Small Molecule Gankyrin Inhibition as a Therapeutic Strategy for Breast and Lung Cancer

Abstract

Gankyrin is an oncoprotein responsible for the development of numerous cancer types. It regulates the expression levels of multiple tumor suppressor proteins (TSPs) in liver cancer, however, gankyrin’s regulation of these TSPs in breast and lung cancer has not been thoroughly investigated. Additionally, no small molecule gankyrin inhibitor has been developed which demonstrates potent antiproliferative activity against gankyrin overexpressing breast and lung cancers. Herein, we are reporting the structure-based design of gankyrin-binding small molecules which potently inhibited the proliferation of gankyrin overexpressing A549 and MDA-MB-231 cancer cells, reduced colony formation, and inhibited the growth of 3D spheroids in an in vitro tumor simulation model. Investigations demonstrated that gankyrin inhibition occurs through either stabilization or destabilization of its 3D structure. These studies shed light on the mechanism of small molecule inhibition of gankyrin and demonstrate that gankyrin is a viable therapeutic target for the treatment of breast and lung cancer.

Table of Contents Graphic

Introduction

Gankyrin is important for regulating numerous oncogenic and inflammatory pathways through its various protein-protein interactions (PPIs).^1–5 Gankyrin is a seven-ankyrin repeat oncoprotein associated with the 19S subunit of the 26S proteasome assembly.^3,6,9 It has been established that gankyrin overexpression increases the degradation of tumor suppressor proteins (TSPs), ultimately resulting in uncontrolled cell proliferation.^4,7 More specifically, gankyrin aids in the hyperphosphorylation of retinoblastoma protein (Rb) by binding to cyclin-dependent kinase 4 (CDK4) and priming Rb for proteasomal degradation.^1,2 In a similar manner, gankyrin binds to the Really Interesting New Gene (RING) E3 ubiquitin ligase mouse double minute 2 homolog (MDM2) to increase its ubiquitylation activity on p53, modifying it for proteolysis through the 26S proteasome.⁸ Additionally, gankyrin is a repeat-protein “chaperone” required for the proper assembly of the 26S proteasome. It binds the S6 ATPase subunit of the 19S regulatory cap, which in turn enhances the targeting of p53, Rb, and other TSPs (e.g., CCAAT/Enhancer-binding protein α (C/EBPα), CUG triplet repeat RNA binding protein 1 (CUGBP1), and hepatocyte nuclear factor 4 α (HNF4α)) to the 26S proteasome for degradation.^3,9 Gankyrin overexpression and reduced TSP levels then results in the onset and progression of multiple cancer types (e.g., lung, breast, and liver).^10–13

TSPs typically function to regulate cellular growth, division, and programmed cell death. Specifically, Rb controls the expression of various genes (e.g., E2F transcription factor family, topoisomerase IIα, cyclin E) required for progression into the S phase of the cell cycle, thereby gatekeeping cell proliferation and growth.^14,15 Additionally, p53 plays a critical role in maintaining the G1 to S phase checkpoint, and degradation of p53 inhibits the DNA damage response, causing an increase in cell proliferation.^16,17 Gankyrin’s regulation of multiple TSPs lends itself as an important regulator of the G0/G1 and S phases of the cell cycle.^7,18 Studies have shown that decreased gankyrin expression levels cause an increase in intracellular TSP levels, which has been associated with reduced cancer cell growth^19,20, illustrating gankyrin’s promise as a therapeutic target. Therefore, inhibiting gankyrin’s ability to facilitate the degradation of various TSPs will arrest the cell cycle and decrease cancer cell proliferation.

Cjoc42 (1) was discovered in 2016 as the first small molecule inhibitor of gankyrin.²¹ A subsequent study demonstrated that cjoc42 increases the levels of multiple TSPs by disrupting the interaction of gankyrin with the 26S proteasome, thereby inhibiting the proliferation of human and murine liver cancer cell lines (HuH6 and hepa1c1c7, respectively).^19,21 Despite these findings, cjoc42 exhibited only modest gankyrin binding and antiproliferative activity in liver cancer, demonstrating its inability to be used by itself as a therapeutic for liver cancer treatment. While part of cjoc42’s inability to effectively inhibit cancer cell proliferation can be attributed to its relatively low affinity for gankyrin, this could also be attributed to its poor drug-like properties (e.g., metabolic stability, Table 4).

Table 4.

In vitro metabolic stability study of cjoc42, as well as compounds 51b, 51c, 51d, 52b and 52d.

Compound	% Metabolic stabilitya
cjoc42	50 ± 5.1
51b	84 ± 9.0
51c	87 ± 3.3
51d	60 ± 11.0
52b	73 ± 0.8
52d	80 ± 3.5

^aMetabolic stability was calculated as the % compound remaining after 60 min of incubation with human liver microsomes.

Initial nuclear magnetic resonance (NMR) spectroscopic analysis and molecular docking studies have suggested that cjoc42 binds gankyrin primarily through interactions with five key amino acid residues in gankyrin (i.e, Y15, R41, W46, S49, W74, and K116) (Figure 1A). From this, it appears that the sulfonate ester group of cjoc42 is involved in a hydrogen bonding interaction with K116. In contrast, its triazole ring seems to engage in hydrogen bonding interactions with Y15 and S49.²¹ Therefore, we hypothesized that bioisosteric replacement of the sulfonate ester and triazole ring would optimize known interactions with gankyrin, as well as probe for potentially new interactions (Figure 1D). Additionally, cjoc42 (1) and its early derivatives (Figure 1D), AFM-1–2 (2) and DK-1-67 (3),^39,44 have found limited use against breast cancer and lung cancer, where small-molecule inhibition of gankyrin and its role in TSP degradation have not been shown. Therefore, we sought to develop gankyrin inhibitors based on the cjoc42 (1) scaffold with enhanced gankyin binding and antiproliferative activity against lung and breast cancer.

Figure 1.

Molecular modeling and structures of cjoc42 (1), AFM-1–2 (2), and DK-1–67 (3). (A) Cjoc42 docked to its proposed gankyrin binding site (PDB: 1QYM). (B) AFM-1–2 (2) docked to its proposed gankyrin binding site (PDB: 1QYM) (C) DK-1–67 (3) docked to its proposed gankyrin binding site (PDB: 1QYM) (D) Chemical structures, thermal shift values, and antiproliferative activity of cjoc42 (1), AFM-1–2 (2), DK-1–67 (3), and targeted cjoc42 derivatives.

Results and Discussion

Synthesis and antiproliferative evaluation

Identifying the optimal replacement of the sulfonate ester was pursued by synthesizing cjoc42 derivatives 20–33 (Scheme 1). Since the triazole of compounds 20–33 could be prepared via a copper-catalyzed alkyne-azide cycloaddition (CuAAc) reaction with methyl 4-azidobenzoate, access to the desired cjoc42 derivatives was achieved by first synthesizing the requisite alkyne intermediates. Specifically, the synthesis of alkyne intermediates 6, 7, 9, and 10 was achieved through an HBTU-mediated ester or amide formation, followed by CuAAc to generate 20, 22, 23, and 25. Amides 20 and 23 then underwent thionation with Lawesson’s reagent to generate compounds 21 and 24, respectively. Urea-containing alkyne intermediate 12 was obtained by a nucleophilic addition reaction of isocyanate 11 with 4-penyn-1-amine, followed by CuAAc to generate urea 26. Compound 26 also underwent thionation with Lawesson’s reagent to afford thiourea 27. N-acyl thioamide intermediate 14 was prepared by a Muikayama reagent-mediated coupling of sulfonamide 13 with 5-hexynoic acid. Triazole (16) and oxadiazole (17) alkyne intermediates were generated by a cycloaddition reaction of compound 15 with either 5-hexynenitrile or 5-hexynoic acid, respectively. Tetrazole alkyne intermediate 19 was generated by a cycloaddition reaction of p-tolunitrile (18) with sodium azide, followed by a Mitsonobu reaction with 4-pentyn-1-ol. Alkyne intermediates 14, 16, 17, and 19 then underwent a CuAAc reaction with methyl 4-azidobenzoate to afford desired cjoc42 derivatives 28–31. Alternatively, compounds 32 and 33, which replaced the sulfonate ester of cjoc42 (1) with either a secondary amine or ether group, were prepared by nucleophilic substitution of the tosyl group of cjoc42 (1) with either p-toluidine or p-cresol, respectively.

Scheme 1.

^a Synthesis of compounds 20–33

^aReagents: (a) Hexynoic acid, HBTU, DIPEA, CH₂Cl₂; (b) 4-pentyn-1-amine or 4-pentyn-1-ol, HBTU, DIPEA, CH₂Cl₂; (c) 4-pentyn-1-amine, DIPEA, CH₂Cl₂; (d) 5-Hexynoic acid, Mukaiyama reagent, DMAP, DIPEA, CH₂Cl₂; (e) 5-Hexynenitrile, K₂CO₃, 1-butanol, 150 °C; (f) 5-Hexynoic acid, POCl₃, reflux; (g) NaN₃, TEA HCl, toluene, reflux; (h) 4-Pentyn-1-ol, PPh₃, diisopropyl azodicarboxylate, THF, reflux; (i) Methyl 4-azidobenzoate, CuSO₄, sodium ascorbate, THF/^tBuOH/H₂O; (j) Lawesson’s reagent, toluene, 90 °C; (k) p-Toluidine, KI, K₂CO₃, DMF, 90 °C; (m) p-cresol, NaH, DMF, 90 °C

Compounds 20–33 were then evaluated for their antiproliferative activity against gankyrin overexpressing A549 (non-small cell lung cancer, NSCLC) and MDA-MB-231 (breast cancer) cell lines, as shown in Table 1. These two cell lines were chosen due to their overexpression of gankyrin, differences in p53 status (A549 expresses wild-type and MDA-MB-231 expresses mutant-type), and both cell lines have been shown multiple times in the literature to be sensitive to gankyrin levels.^{3,8,11,38,45,46,58} Compounds 26 and 27 exhibited modest improvements in antiproliferative activity against A549, while compound 21 demonstrated a substantial improvement (∼10-fold) as compared to cjoc42. Additionally, compounds 20, 21, 26, and 33 demonstrated ∼8–10-fold improvements in antiproliferative activity against MDA-MB-231 cells, while compounds 22, 24, 29, 31, and 32 showed modest or no improvement in antiproliferative activity as compared to cjoc42. These results suggest that replacing the sulfonate ester group of cjoc42 with a hydrogen bond donating group (amide, thioamide, and urea) enhances the antiproliferative activity in both cell lines. This may be due to enhanced binding with gankyrin through a gained interaction with K116 or potentially interacting with the previously unengaged S82 (not shown).

Table 1.

^a-d Antiproliferative evaluation of cjoc42 and compounds 20–33 against A549 and MDA-MB-231.

Compound	Structure	A549IC50(μM)	MDA-MB-231 IC50 (μM)
1: cjoc42		>100	>100
20		75.7 (±5.5)	9.7 (±1.1)
21		7.9 (±11)	8.1 (±11)
22		>100	55.1 (±6.5)
23		>100	>100
24		>100	82.4 (±4.3)
25		>100	>100
26		36.4 (±2.6)	11.6 (±1.0)
27		46.5 (±2.1)	>100
28		>100	>100
29		79.5 (±4.2)	79.1 (±2.1)
30		65.1 (±3.5)	21.5 (±1.2)
31		>100	36.5 (±6.4)
32		>100	33.0 (±7.4)
33		>100	14.2 (±2.3)

^aA549 and MDA-MB-231 cells were incubated for 24 h prior to drug addition.

^bA549 and MDA-MB-231 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.

Replacement of the triazole ring with various 5-membered heterocycles (43–47) was then pursued through a series of cycloaddition reactions followed by tosylation of key alcohol intermediates 35, 37, 39, 41, and 42, as shown in Scheme 2. Specifically, compound 43 was generated from a 1,5-disubstituted triazole intermediate (35), which was prepared through a Ru-catalyzed alkyne azide cycloaddition reaction of 34 with 4-pentyn-1-ol. 1,4-disubstituted triazole intermediate 37 was prepared by a CuAAC reaction of methyl 4-ethynylbenzoate (36) and 3-azido-1-propanol, which was then used to obtain compound 44. Hydrazide 39 was synthesized by an HBTU-mediated amide coupling of monomethyl terepthalate (38) with 4-hydroxybutanohydrazide, which then underwent cyclization and subsequent tosylation in the presence of p-tosyl chloride to afford 45. Compound 46 was obtained from 2,4-disubstituted triazole intermediate 41, which was synthesized by a cycloaddition reaction of methyl 4-cyanobenzoate (40) and 4-hydroxybutanohydrazide. Lastly, tetrazole intermediate 42, required for synthesizing cjoc42 derivative 47, was generated by a cycloaddition reaction of methyl 4-cyanobenzoate (40) with sodium azide, followed by nucleophilic substitution with 3-bromo-1-propanol.

Scheme 2.

^a Synthesis of compounds 43–47

^aReagents: (CpRuCl)₄, 4-pentyn-1-ol, toluene, reflux; (b) 3-Azido-1-propanol, CuSO₄, sodium ascorbate, t-BuOH/THF/H₂O; (c) 4-Hydroxybutanohydrazide, HBTU, DIPEA, DMF; (d) 4-Hydroxybutanohydrazide, MeONa, MeOH, reflux; (e) NaN₃, TEA HCl, toluene, reflux; (f) 3-Bromo-1-propanol, TEA, MeCN, 60 °C; (g) p-Tosylchloride, DMAP, TEA, CH₂Cl₂.

Compounds 43–47 were also evaluated for their antiproliferative activity against gankyrin overexpressing A549 (NSCLC) and MDA-MB-231 (breast cancer) cell lines, as shown in Table 2. Compounds 43-47 exhibited similar antiproliferative activity against A549 (>100 μM) as cjoc42. However, compounds 43 and 44 did demonstrate modest improvements in antiproliferative activity against MDA-MB-231 (IC₅₀ = 58.1 μM and 39.1 μM, respectively). This suggests that the orientation of the triazole ring is vital for gankyrin binding, potentially interacting with Y15, W46, and S49.

Table 2.

^a-d Antiproliferative evaluation of cjoc42, and compounds 43–47 against A549 and MDA-MB-231.

