Synthesis, Self-Assembly, and Biological Activities of Pyrimidine-Based Cationic Amphiphiles

Abstract

Pyrimidine-based cationic amphiphiles (PCAms), i.e., di-trifluoroacetic acid salts of N1-[1′-(1″,3″-diglycinatoxy-propane-2″-yl)-1′,2′,3′-triazole-4′-yl]methyl-N3-alkylpyrimidines have been synthesized utilizing naturally occurring biocompatible precursors, like glycerol, glycine, and uracil/ thymine in good yields. Synthesized PCAms consist of a hydrophilic head group comprising TFA salt of glyceryl 1,3-diglycinate and hydrophobic tail comprising of C-7 and C-12 N3-alkylated uracil or thymine conjugated via a 4-methylene-1,2,3-triazolyl linker. The physicochemical properties of all PCAms, such as critical aggregation concentration, hydrodynamic diameter, shape, and zeta potential (surface charge) were analyzed. These PCAms were also evaluated for their anti-proliferative and anti-tubercular activities. One of the synthesized PCAm exhibited 4- to 75-fold more activity than first-line anti-tubercular drugs streptomycin and isoniazid, respectively, against the multidrug resistant clinical isolate 591 of Mycobacterium tuberculosis.

Introduction

Nanotechnology has found solution for several drawbacks of traditional chemotherapeutics, such as low therapeutic efficacy often caused by poor drug bioavailability and high systemic toxicity.¹⁻³ These issues arise due to instability, insolubility, and poor specificity of hydrophobic drugs.⁴ Nano-aggregates of cationic amphiphilic (CAms) molecules consisting of a core–shell architecture of different forms, like micelles, vesicles, liposomes, and so on, have biomedical applications in drug, gene, and dye delivery.^5,6 Many cationic amphiphiles showing significant applications as drug delivery agents, and they are anti-cancer or anti-microbial agents.^4,7,8 Recently, amino acids have been introduced in cationic amphiphiles as a hydrophilic head through ester linkage because cleavage of such a bond in a biological system through enzyme hydrolysis produces non-toxic substances.^7,9 Cationic amphiphiles bearing glycine as a cationic moiety attached via ester bonds have been developed and found to be potent carriers for various bioactives.¹⁰ Few reports of cationic amphiphiles have been cited constituting naturally occurring nitrogenous bases, especially pyrimidines, which exhibit remarkable self-assembling properties due to their capability of hydrogen bonding and different biological activities.¹¹⁻¹³

Further, cancer cells are loosely packed and carry a net negative charge on their surface due to the pronounced effect of anionic moieties present over it, viz. O-glycosidic linkages,¹⁴ phosphatidylserin,¹⁵ heparin sulphate,¹⁶ and so on. Because of this, cancer cells get attracted by the cationic amphiphiles, while the normal eukaryotic cells having neutral zwitterionic phospholipids and sterols are left unaffected.^1,17,18 Cationic amphiphiles (CAms) have a significant role in fighting drug-resistant bacteria. They are designed to target and alter the structure of the bacterial cell membrane, leading to its rupture and cell death. The hydrophobic chain helps in invasion of the bacterial cell membrane.^8,9c

Herein, we report the synthesis of pyrimidine-based cationic amphiphiles (PCAms) consisting of a hydrophilic head group comprising glyceryl 1,3-diglycinate and a hydrophobic tail comprising a C-7 and C-12 N-alkylated uracil/thymine nucleobase. The C-7 and the C-12 N-alkyl aliphatic chains were incorporated in the synthesized PCAms in order to keep the required balance between hydrophobicity and hydrophilicity.¹⁰ The hydrophilic head group glycerol and the hydrophobic N-alkylated tail of PCAms were conjugated via a 4-methylene-1,2,3-triazolyl linker (Figure 1). In the course of synthesis of cationic amphiphiles, we have used the Cu-catalyzed alkyne azide cycloaddition (CuAAC) reaction, i.e., click reaction to obtain good yields of triazole-linked amphiphiles under greener solvent conditions.¹⁹ The synthesized amphiphiles were fully characterized, and their different physicochemical properties were determined. Further, synthesized cationic amphiphiles were evaluated for their anti-proliferative and anti-tubercular activities against HeLa and multidrug resistant KB-V1 cell lines and against the sensitive reference strain H37Rv and multidrug resistant (MDR) clinical isolate 591 of Mycobacterium tuberculosis.

Figure 1.

Formation of PCAm nano-aggregates via self-assembly in an aqueous medium and their anti-proliferative and anti-tubercular activities.

Results and Discussion

Synthesis of Pyrimidine-Based Cationic Amphiphiles (PCAms)

Two uracil-based cationic amphiphiles 8a and 9a and two thymine-based cationic amphiphiles 8b and 9b have been synthesized in good yields using glycerol, glycine, uracil/thymine, and heptyl and dodecyl bromides (Schemes 1 and 2). The crucial intermediates in the synthesis of cationic amphiphiles 8a-b and 9a-b are 1-[1′-(1″,3″-diacetyloxy-propane-2″-yl)-1′,2′,3′-triazole-4′-yl]methyl-uracil 5a and -thymine 5b. These uracil and thymine derivatives were synthesized by Huisgen cycloaddition (click reaction) between 1,3-diacetoxy-2-azidopropane (2) and 1-propargyluracil (4a) or 1-propargylthymine (4b) in the presence of copper sulfate and sodium ascorbate in THF/t-butanol/water (1:1:1) in 97 and 95% yields, respectively (Scheme 1). In turn, azido compound 2 was synthesized by Novozyme 435-catalyzed selective acetylation of two primary hydroxyl groups of glycerol (1) followed by its mesylation and subsequent S_N²-substitution of mesyl with an azido group in an overall yield of 94% following the literature procedure.²⁰ On the other hand N1-propargylated uracil (4a) and thymine (4b) were synthesized by propargylation of uracil (3a) and thymine (3b) with propargyl bromide in the presence of potassium carbonate in DMF in 75% yield in both cases (Scheme 1).²¹

Scheme 1. Synthesis of 1-[1′-(1″,3″-Diacetyloxy-propane-2″-yl)-1′,2′,3′-triazole-4′-yl]methyl-pyrimidines 5a and 5b.

