N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamide as a New Scaffold that Provides Rapid Access to Antimicrotubule Agents: Synthesis and Evaluation of Antiproliferative Activity Against Select Cancer Cell Lines

Abstract

A series of N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamides was synthesized by copper-catalyzed azide–alkyne cycloaddition (CuAAC) and afforded inhibitors of cancer cell growth. For example, compound 13e had an IC₅₀ of 46 nM against MCF-7 human breast tumor cells. Structure–activity relationship (SAR) studies demonstrated that (i) meta-phenoxy substitution of the N-1-benzyl group is important for antiproliferative activity and (ii) a variety of heterocyclic substitutions for the aryl group of the arylamide are tolerated. In silico COMPARE analysis of antiproliferative activity against the NCI-60 human tumor cell line panel revealed a correlation to clinically useful antimicrotubule agents such as paclitaxel and vincristine. This in silico correlation was supported by (i) in vitro inhibition of tubulin polymerization, (ii) G₂/M-phase arrest in HeLa cells as assessed by flow cytometry, and (iii) perturbation of normal microtubule activity in HeLa cells as observed by confocal microscopy. The results demonstrate that N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamide is a readily accessible small molecule scaffold for compounds that inhibit tubulin polymerization and tumor cell growth.

Introduction

Microtubules, dynamic protein polymers composed of α-tubulin and β-tubulin heterodimers, are a well-established cellular target for anticancer drugs.¹ Dynamic polymerization of tubulin is a necessary and tightly controlled process during mitosis.² Perturbing microtubule dynamics with small molecules blocks the cell cycle in the metaphase/anaphase transition and leads to apoptosis.³ Thus, molecules⁴ that target tubulin halt rapid cell division, a characteristic of cancer cells.⁵ This therapeutic strategy has been validated by the clinical success of antimicrotubule drugs such as paclitaxel, docetaxel, vincristine, and vinblastine. Nonetheless, neurotoxicity and P-glycoprotein-mediated drug resistance limit the clinical utility of these drugs.⁶ New-generation taxoids, vinca alkaloids, and other novel chemotypes that modulate microtubule dynamics have been synthesized in efforts to overcome these limitations.⁷ For example, small molecule modulators of tubulin polymerization that do not elicit neurotoxicity in mice have been identified,⁸ suggesting that neurotoxicity is not intrinsically linked to antimicrotubule agents. Nonetheless, few of these new antimicrotubule agents have produced useful clinical results. The key limitation to the development of new antimicrotubule drugs is a narrow therapeutic window.⁹ A new antimicrotubule scaffold amenable to rapid derivatization and combinatorial library synthesis would provide an exceptional opportunity for the discovery of an efficacious antimicrotubule agent with an improved therapeutic window.

Herein we report the synthesis, in vitro antiproliferative activity against select cancer cell lines, and structure–activity relationships of compounds containing the N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamide scaffold. We also report mode of action studies based on in silico, in vitro and cell culture experiments, which reveal the potent antimicrotubule activity of this scaffold. The discovery of this scaffold stemmed from our work on Mycobactin S (1),¹⁰ a natural product produced by Mycobacterium smegmatis that exhibits anti-tuberculosis activity¹¹ (Chart 1). All synthetic intermediates encountered during our group’s total synthesis of Mycobactin S¹² were screened for biological activity. Surprisingly, benzyl ester 2, a small fragment of the natural product, exhibited anti-tuberculosis activity similar to that of Mycobactin S. Furthermore, in contrast to Mycobactin S, compound 2 provided a scaffold that was amenable to rapid structure–activity relationship studies, which led to the discovery of more potent anti-tuberculosis agents.¹³ While exploring derivatives of 2, we found that 2-phenyl-oxazole-4-carboxamide derivative 3 was also active against M. tuberculosis. 2-Phenyl-oxazole-4-carboxamides are known inhibitors of histone deacetylase,¹⁴ Stat3,¹⁵ phosphodiesterase,¹⁶ phosphatase,¹⁷ thromboxane synthase,¹⁸ and kinase proteins,¹⁹ and known activators of cellular caspase activity.²⁰ We synthesized derivatives of 3 to identify a more potent anti-tuberculosis agent.¹³ One of the derivatives, compound 4e, had weak anti-tuberculosis activity, but broader biological screening serendipitously revealed that 4e and related derivatives have potent antimicrotubule activity in cancer cells, as disclosed in this report.