Compound	Structure	A549 IC50 (μM)	MDA-MB- 231 IC50 (μM)
1: cjoc42		>100	>100
43		>100	58.1 (±6.4)
44		>100	39.1 (±2.1)
45		>100	>100
46		>100	>100
47		>100	>100

^aA549 and MDA-MB-231 cells were incubated for 24 h prior to drug addition.

^bA549 and MDA-MB-231 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 found that a 4-phenyl substitution on the phenyl ring connected to the sulfonate ester of cjoc42, AFM-1–2 (2),⁴⁴ or a 4-trifluoromethyl substitution on the phenyl ring connected to the triazole of cjoc42, DK-1–67 (3),³⁹ improved gankyrin binding and exhibited a modest improvement in antiproliferative activity against certain gankyrin overexpressing cancer cell lines, as compared to cjoc42 (Figure 1D). The current study found that compounds 20, 21, and 44 exhibited substantial improvements in antiproliferative activity against A549 and MDA-MB-231 cells as compared to cjoc42. Therefore, we sought to combine these previously optimized phenyl ring substitutions (AFM-1–2 and DK-1–67) with specific sulfonate ester and triazole replacements explored in this work. This resulted in the synthesis of cjoc42 derivatives 51a-d and 52a-d (Scheme 3). Briefly, amide intermediates 49a and 49b were obtained by an amide coupling reaction of either 48a or 48b with 4-bromobutyryl chloride. Bromo intermediates 49a and 49b then underwent a nucleophilic substitution reaction with sodium azide to provide azido intermediates 50a and 50b. These azido intermediates then underwent a CuAAC reaction in the presence of either methyl 4-ethynylbenzoate or 1-ethynyl-4-(trifluoromethyl)benzene to obtain cjoc42 derivatives 51a-d. Thioamides 52a-d were then generated from 51a-d through treatment with Lawesson’s reagent.

Scheme 3.

^a Synthesis of compounds 51a-d and 52a-d

^aReagents: (a) 4-Bromobutyryl chloride, DIPEA, CH₂Cl₂; (b) NaN₃, DMF, 60 °C; (c) Methyl 4-ethynylbenzoate or 1-ethynyl-4-(trifluoromethyl)benzene; (d) Lawesson’s reagent, toluene, 90 °C.

Compounds 51a-d and 52a-d were also evaluated for their ability to inhibit the proliferation of A549 and MDA-MB-231 cells, as shown in Table 3. These combinations proved fruitful as multiple compounds exhibited IC₅₀ values below 5 μM, which is a significant advance over cjoc42 as well as our previously published cjoc42 derivatives. Interestingly, compound 51c proved to be the most effective at inhibiting the proliferation of A549 cells (IC₅₀ = 0.33 μM), while compounds 52b and 52d also proved to be quite effective against A549 cells (IC₅₀ = 5.5 and 1.0 μM, respectively). Compounds 51b and 51d then demonstrated the most potent antiproliferative activity against MDA-MB-231 cells (IC₅₀ = 2.3 μM and 2.0 μM, respectively). Additionally, compounds 51b and 51d exhibited potent antiproliferative activity (Table S2, IC₅₀ = 3.1 μM and 1.1 μM, respectively) against gankyrin overexpressing HuH6 liver cancer cells, which have been previously shown to be sensitive to cjoc42 treatment.^19,20 These results suggest that combining the most potent sulfonate ester and triazole replacements enhances gankyrin binding and subsequent antiproliferative activity. This enhanced antiproliferative activity may also be due to the improved metabolic stability of compounds 51b, 51c, 51d, 52b, and 52d as compared to cjoc42 (Table 4).

Table 3.

^a-d Antiproliferative evaluation of cjoc42, and compounds 51a-d and 52a-d against A549 and MDA-MB-231.

Compound	Structure	A549 IC50 (μM)	MDA-MB-231 IC50 (μM)
1: cjoc42		>100	>100
2: AFM-1-2		57.3 (±6.3)	>100
3: DK-1-67		>100	>100
51a		>100	2.7 (±0.1)
51b		25.0 (±1.1)	2.3 (±0.1)
51c		0.33 (±0.1)	>100
51d		19.1 (±0.5)	2.0 (±0.1)
52a		50.0 (±2.4)	>100
52b		5.5 (±0.3)	83.2 (±2.8)
52c		42.3 (±2.3)	>100
52d		1.0 (±0.1)	>100

^aA549 and MDA-MB-231 cells were incubated for 24 h prior to drug addition.

^bA549 and MDA-MB-231 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.

Effects on gankyrin conformation

Initial protein thermal shift experiments demonstrated that compounds 51b, 52b, 51d, 51c, and 52d effectively bind gankyrin (Table S1). We also attempted to determine the binding affinity (K_D) of gankyrin for each compound using isothermal titration calorimetry (ITC). Previous studies determined cjoc42 had a K_D of 580 nM with a suggested 1:1 stoichiometry. After replicating this experiment using our in-house bacterially expressed gankyrin (cjoc42 K_D = 532 nM), we performed preliminary ITC screens with each compound. These studies revealed peak broadening and full saturation of ligand to protein. After numerous failed attempts at optimizing these conditions, including smaller injections to mitigate the hypothesized slow kinetics of compound binding to gankryin, we concluded that K_D values using ITC could not be obtained.

Building on this, we hypothesized that the 3D fold of gankyrin might be compromised in the presence of these compounds. To test this hypothesis, we examined the global secondary structural content of gankyrin alone and in the presence of compounds 51b, 51c, 51d, 52b, and 52d using CD. Since the secondary structure of each ankyrin-repeat domain of gankyrin is predominantly α-helical, we would expect to see spectral minima at 208 and 222 nm in full-length gankyrin. Interestingly, many of the compounds exhibited a significant loss of gankyrin secondary structure content with increasing concentrations of the compounds, indicating that many of these new compounds destabilize the 3D fold of gankyrin (Figure 2). For example, the introduction of 100 nM of each compound revealed altered peak values in the expected 208 nm and 222 nm ranges for the CD spectra (Figure 2A). Intriguingly, we did observe an enhancement in the 222 nm spectral minimum for compounds 51b, 52b, and 52d, suggesting that the α-helical content in gankyrin was stabilized and that these compounds may act as structure enhancers of gankyrin. We also observed significant differences in the CD spectra at 208 nm with compounds 52b and 52d. We postulate that some of the α-helical content in gankyrin is still maintained due to increased interactions of critical residues found within gankyrin. This includes the canonical GxTPLHAA motif found in naturally occurring ankyrin repeats that require specific H-bonding and hydrophobic interactions to maintain its characteristic 90° L-shape solenoid fold.⁴¹ The presence of these compounds may be altering the packing and/or proximity of these important residues within gankyrin, limiting its capability to maintain its native fold.

Figure 2.

Circular dichroism (CD) spectral analysis of gankyrin alone and in the presence of compounds 51b, 52b, 51d, 51c, and 52d. (A) Individual samples of gankyrin alone (shown in black) were saturated with compounds 51b, 52b, 51d, 51c, and 52d (darker to lighter color gradients representing an increase in compound concentration) to measure α-helical content in gankyrin. Each compound was assessed at concentrations ranging from 100 nM to 1.5 μM. The CD spectra of gankyrin (black) showed characteristic α-helical minima at 208 and 222 nm. Compounds 51c (light blue) and 51d (muted blue) indicated a weakening of secondary structure content in gankyrin with each increase of compound concentration. Compounds 51b (grey) and 52b (yellow orange) showed signal enhancement. (B) Thermal denaturation of gankyrin monitored at 217 nm at saturated compound concentrations. Colors on this graph reflect identical color schemes used in panel A, highlighting the thermal denaturation of each compound at 1.5 μM. Compounds 52b (yellow), 51b (grey), 51c (light blue) and 52d (green) all showed complete denaturation across the entire measured temperature range (5 – 90 °C). The only exception was compound 51d (muted blue) that maintained gankyrin’s expected two-state transition but unfolded at an accelerated rate compared to gankyrin alone (black). (C) Examples of observed gankyrin precipitation in the presence of 1.5 μM compound. Scoring is denoted with three qualitative levels – “+, little to no precipitation; ++, moderate precipitation; +++, severe precipitation”.

Previous reports have suggested that ankyrin repeat-containing proteins undergo a two-state folding transition, where they show a double sigmoidal-type thermal denaturation curve using CD. ^41–43 To see if this phenomenon holds true for gankyin, we performed CD thermal denaturation experiments to characterize the structural stability of gankyrin in the presence and absence of our compounds. As shown in Figure 2B, we observed that gankyrin does in fact display a two-state folding transition, similar to other ankyrin repeat-containing proteins. In all cases, we noted that gankyrin’s structural integrity was negatively impacted when incubated with our compounds at concentrations as low as 100 nM (Figure 2B). We also performed a visual examination of each CD sample in the presence and absence of our compounds and noted that the samples were predominantly cloudy with white precipitate (Figure 2C), with only a few exceptions (i.e., 51c and 51d), which only showed marginal precipitation. Taken together, our cumulative biophysical studies unequivocally show that these novel compounds promote the unfolding and destabilization of gankyrin’s 3D structure.

Although compounds 51b, 51c, 51d, 52b, and 52d promote the destabilization of gankyrin, the specific sites and subsequent PPIs affected are unclear. This is evident in that compounds 51c, 52b, and 52d proved to be potent against A549 proliferation, while demonstrating little to no efficacy against MDA-MB-231 cells. Additionally, compounds 51b and 51d were effective at inhibiting the proliferation of MDA-MB-231 cells but were relatively ineffective against A549 cells. This suggests that compounds 51c, 52b, and 52d may alter specific PPI binding sites of gankyrin important for A549 proliferation. Similarly, compounds 51b and 51d may alter a different set of gankyrin PPI binding sites more critical to MDA-MB-231 proliferation. Interestingly, only compounds 51b and 51d displayed potent antiproliferative activity against gankyrin overexpressing HuH6 liver cancer cells. This suggests that HuH6 and MDA-MB-231 cell lines are sensitive to a particular conformational change that affects specific gankyrin PPIs, while A549 cells are sensitive to an alternative gankyrin conformational change that may impair different PPIs. The observed efficacy in both A549 and MDA-MB-231 cells when treated with compound 21 suggests it disrupts key PPIs for both cell lines.

Evaluation of TSP Levels

The gankyrin-proteasome interaction facilitates the degradation of TSPs such as p53 and Rb. It was previously shown that cjoc42 alone and in combination with known chemotherapeutics increased the levels of various TSPs, including p53 and Rb, in HuH6 and Hepa1c1c7 cells.^21,24 Therefore, treatment with cjoc42 derivatives 51b, 51c, 51d, 52b, and 52d should also increase the levels of certain TSPs due to disruption of the gankyrin-proteasome interaction.

To determine that compounds 51b and 51d manifest their antiproliferative activity against MDA-MB-231 cells through gankyrin inhibition, western blot analyses were determined (Figure 3B and Figure 3D). Treatment with both compounds resulted in an increase in Rb levels while gankyrin levels remained relatively unchanged, which agrees with previous reports of cjoc42-treated liver cancer cells.²⁰ This suggests that compounds 51b and 51d inhibit gankyrin and disrupt the proteasomal degradation pathway in a similar manner to that observed for cjoc42.^19,20 Additionally, treatment with compounds 51b and 51d resulted in relatively unchanged levels of p53. This result was most likely due to the high expression levels of mutant p53 in MDA-MB-231 cells, which does not possess the typical tumor suppressor activity of wildtype p53.^45,46 Additionally, compound 51d treatment of HuH6 cells (Figure S1) resulted in increased levels of both p53 and Rb, further suggesting its ability to disrupt the proteasomal degradation pathway.

Figure 3.

Western blot analysis of 51b and 51d in MDA-MB-231 cells. (A) Chemical structure and IC₅₀ value of 51b in MDA-MB-231 cells and HEK-293 cells. (B) Western blot analysis of TSP levels from MDA-MB-231 lysate upon treatment with 51b. (C) Chemical structure and IC₅₀ value of 51d in MDA-MB-231 cells and HEK-293 cells. (D) Western blot analysis of TSP levels from MDA-MB-231 lysate upon treatment with 51d.

Compounds 51c, 52b, and 52d proved to be the most effective at inhibiting the proliferation of A549 cells (IC₅₀ = 0.33 μM, 5.5 μM, and 1.0 μM, respectively), as shown in Table 3. Western blot analysis of all three compounds (Figure 4B, Figure 4D and Figure 4F) demonstrated increases in p53 and Rb. Specifically, treatment with compounds 51c, 52b, and 52d all increased Rb levels while gankyrin levels remained relatively unchanged. Interestingly, treatment with compounds 52b and 52d increased p53 levels, while compound 51c resulted in relatively unchanged p53 levels. Additionally, two phosphorylated forms of p53 (Ser6 and Ser15) were also monitored as activation of p53 often occurs through its phosphorylation at multiple sites.^47,48 Phosphorylated p53 (Ser6 and Ser15) was of particular interest as they were previously shown to be relatively unaffected when colon cancer cells are treated with p53/MDM2 inhibitors²⁸, and phosphorylated p53 (Ser6) increased upon treatment with cjoc42¹⁹. This provides preliminary evidence of primarily inhibiting gankyrin’s chaperone activity instead of targeting a related PPI while also agreeing with previous reports utilizing cjoc42. Increased phosphorylated p53 (Ser15) levels were also of interest as this was suggestive of induced apoptosis and cell cycle arrest, a process that gankyrin is known to regulate.²⁹ Compounds 51c, 52b, and 52d all demonstrated an ability to increase the expression levels of phosphorylated p53 (Ser6), while only compounds 51c and 52b also increased phosphorylated p53 (Ser15) expression levels. The elevated levels of both phosphorylated forms of p53 (Ser6 and Ser15), in addition to increased levels of Rb and total p53 is highly suggestive that compounds 51c, 52b, and 52d also inhibit gankyrin and disrupt the proteasomal degradation pathway in A549 cells.

Figure 4.