Scheme 2. Synthesis of Pyrimidine-Based Cationic Amphiphiles 8a-b and 9a-b.

The pyrimidine-based cationic amphiphiles 8a-b and 9a-b were obtained from the above synthesized propyltriazolylmethyl-uracil 5a and -thymine 5b in three steps in an overall yield of 89 and 84% and 86 and 81%, respectively. In the first step, the acetyl protection of the glycerol moiety of 5a and 5b was removed in sodium methoxide-methanol to afford the dihydroxy compounds 6a and 6b in quantitative yields, which on esterification with N-Boc-glycine in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) and N,N-dimethylaminopyridine (DMAP) afforded corresponding Boc-protected glyceryl diglycinate 7a and 7b in 95 and 92% yields, respectively. Subsequently, di-Boc-glycinate ester 7a and 7b were alkylated using heptyl bromide and dodecyl bromide to obtain N3-alkylated-uracil and -thymine, thereafter the crude alkylated products as such were subjected to a de-Boc reaction by treatment with trifluoroacetic acid (TFA) in dichloromethane (DCM) to afford the corresponding di-trifluoroacetic acid salts of N1-[1′-(1″,3″-diglycinatoxy-propane-2″-yl)-1′,2′,3′-triazole-4′-yl]methyl-N3-heptylpyrimidines 8a and 8b in 95 and 94% yields and N1-[1′-(1″,3″-diglycinatoxy-propane-2″-yl)-1′,2′,3′-triazole-4′-yl]methyl-N3-dodecylpyrimidines 9a and 9b in 92 and 90% yields, respectively (Scheme 2).

The structures of all the synthesized compounds 5a-b, 6a-b, 7a-b, 8a-b, and 9a-b were unambiguously established on the basis of their spectral (IR, ¹H NMR, ¹³C NMR, and HRMS) data analysis. The ¹H and the ¹³C NMR spectra of these compounds are given in the Supporting Information. The structure of the known compounds 2 and 4a-b were further confirmed on comparison of their physical and spectral data with those reported in the literature.^20,21

Physico-Chemical Characterization of Amphiphiles 8a-8b and 9a-9b

The physico-chemical properties of N3-heptylpyrimidine amphiphiles 8a-b and N3-dodecylpyrimidine amphiphiles 9a-b, such as aggregation behavior, hydrodynamic diameter (size), surface charge (zeta potential), and shape of the aggregates were studied using UV–visible spectroscopy, dynamic light scattering (DLS), and cryo-TEM measurements.

Critical Aggregation Concentration of Amphiphiles

The aggregation behavior of amphiphiles 8a-b and 9a-b was studied by means of UV–visible spectroscopy employing a fluorescent hydrophobic dye pyrene as a probe in phosphate buffered saline solution (PBS) (pH 7.4, 10 mM, 9.6 mM NaCl).²² The amphiphilic molecules self-assemble to form thermodynamically stable nano-aggregates above a particular concentration in aqueous systems known as the critical aggregation concentration (CAC).²³ In order to avoid undesirable ionic interactions with the synthesized cationic amphiphiles, pyrene, which is a neutral probe, was chosen for the determination of CACs. Pyrene exhibits three peaks, i.e., I₁ (309 nm), I₂ (323 nm), and I₃ (339 nm) in the UV–visible spectrum at their corresponding λ_max values.²⁴ The intersection point where the steep rise in the I₁/I₃ ratio occurs is taken as the point of CAC of the given amphiphile. In an aqueous medium, pyrene exists predominantly at concentrations below the CAC of the given amphiphile and exhibits low absorbance (I₁/I₃ value). As soon as nano-aggregates are formed, it results in preferential encapsulation of the pyrene dye into the hydrophobic interior of the aggregates with a concomitant increase in the I₁/I₃ absorbance ratio (Figure 2). As the concentration of the amphiphiles increases, absorbance was found to be weak at the lowest concentrations, and then it rose sharply. This abrupt increase in the absorbance occurs at or above the CAC of the amphiphile.

Figure 2.

Determination of critical aggregation concentration (CAC) of amphiphiles 8a, 8b, 9a, and 9b in aqueous PBS (pH 7.4).

The CAC was determined by the intersection of the straight line through the absorbance at low amphiphilic concentrations with a straight line through the values in the region of sharp increase in absorbance (Figure 2). The observed CAC values for four cationic amphiphiles are shown in Table 1, which revealed that they aggregated at a micromolar level ranging from 18 to 32 μM. It has been revealed that the amphiphiles having a C-7 alkyl chain, i.e., 8a and 8b, have identical CAC values (18 μM), comparatively lower than the CAC values of amphiphiles having a C-12 alkyl chain, i.e., 9a and 9b (Table 1). Increasing hydrophobicity on amphiphiles by means of increasing the number of carbon atoms in the alkyl chain probably requires more molecules for aggregation due to more hydrophobic interactions and thus leads to the formation of larger aggregates in the aqueous medium.²⁴

Table 1. CAC, Size, PDI, and Zeta Potential of the Synthesized Amphiphiles 8a, 8b, 9a and 9b.

amphiphiles	CACa (μM)	size,a,bdH (nm)	PDI	zeta potential (mV)
8a	18	5.0	0.962	+14.6
8b	18	4.4	0.945	+26.7
9a	28	87.0	0.147	+44.3
9b	32	106.3	1.000	+52.4

^aIn phosphate buffered saline (pH 7.4) at 37 °C.