Chart 1.

Structures of anti-tuberculosis compounds 2 and 3 and antimicrotubule compound 4e, all derived from a fragment of mycobactin S (1). MIC values indicate in vitro anti-tuberculosis activity against M. tuberculosis H37Rv in GAST medium²¹, and IC₅₀ values indicate in vitro antiproliferative activity against human breast cancer cell line MCF-7.

Results and Discussion

Chemistry

2-Phenyl-oxazole-4-carboxylic acid 7 was synthesized according to the protocols shown in Scheme 1.^11,12 Coupling benzoyl chloride to serine benzyl ester hydrochloride afforded β-hydroxy amide 5. Dehydrative cyclization and oxidation of β-hydroxy amide 5 with diethylaminosulfurtrifluoride (DAST) and DBU/BrCCl₃ yielded oxazole 6.²² Catalytic hydrogenolysis of the benzyl ester provided 2-phenyl-oxazole-4-carboxylic acid 7.

Scheme 1.

Synthesis of 2-phenyloxazole-4-carboxylic acid.

Our strategy for exploring the chemical space around the 2-phenyl-oxazole-4-carboxamide fragment employed “click chemistry.”²³ More specifically, we selected the Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) reaction²⁴ because of its wide scope, high efficiency, and recognized utility for drug discovery.²⁵ Following this strategy, N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)-2-phenyl-oxazole-4-carboxamides 4a–e were synthesized as shown in Scheme 2. Coupling propargylamine to freshly prepared 2-phenyloxazole-4-carboxyl chloride (derived from the corresponding carboxylic acid 7) provided alkyne 8. With the terminal alkyne precursor in hand, we turned our attention to the syntheses of azides 10a–e. Benzyl bromides 9a–e were treated with NaN₃ to afford benzyl azides 10a–e.²⁶ Exposing terminal alkyne 8 to benzyl azides 10a–e in the presence of catalytic Cu(I) produced 1,4-disubstituted triazoles 4a–e in high regioselectivity. Aqueous CuAAC conditions (H₂O/t-BuOH, 2:1) facilitated precipitation of the products, which were isolated with high purity.²⁴

Scheme 2.

Synthesis of N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)-2-phenyl-oxazole-4-carboxamide scaffold.

In order to explore the structure–activity relationships of the aryl amide group, a more general N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamide scaffold was synthesized according to the protocols shown in Scheme 3. The simplicity of reaction Scheme 2 and Scheme 3 is anticipated to allow the synthesis of a large library of 1,2,3-triazole-based structures. Moreover, the CuAAC reaction is the convergent synthetic step. Both the arylamide and benzyl groups, attached to opposite sides of the central triazole, are important components of the pharmacophore, as shown by the structure–activity studies described below. The convergence of the synthesis will allow both sides of the scaffold to be systematically varied in future SAR studies.

Scheme 3.

Synthesis of N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamide scaffold.

In Vitro Antiproliferative Activity

The antiproliferative activity of compounds 4a–e against cancer cells was discovered during broad biological screening of a series of compounds originally anticipated to have anti-tuberculosis activity. Although compounds 4a–e had negligible anti-tuberculosis activity, they inhibited the proliferation of cancer cells in vitro. Therefore, we determined the antiproliferative activities (IC₅₀ values) of hit compounds 4a–e against the breast tumor-derived cell line MCF-7 (Table 1). Because the IC₅₀ of 4e (0.56 µM) was significantly lower than those of 4a–d (IC₅₀ = 7.3–16 µM), we conserved meta-phenoxy benzyl substitution at triazole N-1 in the second series of analogs (Scheme 3).