Western blot analysis of 51c, 52b, and 52d in A549 cells. (A) Chemical structure and IC₅₀ value of 51c in A549 cells and HEK-293 cells. (B) Western blot analysis of TSP levels from A549 lysate upon treatment with 51c. (C) Chemical structure and IC₅₀ value of 52b in A549 cells and HEK-293 cells. (D) Western blot analysis of TSP levels from A549 lysate upon treatment with 52b. (E) Chemical structure and IC₅₀ value of 52d in A549 cells and HEK-293 cells. (F) Western blot analysis of TSP levels from A549 lysate upon treatment with 52d.

Clonogenic assays

The ability of compounds 51b and 51d to inhibit the proliferation of MDA-MB-231 was further assessed using a clonogenic assay. The clonogenic assay is an in vitro cell survival assay based on the ability of a single cell to grow into a colony. It is often considered an essential 2D technique to confirm antiproliferative activity previously observed in an MTT assay.^30,31,32 Figure 5A and Figure 5B illustrate the ability of compounds 51b and 51d to inhibit the formation of MDA-MB-231 cell colonies after adding treatment for 48 h followed by a 7 day incubation with fresh media. Treatment with 1.15 μM of compound 51b resulted in 75.3 % growth, and a 2.30 μM treatment resulted in 60.3% growth. Additionally, treatment with 1.0 μM of compound 51d resulted in 72.4% growth, and a 2.0 μM treatment resulted in 46.3% growth. Compounds 51b and 51d significantly reduced colony growth at two different concentrations, demonstrating their long-term efficacy against MDA-MB-231 cells and were chosen for further evaluation in a 3D spheroid model.

Figure 5.

Evaluation of compounds 51b and 51d by clonogenic assay in MDA-MB-231 cells. (A) Images showing the effect of compound 51b on colony formation after staining with crystal violet, and quantification of the effect of compound 51b on colony formation expressed as % colony growth at different concentrations. Cells were incubated for 48 h with each respective compound, followed by incubation in fresh media for an additional 7 days. Data represent the mean ± SD (n = 3). (B) Images showing the effect of compound 51d on colony formation after staining with crystal violet, and quantification of the effect of compound 51d on colony formation expressed as % colony growth at different concentrations. Cells were incubated for 48 h with each respective compound, followed by incubation in fresh media for an additional 7 days. Data represent the mean ± SD (n = 3). Note: *0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, ****0.00001 < p < 0.0001.

The ability of compounds 51c, 52b, and 52dto inhibit the proliferation of A549 cells was also further assessed using a clonogenic assay. Compound 51c treatment (Figure 6A) resulted in 61.9% growth with a 0.15 μM treatment, and 44.6% growth with a 0.3 μM treatment. Compound 52b (Figure 6B) treatment resulted in 32.2% growth with a 2.5 μM treatment and 14.3% growth with a 5 μM treatment. Additionally, treatment with 0.5 μM compound 52d (Figure 6C) resulted in 83.8% growth and 61.4% growth was observed at 1 μM. All three compounds significantly reduced colony growth at the highest concentration used, and so they were also evaluated in a 3D spheroid model.

Figure 6.

Evaluation of compounds 51c, 52b, and 52d by clonogenic assay in A549 cells. (A) Images showing the effect of compound 51c on colony formation after staining with crystal violet, and quantification of the effect of compound 51c on colony formation expressed as % colony growth at different concentrations. Cells were incubated for 48 h with each respective compound, followed by incubation in fresh media for an additional 7 days. Data represent the mean ± SD (n = 3). (B) Images showing the effect of compound 52b on colony formation after staining with crystal violet, and quantification of the effect of compound 52b on colony formation expressed as % colony growth at different concentrations. Cells were incubated for 48 h with each respective compound, followed by incubation in fresh media for an additional 7 days. Data represent the mean ± SD (n = 3). (C) Images showing the effect of compound 51d on colony formation after staining with crystal violet, and quantification of the effect of compound 51d on colony formation expressed as % colony growth at different concentrations. Cells were incubated for 48 h with each respective compound, followed by incubation in fresh media for an additional 7 days. Data represent the mean ± SD (n = 3). Note: *0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, ****0.00001 < p < 0.0001.

3D Spheroid growth assays

The current standard of drug discovery encompasses the screening of compound libraries against 2D cell culture assays. However, many compounds identified through a 2D screen are not effective in clinical trials, and their lack of success is partly due to the limitations of screening cells grown in 2D.^61,62 3D cultures, or spheroids, are an established model that appears to be more clinically predictive and mimics many properties typically present in vivo.^33,34 Therefore, we evaluated compounds 51b and 51d in a 3D spheroid assay utilizing MDA-MB-231 cells to determine the ability of gankyrin inhibitors to exhibit a therapeutic effect which could potentially translate to preclinical models.

Figure 7A shows images of MDA-MB-231 spheroids and the fold-difference in spheroid volume after a single-dose treatment with compound 51b. At a concentration of 1.15 μM, there was a 2.6-fold reduction in spheroid volume, and a 2.30 μM treatment resulted in a 3.1-fold decrease in tumor spheroid volume. Figure 7C then shows images of MDA-MB-231 spheroids and the fold-difference in spheroid volume after a single dose treatment with compound 51d in MDA-MB-231 cells. Compound 51d showed a 2.3-fold decrease in tumor size at 1 μM, and at 2 μM it exhibited a 3.2-fold reduction in tumor size. This is suggestive that both compounds 51b and 51d exhibit significant antitumorigenic activity against MDA-MB-231 cells and that gankyrin inhibition could be a useful therapeutic approach for this cell type.

Figure 7.

3D Spheroid cell culture study for the assessment of antitumorigenic activity of compounds 51b and 51d against MDA-MB-231 cells. (A) Images of spheroid tumor size at day 0, 6, and 15 after a single dose treatment with 51b and quantitative representation of antitumor activity of 51b expressed as a fold-difference in spheroid volume versus time for a single dose treatment. Data represent the mean ± SD (n = 8). (B) Fluorescent images of spheroids treated with 51b subjected to a live-dead assay on day 15 and quantitative assessment of live and dead cells. Data represent the mean ± SD (n = 4). Scalebar = 200 μm. (C) Images of spheroid tumor size at day 0, 6, and 15 after a single dose treatment with 51d and quantitative representation of antitumor activity of 51d expressed as a fold-difference in spheroid volume versus time for a single dose treatment. Data represent the mean ± SD (n = 8). (D) Fluorescent images of spheroids treated with 51d subjected to a live-dead assay on day 15 and quantitative assessment of live and dead cells. Data represent the mean ± SD (n = 4). Scalebar = 200 μm. Note: *0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, ****0.00001 < p < 0.0001.

A live-dead cell assay was also used to monitor MDA-MB-231 spheroid growth. This assay requires two fluorescent probes that detect intracellular esterase activity in live cells and compromised plasma membrane integrity in dead cells. The esterase substrate calcein AM stains live cells green, while the membrane-impermeable DNA dye ethidium homodimer III (EthD-III) stains dead cells red.³⁵ As shown in Figure 7B and Figure 7D, treatment with compound 51b resulted in a decrease in green intensity (live cells) upon treatment with both 1.15 μM and 2.30 μM concentrations, and an increase in red fluorescence intensity (dead cells). Treatment with 1 μM and 2 μM of compound 51d then showed a significant decrease in green fluorescence intensity (live cells) while also exhibiting a significant increase in red fluorescence intensity (dead cells). This data demonstrates that both compounds 51b and 51d increased the number of dead cells and decreased the number of live cells, suggesting that gankyrin inhibitors can effectively reduce tumor size by inhibiting tumor growth, as well as killing cancer cells.

To further illustrate the therapeutic potential of compounds 51c, 52b, and 52d against A549 cells, they were also evaluated in a 3D spheroid assay. Images of A549 spheroids and the fold-difference in spheroid volume after a single dose treatment with compound 51c are shown in Figure 8A. At a concentration of just 0.15 μM, there was a 3.6-fold reduction in tumor spheroid volume, and a 2.4-fold reduction in tumor spheroid volume with a 0.33 μM treatment. Compound 52d (Figure 8C) then exhibited a 3.3-fold reduction in spheroid volume with a 2.5 μM treatment, and a 3.5-fold reduction in tumor size with a 5 μM treatment. Compound 52d was also evaluated for its ability to reduce A549 spheroid volume (Figure 8E). Compound 52d decreased tumor size by 3.8-fold with a 0.5 μM treatment, while reducing tumor size by 4.0-fold with a 1 μM treatment. These findings strongly suggest that gankyrin inhibition by compounds 51c, 52b, and 52d is a novel therapeutic strategy for A549-based tumors.

Figure 8.

3D Spheroid cell culture study for the assessment of antitumorigenic activity of compounds 51c, 52b, and 52d against A549 cells. (A) Images of spheroid tumor size at day 0, 6, and 15 after a single dose treatment with 51c and quantitative representation of antitumor activity of 51c expressed as a fold-difference in spheroid volume versus time for a single dose treatment. Data represent the mean ± SD (n = 8). (B) Fluorescent images of spheroids treated with 51c subjected to a live-dead assay on day 15 and quantitative assessment of live and dead cells. Data represent the mean ± SD (n = 4). Scalebar = 200 μm. (C) Images of spheroid tumor size at day 0, 6, and 15 after a single dose treatment with 52b and quantitative representation of antitumor activity of 52b expressed as a fold-difference in spheroid volume versus time for a single dose treatment. Data represent the mean ± SD (n = 8). (D) Fluorescent images of spheroids treated with 52b subjected to a live-dead assay on day 15 and quantitative assessment of live and dead cells Data represent the mean ± SD (n = 4). Scalebar = 200 μm. (E) Images of spheroid tumor size at day 0, 6, and 15 after a single dose treatment with 52d and quantitative representation of antitumor activity of 52d expressed as a fold-difference in spheroid volume versus time for a single dose treatment. Data represent the mean ± SD (n = 8). (F) Fluorescent images of spheroids treated with 52d subjected to a live-dead assay on day 15 and quantitative assessment of live and dead cells. Data represent the mean ± SD (n = 4). Scalebar = 200 μm. Note: *0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, ****0.00001 < p < 0.0001.

A549 spheroid growth was also monitored by a live-dead cell assay. As shown in Figure 8B, Figure 8D and Figure 8F, the control group shows an intense green color (more viable cells), as compared to treated cells, which are predominantly red in color (more dead cells). Treatment with compound 51c (Figure 8B), compound 52b (Figure 8D) or compound 52d (Figure 8F), resulted in a dose-dependent decrease in green intensity, and an increase in red intensity upon treatment at both concentrations. This data demonstrates that these three compounds increased the number of dead cells while decreasing the number of live cells, suggesting that gankyrin inhibitors can reduce a tumor’s size by inhibiting tumor growth and killing cancer cells.

Cell cycle analysis of compounds 51c and 51d

Since gankyrin plays an important role in controlling the expression levels of cell cycle regulators p53 and Rb, we investigated the effects of compounds 51c and 51d on cell cycle progression (Figure 9). Treatment of A549 cells with compound 51c (0.3 μM) caused a significant increase in the number of cells in the G0/G1 phase while also significantly decreasing the cell population in the S and G2/M phases (Figure 9C). Additionally, treatment of MDA-MB-231 cells with 51d (2 μM) resulted in a significant increase in the number of cells in the G0/G1 phase and a decrease in the cell population in the G2/M phase (Figure 9F). However, the cell population in the S phase was relatively unchanged, and only a modest decrease in the number of cells in the G2/M phase was observed. These findings align with the previously discussed western blot analyses, where treatment with 51c caused an increase in phosphorylated p53 and Rb expression levels, and treatment with 51d caused an increase in Rb expression levels. These findings were as expected due to the ability of p53, phosphorylated p53, and Rb to inhibit the G1/S transition of the cell cycle.^14,40 This also further supports our findings that compounds 51c and 51d inhibit gankyrin and disrupt the proteasomal degradation pathway, leading to cell cycle arrest, and decreased cancer cell proliferation.

Figure 9.

Cell cycle analysis of compounds 51c and 51d in A549 and MDA-MB-231 cells. (A) Cell cycle analysis by flow cytometry of A549 cells treated with DMSO. (B) Cell cycle analysis by flow cytometry of A549 cells treated with compound 51c. (C) Representative histogram data of the cell cycle analysis of A549 cells treated with DMSO and compound 51c. Data represent the mean ± SD (n = 3). (D) Cell cycle analysis by flow cytometry of MDA-MB-231 cells treated with DMSO. (E) Cell cycle analysis by flow cytometry of MDA-MB-231 cells treated with compound 51d. (F) Representative histogram data of the cell cycle analysis of MDA-MB-231 cells treated with DMSO and compound 51d. Data represent the mean ± SD (n = 3). Note: *0.01 < p < 0.05, **0.001 < p < 0.01, ***0.0001 < p < 0.001, ****0.00001 < p < 0.0001.

Further evidence that compounds 51c and 51d bind to and destabilize gankyrin was demonstrated using a cellular thermal shift assay (CETSA) (Figure 10). CETSA is an effective method for determining if a small-molecule effectively binds to a target protein resulting in a change in its thermal stability.⁵⁶ Therefore, we treated A549 and MDA-MB-231 cells with compounds 51c and 51d, respectively. Compound 51c demonstrated a significant ability to decrease gankyrin thermal stability (Figure 10A and Figure 10B) in A549 cells at its respective antiproliferative IC₅₀ (0.3 μM). A similar finding was also observed when MDA-MB-231 cells were treated an IC₅₀ concentration (2 μM) of compound 51d where the thermal stability of gankyrin was significantly decreased. Interestingly, compound 51d proved to be more effective than 51c at destabilizing gankyrin, which agrees with our previously discussed protein thermal shift data (Table S1). Taken together, our cumulative biophysical studies (CD, CD thermal denaturation, protein thermal shift, and CETSA) unequivocally show that these novel compounds bind to and promote the unfolding and destabilization of gankyrin’s 3D structure.

Figure 10.

Cellular thermal shift assay (CETSA) for the binding of 51c and 51d to gankyrin in A549 and MDA-MB-231 cells. (A) Representative blot of gankyrin stability present in A549 cells treated with compound 51c for 48 h. (B) CETSA melt curve for gankyrin from A549 cells in the presence of compound 51c. Data represent the mean ± SD (n = 3). (C) Representative blot of gankyrin stability present in MDA-MB-231 cells treated with compound 51d for 48 h. (D) CETSA melt curve for gankyrin from MDA-MB-231 cells in the presence of compound 51d. Data represent the mean ± SD (n = 3). Note: *0.01 < p < 0.05, **0.001 < p < 0.01.