^bSize-distribution measurement by DLS (by volume).

Hydrodynamic Diameter (Size)

We determined the hydrodynamic diameter (size) of aggregates and their relative size distribution (expressed as polydispersity index, PDI) with dynamic light scattering (DLS) measurements in phosphate buffered saline solution (pH 7.4). As shown in Table 1, cationic amphiphiles 8a and 8b form smaller aggregates in the range of 4.4–5.0 nm, whereas amphiphiles 9a and 9b form larger aggregates in the range of 87.0–106.3 nm (Table 1 and Figure 3).

Figure 3.

Graphs of DLS measurements of PCAms 8a, 8b, 9a, and 9b.

In order to supplement the above described aggregation phenomena in 8a-b and 9a-b by a direct structural analysis and to elucidate the self-assembly behavior, cryo-TEM measurements were performed on aqueous sample solutions at a general concentration of 4.5 mM, which is well above the amphiphiles’ corresponding CAC values. Compound 8a showed a tendency to form small spherical particles in the range of 3–5 nm, whereas compound 9b revealed that it formed comparatively larger vesicular structures in the diameter range of 100–200 nm (Supporting Information, Figures S21 and S22). These results were found to be in corroboration to that obtained with dynamic light scattering experiments.

Furthermore, the above mentioned results regarding the size of the synthesized amphiphiles 8a-b and 9a-b are in concordance with respect to the presence of a larger hydrophobic group C-12 alkyl chain in compounds 9a and 9b, leading to the formation of larger aggregates as compared to the compounds comprising a smaller C-7 alkyl chain in 8a and 8b.

Surface Charge (Zeta Potential) Measurement

The surface charge of the formed nano-aggregates of amphiphiles 8a, 8b, 9a, and 9b was assessed by measuring their zeta potentials (Table 1). All amphiphiles showed positive charges in a range of +14 to +52 mV. The positive charges on amphiphiles 8a and 8b having a C-7 alkyl chain are lower, i.e., +14.6 and +26.7 mV, respectively, as compared to the zeta potentials of the amphiphiles having a C-12 carbon chain 9a and 9b, i.e., +44.3 and +52.4 mV, respectively. The larger-sized aggregates formed from 9a and 9b might result in high zeta potential values due to the involvement of more numbers of monomers.²⁵

Anti-Proliferative Activity of PCAms Nano-Aggregates 8a, 8b, 9a, and 9b

After physicochemical characterization, anti-proliferative activity of synthesized PCAms 8a, 8b, 9a, and 9b was evaluated by MTT assay on the cervical cancer cell line HeLa and its multidrug resistant variant KB-V1. It was expected that the synthesized cationic amphiphiles can interact via their charges with the cell surface of HeLa and KB-V1 cells as it is reported that cancer cells have a negative charge on their cell surface.¹⁵⁻¹⁷ The dose–response relationships shown in Figure 4 revealed that compounds 8a and 8b have an anti-proliferative activity with an IC₅₀ value of about 157 μM on HeLa cells and about 1.7 mM (8a) or ∼300 μM (8b) on KB-V1 cells. Compounds 9a and 9b have the ability to reduce cell viability with an IC₅₀ value of about 4–6 μM on HeLa and about 13–18 μM on the multidrug resistant variant KB-V1 cells. The clear difference between compounds having a C-7 carbon chain 8a-b versus C-12 carbon chain substitutions 9a-b strongly indicates that the length of the carbon chain mediates the increase in anti-proliferative activity. This might be caused by the direct interaction of the longer carbon chain with the cell membrane or simply due to the aggregate size. Further, activity in multidrug resistant cells was shown by comparison of HeLa to KB-V1 cells, where IC₅₀ values are in the same range of concentrations for compounds 9a, 9b, and 8b, while some resistance is seen with 8a having an IC₅₀ value shifted by about one order of magnitude.

Figure 4.

Anti-proliferative activity of PCAms 8a-8b and 9a-9b on the cancer cell line HeLa and its multidrug resistant variant KB-V1 as determined by MTT assay.

Anti-Tuberculosis Activity

In the present study, the synthesized PCAms 8a, 8b, 9a, and 9b were screened for their anti-M. tuberculosis activity against the sensitive reference strain H37Rv and multidrug resistant (MDR) clinical isolate 591 using the broth dilution method, and the results in comparison with first-line anti-tubercular drugs isoniazid, ethambutol, rifampicin, and streptomycin are summarized in Table 2. These compounds were found to be effective at inhibition of the growth of the M. tuberculosis. Amphiphilic compounds 8a, 8b, 9a, and 9b were found to be effective against both the sensitive reference strain H37Rv and multidrug resistant clinical isolate 591 of M. tuberculosis. As shown in Table 2, minimum inhibitory concentration (MIC) values on the sensitive reference strain H37Rv obtained for compound 8a and 8b were found to be between 6 and 8 μg/mL, respectively, whereas compounds 9a and 9b showed an MIC value 10 μg/mL. The MIC value 6 μg/mL of the most active compound 8a against the sensitive reference strain H37Rv of M. tuberculosis was threefold less than the first-line drug streptomycin, whereas compounds 8b and 9a-b were four and five times less active, respectively. These compounds have shown fairly good activity against the drug-susceptible reference strain H37Rv of Mycobacterium, although they are less active than first-line drugs isoniazid, ethambutol, rifampicin, and streptomycin.

Table 2. MIC Results of Anti-M. tuberculosis Activity of PCAms 8a-b and 9a-b against the Sensitive Reference Strain H37Rv and Multidrug Resistant Clinical Isolate 591.