Table 1.

In vitro antiproliferative activity of compounds 4a–e, colchicine, and 2-methoxyestradiol against human breast cancer cell line MCF-7.

		IC50 (µM)
Compound	R1	MCF-7
4a	H	15.9
4b	p-CH3	7.59
4c	p-CF3	7.33
4d	m-OCH3	8.35
4e	m-OPh	0.56
Colchicine		0.013
2-methoxyestradiol		0.84

The structure–activity relationships of the triazole C-4 substituent were investigated by changing the carboxamide group (Table 2). In this SAR study, antiproliferative activity was investigated with the MCF-7 cell line and human lymphoma cell line U937. Addition of electron withdrawing or electron donating groups to the para-position of the 2-phenyloxazole group (13a–c) had a significant effect on antiproliferative activity, revealing an avenue for future optimization. Before performing an extensive SAR study via substitution of the 2-phenyloxazole group, we wanted to know if the 2-phenyloxazole was necessary for antiproliferative activity. If simpler aryl groups could replace the 2-phenyloxazole, the three-step synthesis of the 2-phenyloxazole-4-carboxylic acids could be bypassed by using commercial aryl acids. Thus, we conducted a systematic truncation of the 2-phenyloxazole-4-carboxamide group found in 4e. Removing the 2-phenyl group (13d) did not significantly change the activity. Following this lead, which suggested that we could use simpler aryl groups, we synthesized 2-pyridyl derivative 13e. Against the MCF-7 cell line, the IC₅₀ of 13e (46 nM) was significantly lower than that of 4e (560 nM). Likewise, a significant decrease in IC₅₀ was observed with the U937 cell line. Therefore, replacing the 2-phenyloxazole group with simpler aryl groups represents a significant opportunity to improve antiproliferative activity. Phenyl derivative 13f had improved antiproliferative activity compared to 4e, but was less active than 2-pyridyl derivative 13e. Replacing the aryl group with a methyl group (14) gave a meaningful loss of antiproliferative activity and demonstrated the importance of the aryl carboxamide group.

Table 2.

Antiproliferative activities (IC₅₀) of compounds 4e, 13a–f, 14, colchicine, and 2-methoxyestradiol against human breast cancer cell line MCF-7 and human lymphoma cell line U937.

Compound	R2	IC50 (µM)a
		MCF-7	U937
4e		0.56 ± 0.11	1.40 ± 0.18
13a		0.33 ± 0.045	1.13 ± 0.44
13b		1.9 ± 1.4	nd
13c		0.66 ± 0.31	3.75 ± 1.78
13d		0.64 ± 0.35	2.19 ± 0.58
13e		0.046 ± 0.022	0.58 ± 0.42
13f		0.245 ± 0.007	nd
14		6.4 ± 1.3	28 ± 19
	Colchicine	0.0134	0.008 ± 0.003
	2-methoxyestradiol	0.842 ± 0.090	2.91 ± 1.17

^aIC₅₀ values represent the concentration at which the cell count was inhibited to 50% of that measured in the vehicle control. Error is S.E.M., n≥3.

Time Dependence of In Vitro Cellular Antiproliferative Activity

In order to distinguish cytotoxic activity from cytostatic activity, the time dependence of the effect of 13e on MCF-7 cells was determined (Figure 1). At a concentration of 39 nM, 13e slowed the cell proliferation rate. At higher concentrations (78 nM and 156 nM), cellular proliferation was halted, but 13e did not decrease the number of cells. Thus, at concentrations moderately higher than the IC₅₀, 13e is cytostatic rather than cytotoxic against MCF-7 cells.

Figure 1.