Conclusions

Gankyrin has demonstrated its importance in regulating numerous oncogenic pathways in various cancer types. Gankyrin initially gained interest due to its overexpression in many liver cancers^1,4,5 while also playing a prominent role in their early development. Although most studies have focused on gankyrin’s role and the effects of gankyrin inhibitors in liver cancer, less attention has been focused on gankyrin and its inhibition in breast cancer and NSCLC. Therefore, the design of potent gankyrin inhibitors may provide a valuable tool for studying gankyrin in breast cancer and NSCLC, as well as demonstrating their therapeutic utility going forward.

Using structure-based drug design and aiming to improve the metabolic stability of the cjoc42 scaffold, five compounds were identified as potential leads (51b, 51c, 51d, 52b, and 52d). These 5 compounds exhibited enhanced antiproliferative activity against MDA-MB-231 and A549 cells while also improving their metabolic stability as compared to cjoc42. Western blot analysis established that all 5 compounds could effectively increase levels of p53, phosphorylated p53, and Rb, demonstrating their ability to bind gankyrin and inhibit its facilitation of the proteasomal degradation pathway. Compounds 51c (A549 cells) and 51d (MDA-MB-231 cells) were then able to significantly reduce cell cycle progression, likely due to their effects on Rb and phosphorylated p53. Additionally, CETSA experiments demonstrated that compounds 51c and 51d effectively bind and destabilize gankyrin in A549 and MDA-MB-231 cells, respectively.

Once it was established that these compounds effectively inhibit the proliferation of MDA-MB-231 and A549 cells by inhibiting gankyrin, they were further evaluated in clonogenic and 3D spheroid assays. A single-dose administration of each compound led to significant decreases in colony formation (7 days) and spheroid volume (15 days). Additionally, HEK-293 proliferation was relatively unaffected (IC₅₀ > 100 μM), suggesting a high degree of selectivity for gankyrin overexpressing cancer cell lines.

Our biophysical analysis of gankyrin demonstrated that compounds 51b, 51c, 51d, 52b, and 52d destabilize the 3D fold of gankyrin. Using CD, we could reproducibly measure changes in α-helical content in gankyrin in the presence of different compound concentrations. Interestingly, we observed that while compounds 51c, 51d, and 52d appear to act as gankyrin structure destabilizers, compounds 51b and 52b could potentially serve as gankyrin structure enhancers. This suggests that targeting gankyrin without fully disrupting its folding nature could also be a potential therapeutic approach. Further studies will need to be performed to confirm and/or identify specific gankyrin residues involved in drug binding, as interrupting any of the classic GxTPLH motifs found in ankyrin-repeat containing proteins likely has a significant impact on gankryin’s 3D structure and its subsequent PPIs.

Compounds 51c and 51d appear to manifest the most significant ability to inhibit proliferation, colony formation, and tumor growth of A549 and MDA-MB-231 cells, respectively. Future studies with these two compounds will help determine the roles of gankyrin in these cancer types and shed light on the validity of gankyrin as a therapeutic target.

Experimental Section

Chemistry.

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 (¹H) and carbon (¹³C) NMR spectra were recorded on a 400 MHz spectrometer. Proton chemical shifts are reported in ppm (δ) relative to residual DMSO-d6 (2.49 ppm) or CDCl₃ (7.26 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 (DMSO-d6, 39.5 ppm or CDCl₃, 77.2 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 was performed using reverse-phase C18 column with UV detector. Mass spectrometry data were collected using an Ultra high-performance LC/MS, which was performed on a UPLC/MS instrument equipped with a reverse-phase C18 column (1.7 μm particle size, 2.1 × 50 mm), dual atmospheric pressure chemical ionization (API)/electrospray (ESI) mass spectrometry detector, and photodiode array 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. All final compounds are >95% pure by HPLC or UPLC analysis (Purity Analysis section below). All final compounds were found to not be Pan Assay Interference compounds by visual inspection and the SwissADME web tool.^59,60

Purity analysis

Purity analysis of compounds was carried out using an Agilent 1260 infinity series HPLC system (Agilent, Santa Clara, CA). Purity analysis was performed using Agilent Eclipse plus C18, 3.5 μm, 4.6 mm × 100 mm column and the runs were monitored at 254 nm. Target compounds were analyzed to be > 95% pure (based on major peak area/total area of combined peaks). Methanol and water mixtures were used as mobile phase for purity analysis of compounds. An isocratic run was performed with 90% methanol in water over 5 min. The flow rate was 0.5 mL/min for analysis of compounds 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 43, 47, 51a, 51b, 51c, 51d, 52a, 52b, 52c and 52d. Purity analysis of compounds 31, 44, 45, and 46 was carried out using a Waters Acquity series UPLC (Waters, USA). The column used was an XBridge ® BEH shield RP 2.5 μm (3.0 × 100 mm) (Waters, MA, USA) and the runs were monitored at 254 nm. Acetonitrile (MeCN) and water (0.1% formic acid) mixtures were used as the mobile phase for purity. An isocratic run was performed with 70% MeCN in water for 5 min with a flow rate of 0.5 mL/min. All final compounds were found to be > 95% pure.

General procedure 1 - Amide and ester coupling.

To a stirred solution of carboxylic acid (1 eq) in dichloromethane was added HBTU (1.5–2 eq) and either amine or alcohol (1 eq), followed by the addition of DIPEA (2 eq). 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.

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

Azides (1 eq) were dissolved in a THF/^tBuOH/H₂O (2:3:5) mixture, to which an alkyne (1 eq), copper sulfate (0.3 M, 0.3 eq), and sodium ascorbate (0.1 M, 0.01 eq) were added. 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, and concentrated under reduced pressure.

General procedure 3 – Thioamide and thiourea formation.

To a stirred solution of an amide or urea in anhydrous toluene was added Lawesson’s reagent (1 eq) under an argon atmosphere. The reaction mixture was stirred at 90 °C for 5 h. The reaction mixture was then diluted with ethyl acetate and washed with saturated NaHCO₃ solution 10 times, followed by washing 15 times with water. The organic layer was then dried over sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography.

General procedure 4 - Tosylation.

To a stirred solution of an alcohol intermediate in dichloromethane (20 mL) at 0 °C was added triethylamine (1 eq) and 4-dimethylaminopyridine (0.1 eq). Para-toluene sulfonyl chloride (1.5 eq) 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 desired final compounds were then purified using column chromatography.

General procedure 5 – Acid chloride amide coupling.

To a stirred solution of 4-bromobutyryl chloride and DIPEA in dichloromethane under argon was added 1 eq of either para-toluidine or 4-aminobiphenyl was added at 0 °C. The reaction mixture was then stirred at room temperature for 3–5 h. 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. Desired intermediates 49a and 49b were then purified using column chromatography.

General procedure 6 - Azidation.

To a stirred solution of either mesylate or bromide (1 eq) in DMF was added sodium azide (3 eq) and the mixture was heated at 60 °C for 3 h. Water was then added to the reaction mixture and extracted 3 times with ethyl acetate. The organic layers were combined, dried over sodium sulfate, concentrated, and purified by column chromatography.

General procedure 7 – Tetrazole formation.

To a mixture of nitrile (1 eq) and sodium azide (2 eq) in toluene was added triethylamine hydrochloride (2 eq). The reaction mixture was heated at 90 °C overnight. The reaction mixture was allowed to cool and extracted 3 times with water. The aqueous layers were combined and acidified to pH 1 using 1 M hydrochloric acid. The acidified aqueous layer was stirred in an ice bath for 4 h and then extracted 3 times with ethyl acetate. The organic layers were combined, dried over sodium sulfate, concentrated under reduced pressure, and purified by column chromatography.

N-p-Tolyl-5-hexynamide (6).

General procedure 1. (800 mg, 74%). ¹H NMR (400 MHz, DMSO-d6) δ = 9.83 (s, 1H), 7.47 (d, J = 8.3 Hz, 2H), 7.09 (d, J = 8.2 Hz, 2H), 2.83 (s, 3H), 2.40 (t, J = 7.4 Hz, 2H), 2.25 – 2.19 (m, 3H), 1.79 – 1.71 (m, 2H). ¹³C NMR (100 MHz, DMSO-d6) δ = 173.9, 136.6, 133.3, 129.8 (2C), 119.3 (2C), 83.8, 69.8, 33.93, 23.9, 21.1, 20.2.

p-Tolyl 5-hexynoate (7).

(270 mg, 77%) ¹H NMR (400 MHz, CDCl₃) δ = 8.19 (d, J = 8.7 Hz, 2H), 7.87 – 7.81 (m, 3H), 3.96 (s, 3H), 3.77 (t, J = 6.1 Hz, 2H), 2.95 (t, J = 7.3 Hz, 2H), 2.02 (dt, J = 13.3, 6.6 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ = 172.1, 148.2, 135.8, 129.8 (2C), 115.1 (2C), 83.2, 69.3, 33.0, 23.7, 20.7, 17.6.

N-4-Pentynyl-p-toluamide (9).

General procedure 1. (200 mg, 85%) ¹H NMR (400 MHz, CDCl₃) δ = 7.71 (d, J = 7.7 Hz, 2H), 7.18 (d, J = 7.7 Hz, 2H), 3.58 – 3.32 (m, 2H), 2.36 (s, 3H), 2.27 (t, J = 5.3 Hz, 2H), 2.00 (s, 1H), 1.87 – 1.77 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ = 167.7, 141.6, 131.8, 129.1 (2C), 127.0 (2C), 83.8, 68.9, 39.4, 28.4, 21.4, 16.4.

4-Pentyn-1-yl 4-methylbenzoate (10).

General procedure 1. (100 mg, 88%) ¹H NMR (400 MHz, CDCl₃) δ = 7.92 (d, J = 7.8 Hz, 2H), 7.21 (d, J = 7.8 Hz, 2H), 4.40 (t, J = 6.2 Hz, 2H,), 2.25–2.33 (m, 5H), 1.92–1.86 (m, 3H). ¹³C NMR (100 MHz, CDCl₃) δ = 166.6, 143.6, 129.6 (2C), 129.1; 127.5 (2C), 83.1, 69.2, 63.3, 27.7, 21.6, 15.4.

1-(4-Pentynyl)-3-(p-tolyl) urea (12).

To a stirred solution of 4-pentyn-1-amine hydrochloride (1 eq) and DIPEA (3 eq) in dichloromethane was added p-tolylisocyanate (7, 1 eq), and the reaction mixture was then stirred overnight at room temperature. The reaction mixture was 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 to obtain intermediate 12. (150 mg, 91%) ¹H NMR (400 MHz, DMSO-d6) δ = 8.28 (s, 1H), 7.26 (d, J = 8.2 Hz, 2H), 7.02 (d, J = 8.1 Hz, 2H), 6.13 (t, J = 5.4 Hz, 1H), 3.35 (s, 3H), 3.13 (dd, J = 12.7, 6.5 Hz, 2H), 2.29 – 2.14 (m, 5H), 1.60 (p, J = 6.9 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d6) δ = 156.0,138.3, 130.2, 129.3 (2C), 119.0 (2C), 84.7, 72.4, 38.8, 28.7, 20.7, 15.8.

N-p-Tolylsulfonyl-5-hexynamide (14).

To a stirred solution of 5-hexynoic acid (1 eq) in dichloromethane, Mukaiyama reagent (1.2 eq), para-toluenesulfonamide (13, 1 eq), and 4-dimethylaminopyridine (0.1 eq) were added, followed by the addition of DIPEA (2 eq). The reaction mixture was 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, and concentrated under reduced pressure to obtain intermediate 14.⁵⁴ (130 mg, 70%) ¹H NMR (400 MHz, CDCl₃) δ = 9.15 (s, 1H), 7.88 (d, J = 7.7 Hz, 2H), 7.27 (d, J = 7.8 Hz, 2H), 2.43 – 2.33 (m, 5H), 2.10 (t, J = 6.7 Hz, 2H), 1.85 (s, 1H), 1.71 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ = 170.9, 145.2, 135.6, 129.7 (2C), 128.2 (2C), 83.8, 69.8, 34.7, 23.0, 21.7, 17.3.

3-(4-Pentynyl)-5-(p-tolyl)-4H-1,2,4-triazole (16).

To a stirred solution of 4-methylbenzohydrazide (15, 1 eq) and 5-hexynenitrile (3 eq) in 1-butanol, was added K₂CO₃ (0.5 eq) and the reaction was refluxed at 150 °C for 4 h. The solvent was then removed under high pressure and the resulting residue was purified by column chromatography to obtain intermediate 16. (200 mg, 43%) ¹H NMR (400 MHz, CDCl₃) δ = 7.80 (d, J = 8.0 Hz, 2H), 7.13 (d, J = 7.9 Hz, 2H), 2.85 (t, J = 7.5 Hz, 2H), 2.30 (s, 4H), 2.22 – 2.15 (m, 2H), 1.95 – 1.89 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ = 160.0, 159.7, 139.8, 129.5 (2C), 126.7, 126.4 (2C), 83.3, 69.3, 26.7, 26.0, 21.4, 17.9.

2-(4-Pentynyl) −5-(p-tolyl)-1,3,4-oxadiazole (17).

4-Methylbenzohydrazide (15, 1 eq) and 5-hexynoic acid (1 eq) were refluxed in POCl₃ at 110 °C for 8 h. The reaction mixture was quenched with a saturated aqueous solution of NaHCO₃ and extracted 3 times with ethyl acetate. The organic layers were combined, dried over sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain intermediate 17. (500 mg, 66%) ¹H NMR (400 MHz, CDCl₃) δ = 7.65 (d, J = 8.0 Hz, 2H), 7.10 (d, J = 7.9 Hz, 2H), 2.37 (t, J = 7.4 Hz, 2H), 2.30 (s, 3H), 2.15 (t, J = 6.8 Hz, 2H), 1.89 (s, 1H), 1.81 – 1.71 (m, 2H).¹³C NMR (100 MHz, CDCl₃) δ = 170.9, 164.5, 142.7, 129.4 (2C), 128.2, 126.8 (2C), 83.2, 69.6, 32.6, 23.7, 21.53, 17.7.

