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Inclusion complexes of hydroxypropyl-β-cyclodextrin with novel cytotoxic compounds: Solubility and thermodynamic properties
Fluid Phase Equilibria 384 (2014) 68–72
Contents lists available at ScienceDirect
Fluid Phase Equilibria j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / fl u i d
Inclusion complexes of hydroxypropyl-b-cyclodextrin with novel cytotoxic compounds: Solubility and thermodynamic properties Marina V. Ol’khovich a, *, Angelica V. Sharapova a , Sergey N. Lavrenov b , Svetlana V. Blokhina a , German L. Perlovich a,c a b c
Institute of Solution Chemistry, Russian Academy of Sciences, 1 Akademicheskaya Street, 153045 Ivanovo, Russia Gause Institute of New Antibiotics, Russian Academy of Medical Sciences, 11B Pirogovskaya Street, 11902 Moscow, Russia Institute of Physiologically Active Compounds, Russian Academy of Sciences, 142432 Chernogolovka, Russia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 April 2014 Received in revised form 22 September 2014 Accepted 20 October 2014 Available online 23 October 2014
Complexation processes in the systems of 2-hydroxypropyl-b-cyclodextrin (2HP-b-CD) – pharmacologically active substances within the temperature range of 293–315 K were studied by the UVspectroscopy method. 2HP-b-CD was shown to improve the solubility of the studied drugs in aqueous systems. It was proved that complexation leads to the formation of supramolecular inclusion compounds with a 1:1 component ratio. The obtained complex 2HP-b-CD–methylium, tris(1-pentyl-1H-indol-3-yl)-, chloride was found to be unstable in buffer pH 7.4. Stability constants of the complexes and thermodynamic parameters of the formation of the inclusion compound 2HP-b-CD-1H-indole-3methanol and 1-pentyl-a,a-bis(1-pentyl-1H-indol-3-yl) were calculated. The negative values of the changes in the Gibbs energy and the enthalpy indicate that the inclusion process is spontaneous and exothermic. The excess of the enthalpy term over the entropic one indicates that the inclusion process is enthalpy-determined. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Solubility Inclusion complex Antitumor agent 2-Hydroxypropyl-b-cyclodextrin Thermodynamics
1. Introduction Currently there is active research into new highly efficient antitumor compounds, in particular the so-called “targeted” drugs that block certain target proteins in tumor cells and can, therefore, decrease the cancer mortality rate and improve the life quality. Tumour resistance to traditional chemotherapeutic drugs can be caused by the inhibited start of the programmed cell death – apoptosis. Among the most relevant approaches are development of new original classes of antitumor substances repairing the tumor cell susceptibility to apoptosis induction and finding other targets for triggering the cell death. In the last few years scientists’ attention has been attracted by compounds containing a triphenylmethane fragment [1–5]. Among such compounds they found substances with antiproliferative activity. Researchers [1–4] emphasize the complex role of the triphenylmethyl component in the structure of antitumor compounds. The authors [6] have developed a technique of deriving symmetrical tris(1-alkylindol-3-yl) methanes and by oxidizing them obtained a series of tris(1-alkylindol-3-yl) methylium salts. The cytotoxicity of tris(1-alkylindol-3-yl) methylium salts depends on the length of the substituent at the atom of the heterocycle and
* Corresponding author. Tel.: +7 4932 351545/9605 106787; fax: +7 4932 336246. E-mail address: [email protected] (M.V. Ol’khovich). 0378-3812/$ – see front matter ã 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fluid.2014.10.030
increases from the N-unsubstituted derivative toward N-pentyl derivatives. A further increase in the length of the N-alkyl substituent reduces the compound cytotoxicity. Tris(1-pentyl1H-indol-yl) methilium chloride was found to have the highest cytotoxicity among the obtained salts. The work [7] studies the cytotoxicity of this compound on the cell lines of metastatic melanoma in comparison with different antitumor drugs. The compound showed high cytotoxic activity, also in relation to the metastatic melanoma cells, which are resistant to chemotherapeutic effects, indicating its availability as an antitumor agent. Poor solubility and low bioavailability are the main obstacles for preclinical use of these compounds. One of the ways to increase the bioavailability of such compounds and to make it easier to produce parenteral and liquid peroral drugs is to produce soluble cyclodextrin-based inclusion complexes. Cyclodextrins (CD) are cyclic oligosaccharides containing 6 (a), 7 (b) and 8 (g) glycopyranozic units connected by a-(1,4) bonds. The CD structure consists of an external hydrophilic surface and an internal lipophilic cavity. The internal part of a cyclodextrin molecule is nonpolar due to glycosidic oxygen and methine protons, while the external surface is polar as there are secondary and primary hydroxyl groups along the ring edge [8]. As a result, CDs can form inclusion complexes with a big number of hydrophobic compounds and such complexes are successfully used to increase the chemical stability, solubility and bioavailability of many compounds [9].
