,45

Methyl 4-(3-tosylureido)benzoate (25b)

General procedure 3, purified by trituration, (140 mg, 30%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.96 (s, 1H), 9.25 (s, 1H), 7.86 (d, J = 8.3 Hz, 4H), 7.46 (dd, J = 15.6, 8.4 Hz, 4H), 3.80 (s, 3H), 2.40 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.81, 166.20, 149.81, 144.43, 143.15, 137.38, 131.53, 130.80, 129.98, 129.76, 128.03, 126.08, 118.64, 113.11, 52.35, 51.60. HRMS (ESI+): m/z calculated for C₁₆H₁₆N₂NaO₅S⁺ [M+Na]⁺ 371.0678, found 371.0694. This matches the previously reported spectra.^45,46

4-methyl-N-((4-methylbenzyl)carbamoyl)benzenesulfonamide (25c)

General procedure 3, purified by trituration, (100 mg, 24%). ¹H NMR (400 MHz, DMSO-d₆) δ 7.58 (d, J = 8.2 Hz, 2H), 7.27 (d, J = 8.0 Hz, 2H), 7.22 (d, J = 8.0 Hz, 2H), 7.17 (d, J = 7.9 Hz, 2H), 6.33 (s, 2H), 2.34 (s, 3H), 2.30 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 154.99, 141.51, 141.24, 137.05, 129.37 (3C), 128.89 (2C), 128.43 (2C), 127.38 (2C), 21.38 (2C), 21.17 HRMS (ESI+): m/z calculated for C₁₆H₁₉N₂NaO₃S⁺ [M+Na]⁺ 341.0936 found 341.0948.

Methyl 4-((3-tosylureido)methyl)benzoate (25d)

General procedure 3, purified by trituration, (150 mg, 45%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.89 (s, 1H), 7.92 (d, J = 8.2 Hz, 2H), 7.85 (d, J = 8.2 Hz, 2H), 7.47 (d, J = 8.1 Hz, 2H), 7.30 (d, J = 8.1 Hz, 2H), 7.19 (t, J = 5.7 Hz, 1H), 4.29 (d, J = 5.9 Hz, 2H), 3.90 (s, 3H), 2.46 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 166.52, 152.11, 145.42, 144.14, 137.77, 129.93, 129.62 (2C), 128.61, 127.71 (2C), 127.55 (2C), 52.53, 42.98, 21.48. HRMS (ESI+): m/z calculated for C₁₇H₁₈N₂NaO₅S⁺ [M+Na]⁺ 385.0834, found 385.0834.

N-((4-methoxybenzyl)carbamoyl)-4-methylbenzenesulfonamide (25e)

General procedure 3, purified by trituration, (90 mg, 18%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.64 (s, 1H), 7.78 (d, J = 8.1 Hz, 2H), 7.41 (d, J = 8.1 Hz, 2H), 7.07 (d, J = 8.4 Hz, 2H), 6.91 (s, 1H), 6.83 (d, J 8.3 Hz, 2H), 4.07 (d, J = 5.7 Hz, 2H), 3.72 (s, 3H), 2.40 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 158.83, 151.86, 144.08, 137.84, 131.51, 129.91 (2C), 128.93 (2C), 127.71 (2C), 114.12 (2C), 55.50, 42.64, 21.50. HRMS (ESI+): m/z calculated for C₁₆H₁₈N₂NaO₄S⁺ [M+Na]⁺ 357.0885, found 357.0888.

Cell Culture

HuH6 cells,²⁹ HuH7 (XenoTech JCRB0403) cells and HEK-293 cells (ATCC CRL-1573) were grown in Dulbecco’s Modified Eagle Medium (VWR #45000-304) supplemented with 10% FBS and 1% penicillin/streptomycin. HepG2 (ATCC HB-8065) were grown in RPMI-1640 Medium (VWR #10-040 CV) supplemented with 10% FBS, 1% sodium pyruvate and 1% penicillin/streptomycin. Hep3B cells (ATCC HB-8064) were grown in Eagle's Minimum Essential Medium (EMEM) (ATCC #30-2003) supplemented with 10% FBS and 1% penicillin/streptomycin.