2-(4-Pentynyl)-5-(p-tolyl)-2H-1,2,3,4-tetrazole (19).

General procedure 7 to generate 5-(p-Tolyl)-2H-1,2,3,4-tetrazole intermediate. (500 mg, 80%) ¹H NMR (400 MHz, DMSO-d6) δ = 8.26 – 8.11 (m, 4H), 3.90 (s, 3H). ¹³C NMR (100 MHz, DMSO-d6) δ = 155.4, 141.7, 130.4 (2C), 127.4 (2C), 121.7, 21.5.

To a stirred solution of the 5-(p-Tolyl)-2H-1,2,3,4-tetrazole intermediate (1 eq), 4-pentyn-1-ol (2 eq), and PPh₃ (2 eq), was added diisopropyl azodicarboxylate (2 eq) under a nitrogen atmosphere at room temperature. The reaction mixture was then heated at 80 °C overnight. The solvent was removed under reduced pressure and the crude was purified by column chromatography to obtain intermediate 19. (565 mg, 66%) ¹H NMR (400 MHz, CDCl₃) δ = 8.25 (d, J = 8.3 Hz, 2H), 8.18 (d, J = 8.3 Hz, 2H), 4.86 (t, J = 7.0 Hz, 2H), 4.77 (t, J = 4.9 Hz, 1H), 3.94 (s, 3H), 3.53 (dd, J = 11.1, 5.7 Hz, 2H), 2.18 (p, J = 6.5 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ = 164.2, 139.4, 128.5 (2C), 125.7(2C), 123.6, 80.9, 69.0, 50.7, 27.0, 20.4, 14.7.

Methyl p-{4-[3-(N-p-tolylcarbamoyl)propyl]-1H-1,2,3-triazol-1-yl}benzoate (20).

General procedure 2. (330 mg, 67%) ¹H NMR (400 MHz, DMSO-d6) δ = 9.82 (s, 1H), 8.77 (s, 1H), 8.16 (d, J = 8.8 Hz, 2H), 8.07 (d, J = 8.8 Hz, 2H), 7.47 (d, J = 8.4 Hz, 2H), 7.08 (d, J = 8.4 Hz, 2H), 3.90 (s, 3H), 2.78 (t, J = 7.5 Hz, 2H), 2.39 (t, J = 7.4 Hz, 2H), 2.24 (s, 3H), 2.00 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ = 171.0, 165.9, 148.4, 140.2, 135.6, 133.7, 131.3 (2C), 124.9, 130.0, 129.4 (2C), 119.9 (2C), 119.8 (2C), 52.5, 36.1, 25.0, 24.5, 20.9. HRMS (ESI+): m/z calculated for C₂₁H₂₂N₄O₃ [M+H]⁺ 379.1692 found 379.1804. HPLC purity (t_R = 1.30 min, 99.9%).

Methyl p-{4-[3-(p-tolylcarbamthioyl)propyl]-1H-1,2,3-triazol-1-yl}benzoate (21).

General procedure 3. (60 mg, 35%) ¹H NMR (400 MHz, CDCl₃) δ =8.41 (s, 1H), 7.92 (d, J = 7.5 Hz, 2H), 7.65 (s, 1H), 7.59 (d, J = 7.5 Hz, 2H), 7.32 (d, J = 7.5 Hz, 2H), 7.20 (d, J = 7.5 Hz, 2H), 3.97 (s, 3H), 2.81 (t, J = 5.4 Hz, 2H), 2.74 (t, J = 5.5 Hz, 2H), 2.42 (s, 3H), 1.90 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ = 203.5, 167.4, 154.6, 138.1, 136.3, 133.2, 128.5 (2C), 131.5, 130.6, 129.6 (2C), 123.7 (2C), 120.0 (2C), 53.5, 52.7, 45.7, 29.2, 20.8. HRMS (ESI+): m/z calculated for C₂₁H₂₂N₄O₂S [M+H]⁺ 395.1463, found 395.1563. HPLC purity (t_R = 1.46 min, 97.7%).

Methyl p-{4-[3-(p-tolyloxycarbonyl)propyl]-1H-1,2,3-triazol-1-yl}benzoate (22).

General procedure 2. (160 mg, 79%) ¹H NMR (400 MHz, CDCl₃) δ = 8.17 (d, J = 8.5 Hz, 2H), 7.90 (s, 1H), 7.82 (d, J = 8.5 Hz, 2H), 7.14 (d, J = 8.1 Hz, 2H), 6.94 (d, J = 8.2 Hz, 2H), 3.94 (s, 3H), 2.94 (t, J = 7.4 Hz, 2H), 2.67 (t, J = 7.3 Hz, 2H), 2.31 (s, 3H), 2.21 (dd, J = 14.9, 7.5 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ = 171.7, 165.7, 148.6, 148.1, 140.2, 135.5, 131.3 (2C), 129.9 (3C), 120.9 (2C), 119.9 (2C), 119.2, 52.3, 33.3, 24.8, 24.4, 20.8. HRMS (ESI+): m/z calculated for C₂₁H₂₁N₃O₄ [M+H]⁺ 380.1532 found 380.1649. HPLC purity (t_R = 1.74 min, 97.6%).

Methyl p-{4-[3-(p-toluoylamino)propyl]-1H-1,2,3-triazol-1-yl}benzoate (23).

General procedure 1. (200 mg, 80%) ¹H NMR (400 MHz, DMSO-d6) δ = 8.75 (s, 1H), 8.15 (d, J = 8.9 Hz, 2H), 8.06 (d, J = 8.9 Hz, 2H), 7.75 (d, J = 8.2 Hz, 2H), 7.26 (d, J = 7.9 Hz, 2H), 3.90 (s, 3H), 2.78 (t, J = 7.6 Hz, 2H), 2.34 (s, 3H), 2.00 – 1.88 (m, 2H). ¹³C NMR (100 MHz, DMSO-d6) δ = 166.6, 165.9, 148.6, 141.3, 140.5, 132.4, 131.5 (2C), 125.0, 129.2 (2C), 127.6 (2C), 120.9, 120.0 (2C), 52.9, 29.2, 23.1, 21.4, 20.9. HRMS (ESI+): m/z calculated for C₂₁H₂₂N₄O₃ [M+H]⁺ 379.1692, found 379.1789. HPLC purity (t_R = 1.19 min, 99.9%).

Methyl p-{4-[3-(p-tolylcarbamthioyl)propyl]-1H-1,2,3-triazol-1-yl}benzoate (24).

General procedure 3. (35 mg, 41%) ¹H NMR (400 MHz, CDCl₃) δ = 8.12 (d, J = 8.7 Hz, 2H), 7.87 (s, 1H), 7.73 (d, J = 8.7 Hz, 2H), 7.62 (d, J = 8.1 Hz, 2H), 7.09 (d, J = 8.0 Hz, 2H), 3.99 – 3.85 (m, 5H), 2.90 (t, J = 6.8 Hz, 2H), 2.28 (s, 3H), 2.24 – 2.15 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ = 197.8, 164.9, 157.9, 147.0, 140.6, 137.7, 130.3 (2C), 129.1, 128.1 (2C), 125.7 (2C), 118.8 (2C), 118.3, 51.4, 45.0, 25.8, 22.1, 20.3. HRMS (ESI+): m/z calculated for C₂₁H₂₂N₄O₂S [M+H]⁺ 395.1463 found 395.1553. HPLC purity (t_R = 1.47 min, 96.0%).

Methyl p-{4-[3-(p-toluoxy)propyl]-1H-1,2,3-triazol-1-yl}benzoate (25).

General procedure 1. (110 mg, 78%) ¹H NMR (400 MHz, CDCl₃) δ = 8.11 (d, J = 8.5 Hz, 2H), 7.94 – 7.83 (m, 3H), 7.76 (d, J = 8.6 Hz, 2H), 7.16 (d, J = 8.0 Hz, 2H), 4.37 (t, J = 6.2 Hz, 2H), 3.90 (s, 3H), 2.94 (t, J = 7.4 Hz, 2H), 2.33 (s, 3H), 2.28 – 2.16 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ = 166.6, 165.7, 148.2, 143.7, 140.1, 131.1 (2C), 131.0, 129.6 (2C), 129.4, 127.6, 119.7 (2C), 119.3 (2C), 62.6, 55.3, 52.6, 28.3, 22.6. HRMS (ESI+): m/z calculated for C₂₁H₂₁N₃O₄ [M+H]⁺ 380.1532, found 380.1549. HPLC purity (t_R = 1.91 min, 98.5%).

Methyl p-(4-{3-[3-(p-tolyl)ureido]propyl}−1H-1,2,3-triazol-1-yl)benzoate (26).

General procedure 2. (160 mg, 73%) ¹H NMR (400 MHz, DMSO-d6) δ = 8.76 (s, 1H), 8.50 (s, 1H), 8.33 (s, 1H), 8.16 (d, J = 8.6 Hz, 2H), 8.07 (d, J = 8.7 Hz, 2H), 7.32 (d, J = 8.1 Hz, 2H), 7.27 (d, J = 8.2 Hz, 2H), 7.08 (d, J = 8.2 Hz, 2H), 7.01 (d, J = 8.1 Hz, 2H), 3.90 (s, 3H), 3.17 (q, J = 6.4 Hz, 2H), 2.76 (t, J = 7.5 Hz, 2H), 1.93 – 1.77 (m, 2H). ¹³C NMR (100 MHz, DMSO-d6) δ = 170.9, 165.7, 156.1, 148.7, 140.2, 138.5, 131.7 (2C), 130.2, 129.4 (2C), 120.8, 120.0 (2C), 118.5 (2C), 60.2, 55.4, 52.6, 20.8, 14.7. HRMS (ESI+): m/z calculated for C₂₁H₂₃N₅O₃ [M+H]⁺ 394.1801 found 394.1910. HPLC purity (t_R = 1.42 min, 95.4%).

Methyl p-(4-{3-[3-(p-tolyl)thioureido]propyl}−1H-1,2,3-triazol-1-yl)benzoate (27).

General procedure 3. (280 mg, 79%) ¹H NMR (400 MHz, DMSO-d6) δ = 9.42 (s, 1H), 8.77 (s, 1H), 8.16 (d, J = 8.2 Hz, 2H), 8.07 (d, J = 8.3 Hz, 2H), 7.72 (s, 1H), 7.24 (d, J = 7.5 Hz, 2H), 7.13 (d, J = 7.7 Hz, 2H), 3.90 (s, 3H), 3.56 (t, J = 15.2 Hz, 2H), 2.83 – 2.70 (m, 2H), 2.27 (s, 3H), 2.03 – 1.88 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ = 180.6, 166.3, 148.0, 140.6, 131.4 (2C), 130.9, 130.2, 130.0, 125.6 (2C), 125.6, 119.7 (2C), 119.3 (2C), 52.6, 49.7, 28.7, 22.4, 21.2. HRMS (ESI+): m/z calculated for C₂₁H₂₃N₅O₂S [M+H]⁺ 410.1572 found 410.1691. HPLC purity (t_R = 1.33 min, 95.5%).

Methyl p-{4-[3-(N-p-tolylsulfonylcarbamoyl) propyl]-1H-1,2,3-triazol-1-yl}benzoate (28).

General procedure 2. (91 mg, 67%) ¹H NMR (400 MHz, DMSO-d6) δ = 8.70 (s, 1H), 8.15 (d, J = 8.6 Hz, 2H), 8.06 (d, J = 8.6 Hz, 2H), 7.81 (d, J = 8.1 Hz, 2H), 7.42 (d, J = 8.0 Hz, 2H), 3.89 (s, 3H), 2.64 (t, J = 7.4 Hz, 2H), 2.37 (s, 3H), 2.32 (t, J = 7.3 Hz, 2H), 1.83 (dd, J = 14.6, 7.3 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d6) δ = 171.9, 165.8, 148.2, 144.7, 140.4, 137.0, 131.4 (2C), 1230.0 (2C), 129.5, 128.0 (2C), 120.9, 120.0 (2C), 52.9, 35.1, 24.7, 23.9, 21.5. HRMS (ESI+): m/z calculated for C₂₁H₂₂N₄O₅S [M+H]⁺ 443.1311 found 443.1398. HPLC purity (t_R = 1.26 min, 96.0%).

Methyl p-(4-{3-[5-(p-tolyl)-4H-1,2,4-triazol-3-yl]propyl}−1H-1,2,3-triazol-1-yl)benzoate (29).

General procedure 2. (50 mg, 28%) ¹H NMR (400 MHz, CDCl₃) δ = 8.09 (d, J = 8.5 Hz, 2H), 7.87 (d, J = 7.6 Hz, 2H), 7.80 (s, 1H), 7.71 (d, J = 8.5 Hz, 2H), 7.16 (d, J = 7.6 Hz, 2H), 3.91 – 3.73 (m, 3H), 2.86 – 2.78 (m, 2H), 2.31 (s, 3H), 2.21 – 2.12 (m, 2H). ¹³C NMR (100 MHz, DMSO-d6) δ = 165.8, 148.6, 140.2, 139.1, 131.5 (2C), 130.7, 129.8 (2C), 129.7, 129.3, 126.2 (2C), 125.3, 121.0, 119.6 (2C), 55.5, 52.8, 27.5, 25.2, 21.2. HRMS (ESI+): m/z calculated for C₂₂H₂₂N₆O₂ [M+H]⁺ 403.1804 found 403.1920. HPLC purity (t_R = 1.47 min, 95.1 %).

Methyl p-(4-{3-[5-(p-tolyl)-1,3,4-oxadiazol-2-yl]propyl}−1H-1,2,3-triazol-1-yl)benzoate (30).

General procedure 2. (150 mg, 42%) ¹H NMR (400 MHz, CDCl₃) δ = 8.09 (d, J = 8.4 Hz, 2H), 7.89 – 7.77 (m, 3H), 7.73 (d, J = 8.4 Hz, 2H), 7.19 (d, J = 7.9 Hz, 2H), 3.87 (s, 3H), 2.96 (t, J = 7.2 Hz, 2H), 2.89 (t, J = 7.2 Hz, 2H), 2.33 (s, 3H), 2.30 – 2.22 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ = 166.0, 165.9, 165.0, 147.9, 142.11, 140.1, 131.3 (2C), 130.0, 129.7 (2C), 126.7 (2C), 121.1, 119.7 (2C), 119.4, 52.4, 26.2, 24.7, 24.6, 21.5. HRMS (ESI+): m/z calculated for C₂₂H₂₁N₅O₃ [M+H]⁺ 404.1644 found 404.1763. HPLC purity (t_R = 1.33 min, 97.6%).