M.V. Ol’khovich et al. / Fluid Phase Equilibria 384 (2014) 68–72
69
Table 1 Characteristics of chemicals used in this study. Chemical name
CAS number
Source
1H-indole-3-methanol, 1-pentyl-a,a-bis(1-pentyl1H-indol-3-yl)Methylium, tris(1-pentyl-1H-indol-3-yl)-, chloride (1:1) 2-Hydroxypropyl-b-cyclodextrin
1225221-24-8
Synthesis –
Recrystallization 0.99
HPLCa 1H NMRb
1362426-61-6
Synthesis –
Recrystallization 0.99
HPLC 1H NMR
128446-35-5
Aldrich
–
–
a b
Initial mass fraction purity
0.97
Purification method
Final mass fraction purity
–
Analysis method
High performance liquid chromatography. Hydrogen-1 nuclear magnetic resonance.
Native CDs are known to interact with human tissues and to extract cholesterol and other cell membrane components, particularly if renal tubules are accumulated in the cells, thus having a nephrotoxic effect [10]. Chemical modification of original CDs allowed us to obtain safer derivatives with the same or better complexing ability. Among the numerous cyclodextrin derivatives 2-hydroxypropyl-b-cyclodextrin (2HPb-CD) has the highest solubility in water and bioavailability [11]. In works [12–16], the authors used 2HP-b-CD to increase the solubility of triclosan, glibenclamide, ketoconazole, ganciclovir ether acyl prodrugs. Besides, some toxicological studies have revealed that 2HP-b-CD is well tolerated by people when taken orally and intravenously [17]. The goal of the present study is to obtain stable inclusion complexes of 1H-indole-3-methanol, 1-pentyl-a,a-bis(1-pentyl1H-indol-3-yl) (I) and methylium, tris(1-pentyl-1H-indol-3-yl)-, chloride with 2-hydroxypropyl-b-cyclodextrin with improved solubility and to investigate thermodynamics of the complexation process. 2. Experimental 2.1. Materials The compounds 1H-indole-3-methanol, 1-pentyl-a,a-bis(1pentyl-1H-indol-3-yl) (I) and methylium, tris(1-pentyl-1H-indol3-yl)-, chloride (1:1) (II) were synthesized by the method described in [6]. 2-Hydroxypropyl-b-cyclodextrin was received from Aldrich. The major information of the used chemicals is given in Table 1. As the water phase we used a phosphate buffer pH 7.4 modeling a vascular system medium. Phosphate buffer (0.06 M,
N
pH 7.4) was prepared combining the K2HPO4 (9.1 g in 1 L) and NaH2PO412H2O (23.6 g in 1 L) salts [18]. Salts K2HPO4 and NaH2PO412H2O were supplied by Merck (99% purity). Ionic strength was adjusted by adding potassium chloride. All chemicals were of AR grade. The pH values were measured by using Electroanalytical Analyser, Type OP-300, Radelkis, Budapest standardized with pH 1.68, 6.86 and 9.22 solutions. 2.2. Solubility measurements The study of inclusion complex solubility consisted in quantitative determination of the “guest” compound solubility (the substance under consideration) at different cyclodextrin concentrations. Based on the obtained data we plotted “guest” phase solubility diagrams of different cyclodextrin concentrations [19]. An excessive amount of the compound under study was added to the phosphate buffer solution with 2HP-b-CD. The 2HPb-CD concentration varied from 0 to 0.2 mol/L. The prepared solutions were placed into glass test tubes and mixed in a temperature-controlled air bath within the temperature range of 293–315 K for 72 h. After the balance was reached, the solutions were centrifuged and filtered out through a 0.20 mm Millex1 HA filter (Ireland). The concentration of the dissolved compounds was determined by the spectrophotometer Cary 50 (Varian) using a 1 cm quartz cell. The complexes stoichiometry was measured by the isomolar series method (Job’s method) [20]. The measurements were conducted based on a series of solutions in which the total amount of molar concentration of cyclodextrin and the compounds under study was constant, and their ratio was changing continuously.
