Complexation of water-soluble phosphorylated calixarenes with uracils. Stability constants and DFT study of the supramolecular complexes

Modification of the upper rim of the lipophilic cone-shaped tetrapropoxycalix [4]arene with hydrophilic phosphine oxide groups or phosphinic acid groups yielded nano-sized water-soluble calixarenes that form supramolecular complexes with uracils, including active pharmaceutical ingredients of 5-Fluorouracil and 5-Methyluracil drugs. Stability constants of the formed complexes in an aqueous-organic medium were determined by the HPLC method. The most favored structures of the calixarenes and their uracil complexes were optimized at the DFT level of approximation. In the most favored structures of all adducts, the uracil molecules coordinate via hydrogen bonding with the phosphorus-containing groups on the upper rim of the calixarene ligands.

Stable fluorescent calixarene based nanoparticles with a diameter of 7 nm and a fluorescence maximum of 590 nm easily pass through biological membranes and selectively stain certain areas inside cells.These brightly colored biocompatible nanoparticles can serve as marking vectors for the delivery of biologically active substances [48].
Prospective nanovectors for drug delivery are calixarenes, functionalized on the upper or lower rim of the macrocycle by hydrophilic organophosphorus groups.Phosphorus is a biologically friendly element and a number of medicines have been created on the basis of natural and synthetic organophosphorus compounds [49].Phosphoruscontaining calixarenes are also characterized by various biological activities [50][51][52].
Calixarene diphosphoric acid (CDPA) has a significant biomedical potential.CDPA binds to amino acids and DNA in the presence of zinc cations [53,54].The acid, with low toxicity to erythrocytes and leukocytes, shows high activity in relation to ordinary and multi drug-resistant cancer cells [55].CDPA activates the transfer of polyarginine cellpenetrating peptides through biological membranes [56] and forms supramolecular complexes with water-insoluble active pharmaceutical ingredients of drugs [57,58].
Long alkyl groups on the lower rim and polar P(O)(OH) 2 groups on the upper rim of the macrocycle ensure the selfassembly of calixarene phosphonic acids in the crystalline state and in aqueous solutions due to hydrophobic interactions, intermolecular electrostatic interactions and hydrogen bonds P(O)-OH…O = P [67,68].Such acids form self-assembled nanocapsules, porous structures, nanofibers, micelles, vesicles, lipid nanoparticles suitable as vectors for drug delivery [69,70].
The authors [71] have demonstrated the ability of amphiphilic calixarene methylphosphonic acid to form three-component nanoparticles with the well-known antitumor drugs Carboplatin and Taxol.In this three-component complex, Carboplatin enters the molecular cavity of calixarene, and Taxol is placed among the alkyl substituents of the formed micellar structure.The authors investigated the antitumor activity of the created nanoparticles on HT-29 and Caco-2 colon cancer cells.On these cancer cells, the nanocomposite showed higher cytotoxicity than a simple mixture of two drugs.This cytotoxicity is associated with the induction of apoptosis, cell cycle arrest, and inhibition of HT-29 invasion and migration.
Amphiphilic calixarene hydroxymethylene-bis-phosphonic acids form self-assembled anionic micelles in aqueous solutions [72].In turn, the micelles form nanoscale supramolecular complexes with fluorescently labeled polylysine and HIV-1 nucleocapsid due to electrostatic interactions.Such nanocomplexes cross biological membranes and deliver therapeutically important proteins into biological cells.
In this paper, the nano-sized water-soluble cone-shaped tetrapropoxycalix [4]arenes modified on the upper rim with hydrophilic phosphine oxide groups (CPO) or phosphinic acid groups (CPA) were synthesized (Scheme 1) and their complexation with eleven uracils (2,4-dioxopyrimidines), including APIs of 5-Fluorouracil and 5-Methyluracil drugs (Fig. 1), were investigated by HPLC and DFT calculation methods in the context of drug delivery.