Methyl p-(4-{3-[5-(p-tolyl)-2H-1,2,3,4-tetraazol-2-yl]propyl}−1H-1,2,3-triazol-1-yl)benzoate (31).

General procedure 2. (380 mg, 72%) ¹H NMR (400 MHz, CDCl₃) δ = 8.04 (d, J = 8.6 Hz, 2H), 7.89 (d, J = 8.0 Hz, 2H), 7.78 (s, 1H), 7.69 (d, J = 8.6 Hz, 2H), 7.16 (d, J = 7.8 Hz, 2H), 4.64 (t, J = 6.6 Hz, 2H), 3.84 (s, 3H), 2.78 (t, J = 7.1 Hz, 2H), 2.53 – 2.38 (m, 2H), 2.29 (s, 4H). ¹³C NMR (100 MHz, CDCl₃) δ = 165.8, 165.2, 146.9, 140.4, 140.2, 131.2 (2C), 130.1, 129.5 (2C), 126.9 (2C), 124.6, 119.8 (2C), 119.4, 52.3, 52.1, 28.5, 22.4, 21.4. HRMS (ESI+): m/z calculated for C₂₁H₂₁N₇O₂ [M+H]⁺ 404.1757 found 404.1850. UPLC purity (t_R = 0.73 min, 96.1%).

Methyl p-{4-[3-(p-toluidino)propyl]-1H-1,2,3-triazol-1-yl}benzoate (32).

To a stirred solution of cjoc42 (1 eq), K₂CO₃ (2 eq), and KI (0.1 eq) in DMF, was added para-toluidine (1.5 eq) and the reaction mixture was stirred overnight at 90 °C. Upon completion, aqueous saturated NH₄Cl was added and extracted 3 times with ethyl acetate. The organic layers were combined and dried over sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to obtain 32. (30 mg, 17%) ¹H NMR (400 MHz, CDCl₃) δ = 8.12 (d, J = 8.5 Hz, 2H), 7.74 (d, J = 8.6 Hz, 3H), 6.91 (d, J = 7.9 Hz, 2H), 6.47 (d, J = 8.1 Hz, 2H), 3.88 (s, 3H), 3.15 (t, J = 6.6 Hz, 2H), 2.86 (t, J = 7.3 Hz, 2H), 2.16 (s, 3H), 2.07 – 1.92 (m, 2H).¹³C NMR (100 MHz, CDCl₃) δ = 166.0 148.7, 145.9, 140.2, 131.3 (2C), 130.0, 129.8 (2C), 126.6, 119.7 (2C), 118.9, 113.0 (2C), 52.4, 43.5, 29.0, 23.2, 20.4. HRMS (ESI+): m/z calculated for C₂₀H₂₂N₄O₂ [M+H]⁺ 351.1743 found 351.1849. HPLC purity (t_R = 1.39 min, 99.3%).

Methyl p-{4-[3-(p-tolyloxy)propyl]-1H-1,2,3-triazol-1-yl}benzoate (33).

To a stirred solution of cjoc42 (1 eq) and NaH (1 eq) in DMF was added para-cresol (1.5 eq) and the reaction mixture was stirred overnight at 90 °C. The reaction mixture was then quenched with water and extracted 3 times with ethyl acetate. The organic layers were combined and dried over sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to obtain compound 33. (105 mg, 62%) ¹H NMR (400 MHz, CDCl₃) δ = 8.11 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 7.7 Hz, 3H), 7.00 (d, J = 8.0 Hz, 2H), 6.73 (d, J = 8.2 Hz, 2H), 3.94 (t, J = 5.9 Hz, 3H), 3.88 (s, 2H), 2.94 (t, J = 7.4 Hz, 2H), 2.34 – 2.08 (m, 5H). ¹³C NMR (100 MHz, CDCl₃) δ = 166.0, 156.8, 148.5, 140.2, 131.3 (2C), 129.97, 129.96, 126.9 (2C), 119.7 (2C), 119.1, 114.4 (2C), 52.4, 43.5, 29.0, 23.2, 20.4. HRMS (ESI+): m/z calculated for C₂₀H₂₁N₃O₃ [M+H]⁺ 351.1583 found 351.1815. HPLC purity (t_R = 1.94 min, 97.0 %).

Methyl p-[5-(3-hydroxypropyl)-1H-1,2,3-triazol-1-yl]benzoate (35).

To a stirred solution of methyl 4-azidobenzoate (34, 1 eq) and 4-pentyn-1-ol (1 eq) in toluene was added 3 mol % of [Cp*RuCl]₄ under a nitrogen atmosphere. Then reaction mixture was heated to 110 °C for 10 h. The reaction mixture was then diluted with ethyl acetate and washed 3 times with water. The organic layer was dried over sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography to obtain intermediate 35.⁵⁵ (120 mg, 35%) ¹H NMR (400 MHz, CDCl₃) δ = 7.77 (d, J = 8.6 Hz, 2H), 6.56 (d, J = 8.6 Hz, 2H), 3.77 (s, 3H), 3.68 (t, J = 6.1 Hz, 2H), 2.24 (t, J = 8.3 Hz, 2H), 1.70 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ = 167.4, 136.9, 136.2, 135.8, 130.1, 127.9 (2C), 123.6 (2C), 62.8, 52.1, 31.2, 23.7.

Methyl p-[1-(3-hydroxypropyl)-1H-1,2,3-triazol-4-yl]benzoate (37).

General procedure 2. (150 mg, 45%) ¹H NMR (400 MHz, CDCl₃) δ = 8.13 (d, J = 6.2 Hz, 2H), 7.96 (d, J = 5.2 Hz, 2H), 4.64 (t, J = 6.6 Hz, 2H), 3.96 (s, 3H), 3.73 (t, J = 5.6 Hz, 2H), 2.29 – 2.18 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ = 166.8, 135.0, 132.2, 130.1 (2C), 129.9, 128.6, 125.3 (2C), 59.1, 52.6, 47.2, 32.4.

Methyl p-{[2-(4-hydroxybutyryl) hydrazino]carbonyl}benzoate (39).

To a stirred solution of monomethyl terepthalate (38, 1 eq), HBTU (1.5 eq), and triethylamine (4 eq) in DMF was added 4-hydroxybutanohydrazide (1.5eq) and stirred at room temperature overnight. The reaction mixture was diluted with water and extracted 3 times with ethyl acetate. The organic layers were combined, dried over sodium sulfate, concentrated under reduced pressure, and purified using column chromatography to obtain intermediate 39. (500 mg, 64%) ¹H NMR (400 MHz, DMSO-d6) δ = 10.48 (s, 1H), 9.92 (s, 1H), 8.07 (d, J = 8.2 Hz, 2H), 7.99 (d, J = 8.2 Hz, 2H), 4.50 (s, 1H), 3.89 (s, 3H), 3.45 (s, 2H), 2.24 (t, J = 7.5 Hz, 2H), 1.79 – 1.64 (m, 2H). ¹³C NMR (100 MHz, DMSO-d6) δ = 172.1, 166.1, 165.2, 137.1, 132.7, 129.7 (2C), 128.3 (2C), 60.6, 52.9, 30.5 28.9.

Methyl p-[5-(3-hydroxypropyl)-4H-1,2,4-triazol-3-yl]benzoate (41).

To a stirred solution of methyl 4-cyanobenzoate (40, 1eq) in methanol was added sodium methoxide (0.5 eq) and the reaction was stirred at room temperature overnight. 4-hydroxybuthanohydrazide (1 eq) was then added to the reaction mixture and refluxed overnight. The solvent was then evaporated, and the crude material was partitioned between ethyl acetate and water. The organic layer was dried over sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain intermediate 41. (300 mg, 37%) ¹H NMR (400 MHz, DMSO-d6) δ = 8.15 (d, J = 8.2 Hz, 2H), 8.05 (d, J = 8.2 Hz, 2H), 3.86 (s, 3H), 3.48 (t, J = 6.1 Hz, 2H), 2.88 (t, J = 7.6 Hz, 2H), 2.51 (s, 1H), 1.95 – 1.84 (m, 2H).¹³C NMR (100 MHz, DMSO-d6) δ = 176.2, 166.3, 158.6, 157.8, 134.2, 130.2 (2C), 126.6 (2C), 60.3, 52.7, 30.8, 22.9.

Methyl p-[2-(3-hydroxypropyl)-2H-1,2,3,4-tetraazol-5-yl]benzoate (42).

General procedure 7 to generate methyl p-(2H-1,2,3,4-tetraazol-5-yl)benzoate intermediate. (800 mg, 58%) ¹H NMR (400 MHz, DMSO-d6) δ = 7.94 (d, J = 8.1 Hz, 2H), 7.42 (d, J = 7.9 Hz, 2H), 2.40 (s, 3H). ¹³C NMR (100 MHz, DMSO-d6) δ = 166.0, 155.9, 132.1, 130.6 (2C), 129.1, 127.7 (2C), 52.9. To a stirred suspension of the methyl p-(2H-1,2,3,4-tetraazol-5-yl)benzoate intermediate (1 eq) in MeCN was added TEA (1 eq) to create a clear solution. 3-Bromopropanol (1 eq) was added, and the reaction mixture was stirred at room temperature overnight. The solvent was then removed under reduced pressure and purified by column chromatography to obtain intermediate 42. (300 mg, 42%) ¹H NMR (400 MHz, CDCl₃) δ = 7.93 (d, J = 8.1 Hz, 2H), 7.19 (d, J = 8.1 Hz, 2H), 4.67 (t, J = 6.6 Hz, 2H), 2.30 (s, 3H), 2.26 – 2.14 (m, 4H), 1.96 (s, 1H). ¹³C NMR (100 MHz, DMSO-d6) δ = 166.1, 163.6, 131.6, 131.6, 130.6 (2C), 127.1 (2C), 57.8, 52.8, 50.8, 32.4.

Methyl p-{5-[3-(p-tolylsulfonyloxy)propyl]-1H-1,2,3-triazol-1-yl}benzoate (43).

General procedure 4. (60 mg, 37%) ¹H NMR (400 MHz, CDCl₃) δ = 8.12 (d, J = 8.7 Hz, 2H), 7.83 – 7.65 (m, 5H), 7.25 (d, J = 8.1 Hz, 2H), 4.03 (t, J = 6.0 Hz, 2H), 3.88 (s, 3H), 2.82 (t, J = 7.2 Hz, 2H), 2.34 (s, 3H), 2.14 – 2.00 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ = 165.9, 147.1, 144.9, 140.1, 132.9, 131.3 (2C), 130.1, 129.9 (2C), 127.9 (2C), 119.8 (2C), 119.6, 69.3, 52.4, 28.2, 21.6, 21.4. HRMS (ESI+): m/z calculated for C₂₀H₂₁N₃O₅S [M+H]⁺ 416.1202 found 416.1328. HPLC purity (t_R = 1.39 min, 99.4%).

Methyl p-{1-[3-(p-tolylsulfonyloxy) propyl]-1H-1,2,3-triazol-4-yl}benzoate (44).

General procedure 4. (75 mg, 47%) ¹H NMR (400 MHz, CDCl₃) δ = 8.00 (d, J = 8.1 Hz, 2H), 7.79 (d, J = 8.0 Hz, 3H), 7.67 (d, J = 8.3 Hz, 2H), 7.22 (d, J = 8.0 Hz, 2H), 4.43 (t, J = 6.6 Hz, 2H), 3.96 (t, J = 5.7 Hz, 2H), 3.85 (s, 3H), 2.33 – 2.24 (m, 5H). ¹³C NMR (100 MHz, CDCl₃) δ = 166.7, 146.7, 145.3, 134.7, 132.4, 130.2 (2C), 130.0 (2C), 129.7, 127.9 (2C), 125.5 (2C), 121.5, 66.5, 52.2, 46.4, 29.4, 21.6. HRMS (ESI+): m/z calculated for C₂₀H₂₁N₃O₅S [M+H]⁺ 416.1202 found 416.1327. UPLC purity (t_R = 0.8 min, 99.9%).

Methyl p-{5-[3-(p-tolylsulfonyloxy)propyl]-1,3,4-oxadiazol-2-yl}benzoate (45).

General procedure 4. (203 mg, 70%) ¹H NMR (400 MHz, CDCl₃) δ = 8.08 (d, J = 8.4 Hz, 2H), 8.00 (d, J = 8.4 Hz, 2H), 7.69 (d, J = 8.2 Hz, 2H), 7.23 (d, J = 8.1 Hz, 2H), 4.14 (t, J = 5.9 Hz, 2H), 3.88 (s, 3H), 2.95 (t, J = 7.3 Hz, 2H), 2.31 (s, 3H), 2.23 – 2.12 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ = 166.0, 165.8, 164.0, 145.0, 132.7, 132.6, 130.1 (2C), 129.9 (2C), 127.8 (2C), 127.5, 126.6 (2C), 68.7, 52.4, 25.5, 21.6, 21.5. HRMS (ESI+): m/z calculated for C₂₀H₂₀N₂O₆S [M+H]⁺ 417.1042 found 417.1186. UPLC purity (t_R = 0.77 min, 99.9%).

Methyl p-{5-[3-(p-tolylsulfonyloxy)propyl]-4H-1,2,4-triazol-3-yl}benzoate (46).

General procedure 4. (150 mg, 49%) ¹H NMR (400 MHz, CDCl₃) δ = 8.07 (s,1H), 7.98 (d, J = 8.2 Hz, 2H), 7.72 (d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 7.22 (d, J = 7.9 Hz, 2H), 4.25 (t, J = 5.8 Hz, 2H), 3.92 (s, 3H), 3.23 (t, J = 7.0 Hz, 2H), 2.42 (s, 1H), 2.33 (s, 3H), 2.29 – 2.23 (m, 2H).¹³C NMR (100 MHz, CDCl₃) δ = 166.5, 158.8, 144.7, 133.8, 133.4, 132.9, 131.5, 130.3 (2C), 128.5 (2C), 127.8 (2C), 126.8 (2C), 68.9, 52.2, 26.2, 23.7, 21.8. HRMS (ESI+): m/z calculated for C₂₀H₂₁N₃O₅S [M+H]⁺ 416.1202 found 416.1298. UPLC purity (t_R = 0.81 min, 95.1%).