OH N
C N
N
N
N
Cl
I
II Fig. 1. Structural formulas of the compounds I and II.
O
70
M.V. Ol’khovich et al. / Fluid Phase Equilibria 384 (2014) 68–72
Fig. 2. UV–vis absorption spectra for compounds I and II in buffer solution at different concentrations 2HP-b-CD.
3. Results and discussion As works [21–23] show, the substitution of OH-groups surrounding the CD cavity leads to a significant increase in the stability of the forming inclusion complexes. The introduced substituents increase the size of the macrocyclic cavity, change its hydrophobicity and affect the flexibility of the CD molecule allowing it to get involved in additional interactions with the guest molecule and thus retaining it inside the complex. Such results were obtained by studying 2HPb-CD complexes with diclofenac [21], nabumetone [22], nitrophenol [23]. Therefore, it is of interest to investigate 2HP-b-CD interactions with 1H-indole-3-methanol, 1-pentyl-a,a-bis(1-pentyl-1H-indol3-yl) (I) and its derivative chloride (II), the structural formulas of which are presented in Fig. 1. The ability of CDs to form inclusion complexes with hydrophobic compounds is the basis for their application as solubilizing agents [24]. Permeation of the molecules of biologically active substances into a macrocyclic cavity and their arrangement within it are provided by nonvalent interactions (van der Waals, Table 2 The solubilities of compounds studied in HP-b-CD buffer solutions (pH 7.4) at T = 298.15 K and pressure p = 0.1 MPa. SCD (mol/L)
SDrug (104 mol/L)
I 0.000 0.005 0.029 0.046 0.051 0.075 0.087 0.098 0.113 0.179
0.06 0.12 0.50 0.76 0.83 0.12 0.14 0.16 0.18 0.27
II 0.000 0.001 0.005 0.007 0.010 0.017 0.022 0.025 0.031 0.042 0.051
0.21 0.43 2.13 3.51 5.11 8.21 11.03 13.20 16.01 21.93 29.01
Standard uncertainties are u(T) = 0.05 K, ur(S) = 0.04, u(p) = 0.05.
Fig. 3. Phase-solubility diagram of compounds I and II in the presence of 2HP-b-CD in buffer at 298 K.
hydrophobic, electrostatic, hydrogen bonding and steric effects). Besides, if the “guest” molecule has hydrophobic and hydrophilic fragments of a similar size, CDs prefer bonding with the former. The hydrophobic fragments in the compounds under study are alkyl chains and aromatic rings. UV–vis absorption spectra for the pure compounds and at the addition different concentrations of cyclodextrin have been studied in buffer pH 7.4 (Fig. 2). As it can be seen from Fig. 2, the results of UV–vis spectra how the absorption spectrum of inclusion complexes aqueous solutions coincides with the pure compounds, the maximum peaks appear at 476 nm. A solution increases with the addition of 2HP-b-CD. The similarity of the changes reveals that the inclusion complexes have been formed between b-CD and compounds I–II [25]. To study the dissolution processes of compounds I and II with 2HP-b-CD, we measured solubility of these substances in buffer solutions with different concentrations of cyclodextrin (Table 2). Fig. 3 shows the phase solubility diagrams of the compounds under consideration at the temperature of 298 K. It was determined that the maximum solubility of compound I – 2.74 104 mol/L is reached in the solution with the concentration of 2HP-b-CD 0.179 mol/L. The resulting value of solubility is greater than the initial concentration in buffer 6.32106 mol/L in 43 times. The solubility of compound II in buffer 2.12105 mol/L by adding the 2HP-b-CD solution (0.051 mol/L) increased 137 time and reached
Fig. 4. Job’s curves for complex formation of 2HP-b-CD with compounds I and II at in buffer at 298 K.