S. no.	test compounds	MIC against M. tuberculosis H37Rv (μg/mL)	MIC against MDR clinical isolate 591 (μg/mL)
1	8a	6	4
2	8b	8	8
3	9a	10	10
4	9b	10	10
first-line drugs of TB	isoniazid	0.03	>300
	rifampicin	0.015	>125
	ethambutol	0.25	40
	streptomycin	2	15

The anti-M. tuberculosis activity of PCAms 8a and 8b against the multidrug resistant clinical isolate 591 was found to be very high in comparison to the first-line anti-tubercular drugs isoniazid, ethambutol, rifampicin, and streptomycin (Table 2). Thus, PCAms 8a and 8b exhibited MIC values of 4 and 8 μg/mL, respectively, whereas PCAms 9a and 9b exhibited an MIC value of 10 μg/mL against the multidrug resistant clinical isolate 591 of M. tuberculosis. Although, anti-tubercular activities of all four PCAms against the MDR clinical isolate were much higher compared to the first-line drugs, PCAm 8a exhibited 4–75 times better activity than streptomycin and isoniazid, respectively.

Conclusions

We have successfully synthesized uracil-/thymine-based cationic amphiphiles utilizing naturally occurring, cheap, commercially available, and biocompatible starting materials in overall good yields. The physicochemical characterization of nano-aggregates of cationic amphiphiles in aqueous buffer revealed that aggregates derived from amphiphiles having higher hydrophobicity due to the C-12 alkyl chain, i.e., aggregates of compounds 9a and 9b have a bigger size, higher positive zeta potential, and higher CAC, compared to the less hydrophobic PCAms 8a and 8b containing C-7 alkyl chains. These features of cationic amphiphiles have influence on their anti-proliferative activity on HeLa and its multidrug resistant variant KB-V1 cancer cell lines; compounds 8a-b have shown better viability profiles as compared to 9a-b owing to their smaller alkyl chain length/ smaller nano-aggregates. Furthermore, anti-tubercular activity evaluation of synthesized cationic amphiphiles against the M. tuberculosis sensitive reference strain H37Rv and MDR clinical isolate 591 revealed that they are moderately active against the reference strain, but they are highly active against the MDR clinical isolate 591. The most active compound 8a against the MDR clinical isolate 591 exhibited 4- and 75-fold more activity with respect to first-line drugs streptomycin and isoniazid, respectively.

Experimental Section

General Methods

Melting points were determined on a Buchi M-560 instrument. The IR spectra were recorded on a Perkin-Elmer model 2000 FT-IR spectrometer by making a KBr disc for solid samples and a thin film for oils. The ¹H and ¹³C NMR spectra were recorded at a Jeol alpha-400 spectrometer at 400 and 100.6 MHz, respectively, using TMS as the internal standard. The chemical shift values are on a δ scale, and the coupling constants (J) are in Hz. The mass spectrum recordings have been done on a micro-TOF-Q instrument from Bruker Daltonics, Bremen and a 6520 Q-TOF instrument from Agilent Technologies on ESI positive mode. Millipore water was used for encapsulation and physicochemical characterization experiments. Analytical TLCs were performed on pre-coated Merck silica-gel 60F₂₅₄ plates; the spots were detected either under UV light or in an iodine chamber. Novozyme-435 was purchased from Sigma-Aldrich Chemicals Pvt. Limited, India. Silica gel (100–200 mesh) has been used for column chromatography. The critical aggregation concentrations were determined through UV–vis spectroscopy using a Shimadzu UV-1601 UV–vis spectrophotometer cell (1 cm). The size and the zeta potential measurements were conducted using a Zetasizer Nano ZS analyzer (Malvern Instruments Ltd., U.K). Cryo-TEM images obtained by a Tecnai F20 FEG transmission electron microscope (FEI Company, Oregon, USA) using the Gatan (Gatan Inc., California, USA) cryoholder and cryostage (model 626).

General Procedure for the Synthesis of 1-[1′-(1″,3″-Diacetyloxy-propane-2″-yl)-1′,2′,3′-triazole-4′-yl]methyl-uracil (5a) and -thymine (5b)

In a mixture of 1,3-diacetoxy-2-azidopropane (2, 0.67 mmol) and N1-propargyl-uracil/-thymine (4a/4b, 0.67 mmol) in THF/t-butanol/water (10 mL, 1:1:1) was added CuSO₄·5H₂O (0.27 mmol) and sodium ascorbate (0.54 mmol), and the reaction mixture was stirred at 30 °C for 12 h. Completion of the reaction was monitored by TLC examination (solvent 4% methanol–chloroform). After completion, the reaction mixture was dried over high vacuum, and the crude product thus obtained was purified via silica gel column chromatography using 2% methanol–chloroform as the eluent to afford pure targeted compound 5a and 5b.

1-[1′-(1″,3″-Diacetyloxy-propane-2″-yl)-1′,2′,3′-triazole-4′-yl]methyl-uracil (5a)

It was obtained as a white solid (1.13 g) in 97% yield, M. Pt. =132–134 °C. Rf = 0.40 (4% MeOH–CHCl₃); IR (KBr, cm^–1): 3147, 1745, 1681, 1631, 1213, 1049, and 777; ¹H NMR (400 MHz, CDCl₃): δ 9.62 (s, 1H,), 7.88 (s, 1H), 7.54 (d, 1H, J = 8 Hz), 5.73 (d, 1H, J = 8 Hz), 5.07 ( p, 1H, J = 4 Hz), 5.00 (s, 2H), 4.52 (d, 4H, J = 8 Hz), and 2.05 (s, 6H); ¹³C NMR (100.6 MHz, CDCl₃): δ 170.1, 163.5, 150.9, 144.3, 141.8, 123.6, 102.7, 62.3, 58.7, 43.1, and 20.5; HRMS (ESI) m/z: calcd. for C₁₄H₁₈N₅O₆⁺ [M + H]⁺ 352.1252, found 352.1254.