Reagents and materials
Uracils (Fig. 1) were purchased from Sigma-Aldrich and used without further purification.
Organic solvents were obtained from UOSLAB (Kyiv, Ukraine).NMR spectra were recorded on Varian Mercury Plus NMR spectrometer with operating frequencies of 301.546MHz ( 1 H), and 80.95 MHz ( 31 P) in deuterochloroform. 1 H NMR chemical shifts are referred to tetramethylsilane as internal standard, and 31 P chemical shifts are referred to 85% orthophosphate acid.All reactions were performed under dry conditions under argon atmosphere using the Schlenk technique.

Synthesis of calixarenes
The main method for phosphorylation of the calixarenes upper rim is the Arbuzov reaction of their chloromethyl derivatives with alkyl esters of trivalent phosphorus acids [79].Calixarenes CPO was synthesized in one step by the Arbuzov reaction of chloromethylpropoxycalix [4]arene ChC with i-PrOPEt A solution of CPAPr (1.2 g, 1 mmol) and trimethylbromosilane (5 mL, 37.88 mmol) in chloroform (20 mL) was stirred at room temperature for 48 h.Volatile components were removed in vacuum (10 mm Hg).The residue was dissolved in methanol (40 ml) and refluxed for 6 h.Water (15 ml) was added to the clear solution of the formed CPASi.The solid residue of CPA formed was filtered off and dried under vacuum (0.01 mm Hg) to constant weight.Yield 0.89 g (88%).White powder.M.p. 256-258 o C. 1

RP HPLC analysis
The RP HPLC analysis was performed using the liquid chromatographic system Laboratorni Pristroje (Czech Republic) equipped with the Separon SGX NH 2 (250 × 4.6 mm) column.The stability constants of calixarene complexes with uracil derivatives were determined in a four-component solution of methanol/acetonitrile/tetrahydrofuran/water (15/10/5/70, v/v), which was used as a mobile phase.The mobile phase contained calixarene additives in concentrations from 4 × 10 − 5 to 1.2 × 10 -4 M. Uracil samples for analysis were prepared in a solvent identical in composition to the mobile phase (C = 1 × 10 -5 M).The volume of the sample introduced into the chromatographic column was 25 mkl.Each sample was analyzed three times.All chromatograms were obtained at a temperature of 31 o C. The flow rate of the mobile phase was 0.6 ml/min, the wavelength of the UV detector was 254 nm.Experiments were performed under isocratic conditions.
A conical conformation of calixarenes CPO, CPAPr, and CPA were confirmed in 1 H NMR spectra by the presence of two doublets of the AB spin system of axial and equatorial protons of Ar-CH 2 -Ar methylene groups with spin-spin interaction constants 2 J HH 12.6-13.4Hz and the difference in chemical shifts Δδ 1.19-1.29 ppm [81] (Fig. S1 and S2 in ESI).

Determination of stability constants of the complexes
The complex formation of calixarenes СРО and СРА with uracils was investigated by the RP HPLC method in the flow of the mobile phase methanol/acetonitrile/tetrahydrofuran/water (15/10/5/70, v/v), according to the method described in the paper [103].Such a four-component phase was required for simultaneous solubility of calixarenes and uracils in it.Сalixarene СРО and СРА appeared on the chromatograms as peaks with retention times t R 1.52 and 2.17 min, respectively (Fig. S3, S4 in ESI).Under these conditions of analysis, uracils appeared as peaks with retention times ranging from 1.50 to 4.00 min.
Calixarenes СРО and СРА have linear adsorption isotherms under the conditions of analysis (Figs. 3 and 4), which indicates their reversible adsorption on the surface of the column support.
Calixarene additives to the mobile phase reduce the value of the capacity coefficient k' of the uracil derivatives due to the formation of the guest-host complexes.Linear dependences of 1/k' on the concentrations of CPO or CPA (Fig. S5-S8 in ESI) confirm the formation of the complexes with a stoichiometry of 1:1.The reversible sorption of CPO or CPA and the 1:1 stoichiometry of the formed complexes allows us to use Eq. ( 1) to calculate their stability constants of К А : where, k 0 ′ and k′ are the capacity coefficients of uracil derivatives determined in the absence and presence of CPO or CPA in the mobile phase.
[CA] is the calixarene concentration in the mobile phase.
Based on the linear dependences of 1/k' of uracils on the concentrations of calixarenes in the mobile phase, the stability constants of their supramolecular complexes K A were determined and Gibbs free energies were calculated (ΔG = -RT⋅lnK A ) (Table 1).
The dependence of stability constants (Table 1) of the complexes on the structure of calixarenes and uracils is complicated.The stability complexes can be determined by all cases to increase calculation speed and efficiency.Solvents effects (water, ε = 80.1) were taken into account using the COSMO routine [92,93] by single-point energy calculation at the same level of theory used for geometry optimization.Vibration frequencies and corrections for calculation of relative energy (ΔE), enthalpy (ΔH) and Gibbs free energies (ΔG) values were derived analytically.For all optimized structures, no imaginary frequencies were detected by vibration analysis.The Jmol [94,95] program was used for the graphical presentation of structures.