M.V. Ol’khovich et al. / Fluid Phase Equilibria 384 (2014) 68–72
Fig. 5. Kinetic dependences of solubility for compounds I and II in the presence of various concentrations of 2HP-b-CD. 3
2.91 10 mol/L. The considerable solubility increase proves that the 2HP-b-CD and the drugs interact with each other and form complexes. For all of the obtained inclusion complexes the solubility increased linearly as the 2HP-b-CD concentration grew, therefore, the plotted phase diagrams can be viewed as AL-type charts [19]. The linearity of these dependences indicates that 1:1 component ratio complexes are formed between the compounds under study and 2HP-b-CD. The stoichiometric parameters of the obtained 1:1 inclusion complexes were confirmed by the Job’s method. To do this, we plotted a diagram showing the dependence of the measured solution optical density (D), proportional to the complex formation, on the mole fractions of both components. Fig. 4 represents Job’s curves of the studied systems with the minimum solubility values observed if the mole ration is equal to 0.5 which corresponds to 1:1 complex formation. The stability constant of the 1:1 inclusion complexes were calculated based on the solubility diagram by equation: K¼
slope S0 ð1 slopeÞ
(1)
where K is stability constant; S0 is solubility of compound studied in buffer. The inclusion complex stability constants at 298 K for compounds I and II were equal to 252.94 2.78 L/mol and 2404.56 5.40 L/mol, respectively. To check the stability of the obtained complexes we studied the solubility kinetics of compounds I and II. The solubility kinetic dependences are represented in Fig. 5. As Fig. 5 shows, the inclusion complex of compound I with 2HP-b-CD is remained stable for a long time. The solubility of compound II is decreased in time in the presence of 2HP-b-CD. It was estimated that in buffer solution pH 7.4 individual compound II is hydrolyzing during 60 h Table 3 The values of the parameters of Eq. (1) and the stability constants of the complex 2HP-b-CD with a compound I in buffer pH 7.4 at pressure p = 0.1 MPa.
G (K)
S0 106 (mol/L)
Slope 103
K (L/mol)
Correlation coefficient
293.15 298.15 303.15 311.15 315.15
2.50 6.32 11.30 23.68 39.47
1.533 0.018 1.596 0.017 1.637 0.018 1.601 0.015 1.748 0.014
614.13 7.36 252.94 2.78 145.12 1.59 67.72 0.63 44.28 0.35
0.9996 0.9998 0.9994 0.9991 0.9987
Standard uncertainties are u(T) = 0.05 K, ur(S0) = 0.04, u(p) = 0.05.
71
Fig. 6. Logarithmic dependence of the stability constants of the inclusion complex 2HP-b-CD–compound I on the reciprocal temperature (R = 0.9972).