Anti-Proliferative Assay

Compounds 4a-b, 7a-c, 10a-h, 13a-f, 17a-l, 21a-c, 23a-b, 24a-c, 26a-b, and 27a-c were initially dissolved in 100% DMSO. HuH6, HuH7, and HEK-293 cells were all seeded in 96-well plates at 2.5 x 10⁴ cells/well. HepG2 and Hep3B cells were all seeded in 96-well plates at 3.5 x 10⁴ cells/well. Cells were treated with increasing concentrations of all compounds for 72 h at 37 °C in a CO₂ incubator. The MTT assay was performed with thiazolyl blue tetrazolium bromide (MTT, VWR #97062-380) dye as follows: 20 μL of MTT dye at a concentration of 5.4 mg /mL was added to each well, and then plates were incubated for 3 h at 37 °C in a CO₂ incubator. Media was then removed and 100 μL of DMSO was added. Plates were then shaken at room temperature for 10 min to dissolve the purple-colored formazan crystals. After 10 min, the absorbance was measured using a Tecan SPARK10M spectrophotometer (Tecan Group Ltd., Mannedorf, Switzerland) at 570 nm. Each experiment had 6 repeats per treatment. Analysis and IC₅₀ determination were performed using GraphPad Prism v6.0.

CETSA Analysis

HuH6 cells were seeded in a T175 flask and treated with an IC₅₀ concentration of compound 13d in DMSO (0.25% DMSO final concentration), or 0.25% DMSO only for 5 h. Cells were harvested and divided into 6 aliquots, these individual aliquots were then subjected to heat treatment at either 50, 53, 56, 59, 62 or 65 °C. After heat treatment, cells were lysed with Radioimmunoprecipitation assay (RIPA) buffer and a protease/phosphatase inhibitor cocktail (ThermoFisher) on ice for 60 min at 4 °C. Protein concentrations were determined with the DC Protein Assay Kit (BioRad) per the manufacturer’s instructions. Equal amounts of protein (15 μg) were electrophoresed under reducing conditions, transferred to a PVDF membrane, and immunoblotted with the corresponding primary antibodies: anti-β-actin (Cell Signaling) and anti-Gankyrin (Cell Signaling). Membranes were incubated with an appropriate horseradish peroxidase-labeled secondary antibody (anti-Rabbit, Invitrogen), developed with WesternBright ECL chemiluminescent kit (Gel Company, San Francisco, CA), and visualized with an Omega LumG imaging system (Gel Company, San Francisco, CA).

Western blot analysis

HuH6 cells were seeded in 10 cm dishes and treated with varying concentrations of compounds 13d in DMSO (0.25% DMSO final concentration), or 0.25% DMSO only for 48 h. Cells were harvested in cold PBS and lysed with Radioimmunoprecipitation assay (RIPA) buffer and a protease/phosphatase inhibitor cocktail (ThermoFisher) on ice for 60 min at 4 °C. Protein concentrations were determined with the DC Protein Assay Kit (BioRad) per the manufacturer’s instructions. Equal amounts of protein (15 μg) were electrophoresed under reducing conditions, transferred to a PVDF membrane, and immunoblotted with the corresponding primary antibodies: anti-p53 (Cell Signaling), anti-Rb (Cell Signaling), anti-Cyclophilin (Cell Signaling), anti-Gankyrin (Cell Signaling). Membranes were incubated with an appropriate horseradish peroxidase-labeled secondary antibody (anti-Mouse, ThermoFisher Scientific; anti-Rabbit, Invitrogen), developed with WesternBright ECL chemiluminescent kit (Gel Company, San Francisco, CA), and visualized with an Omega LumG imaging system (Gel Company, San Francisco, CA).

Cell cycle analysis

HuH6 cells were seeded in 10 cm dishes and treated with 1.3 μM of compound 13d, in DMSO (0.25% DMSO final concentration), or 0.25% DMSO only for 72 h. Cells were harvested in cold PBS and normalized. The cells were then fixed in 70% ethanol and incubated at −20 °C overnight. Cells were then centrifuged, and the pellet was resuspended in 50 μL of PI/RNase kit (BD). The cells were then incubated for 30 min at 37 °C before performing flow and imaging cytometry analysis. Results were analyzed using FlowJo^™ v10.8. One-way ANOVA was used to determine the statistical significance. A P-value < 0.05 was considered statistically significant.