Results and discussion
Previously, it was shown that para-unsubstituted propoxycalixarene C, isopropyl ester of propoxycalixarene phosphonic acid CPAPr2 (Fig. 2), and their analogues form supramolecular inclusion complexes with uracils and adenines in aqueous-organic solutions [96,97].However, these compounds are characterized by very low solubility in water and have no prospect for use as carrier compounds for drug delivery.
Water-soluble calixarenes have been prepared by introducing various anionic, cationic, or neutral hydrophilic substituents on the upper or lower rim of the macrocycle [98].Four polar diethylphosphine oxide or ethylphosphinic acid groups provide high solubility of CPO and CPA calixarenes (Fig. 2) in water (> 100 mg/g).
Similar approaches are also used in medicinal chemistry to improve the solubility of drugs in water.Recently, polar low molecular phosphine oxide and related phosphoruscontaining functional groups, both neutral and charged, has been considered as valuable structural motifs in drug design H,π-interactions with a guest molecule.In order to decrease the conformational diversity in the model structures that mimic the experimentally studied of CPO and CPA calixarene ligands (structures 1 and 2, respectively), ethyl groups were replaced with methyl substituents.
Structures of calixarenes and their nanoadducts were optimized using a novel and efficient Grimme's RI-B97-3c DFT functional [91] that reproduces well electron dispersion effects and, on the other hand, is suitable for treating large molecules.The solvent effects were taken into account by means of the COSMO routine [92,93] at the final singlepoint energy calculation (water was chosen as solvent).The found most favorable conformations for model ligands 1 and 2 with a large number of hydrogen bonds are shown in Fig. 5.The structures of calixarene-ligand 1 is a slightly flattened cone and stabilized by the weak hydrogen bonding between oxygen atoms of the P = O moieties and methyl hydrogen bonds, van der Waals forces, solvatophobic and other non-valent interactions.