and reveals the solubility decrease from 2.12 105 mol/L to 6.32 106 mol/L. The enhanced solubility of compound II in the presence of 2HP-b-CD remains constant during a long period and does not decrease lower than that of the individual one that can be explained by the CD solubilizing action. Diagrams of phase solubility at different temperatures were plotted for the stable inclusion complex of compound I. It was determined that the compound solubility in the presence of 2HPb-CD increases as the temperature rises. This may be caused by the release of water molecules bound in the cyclodextrin cavity [26]. The values of parameters of Eq. (1) and stability constants of the obtained inclusion complexes at different temperatures are presented in Table 3. It has been determined that the stability constants decrease as the temperatures grow which is typical of the exothermic process of complexation [27,28]. The thermodynamic and structural characteristics of complexation allow us to evaluate the solubilizing and stabilizing action of cyclodextrin and the degree of changing of the physico-chemical properties of guest-molecules introduced into the cyclodextrin cavity. The thermodynamic parameters of forming the inclusion complex 2HP-b-CD – compound I were obtained from the logarithmic dependence of the stability constant on the reciprocal temperature (Fig. 6). To calculate the complexing enthalpy and entropy values, we used the integral form of the van Hoff’s equation [29]:
DH 0
lnK ¼
RT
þ
DS 0
(2)
R
where DH and DS are the enthalpy and entropy of complex formation, respectively, T is the temperature, R is the gas constant. The Gibbs energy change was calculated by equation:
DG0 ¼ RTlnK
(3)
The obtained thermodynamic parameters are shown in Table 4. The negative values of the changes in the Gibbs energy and the enthalpy indicate that the interactions of compound I and 2HPTable 4 Thermodynamic parameters of the formation complex 2HP-b-CD with a compound I in buffer pH 7.4 at 298 K.
DG (kJ mol1) DH (kJ mol1) TDS (kJ mol1)
Complex
K (L/mol)
2HP-b-CD– I
252.94 2.78 13.70 0.15
90.7 5.2
76.9 4.4
72
M.V. Ol’khovich et al. / Fluid Phase Equilibria 384 (2014) 68–72
b-CD are spontaneous and exothermic. The negative DH values indicate of quite strong molecular interactions caused by both van der Waals forces and hydrogen bond formation [30]. As the authors state [28] these interactions are thought to result from the hydrophobic guest permeation into the cyclodextrin cavity as well as the dehydration of the guest molecule. The negative changes of the complexation entropy can be explained by the reduction of the translational and rotational degrees of freedom of the guest molecule when the latter is introduced into the cyclodextrin cavity and by the fact that a more ordered system is created. The excess (in absolute magnitude) of the enthalpy term over the entropic one indicates that the inclusion process is enthalpy-determined. 4. Conclusion By using UV-spectroscopy we have studied the behavior of pharmacologically active substances in a buffer solution containing different 2HP-b-CD concentrations. The phase solubility diagrams of the studied compounds showed that drug solubility rises linearly as the 2HP-b-CD concentration increases. The linear solubility growth can be explained to the formation of inclusion complexes between compounds I, II and 2HP-b-CD with a 1:1 stoichiometric ratio. It has been found that the obtained 2HP-b-CD–compound II complex is unstable in a pH 7.4 buffer solution. The stability constants of the inclusion complex of compound I and the main thermodynamic parameters DG , DH and DS are calculated. The conclusion is made that the formation of a supramolecular 2HP-b-CD–compound I complex is an exothermal enthalpy-determined process driven by hydrogen bond formation and van der Waals interactions, particularly the dispersion attraction of the aromatic ring and alkyl chains of the molecule to the internal hydrophobic cavity of the 2HP-b-CD molecule. The research results have shown that it is possible to use 2HP-b-CD to significantly increase the solubility of the studied drugs in aqueous solutions that would reduce the necessary dosage, and hence toxicity of the proposed drugs.