Methyl p-{2-[3-(p-tolylsulfonyloxy)propyl]-2H-1,2,3,4-tetraazol-5-yl}benzoate (47).

General procedure 4. (120 mg, 33%) ¹H NMR (400 MHz, CDCl₃) δ = 8.04 (d, J = 8.6 Hz, 2H), 7.89 (d, J = 8.0 Hz, 2H), 7.78 (s, 1H), 7.69 (d, J = 8.6 Hz, 2H), 7.16 (d, J = 7.8 Hz, 2H), 4.64 (t, J = 6.6 Hz, 2H), 3.84 (s, 3H), 2.78 (t, J = 7.1 Hz, 2H), 2.53 – 2.38 (m, 2H), 2.29 (s, 4H). ¹³C NMR (100 MHz, CDCl₃) δ = 166.4, 164.2, 145.2, 132.4, 131.7, 131.3, 130.1 (2C), 130.0 (2C), 127.9 (2C), 126.7 (2C), 66.4, 52.3, 49.3, 28.6, 21.6. HRMS (ESI+): m/z calculated for C₁₉H₂₀N₄O₅S [M+H]⁺ 417.1154 found 417.1244. HPLC purity (t_R = 1.4 min, 96.1%).

N-p-Tolyl4-bromobutyramide (49a).

General procedure 5. (680 mg, 63%) ¹H NMR (400 MHz, CDCl₃) δ = 7.38 (d, J = 8.2 Hz, 2H), 7.31 (s, 1H), 7.12 (d, J = 8.1 Hz, 2H), 3.65 (t, J = 6.1 Hz, 2H), 2.53 (t, J = 7.0 Hz, 2H), 2.31 (s, 3H), 2.24 – 2.13 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ = 173.9, 136.6, 133.3 (2C), 129.8, 119.3 (2C), 35.1, 32.3, 28.8, 21.1.

N-4-Biphenylyl4-bromobutyramide (49b).

General procedure 5. (650 mg, 68%) ¹H NMR (400 MHz, CDCl₃) δ = 7.60 (s, 1H), 7.59 – 7.53 (m, 6H), 7.42 (t, J = 7.5 Hz, 3H), 7.32 (t, J = 7.2 Hz, 1H), 3.67 (t, J = 6.1 Hz, 2H), 2.58 (t, J = 7.0 Hz, 2H), 2.27 – 2.14 (m, 2H). ¹³C NMR (100 MHz, CDCl3) δ = 170.1, 140.4, 137.5, 137.0, 128.9 (2C), 127.6 (2C), 127.1, 126.8 (2C), 120.2 (2C), 44.5, 34.2, 27.9.

N-p-Tolyl4-azidobutyramide (50a).

General procedure 6. (600 mg, 89%) ¹H NMR (400 MHz, CDCl₃) δ 8.16 (s, 1H), 7.27 (d, J = 8.2 Hz, 2H), 6.98 (d, J = 8.1 Hz, 2H), 3.22 (d, J = 6.6 Hz, 2H), 2.29 (t, J = 7.3 Hz, 2H), 2.20 (s, 4H), 1.83 (p, J = 6.9 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ = 170.1, 135.2, 134.1, 129.8 (2C), 119.8 (2C), 50.8, 34.0, 24.7, 20.9.

N-4-Biphenylyl4-azidobutyramide (50b).

General procedure 6. (570 mg, 89%) ¹H NMR (400 MHz, CDCl₃) δ 7.57 (m, 6H), 7.43 (t, J = 7.5 Hz, 2H), 7.34 (d, J = 7.5 Hz, 2H), 3.44 (t, J = 6.4 Hz, 2H), 2.49 (t, J = 7.1 Hz, 2H), 2.12 – 1.96 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ = 170.0, 140.4, 137.3, 136.9, 128.8 (2C), 127.7 (2C), 127.2, 126.9 (2C), 120.1 (2C), 50.7, 34.2, 24.6.

Methyl p-{1-[3-(N-p-tolylcarbamoyl) propyl]-1H-1,2,3-triazol-4-yl}benzoate (51a).

General procedure 2. (160 mg, 75%) ¹H NMR (400 MHz, DMSO-d6) δ 9.84 (s, 1H), 8.79 (s, 1H), 8.03 (q, J = 8.4 Hz, 5H), 7.45 (d, J = 8.2 Hz, 2H), 7.08 (d, J = 8.1 Hz, 2H), 4.50 (t, J = 6.7 Hz, 2H), 3.87 (s, 4H), 2.34 (t, J = 7.2 Hz, 2H), 2.28 – 2.16 (m, 5H). ¹³C NMR (100 MHz, DMSO-d6) δ = 170.2, 166.3, 146.3, 137.1, 135.8, 132.0, 130.5 (2C), 129.5 (2C), 129.2, 125.6 (2C), 123.1, 119.3 (2C), 52.5, 49.5, 33.4, 26.0, 14.5. HRMS (ESI+): m/z calculated for C₂₁H₂₂N₄O₃ [M+H]⁺ 379.1511 found 379.1788. HPLC purity (t_R = 1.4 min, 98.9 %).

N-p-Tolyl4-{4-[p-(trifluoromethyl)phenyl]-1H-1,2,3-triazol-1-yl}butyramide (51b).

General procedure 2. (120 mg, 75%) ¹H NMR (400 MHz, DMSO-d6) δ = 9.85 (s, 1H), 8.79 (s, 1H), 8.07 (d, J = 8.0 Hz, 2H), 7.82 (d, J = 8.1 Hz, 2H), 7.45 (d, J = 8.2 Hz, 2H), 7.07 (d, J = 8.1 Hz, 2H), 4.50 (t, J = 6.7 Hz, 2H), 2.35 (t, J = 7.1 Hz, 2H), 2.28 – 2.10 (m, 5H). ¹³C NMR (100 MHz, DMSO-d6) δ = 170.2, 145.5, 137.1, 135.4, 132.4, 129.5 (2C), 128.2 (q, J = 28.2 Hz), 126.4 (q, J = 7.4 Hz) (2C), 126.3 (q, J = 262.4 Hz), 126.1 (q, J = 7.5 Hz) (2C), 123.1, 119.5 (2C), 49.7, 33.2, 25.9, 20.9. HRMS (ESI+): m/z calculated for C₂₀H₁₉F₃N₄O [M+H]⁺ 389.1692 found 389.1617. HPLC purity (t_R = 1.5 min, 97.3 %).

Methyl p-{1-[3-(N-4-biphenylylcarbamoyl)propyl]-1H-1,2,3-triazol-4-yl}benzoate (51c).

General procedure 2. (80 mg, 34%) ¹H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H), 8.82 (s, 1H), 8.02 (q, J = 8.3 Hz, 4H), 7.68 (d, J = 8.5 Hz, 2H), 7.61 (dd, J = 11.9, 8.3 Hz, 5H), 7.43 (t, J = 7.5 Hz, 2H), 7.32 (t, J = 7.2 Hz, 1H), 4.52 (t, J = 6.8 Hz, 2H), 3.87 (s, 3H), 2.40 (t, J = 7.2 Hz, 3H), 2.26 – 2.16 (m, 2H). ¹³C NMR (100 MHz, DMSO-d6) δ = 170.5, 166.1, 145.3, 140.2, 139.2, 135.8, 135.2, 130.3 (2C), 129.4 (2C), 129.1, 127.6, 127.2 (2C), 126.8 (2C), 125.7 (2C), 123.5, 119.6 (2C), 53.0, 49.5, 33.8, 25.7. HRMS (ESI+): m/z calculated for C₂₆H₂₄N₄O₃ [M+H]⁺ 441.1848 found 441.1949. HPLC purity (t_R = 1.6 min, 96.0 %).

N-4-Biphenylyl4-{4-[p-(trifluoromethyl)phenyl]-1H-1,2,3-triazol-1-yl}butyramide (51d).

General procedure 2. (210 mg, 71%) ¹H NMR (400 MHz, DMSO-d6) δ 9.92 (s, 1H), 8.68 (s, 1H), 7.95 (d, J = 7.9 Hz, 2H), 7.69 (d, J = 8.0 Hz, 2H), 7.55 (d, J = 8.2 Hz, 3H), 7.48 (t, J = 9.0 Hz, 4H), 7.30 (t, J = 7.4 Hz, 2H), 7.19 (t, J = 7.1 Hz, 1H), 4.39 (t, J = 6.5 Hz, 2H), 2.28 (t, J = 6.9 Hz, 2H), 2.14 – 2.05 (m, 2H). ¹³C NMR (100 MHz, DMSO-d6) δ = 170.6, 145.5, 140.2, 139.1, 135.2, 129.4 (2C), 128.4 (q, J = 21.6 Hz) 127.4, 127.3 (2C), 126.7 (2C), 126.4 (q, J = 7.5 Hz) (2C), 126.4 (q, J = 262.4 Hz), 126.1 (2C), 124.5, 123.2, 119.9 (2C), 55.6, 33.3, 25.9. HRMS (ESI+): m/z calculated for C₂₅H₂₁F₃N₄O [M+H]⁺ 451.1667 found 451.1768. HPLC purity (t_R = 1.7 min, 96.6%).

Methyl p-{1-[3-(p-tolylcarbamthioyl)propyl]-1H-1,2,3-triazol-4-yl}benzoate (52a).

General procedure 3. (95 mg, 52%) ¹H NMR (400 MHz, DMSO-d6) δ 11.49 (s, 1H), 8.80 (s, 1H), 8.03 (q, J = 8.3 Hz, 5H), 7.63 (d, J = 8.2 Hz, 2H), 7.19 (d, J = 8.3 Hz, 2H), 4.52 (t, J = 6.8 Hz, 3H), 3.87 (s, 5H), 2.84 – 2.68 (m, 4H), 2.43 – 2.35 (m, 3H), 2.29 (s, 3H). ¹³C NMR (100 MHz, DMSO-d6) δ 202.1, 166.3, 145.9, 137.6, 135.9, 135.8, 130.3 (2C), 129.5 (2C), 129.0, 125.9 (2C), 123.9 (2C), 123.3, 52.6, 49.6, 43.3, 30.0, 21.3. HRMS (ESI+): m/z calculated for C₂₁H₂₂N₄O₂S [M+H]⁺ 395.1463, found 395.1575. HPLC purity (t_R = 1.4 min, 97.6 %).

(3-{4-[p-(Trifluoromethyl)phenyl]-1H-1,2,3-triazol-1-yl}propyl)-p-toluenecarbothioamide (52b).

General procedure 3. (130 mg, 62%) ¹H NMR (400 MHz, DMSO-d6) δ = 11.50 (s, 1H), 8.82 (s, 1H), 8.08 (d, J = 8.1 Hz, 2H), 7.83 (d, J = 8.1 Hz, 2H), 7.63 (d, J = 8.1 Hz, 2H), 7.19 (d, J = 8.2 Hz, 2H), 4.53 (t, J = 6.8 Hz, 2H), 2.76 (t, J = 7.4 Hz, 3H), 2.44 – 2.35 (m, 3H), 2.29 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ = 202.0, 146.6, 136.9, 136.1, 132.7 (2C), 130.4 (q, J = 27.7 Hz), 129.3 (2C), 126.2, 126.0 (q, J = 262.3 Hz), 126.0 (q, J = 6.6 Hz) (2C), 123.8, 113.8 (q, J = 7.5 Hz) (2C), 55.6, 49.7, 43.7, 29.2. HRMS (ESI+): m/z calculated for C₂₀H₁₉F₃N₄S [M+H]⁺ 405.1283, found 405.1371. HPLC purity (t_R = 1.6 min, 98.9 %).

Methyl p-{1-[3-(4-biphenylylcarbamthioyl)propyl]-1H-1,2,3-triazol-4-yl}benzoate (52c).

General procedure 3. (20 mg, 59%) ¹H NMR (400 MHz, DMSO-d6) δ 11.66 (s, 1H), 8.81 (s, 1H), 8.03 (dd, J = 13.9, 8.2 Hz, 5H), 7.91 (d, J = 8.2 Hz, 2H), 7.70 (t, J = 8.1 Hz, 5H), 7.47 (t, J = 7.4 Hz, 3H), 7.37 (t, J = 7.2 Hz, 1H), 4.55 (t, J = 6.7 Hz, 2H), 3.87 (s, 3H), 2.85 – 2.75 (m, 2H), 2.43 (m, 2H).¹³C NMR (100 MHz, DMSO-d6) δ = 203.5, 166.4, 150.8, 145.8, 139.9, 139.3, 138.1, 135.8, 130.4 (2C), 129.4 (2C), 127.9, 127.1 (2C), 127.0 (2C), 125.7 (2C), 124.1 (2C), 123.2, 52.1, 51.6, 40.9, 22.6. HRMS (ESI+): m/z calculated for C₂₆H₂₄N₄O₂S [M+H]⁺ 457.1629, found 457.1567. HPLC purity (t_R = 1.8 min, 97.2 %).

(3-{4-[p-(Trifluoromethyl) phenyl]-1H-1,2,3-triazol-1-yl} propyl)-4biphenylcarbothioamide (52d).

General procedure 3. (120 mg, 53%) ¹H NMR (400 MHz, DMSO-d6) δ 11.66 (s, 1H), 8.83 (s, 1H), 8.09 (d, J = 8.0 Hz, 2H), 7.92 (d, J = 8.5 Hz, 3H), 7.83 (d, J = 8.1 Hz, 3H), 7.73 – 7.63 (m, 5H), 7.47 (t, J = 7.6 Hz, 3H), 7.38 (d, J = 7.3 Hz, 1H), 4.56 (t, J = 6.8 Hz, 2H), 2.86 – 2.77 (m, 2H), 2.51 (s, 3H), 2.44 (dd, J = 14.3, 7.1 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d6) δ = 202.3, 145.6, 139.9, 137.8, 139.4, 135.2, 128.9 (2C), 128.6, 127.9 (q, J = 27.6 Hz), 127.1 (2C), 127.0 (2C), 126.4 (q, J = 7.6 Hz) (2C), 126.3 (q, J = 260.4 Hz), 126.1 (q, J = 3.7 Hz) (2C), 124.2 (2C), 123.2, 49.6, 43.5, 29.8. HRMS (ESI+): m/z calculated for C₂₅H₂₁F₃N₄S [M+H]⁺ 467.1439, found 467.1539. HPLC purity (t_R = 2.0 min, 97.2 %).