1-[1′-(1″,3″-Diacetyloxy-propane-2″-yl)-1′,2′,3′-triazole-4′-yl]methyl-thymine (5b)

It was obtained as a white solid (1.11 g) in 95% yield, M. Pt. = 153–155 °C. Rf = 0.45 (4% MeOH–CHCl₃); IR (KBr cm^–1): 3026, 1741, 1680, 1462, 1219, and 1039; ¹H NMR (400 MHz, CDCl₃): δ 9.34 (brs, 1H), 7.82 (s, 1H), 7.30 (s, 1H), 5.00 (p, 1H, J = 8 Hz), 4.92(s, 2H), 4.46 (d, 4H, J = 4 Hz), 1.96 (s, 6H), and 1.85 (s, 3H); ¹³C NMR (100.6 MHz, CDCl₃): δ 170.1, 164.2, 151.0, 142.1, 140.1, 123.6, 111.3, 62.3, 58.7, 42.9, 20.5, and 12.2; HRMS (ESI) m/z: calcd. for C₁₅H₂₀N₅O₆⁺ [M + H]⁺ 366.1408, found 366.1411.

General Procedure for the Synthesis of 1-[1′-(1″,3″-Dihydroxy-propane-2″-yl)-1′,2′,3′-triazole-4′-yl]methyl-uracil (6a) and -thymine (6b)

To a stirred solution of compound 5a/5b (0.28 mmol) in 10 mL of methanol was added sodium methoxide (0.57 mmol), and the reaction was stirred for 1 h at 30 °C. After completion as indicated by TLC (10% methanol–chloroform) examination, Seralite resin (H⁺ resin) was added into the reaction mixture to neutralize sodium methoxide, and stirring continued for another 5 min. The reaction mixture was filtered and dried over high vacuum. The crude product thus obtained was then purified via silica gel column chromatography using 5% methanol–chloroform as the eluent to obtain pure targeted compound 6a-b.

1-[1′-(1″,3″-Dihydroxy-propane-2″-yl)-1′,2′,3′-triazole-4′-yl]methyl-uracil (6a)

The compound was obtained as a white solid (760 mg) in 99% yield, M. Pt. = 165–167 °C. Rf = 0.50 (5% MeOH–CHCl₃); IR (KBr cm^–1): 3448, 2773, 1672, 1598, 1355, and 1035; ¹H NMR (400 MHz, CD₃OD): δ 8.43 (s, 1H), 7.91 (s, 1H), 7.60(d, 1H, J = 8 Hz), 5.57 (d, 1H, J = 8 Hz), 4.92 (s, 2H), 4.60 (p, 1H, J = 8 Hz), and 3.83–3.88 (m, 4H); ¹³C NMR (100.6 MHz, CD₃OD): δ 169.9, 152.7, 146.9, 143.2, 125.1, 102.6, 66.8, 62.2, and 43.9; HRMS (ESI) m/z: calcd. for C₁₀H₁₄N₅O₄⁺ [M + H]⁺ 268.1040, found 268.1036.

1-[1′-(1″,3″-Dihydroxy-propane-2″-yl)-1′,2′,3′-triazole-4′-yl]methyl-thymine (6b)

The compound was obtained as a white solid (773 mg) in 99% yield, M. Pt. = 181–183 °C. Rf = 0.55 (5% MeOH–CHCl₃); IR (KBr cm^–1): 3452, 3128, 1662, 1460, 1325, 1087, and 779; ¹H NMR (400 MHz, CD₃OD): δ 8.28 (s, 1H), 7.95 (s, 1H), 7.45 (s, 1H), 4.89 (s, 2H), 4.59 (p, 1H, J = 8 Hz), 3.84–3.86 (m, 4H), and 1.76 (s, 3H); ¹³C NMR (100.6 MHz, CD₃OD): δ 166.8, 152.7, 143.4, 142.6, 125.0, 111.5, 66.8, 62.1, 43.7, and 12.2; HRMS (ESI) m/z: calcd. for C₁₁H₁₆N₅O₄⁺ [M + H]⁺ 282.1197, found 282.1198.

General Procedure for the Synthesis of 1-[1′-(1″,3″-Di-Boc-glycinatoxy-propane-2″-yl)-1′,2′,3′-triazole-4′-yl]methyl-uracil (7a) and -thymine (7b)

To a stirred solution of compound 6a/6b (0.561 mmol) and Boc-glycine (1.68 mmol) in DMF at 0 °C was added 4-dimethyl amino pyridine (0.67 mmol). The reaction mixture was vigorously stirred for 10 min, and then 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (1.68 mmol) was added at 0 °C. The reaction mixture was kept on further stirring for 12 h in an inert atmosphere at 30 °C. On completion of the reaction as observed by TLC (10% methanol–chloroform) examination, the compound was extracted from the reaction mixture with dichloromethane (3 × 50 mL). The combined organic layer thus obtained was dried over anhydrous sodium sulfate and evaporated under high vacuum. The pure targeted compounds 7a and 7b were obtained after purification of the crude product on silica gel column chromatography using 5% methanol–chloroform as the eluting system.