DFT calculations
The implementation of the efficient linear scaling procedures, such as RI [87][88][89][90] and Multipole Accelerated Resolution of Identity (MARIJ) [91] has previously allowed us calculating successfully such large structures as calixarenes [104][105][106][107]. Conformational flexibility of the structures requires analysis of a large number of conformers in order to find and locate the one with the lowest total energy.One of the criteria for the search of the conformation is the number of hydrogen bonds, which obviously play an important role in the formation and stabilization of the adduct modified calixarene-uracil.Another binding property of calixarenes requiring our attention are hydrophobic, π,π-and Fig. 5 Different Jmol [94,95] views of equilibrium (B97-3c/ TZVP) structures of model calixarenes 1 (A) and 2 (B).In the structures on the left side hydrogen atoms are omitted for clarity to the outside of the cavity have higher energy (see ESI).Adduct 2*UR (Fig. 6, B) is additionally stabilized by two hydrogen bonds between P = O oxygen atom and OH group of the P(= O)(Me)OH substituent at the neighboring aromatic moiety.In the equilibrium structure of complex 1*UR (Fig. 6, A) effective hydrogen bonding within the calixarene ligand is substituted by weak CH⋅⋅⋅O = P interactions.The alternative structures of the complexes with the uracil molecule immersed into the cavity of the calixarene ligand possess much higher total energy and will not be considered for the substituted uracils (see ESI for more detail).
The structures of uracil derivative adducts are shown in Fig. 7.As the substituents in the uracil moiety do not take part in the host-guest interaction, its nature does not play a significant role in the adduct stabilization.It was then logic to suggest for the adducts the structure similar to 1*UR and 2*UR.Interestingly, in the case of 2*5-AmUR, no additional interaction of P-OH hydrogen with occurs with the amino group at position 5.At the used DFT level of approximation, groups (Fig. 5A).In the more flattened structure of free ligand 2, P = O and OH groups of the phosphorus-containing substituents at the neighboring aromatic ring interact with each other (Fig. 5B).
Principal structures of the calixarene nanoadducts were investigated on the example of uracyl-based adducts, 1*UR and 2*UR (Fig. 6).We decided to start our study comparing the adducts with uracil interacting only with the phosphorus-containing substituents on the top rim of the calixarene scaffold with the complexes, in which the guest is deeply immersed into the cavity of the host (see the ESI for more detail).The former structures possessed the lowest total energy.Important factors stabilizing the adducts, are two hydrogen NH⋅⋅⋅O = P bonds and (in the case of 2*UR) POH⋅⋅⋅O = C bonding.In all adducts, protons of two CH 2 groups of the substituents on the opposing aromatic moieties are directed to the middle of the cavity and two remained ones are oriented to the outside of the cavity.The isomeric structures with all CH 2 groups pointing It included geometry optimization in the gas phase, overestimating intramolecular hydrogen bonding in the free ligands, which cannot be compensated by modelling solvent effects for the final geometry with the empirical COSMO procedure.Therefore, while the experimentally observed highest stability of the 5-fluorouracil adducts and the lowest one for 5-nitrouracil is kept for the calculated 2*AmUR and 2*NUR adducts, stability of 1*NUR turns out to be unexpectedly overestimated at the DFT approximation level (see Table 2).The used theoretical approach can be also too rough for describing such subtle effects [108].Interestingly, calculated enthalpy values referred to the formation of the adducts are definitely negative, whereas free Gibbs energy ranges from small negative values to slightly positive magnitudes (see Table 2).At the first sight, this is in some contradiction with the complexation energy values obtained in the experiment.However, in contrast to DFT calculations, the experimental ΔG were determined in the indirect way.On the other hand, in addition to generally known inexactness of DFT calculations, the calculated free Gibbs energy for the complexation reaction can be underestimated due to an approximated character of the used theoretical approach.Calculated at the COSMO//RI-B97-3c/TZVP level of theory as the final single-point energy calculation.Other values were derived at the same theoretical level using the gas-phase approximation the predicted energy values for complexation of the ethylsubstituted calixarenes 3 and 4 (see ESI) are slightly more negative and hence closer to the experimental magnitudes.One of the possible explanations is some destabilization of hydrogen bonding in the more sterically crowded free ligands.Anyway, the theoretically predicted ΔG energy for the host-guest interaction in the uracil adducts is small (few kcal⋅mol − 1 ), which is in agreement with the results of the chromatographic investigations described above.

Conclusion
Water-soluble phosphorylated tetrapropoxycalix [4]arenes CPO and CPA were obtained by introduction of hydrophilic diethylphosphine oxide groups or ethylhydroxyphosphonic groups respectively on the upper rim of mother tetrapropoxycalix [4]arenes.These nano-sized cup-shaped macrocycles form supramolecular complexes with uracil derivatives, including 5-fluorouracil, and 5-methyluracil, which are active pharmaceutical ingredients of widely used medicines.DFT calculations of the structures provide the structure of the adducts with uracil molecules forming hydrogen bonds with the phosphorus-containing substituents at the upper rim of calixarene.
The obtained results allow proposing the water-soluble calixarenes CPO and CPA as promising compounds in the development of nanovectors for drug delivery of bio-active uracil derivatives.