Cell Culture.

A549 cells (ATCC CCL-185) were grown in RPMI 1640 media (VWR #45000–396) supplemented with 10% FBS, 1% sodium pyruvate, and 1% penicillin/streptomycin. MDA-MB-231 cells (ATCC HTB-26) and HuH6 cells (kind gift from Dr. Thomas Pietschmann) were grown in Dulbecco’s Modified Eagle Medium (VWR #45000–304) supplemented with 10% FBS and 1% penicillin/streptomycin. 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. Cells were grown and maintained at 37 °C in a CO₂ incubator.

Antiproliferative Assay.

A549, MDA-MB-231, HuH6, and HEK-293 cells were all seeded in 96-well plates at 2.5 × 10⁴ cells/well. Compounds 20–33, 43–47, 51a-d and 52a-d were initially dissolved in 100% DMSO. Cells were treated with increasing concentrations of all compounds at a final dilution of 0.5% DMSO 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: media was removed, 100 μL of MTT dye was diluted in complete media to a final concentration of 1 mg/mL and was added to each well, and then plates were incubated for 2 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 30 min to dissolve the purple-colored formazan crystals. After 30 min, the absorbance was measured using a Tecan SPARK10M spectrophotometer (Tecan Group Ltd., Männedorf, Switzerland) at 570 nm. Each experiment had 6–8 repeats per treatment. Analysis and IC₅₀ determination were performed using GraphPad Prism v6.0.

Western blot analysis.

A549, MDA-MB-231, and HuH6 cells were seeded in 10 cm dishes and treated with varying concentrations of compounds 51b, 51c, 51d, 52b and 52d in DMSO (0.5% DMSO final concentration), or DMSO only for 48 h. Cells were harvested in cold PBS and lysed with Radioimmunoprecipitation assay (RIPA) buffer and a protease/phosphatase inhibitor cocktail (Thermo-Fisher) 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-phosphorylated p53 S6 (Cell Signaling), anti-phosphorylated p53 S15 (ThermoFisher Scientific), 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).

Clonogenic assay.

MDA-MB-231 and A549 cells were seeded (125 cells/mL) in a 6-well tissue culture treated plate and incubated overnight for attachment. The MDA-MB-231 cells were treated with compounds 51b and 51d at IC₅₀ and half IC₅₀ concentrations for 48 h at 37 °C. A549 cells were treated with compounds 51c, 52b, and 52d at IC₅₀ and half IC₅₀ concentrations for 48 h at 37 °C. The treatments were then aspirated, fresh media was replaced every alternate day in each well, and the cells were incubated for an additional 7 days to allow colony growth. Cells were then washed twice with ice-cold PBS (1X) and fixed using 4% paraformaldehyde (prepared in PBS). The cells were washed with PBS and stained by incubating with crystal violet (0.2%) for 1 h. Colonies containing more than 50 cells were counted using Open CFU software (Version 4.8) and imaged using a digital camera. The % growth of the colonies for each treatment group was calculated relative to the number of colonies observed for the control group.^49,50 All data are presented as mean ± SD or SEM (n = 3 to 8). Results represent the average of 3 independent trials (n = 6 for each trial). Unpaired student’s t-test was used to compare two groups. A P-value < 0.05 was considered statistically significant.

In-vitro tumor simulation studies (3D spheroids).

MDA-MB-231 and A549 cells were grown at a density of 2000 cells/well in an ultra-low attachment 96-well U bottom plate (Nuclon® Sphera, Thermo-Fisher Scientific, Waltham, MA, USA) and incubated for 3 days. Then MDA-MB-231 cells were treated with compounds 51b and 51d at IC₅₀ and half IC₅₀ concentrations. A549 cells were treated with compounds 51c, 52b, and 52d at IC₅₀ and half IC₅₀ concentrations. A single dose treatment was provided on day 3 of the experiment, then half the volume of media was replenished every 3 days, and images were taken. Optical imaging was performed using an LMI-6000 inverted microscope (LAXCO, Bothell, WA, USA), and transmitted light microscope images were collected using SEBA view software. Changes in the morphology of spheroids, such as spheroid diameter and spheroid volume, were calculated using Image J software version 2.2.0.^49,50 All data are presented as mean ± SD or SEM (n = 3 to 8). Results represent the average of 3 independent trials (n = 6 for each trial). Unpaired student’s t-test was used to compare two groups. A P-value < 0.05 was considered statistically significant.

Live-Dead cell assay.

The live-dead cell assay was performed with the spheroids developed for the in-vitro tumor simulation studies (after the 15^th day) using a live-dead assay kit (Biotium, Fremont, CA, USA). The assay distinguishes between live and dead cells using two dyes: calcein AM and EthD-III, respectively. Briefly, media was replaced by 100 μL of kit reagent followed by incubation in the dark for 30 min at room temperature. The labeled spheroid cells were imaged at 4X using a florescence microscope EVOS FL cell imaging system (ThermoFisher Scientific, Waltham, MA, USA).⁴⁹ All data are presented as mean ± SD or SEM (n = 3 to 8). Results represent the average of 3 independent trials (n = 6 for each trial). Unpaired student’s t-test was used to compare two groups. A P-value < 0.05 was considered statistically significant.

Cell cycle analysis.

A549 and MDA-MB-231 cells were seeded in 10 cm dishes and treated with 0.3 μM and 2 μM of compounds 51c and 51d, respectively, in DMSO (0.1% DMSO final concentration), or 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 150 μL of PI kit (Nexcelom Bioscience). The cells were then incubated for 40 min at 37 °C before performing flow and imaging cytometry analysis. Results were analyzed using FCS Express 4 software (De Novo Flow Cytometry Software).⁵¹ One-way ANOVA was used to determine statistical significance. A P-value < 0.05 was considered statistically significant.

Protein Expression and Purification of recombinant Gankyrin.

The pQTEV-PSMD10 bacterial expression vector was obtained from Addgene (Cambridge, MA; plasmid #31332).⁵² The plasmid was transformed into BL21(DE3) Codon +RIL Escherichia coli and streaked out on Luria Burtani (LB) agar plates supplemented with 100 μg/mL ampicillin and 34 μg/mL chloramphenicol and incubated at 37 °C for approximately 16 h. Colonies were then aseptically transferred into a sterile 250 mL baffled flask with 50 mL of autoclaved LB broth supplemented with 100 μg/mL ampicillin and 34 μg/mL chloramphenicol. Starter cultures were then incubated at 37 °C and 200 rpm for approximately 16 h. Large scale production cultures with 1 L LB Broth and the appropriate antibiotics as previously described were inoculated at a 1:200 passage and grown at 37 °C until the OD₆₀₀ reached 0.6–0.8. The cultures were then cooled to 25 °C, induced with 1 mM isopropyl thiogalactoside (IPTG), and grown for approximately 5 h. Cells were harvested by centrifugation with a Sorvall LYNX 4000 Superspeed Centrifuge (Thermo Fisher) at 10,000 rpm for 10 min at 4 °C. The cell pellets were resuspended at a 1:4 ratio in 50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0 and 100 μL/mL ProBlock™ Protease Inhibitor Cocktail (GoldBio) to mitigate protein degradation. The cells were lysed using an Avestin EmulsiFlex-C5 homogenizer, and the cell extract centrifuged with a Beckman Coulter Optima L-100XP Preparative Ultracentrifuge (Beckman Coulter) at 41,000 rpm for 40 min at 4 °C. The clarified supernatant was then collected and passed through a 5.0 μm sterile filter (Thermo Fisher) then loaded onto a Nickel Immobilized Metal Affinity Chromatography (Ni²⁺-IMAC) resin column (GoldBio). The resin was washed extensively with 50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0 until the Better Bradford reagent (Thermo Fisher) indicated the absence of protein. Gankyrin was eluted with 50 mM Tris-HCl, 300 mM NaCl, 250 mM imidazole, pH 8.0. The N-terminal polyhistidine tag (His₆) removed with in-house generated TEV protease (40 μg/mL) and incubated for 1 h at room temperature. The sample was dialyzed to remove excess imidazole with 3.5 MWCO Spectra/Por 3 Dialysis Tubing (Thermo Fisher) in 50 mM Tris-HCl, 300 mM NaCl, pH 8.0 at 4 °C with gently mixing overnight. The sample was then passed through Ni²⁺-IMAC) resin again to remove the His₆-tag and TEV protease from the sample. Any remaining protein contaminants were removed through gel filtration chromatography using a Superdex 75 HiLoad column (Cytivia Life Sciences) equilibrated with 50 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, pH 7.4 on an ÄKTA pure 25 L FPLC system (Cytivia Life Sciences). 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.

Circular Dichroism Spectra and Thermal Denaturation.

To confirm biochemically pure gankyrin was produced and detect its native fold, circular dichroism (CD) and temperature denaturation experiments were collected on a Jasco J-815 Circular Dichroism Spectropolarimeter (JASCO Inc., Japan). CD is a robust biophysical method routinely used to characterize the secondary structure of a protein sample.⁵³ With gankyrin having solely seven ankyrin repeat domain, spectral minima at 208 nm and 222 nm were expected to confirm the presence of α-helical content. Samples containing gankyrin alone and in the presence of compounds were measured in 1x PBS, 0.5% DMSO, pH 7.4 in a 1 mm quartz cuvette. CD spectra were collected from 190–260 nm, with six accumulation trials. The thermal denaturation of gankyrin alone and in the presence of compounds were measured at 217 nm from 5 to 90 °C at a rate of 1 °C/min, 1.0 nm bandwidth, 100 mdeg sensitivity with a 4-second response.

Cellular Thermal Shift Assay.⁵⁶

A549 and MDA-MB-231 cells were seeded in 10 cm dishes and treated with 0.3 μM of compound 51c and 2 μM of compound 51d in DMSO (0.5% DMSO final concentration), or DMSO only for 48 h. Cells were harvested and equally divided in PCR tubes. Cells were then heated at 6 different temperatures (50, 53, 56, 59, 62, and 65 °C) separately. Heated samples were then collected and lysed in RIPA buffer on ice for 60 min. Lysates were cleared at 15,000 rpm for 15 min at 4 °C. Protein concentrations were determined with the DC Protein Assay Kit (BioRad) per the manufacturer’s instructions. Equal amounts of protein were electrophoresed under reducing conditions, transferred to a PVDF membrane, and immunoblotted with either anti-actin or anti-gankyrin primary antibodies. 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). Images were quantified using ImageJ software. Results represent the average of 3 independent trials. Unpaired student’s t-test was used to compare two groups. A P-value < 0.05 was considered statistically significant.

Metabolic stability study

A reaction mixture containing microsomal proteins (0.5 mg/mL), NADPH (3 mM), MgCl₂ (3 mM), and 100 μM test compound (51b, 51c, 51d, 52b, and 52d) in 1X PBS was used. The final incubation volume was 250 μL. The incubation was carried out aerobically at 37 °C. The mixture was pre-incubated without NADPH for 10 min at 37 °C, and NADPH was added to initiate the reaction. At 0 min and 60 min an aliquot (50 μL) of each incubation mixture was taken and mixed with 500 μL of ice-cold acetonitrile to terminate the reaction. The sample was then precipitated by centrifugation (15000 rpm) at room temperature. The resulting supernatant was analyzed by HPLC (LC, Agilent 1260 Infinity; column, Eclipse Plus C18; column temperature, 25 °C; mobile phase, solvent A, methanol, solvent B water, gradient elution, 90% solvent A; flow rate, 1 mL/min; UV signals were recorded at 254 nm) for each compound to determine their metabolic stability.⁵⁷

ABBREVIATIONS USED

δ

chemical shift in parts per million

μ

micro

°C

degrees Celsius

3D

three-dimensional

aq

aqueous

CD

Circular dichroism

CDK4

Cyclin-dependent kinase 4

C/EBPα

CCAAT/Enhancer-binding protein α

CETSA

cellular thermal shift assay

CuAAc

copper-catalyzed alkyne-azide cycloaddition

CUGBP1

CUG triplet repeat RNA binding protein 1

d

doublet (spectral)

DIPEA

N,N-Diisopropylethylamine

DMAP

4-Dimethylaminopyridine

DMF

Dimethylformamide

DMSO

Dimethyl sulfoxide

EthD-III

ethidium homodimer III

ESI

Electrospray ionization

eq

Equivalent

FBS

Fetal bovine serum

g

Gram(s)

h

Hour(s)

HBTU

(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

HNF4α

Hepatocyte nuclear factor 4 α

HPLC

High-performance liquid chromatography

HRMS

high-resolution mass spectrometry

ITC

Isothermal titration calorimetry

j

coupling constant (in NMR spectrometry)

L

Liters

m

Multiplet (spectral)

M

Molar

MDM2

Mouse double minute 2 homolog

MHz

megahertz

mL

Milliliter(s)

mm

Millimeters

mM

Millimolar

min

Minute(s)

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide

m/z

mass-charge-ratio

NADPH

Nicotinamide adenine dinucleotide

NMR

Nuclear magnetic resonance

NSCLC

Non-small cell lung cancer

PAGE

Polyacrylamide gel electrophoresis

PBS

Phosphate buffered saline

PPI

Protein-protein interaction

PVDF

polyvinylidene fluorine

Rb

Retinoblastoma protein

RING

Really interesting new gene

RIPA

Radioimmunoprecipitation assay buffer

rt

Room temperature

s

singlet (spectral)

SD

Standard deviation

SDS

Sodium dodecyl sulfate

t

Triplet (spectral)

TEA

Triethylamine

THF

tetrahydrofuran

TLC

Thin layer chromatography

T_m

melting temperature or point of sigmoidal inflection

TSP

Tumor suppressor protein