1-[1′-(1″,3″-Di-Boc-glycinatoxy-propane-2″-yl)-1′,2′,3′-triazole-4′-yl]methyl-uracil (7a)

The compound was obtained as a white semi-solid (1.57 g) in 95% yield. Rf = 0.60 (10% MeOH–CHCl₃); IR (thin film, cm^–1): 2927, 1695, 1664, 1633, 1031, and 750; ¹H NMR (400 MHz, CDCl₃): δ 9.17 (s, 1H), 7.85 (s, 1H), 7.44 (d, 1H, J = 8 Hz), 5.66 (d, 1H, J = 8 Hz), 5.04, (p, 1H, J = 8 Hz), 4.90 (s, 2H), 4.53 (d, 4H, J = 8 Hz), 3.81–3.83 (m, 4H), 1.67 (s, 2H), and 1.37 (s, 18H); ¹³C NMR (100.6 MHz, CDCl₃): δ 170.0, 163.4, 155.9, 144.5, 141.7, 124.3, 103.2, 80.5, 62.9, 58.7, 42.3, and 28.4; HRMS (ESI) m/z: calcd. for C₂₄H₃₆N₇O₁₀⁺ [M + H]⁺ 582.2518, found 582.2512.

1-[1′-(1″,3″-Di-Boc-glycinatoxy-propane-2″-yl)-1′,2′,3′-triazole-4′-yl]methyl-thymine (7b)

The compound was obtained as a white semi-solid (1.49 g) in 92% yield. Rf = 0.55 (10% MeOH–CHCl₃); IR (thin film, cm^–1): 2963, 1686, 1661, 1320, 1029, and 771; ¹H NMR (400 MHz, CDCl₃): δ 8.85 (s, 1H), 7.81 (s, 1H), 7.20 (s, 1H), 5.04 (p, 1H, J = 8 Hz), 4.86 (s, 2H), 4.52 (d, 4H, J = 5.2 Hz), 3.82 (d, 4H, J = 5.2 Hz), 1.84 (s, 3H), 1.67 (brs, 2H), and 1.38 (s, 18H); ¹³C NMR (100.6 MHz, CDCl₃): δ 169.8, 164.3, 155.8, 142.0, 140.3, 124.0, 111.3, 80.3, 62.7, 58.3, 42.1, 29.6, 28.2, and 12.2; HRMS (ESI) m/z: calcd. for C₂₅H₃₈N₇O₁₀⁺ [M + H]⁺ 596.2675, found 596.2700.

General Procedure for the Synthesis of Di-trifluoroacetic Acid Salts of N1-[1′-(1″,3″-Di-glycinatoxy-propane-2″-yl]-1′,2′,3′-triazole-4′-yl)methyl-N3-heptylpyrimidines 8a and 8b

A mixture of compound 7a/7b (0.17 mmol), potassium carbonate (0.17 mmol), and 1-bromohexane (0.21 mmol) was taken in DMF (10 mL) in a round-bottom flask, and the reaction mixture was stirred for 12 h at 28–30 °C. The progress of the reaction was monitored through TLC. After complete consumption of starting compound 7a/7b, reaction mixture was filtered and dried over reduced pressure, water (30 mL) was added, and then the compound was extracted using DCM (2 × 25 mL). The combined organic layer was dried over anhydrous Na₂SO₄ and evaporated under reduced pressure. The crude product thus obtained was washed with n-hexane (3 × 10 mL) to remove excess 1-bromohexane and dried over a rotavap to obtain almost pure alkylated product. The alkylated product thus obtained was dissolved in DCM (10 mL) followed by addition of trifluoroacetic acid (7.06 mmol), and then the reaction mixture was stirred at ambient temperature for 1 h. After completion of the reaction as indicated by TLC examination, it was dried over high vacuum and washed with n-hexane (3 × 10 mL) and diethyl ether (3 × 10 mL) to obtain pure targeted compounds 8a and 8b in pure form.

Di-trifluoroacetic Acid Salts of N1-[1′-(1″,3″-Di-glycinatoxy-propane-2″-yl]-1′,2′,3′-triazole-4′-yl)methyl-N3-heptyluracil (8a)

The pure compound was obtained as a semi-solid (1.50 g) in 95% yield. Rf = 0.30 (10% MeOH–CHCl₃); IR (thin film, cm^–1): 3394, 2927, 2360, 1701, 1656, 1460, 1201, 1134, and 721; ¹H NMR (400 MHz, CD₃OD): δ 8.04 (s, 1H), 7.60 (d, 1H, J = 8 Hz), 5.64 (d, 1H, J = 8 Hz), 4.96 (s, 2H), 4.60–4.62 (m, 1H), 3.85–3.86 (m, 4H), 3.74–3.81 (m, 4H), 3.60 (s, 2H), 1.48 (s, 2H), 1.20–1.29 (m, 10H), and 0.78–0.83 (m, 3H); ¹³C NMR (100.6 MHz, CD₃OD): δ 169.0, 168.1, 165.3, 152.5, 152.5, 144.9, 125.3, 102.1, 66.9, 65.4, 62.1, 45.0, 42.2, 40.6, 32.8, 30.0, 28.4, 27.9, 23.6, and 14.4; HRMS (ESI) m/z: calcd. for C₂₁H₃₄N₇O₆⁺ [M + H]⁺ 480.2565, found 480.2558.

Di-trifluoroacetic Acid Salts of N1-[1′-(1″,3″-Di-glycinatoxy-propane-2″-yl]-1′,2′,3′-triazole-4′-yl)methyl-N3-heptylthymine (8b)

The pure compound was obtained as a semi-solid (1.39 g) in 94% yield. Rf = 0.35 (10% MeOH–CHCl₃); IR (thin film, cm^–1): 3389, 2924, 2360, 1755, 1668, 1631, 1467, 1201, 1132, and 723; ¹H NMR (400 MHz, CD₃OD): δ 8.17 (s, 1H), 7.56 (s, 1H), 5.28 (p, 1H, J = 8 Hz), 5.01 (s, 2H), 4.76 (d, 4H, J = 8 Hz), 3.89 (t, 2H, J = 8 Hz), 3.82 (d, 4H, J = 6 Hz), 1.89 (s, 3H), 1.56–1.59 (m, 2H), 1.25–1.31 (m, 8H), and 0.88 (t, 3H, J = 8 Hz); ¹³C NMR (100.6 MHz, CD₃OD): δ 169.0, 165.4, 140.6, 125.0, 110.8, 65.4, 63.1, 61.8, 61.6, 45.1, 42.4, 40.8, 32.8, 30.3, 28.4, 27.9, 23.5, 14.3, and 12.9; HRMS (ESI) m/z: calcd. for C₂₂H₃₆N₇O₆⁺ [M + H]⁺494.2722, found 494.2724.