Protein Expression and Purification of recombinant gankyrin and ANKRA2

The pQTEV-PSMD10 bacterial expression vector for gankyrin was obtained from Addgene (plasmid #31332). The plasmid was transformed into LOBSTR BL21 (DE3) Escherichia coli cell line on LB agar plates supplemented with 100 μg/mL ampicillin. Colonies were aseptically transferred into a sterile 250 mL baffled flask with 50 mL of autoclaved LB broth supplemented with 100 μg/mL ampicillin and placed in an incubator at 37 °C and 200 rpm overnight. Large scale production cultures with 1 L LB Broth and the appropriate antibiotics were inoculated with 25 mL of starter culture and grown at 37 °C until the OD₆₀₀ reached 0.6–0.8. The cultures were then induced with 0.5 mM isopropyl thiogalactoside (IPTG) and grown at 37 °C and 200 rpm for 16 h. Cells were harvested by centrifugation with a Sorvall LYNX 4000 Superspeed Centrifuge with a Fiberlite F10-4x1000 LEX rotor (Thermo Fisher) at 6,000 rpm for 15 min at 4 °C. The cell pellets were then resuspended in wash buffer (25 mM Tris, 200 mM NaCl, 10 mM Imidazole, pH 8) supplemented with ProBlock^™ Protease Inhibitor Cocktail (GoldBio) at 100 μL/mL. The cells were lysed using an EmulsiFlex-C5 homogenizer (Avestin), and the cell extract was clarified using a Beckman Coulter Optima L-100XP Preparative Ultracentrifuge with a Ti 70.1 rotor (Beckman Coulter) at 41,000 rpm for 40 min at 4 °C. The clarified supernatant was collected and passed through a 5.0 μm syringe filter (Thermo Fisher) before being loaded onto a 5 mL HisTrap FF column (Cytiva) on an ÄKTA Start FPLC (Cytiva). After 20 column volumes of wash buffer, gankyrin was eluted with elution buffer (25 mM Tris, 200 mM NaCl, 200 mM Imidazole, pH 8). The N-terminal poly-histidine tag was removed with TEV protease (40 μg/mL) and incubated for 1 h at room temperature on a platform shaker. The sample was then dialyzed to remove excess imidazole with 3.5 MWCO Spectra/Por 3 Dialysis Tubing (Thermo Fisher) in wash buffer at 4 °C gently mixing overnight. The sample was then passed through 5 mL HisTrap FF again to remove the His6-tag and TEV protease from the sample. If any protein contaminants remained, gel filtration chromatography using a Superdex 75 HiLoad column (Cytiva) was performed on an AKTA pure 25L FPLC system (Cytiva) equilibrated with wash buffer. Fractions containing pure gankyrin protein as assessed by SDS-PAGE were pooled, quantified by absorbance, aliquoted, flash frozen on liquid nitrogen, and stored at −80 °C.

The ANKRA2 bacterial expression vector was obtained from Addgene (plasmid #36904). The plasmid was transformed into transformed in a LOBSTR BL21 (DE3) Escherichia coli cell line on LB agar plates supplemented with 30 μg/mL kanamycin. The remainder of the ANKRA2 overexpression and purification was performed the same as described for gankyrin except for using kanamycin for selection during culture growth and protein overexpression.

Circular Dichroism

Circular dichroism (CD) experiments were collected on a Jasco J-815 Circular Dichroism Spectropolarimeter (JASCO Inc.). Each sample contained 10 μM of purified gankyrin or ANKRA2 in CD buffer (25 mM Tris, 50 mM NaCl, pH 7.4) alone or in the presence of 1 μM, 10 μM, and 100 μM compounds 10d or 13d. Samples were measured in a 1 mm quartz cuvette. CD spectra were collected from 195–260 nm, with six accumulation trials at 25 °C. All CD samples were performed in triplicate to ensure reproducibility.

Figure 6. Circular dichroism spectral analysis of gankyrin and ANKRA2 in the presence of compounds 10d (blue) and 13d (orange).

Protein alone samples (black) were saturated with increasing concentrations of compounds 10d and 13d (represented by a dark to light colorimetric scheme) to measure changes in α-helical content. Each protein was measured at 10 μM and introduced to compound concentrations ranging from 1 μM to 100 μM. Gankyrin samples are represented by circular markers while ANKRA2 samples are triangular markers. The CD spectra for each protein alone showed characteristic α-helical spectral minima at 208 and 222 nm. (A) In the presence of compound 10d, the CD spectra of gankyrin (left) depicted a reduction in secondary structure content as the drug concentration increased. Conversely, for ANKRA2 (right), the CD spectra showed no changes in secondary structure content. (B) For compound 13d, the CD spectra of gankyrin (left) indicated a decline in secondary structure content with increasing drug concentration. In contrast, the CD spectra of ANKRA2 (right) revealed no alterations in secondary structure content. Data shown is representative of triplicate runs.

Highlights.

• Potent antiproliferative activity against certain liver cancers was exhibited

• Minimal impact on potency from only replacing the propyl linker of cjoc42

• Gankyrin stabilization was observed as a mechanism of inhibition

• Gankyrin specific binding was demonstrated

• Disruption of the proteasomal degradation pathway was demonstrated

Data Availability

Data will be made available upon reasonable request.