Time and concentration dependence of the antiproliferative activity of 13e against MCF-7 tumor cells. Time is in terms of time elapsed after addition of the compound. Cell counts are shown relative to the cell count observed in the vehicle control 96 h after addition of the 0.5% DMSO solution.

Broad-Spectrum In Vitro Antiproliferative Activity

The NCI-60 anticancer drug screen is an in vitro assay consisting of 60 different human tumor cell lines.²⁷ Organized by disease type, the NCI-60 panel includes various leukemia cell lines and cell lines derived from solid tumor sources. Cell line selectivity guides further biological evaluation. In the NCI-60 panel, compounds 4b, 4c, 4d, and 4e induced broad-spectrum antiproliferative activity against tumor cell lines derived from leukemia, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer (see Supplemental Information). Of these four compounds, 4e (NCI-60 mean GI₅₀ = 870 nM) was more than twenty times more potent than the other three compounds (Table 3). This result is consistent with the results from our MCF-7 and U937 assays and further emphasizes the importance of meta-phenoxy benzyl substitution of triazole N-1 for optimal activity within this scaffold. With respect to selectivity among the 60 cell lines in the NCI-60 panel, all four compounds tested in the panel showed greater than average potency against human leukemia cell line HL-60, breast cancer cell line MCF-7, and melanoma²⁸ cell line MDA-MB-435 (Table 3).

Table 3.

Antiproliferative activity of compounds 4b, 4c, 4d, and 4e against selected cell lines in the NCI-60 screen.

	GI50 (µM)
compound	mean	HL-60	MCF-7	MDA-MB-435
4b	21.9	1.53	8.93	3.71
4c	16.6	2.34	3.83	1.94
4d	17.8	2.37	4.16	2.12
4e	0.87	0.39	0.36	0.18

COMPARE Analysis Revealed a Correlation to Antimicrotubule Drugs

For a given compound, the antiproliferative activity measured in the NCI-60 differs by cell line. Furthermore, antitumor agents with similar mechanisms of action can produce similar patterns of differential antiproliferative data. We used the matrix COMPARE algorithm²⁹ to measure the correlations between compounds 4b, 4c, 4d, and 4e with respect to differential antiproliferative activity. The matrix produced by the analysis showed that 4c and 4d have highly correlated activities (r = 0.968) (Table 4). On the contrary, 4e (the most potent compound tested in the NCI-60) had low correlations (r < 0.5) with the other three compounds. These matrix COMPARE results suggest that 4c and 4d have the same mechanism of action, but the mechanism of 4e is distinct. Future studies will further investigate the importance of the meta-phenoxy substituent found in 4e for its mechanism action. In the studies reported here, we focused on 4e and related analogs, which also have the meta-phenoxy substituent, because of their superior potency.

Table 4.

Matrix COMPARE analysis of 4b, 4c, 4d, and 4e. Matrix values (r values) are Pearson’s correlation coefficients.³⁰

compound	4b	4c	4d	4e
4b	1.000	0.600	0.583	0.489
4c	0.600	1.000	0.968	0.475
4d	0.583	0.968	1.000	0.491
4e	0.489	0.475	0.491	1.000

The COMPARE algorithm can also compare the differential antiproliferative activity of a new compound to those of compounds with known mechanisms of action in the NCI Standard Agent Database.³¹ Standard COMPARE analysis has been used previously to identify the cellular targets of antitumor agents.³² Thus, the pattern of differential antiproliferative activity of 4e was used to probe the NCI Standard Agent Database for correlations. We used all three measures of activity provided by the NCI-60 screen (GI₅₀, 50% growth inhibition; TGI, total growth inhibition; and LC₅₀, 50% lethal concentration). A standard COMPARE analysis of 4e (Table 5) showed correlations to paclitaxel, maytansine, vincristine, vinblastine, and rhizoxin, all of which affect microtubule polymerization. Given this in silico result, we hypothesized that 4e targets microtubules and directly tested this hypothesis in vitro. In contrast, the first-ranked COMPARE hits for 4b–d did not include antimicrotubule agents (see Supplemental Information).