General Procedure for the Synthesis of Di-trifluoroacetic Acid Salts of N1-[1′-(1″,3″-Di-glycinatoxy-propane-2″-yl]-1′,2′,3′-triazole-4′-yl)methyl-N3-dodecylpyrimidines 9a and 9b

A mixture of compound 7a/7b (0.20 mmol), potassium carbonate (0.20 mmol), and 1-bromododecane (0.22 mmol) was dissolved in DMF (10 mL) in a round-bottom flask, and the reaction mixture was stirred for 12 h at 28–30 °C. The progress of the reaction was monitored through TLC. After complete consumption of starting compound 7a/7b, the reaction mixture was filtered and dried over reduced pressure, water (30 mL) was added, and then the compound was extracted using DCM (2 × 25 mL). The combined organic layer was dried over anhydrous Na₂SO₄ and evaporated under reduced pressure. The crude product thus obtained was washed with n-hexane (3 × 10 mL) to remove excess 1-bromododecene and dried over a rotavap to obtain an almost pure alkylated product. The alkylated product thus obtained was dissolved in DCM (10 mL) followed by addition of trifluoroacetic acid (7.84 mmol), and then the reaction mixture was stirred at ambient temperature for 1 h. After completion of the reaction as indicated by TLC examination, it was dried over high vacuum and washed with n-hexane (3 × 10 mL) and diethyl ether (3 × 10 mL) to obtain pure targeted compounds 9a and 9b in pure form.

Di-trifluoroacetic Acid Salts of N1-[1′-(1″,3″-Di-glycinatoxy-propane-2″-yl]-1′,2′,3′-triazole-4′-yl)methyl-N3-dodecyluracil (9a)

The pure compound was obtained as a semi-solid (1.60 g) in 92% yield. Rf = 0.40 (10% MeOH–CHCl₃); IR (thin film, cm^–1): 3350, 2924, 2360, 1658, 1460, 1199, 1134, and 758; ¹H NMR (400 MHz, CD₃OD): δ 8.08 (1H, s), 7.60 (d, 1H, J = 8 Hz), 5.64 (d, 1H, J = 8 Hz), 4.96 (s, 2H), 4.56–4.62 (m, 1H), 3.86–3.91 (m, 4H), 3.78–3.81 (m, 4H), 3.53 (s, 2H), 1.49 (s, 2H), 1.18–1.12 (m, 18H), and 0.80 (t, 3H, J = 8 Hz); ¹³C NMR (100.6 MHz, CD₃OD): δ 168.1, 166.3, 165.3, 150.6, 145.1, 125.1, 102.3, 65.3, 64.8, 61.8, 60.2, 45.3, 42.3, 40.8, 33.0, 30.7, 30.4, 30.3, 30.1, 28.4, 27.9, 27.3, 23.6, and 14.4; HRMS (ESI) m/z: calcd. for C₂₆H₄₄N₇O₆⁺ [M + H]⁺ 550.3348, found 550.3394.

Di-trifluoroacetic Acid Salts of N1-[1′-(1″,3″-Di-glycinatoxy-propane-2″-yl]-1′,2′,3′-triazole-4′-yl)methyl-N3-dodecylthymine (9b)

The compound was obtained as a semi-solid (1.55 g) in 90% yield. Rf = 0.45 (10% MeOH–CHCl₃); IR (thin film, cm^–1): 3362, 2924, 2360, 1666, 1633, 1465, 1201, 1136, 1055, and 723. ¹H NMR (400 MHz, CD₃OD): δ 8.44 (s, 1H), 7.48 (s, 1H), 4.89–4.97 (m, 1H), 4.69–4.63 (m, 2H), 3.80–3.89 (m, 10H), 1.81 (s, 3H), 1.42–1.55 (m, 2H), 1.18–1.22 (m, 18H), and 0.80 (t, 3H, J = 8 Hz); ¹³C NMR (100.6 MHz, CD₃OD): δ 168.2, 166.0, 162.0, 152.5, 143.5, 124.2, 113.5, 67.9, 66.2, 64.6, 45.1, 42.8, 41.1, 35.6, 33.2, 33.0, 31.2, 30.6, 26.1, 17.2, and 15.6; HRMS (ESI) m/z: calcd. for C₂₇H₄₆N₇O₆⁺ [M + H]⁺ 564.3504, found 564.3528.

Procedure for Determination of the Critical Aggregation Concentration (CAC) of Synthesized Pyrimidine-Based Cationic Amphiphiles (PCAms) 8a, 8b, 9a, and 9b

Absorption spectra were recorded at a range of 250–700 nm using a Shimadzu UV-1601 UV–vis spectrophotometer cell (1 cm). In the present study, pyrene dye was used as a hydrophobic probe for the determination of critical micelle concentrations (CACs) of pyrimidine-based cationic amphiphiles (PCAms) in buffered aqueous solution (10 mM phosphate buffer saline, pH 7.4). In order to determine the CACs, the absorbance I₁ (309 nm)/I₃ (339 nm) of the pyrene in UV spectra was plotted against the log of concentration PCAms. Independent linear regressions were performed on the observed data points above and below the evident CAC. Finally, the CACs were derived from the intersection points of the independent linear regressions. Prior to the measurements, a pyrene stock solution of 0.2 μM (in phosphate buffer saline) was freshly prepared by dissolving the probe in THF. Increasing concentrations of amphiphiles (1 μM to 100 μM) were then added to pyrene (0.2 μM) from a stock solution, and absorption spectra were recorded at different amphiphile concentrations. All samples were stirred thoroughly by using a laboratory vortex shaker to ensure proper mixing and dissolution of the compounds. The samples were then incubated for 12 h at room temperature. All measurements were carried out at 25 ± 2 °C and taken in triplicate and averaged. Data analysis was performed using Origin 6.0 professional software.