Table 5.

Standard COMPARE analysis of 4e. The Target Set was the Standard Agent Database and the Target Set Endpoints were set equal to the Seed Endpoints (GI₅₀, TGI, and LC₅₀). Correlation values (r) are Pearson’s correlation coefficients. Some hits appear multiple times because they were tested by the NCI for the Standard Agent Database at multiple concentration ranges.

rank	compound	r
Based on GI50 Mean Graph
1	paclitaxel	0.541
2	maytansine	0.534
3	vincristine sulfate	0.500
4	trimetrexate	0.480
5	soluble Baker's Antifol	0.460
Based on TGI Mean Graph
1	paclitaxel (hiConc = 10−5 M)	0.654
2	paclitaxel (hiConc = 10−6 M)	0.646
3	paclitaxel (hiConc = 10−4.6 M)	0.642
4	maytansine	0.621
5	vinblastine sulfate	0.606
Based on LG50 Mean Graph
1	rhizoxin (hiConc = 10−9 M)	0.681
2	rhizoxin (hiConc = 10−4 M)	0.650
3	vinblastine sulfate (hiConc = 10−7.6 M)	0.558
4	α-2'-deoxythioguanosine	0.545
5	vinblastine sulfate (hiConc = 10−4 M)	0.540

Inhibition of Tubulin Polymerization In Vitro

The polymerization of microtubules from purified tubulin can be monitored in vitro by measuring an increase in light scattering. This in vitro experiment removes complicating factors, such as microtubule-associated proteins (MAPs), which might be part of a putative target that leads to disruption of microtubules as observed with microscopy. In order to test our hypothesis that the target of the N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamide scaffold is tubulin, and not a MAP, we monitored the polymerization of tubulin after treatment with 4e, 13a, and 13e (Figure 2). In this experiment, paclitaxel, a microtubule stabilizer, enhanced the rate of tubulin polymerization, while nocodazole, a microtubule destabilizer, prevented the polymerization of tubulin. Similar to nocodazole, 4e, 13a, and 13e completely inhibited tubulin polymerization at 10 µM. Thus, the triazole-based compounds prevent the formation of microtubules in vitro. We followed this in vitro assay with cell culture experiments to see if microtubules are the primary cellular target.

Figure 2.

Inhibition of tubulin assembly by 4e, 13a, and 13e in vitro. All compounds were tested at a concentration of 10 µM. Effects of compounds on tubulin polymerization were assessed by monitoring the increase in light scattering, measured as optical density (O.D.), at 340 nm. Standards of 10 µM nocodazole (a tubulin assembly inhibitor) and 10 µM paclitaxel (a tubulin assembly promoter) were used for direct comparison.

Cell Cycle Analysis Demonstrated G₂/M-Phase Arrest

Antimicrotubule agents induce M-phase arrest. Flow cytometry can quantitatively determine the population of cells in each phase of the cell cycle by measuring the DNA content of individual cells. Cells in G₂-phase or M-phase have twice as much DNA as cells in G₁-phase. Thus, we conducted flow cytometric cell cycle analysis of HeLa cells treated with 4e, 13a, 13d, 13e, and 14. Consistent with the hypothesized mechanism of action, compounds 4e, 13a, 13d, and 13e significantly increased the population of cells in G₂/M-phase (Figure 3). Upon treatment with these four compounds, the population of G₂/M-phase cells increased from 13% in the control to over 90%. Compound 14, however, did not induce significant G₂/M-phase arrest, which suggested that the arylamide moiety is important for the antimitotic activity of the N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamide scaffold.

Figure 3.

Effects of 4e, 13a, 13d, 13e, and 14 on the cell cycle distribution of HeLa cells as measured by propidium iodide staining and flow cytometry. HeLa cells were treated with 5 µM compound for 18 h in triplicate.

Confocal Microscopy Showed M-phase Arrest and Disruption of Microtubules

Visual evidence for M-phase arrest can be obtained with confocal microscopy due to DNA condensation resulting in enhanced staining by propidium iodide. This outcome is in contrast to the diffuse staining of DNA in interphase cells. Visual evidence for the disruption of microtubules can be obtained concurrently using a fluorescein isothiocyanate-conjugated anti-tubulin antibody. We therefore used confocal microscopy to examine HeLa cancer cells treated with compounds 4e and 13e. Both DNA condensation and disruption of microtubules were observed at 5 µM of 4e or 13e (Figure 4). Together, these images show that 4e and 13e induce M-phase arrest and interfere with microtubule formation in whole cells.

Figure 4.

Confocal microscopy images of HeLa cells after 18 h incubation in the presence of 5 µM compound. Nocodazole is a known tubulin polymerization inhibitor. Nuclear DNA was stained with propidium iodide (red channel) and tubulin was stained with FITC-conjugated anti-α-tubulin antibody (green channel). Compounds 4e and 13e disrupted normal microtubule structures, caused fragmentation of mitotic spindles, and induced M-phase arrest.

Conclusion

In summary, we identified N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamide as a novel and proprietary³³ small molecule scaffold for potential antitumor agents. Elucidating structure–activity relationships by subtraction from initial hit compound 4e (MCF-7 IC₅₀ = 560 nM) led to the discovery of 13e (MCF-7 IC₅₀ = 46 nM), a foundational compound for further study. Compound 13e (and related compounds) induced M-phase arrest in HeLa cells at 5 µM and inhibited tubulin polymerization in vitro at 10 µM, providing strong support for antimicrotubule activity as the primary mechanism of action. The NCI-60 screen demonstrated broad-spectrum antitumor activity and prompted further biological evaluation. Compound 13c was recently evaluated by the National Cancer Institute Developmental Therapeutics Program (NCI DTP) for acute toxicity in vivo, and 100, 200 and 400 mg/kg intraperitoneal (IP) doses were well tolerated in non-tumor bearing mice. Ongoing studies in collaboration with the NCI DTP will evaluate in vivo efficacy in hollow fiber assays.³⁴ Extensive SAR studies and the development of a combinatorial library are accessible because compounds based on the N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)arylamide scaffold are readily synthesized with the CuAAC reaction. Our findings will facilitate the design and optimization of potent, cell-permeable antimicrotubule agents.

Experimental Section

2-(4-methoxyphenyl)-N-((1-(3-phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)oxazole-4-carboxamide (C₂₇H₂₃N₅O₄, 13c)

2-(4-Methoxyphenyl)oxazole-4-carboxylic acid (11c, 0.951 g, 4.3 mmol) was suspended in anhydrous CH₂Cl₂ (12 mL) under argon. Oxalyl chloride (0.45 mL, 5.2 mmol) and N,N-dimethylformamide (20 µL) were added carefully to the mixture because of gas evolution. The reaction slowly turned to a light yellow homogeneous solution over 3 h. The solution was concentrated in vacuo to give 2-(4-methoxyphenyl)oxazole-4-carbonyl chloride (1.0 grams, 97%) as an yellow solid, which was used immediately in the next reaction without characterization.