Dynamic Light Scattering (DLS) Studies and Zeta Potential Measurements of Synthesized PCAms 8a, 8b, 9a, and 9b

The size and the zeta potential measurements were conducted at 25 °C using a Zetasizer Nano ZS analyzer with integrated 4 mW He–Ne laser, λ = 633 nm (Malvern Instruments Ltd., U.K.). The PCAms were measured in phosphate buffered saline at a pH of 7.4.

Cryo-Transmission Electron Microscopy (Cryo-TEM)

For all sample preparations, aqueous amphiphile solutions were used at a concentration of 4.5 × 10^–3 M. Droplets of the corresponding sample solution (5 μL) were applied to perforated (1 μm hole diameter) carbon film-covered 200 mesh copper grids (R1/4batch of Quantifoil Micro Tools GmbH, Jena, Germany), which had been hydrophilized prior to use by 60 s plasma treatment at 8 W in a BALTEC MED 020 device. The supernatant fluid was removed with a filter paper until an ultra-thin layer spanning the holes of the carbon film was obtained. The samples were immediately vitrified by propelling the grids into liquid ethane at its freezing point (90 K) and by operating a guillotine-like plunging device. The vitrified samples were transferred under liquid nitrogen cooling into a Tecnai F20 FEG transmission electron microscope (FEI Company, Oregon, USA) using the Gatan (Gatan Inc., California, USA) cryoholder and cryostage (model 626). Microscopy was carried out at a 94 K sample temperature using the microscope’s low-dose protocol at a calibrated primary magnification of 50,000 and an accelerating voltage of 160 kV (FEG-illumination). Images were recorded using an EAGLE 4 k-CCD camera (FEI Company, Oregon, USA) operated with binning factor 2 (2048 by 2048 pixel). The defocus was chosen in all cases to be 2 μ. It is important to note that although the determined diameter measurements were prone to error due to the very small size of the assemblies, more reliable diameter values could be derived from sample areas where micelles were densely packed. Fourier transforms of corresponding images revealed a diffraction pattern, which indicates repetitive distances, which can be said to correlate with the diameter of the micelles.

Procedure for Evaluation of Anti-Proliferative Activity of PCAms 8a, 8b, 9a, and 9b

HeLa and KB-V1 cells were obtained from Leibnitz Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH. HeLa cells were routinely cultured in RPMI medium supplemented with 10% FBS (FBS Superior Merck), 1% penicillin/streptomycin, and 1% MEM non-essential amino acids at 37 °C and 5% CO₂. KB-V1 cells were cultured in DMEM supplemented with 15% FBS and 1% penicillin/streptomycin. Cells were seeded into 96-well plates at a density of 100,000 cells/mL in 0.1 mL/well and grown over night. The next day, the cell culture medium was replaced with fresh medium containing 10-fold serial dilutions of the test compounds. After 48 h of incubation at 37 °C and 5% CO₂, the medium was discarded and replaced with 100 μL/well fresh medium containing 10 μL of MTT ((3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide), 5 mg/mL stock solution in PBS, Merck). After 4 h of incubation at 37 °C, the supernatant was removed and formazan crystals were dissolved by adding 100 μL/well of isopropanol containing 0.04 M HCl. Plates were shaken for 5 min, and then absorbance was read in a Tecan Infinite M200 Pro microplate reader at 590 nm. Relative viabilities were calculated by dividing average absorbance values of duplicate wells containing treated cells by values of wells containing untreated cells (100%). All experiments were performed three times independently in duplicate. Errors are given as +/– S.E.M. in the graphic representation of the data.

Procedure for Anti-Tubercular Activity Evaluation of PCAms 8a, 8b, 9a, and 9b: Determination of the Minimum Inhibitory Concentrations (MICs)

The reference strain M. tuberculosis H37Rv was obtained from the Department of Microbiology, V.P. Chest Institute, University of Delhi, India. The culture was maintained on a Middlebrook 7H9 medium (Difco Laboratories, MI, USA). The MICs of the test compounds 8a-d and 9a-d and the first-line drugs isoniazid, rifampicin, ethambutol, and streptomycin taken as reference standards were determined using the micro-plate Alamar blue assay (MABA).²⁶ The required amount of 7H9 medium, the calculated amount of the drug, and 1 × 10⁶ cells from the single-cell suspension prepared from a log-phase culture of M. tuberculosis H37Rv were added to each well so as to make up the volume to 200 mL. The plates were then sealed with Parafilm and incubated aerobically at 37 °C. After 10–12 days of incubation, 30 mL of 0.02% resazurin solution was added to each well, and the plates were again incubated overnight at 37 °C before being observed for color change. A color change from blue to pink was considered as growth, and the MIC value was recorded as the lowest drug concentration that prevented visible growth/color change (Table 2). MICs and each concentration were checked in triplicate in a micro-plate, with the entire procedure being repeated a minimum of three times.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c03623.

• The syntheses and characterization data of all the new compounds (PDF)

Author Present Address

^# Present Address: INFIQC–CONICET-UNC, Departamento de Química Orgánica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, X5000HUA Córdoba, Argentina

The authors declare no competing financial interest.