2-(4-Methoxyphenyl)oxazole-4-carbonyl chloride (1.0 g, 4.0 mmol) was dissolved in anhydrous CH₂Cl₂ (15 mL) under argon and cooled to 0 °C (ice bath). Propargyl amine hydrochloride (0.443 g, 4.8 mmol) and N,N-diisopropylethylamine (2.1 mL, 12.0 mmol) were added with stirring. The reaction was allowed to warm to room temperature. After stirring for 20 h, TLC analysis indicated completion of the reaction. The mixture was poured into a solution of 10% aqueous NaHCO₃ and extracted with CH₂Cl₂ (2x). The organic layer was separated, washed with 10% aqueous NaHCO₃ and brine, dried with Na₂SO₄, filtered, and concentrated in vacuo. The resultant crude material was purified by column chromatography (SiO₂, EtOAc/CH₂Cl₂ stepwise elution, 1:1 to 10:1) to give 2-(4-Methoxyphenyl)-N-(prop-2-ynyl)oxazole-4-carboxamide (12c) as an off white solid (0.788 g, 77%). mp 151–152 °C; ¹H NMR (300 MHz, CDCl₃) δ 8.06 (s, 1H), 7.82 (d, J = 8.5 Hz, 2H), 7.13 (bs, NH, 1H), 6.84 (d, J = 8.5 Hz, 2H), 4.15−4.07 (m, 2H), 3.72 (s, 3H), 2.15 (s, 1H); ¹³C NMR (126 MHz, CDCl₃) δ 161.82, 161.57, 160.40, 140.49, 136.44, 128.83, 128.29, 119.13, 114.26, 113.69, 79.17, 71.69, 55.37, 28.66; HRMS–FAB (m/z) [M+H]⁺ calcd for C₁₄H₁₂N₂O₃, 257.0921; found, 257.0930.

2-(4-Methoxyphenyl)-N-(prop-2-ynyl)oxazole-4-carboxamide (12c, 300 mg, 1.17 mmol) and 1-(azidomethyl)-3-phenoxybenzene (290 mg, 1.29 mmol) were suspended in a 2:1 mixture of water and tert-butyl alcohol (4.7 mL total volume). Sodium ascorbate (0.12 mmol, 0.12 mL, 1 M) and copper(II) sulfate (0.012 mmol, 0.12 mL, 0.1 M) were added sequentially. After stirring for 4 days at room temperature, TLC analysis indicated complete consumption of the reactants. The reaction mixture was diluted with water (5 mL) and cooled on ice. The white precipitate was isolated by vacuum filtration and washed with cold water (3 × 5 mL) and cold diethyl ether (3 × 3 mL) to afford 506 mg (90%) of pure product (13c) as a white powder. TLC R_f = 0.42 (EtOAc); HPLC t_r = 6.82 min (9:1 hexanes/2-propanol); m.p. 119.1 – 119.4 °C; ¹H NMR (300 MHz, CDCl₃) δ 8.18 (s, 1 H), 7.99 − 7.93 (m, 2 H), 7.60 (bs, NH, 1 H), 7.55 (s, 1 H), 7.39 − 7.28 (m, 3 H), 7.16 − 7.09 (m, 1 H), 7.03 − 6.90 (m, 7 H), 5.47 (s, 2 H), 4.72 (d, J=6.1 Hz, 2 H), 3.88 (s, 3 H); ¹³C NMR (151 MHz, CDCl₃) δ 161.83, 161.56, 160.84, 158.04, 156.36, 145.05, 140.26, 136.70, 136.29, 130.46, 129.85, 128.31, 123.79, 122.46, 122.27, 119.26, 119.18, 118.56, 118.04, 114.28, 55.40, 53.84, 34.46. HRMS–FAB (m/z) [M+H]⁺ calcd for C₂₇H₂₃N₅O₄, 482.1823; found, 482.1803.

Abbreviations

CuAAC

copper-catalyzed azide–alkyne cycloaddition

MIC

minimum inhibition concentration to kill 90% of the bacterium

GI₅₀

50% growth inhibition

TGI

total growth inhibition

LC₅₀

50% lethal concentration, IC₅₀, half maximal inhibitory concentration

TB

Mycobacterium tuberculosis

GAST

medium of glycerol-alanine-salts-Tween 80 without added iron

DBU

1,8-Diazabicyclo[5.4.0]undec-7-ene

S.E.M.

standard error of the mean