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Contents lists available at ScienceDirect
Fluid Phase Equilibria j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / fl u i d
Inclusion complexes of hydroxypropyl-b-cyclodextrin with novel cytotoxic compounds: Solubility and thermodynamic properties Marina V. Ol’khovich a, *, Angelica V. Sharapova a , Sergey N. Lavrenov b , Svetlana V. Blokhina a , German L. Perlovich a,c a b c
Institute of Solution Chemistry, Russian Academy of Sciences, 1 Akademicheskaya Street, 153045 Ivanovo, Russia Gause Institute of New Antibiotics, Russian Academy of Medical Sciences, 11B Pirogovskaya Street, 11902 Moscow, Russia Institute of Physiologically Active Compounds, Russian Academy of Sciences, 142432 Chernogolovka, Russia
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 April 2014 Received in revised form 22 September 2014 Accepted 20 October 2014 Available online 23 October 2014
Complexation processes in the systems of 2-hydroxypropyl-b-cyclodextrin (2HP-b-CD) – pharmacologically active substances within the temperature range of 293–315 K were studied by the UVspectroscopy method. 2HP-b-CD was shown to improve the solubility of the studied drugs in aqueous systems. It was proved that complexation leads to the formation of supramolecular inclusion compounds with a 1:1 component ratio. The obtained complex 2HP-b-CD–methylium, tris(1-pentyl-1H-indol-3-yl)-, chloride was found to be unstable in buffer pH 7.4. Stability constants of the complexes and thermodynamic parameters of the formation of the inclusion compound 2HP-b-CD-1H-indole-3methanol and 1-pentyl-a,a-bis(1-pentyl-1H-indol-3-yl) were calculated. The negative values of the changes in the Gibbs energy and the enthalpy indicate that the inclusion process is spontaneous and exothermic. The excess of the enthalpy term over the entropic one indicates that the inclusion process is enthalpy-determined. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Solubility Inclusion complex Antitumor agent 2-Hydroxypropyl-b-cyclodextrin Thermodynamics
1. Introduction Currently there is active research into new highly efficient antitumor compounds, in particular the so-called “targeted” drugs that block certain target proteins in tumor cells and can, therefore, decrease the cancer mortality rate and improve the life quality. Tumour resistance to traditional chemotherapeutic drugs can be caused by the inhibited start of the programmed cell death – apoptosis. Among the most relevant approaches are development of new original classes of antitumor substances repairing the tumor cell susceptibility to apoptosis induction and finding other targets for triggering the cell death. In the last few years scientists’ attention has been attracted by compounds containing a triphenylmethane fragment [1–5]. Among such compounds they found substances with antiproliferative activity. Researchers [1–4] emphasize the complex role of the triphenylmethyl component in the structure of antitumor compounds. The authors [6] have developed a technique of deriving symmetrical tris(1-alkylindol-3-yl) methanes and by oxidizing them obtained a series of tris(1-alkylindol-3-yl) methylium salts. The cytotoxicity of tris(1-alkylindol-3-yl) methylium salts depends on the length of the substituent at the atom of the heterocycle and
* Corresponding author. Tel.: +7 4932 351545/9605 106787; fax: +7 4932 336246. E-mail address: [email protected] (M.V. Ol’khovich). 0378-3812/$ – see front matter ã 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fluid.2014.10.030
increases from the N-unsubstituted derivative toward N-pentyl derivatives. A further increase in the length of the N-alkyl substituent reduces the compound cytotoxicity. Tris(1-pentyl1H-indol-yl) methilium chloride was found to have the highest cytotoxicity among the obtained salts. The work [7] studies the cytotoxicity of this compound on the cell lines of metastatic melanoma in comparison with different antitumor drugs. The compound showed high cytotoxic activity, also in relation to the metastatic melanoma cells, which are resistant to chemotherapeutic effects, indicating its availability as an antitumor agent. Poor solubility and low bioavailability are the main obstacles for preclinical use of these compounds. One of the ways to increase the bioavailability of such compounds and to make it easier to produce parenteral and liquid peroral drugs is to produce soluble cyclodextrin-based inclusion complexes. Cyclodextrins (CD) are cyclic oligosaccharides containing 6 (a), 7 (b) and 8 (g) glycopyranozic units connected by a-(1,4) bonds. The CD structure consists of an external hydrophilic surface and an internal lipophilic cavity. The internal part of a cyclodextrin molecule is nonpolar due to glycosidic oxygen and methine protons, while the external surface is polar as there are secondary and primary hydroxyl groups along the ring edge [8]. As a result, CDs can form inclusion complexes with a big number of hydrophobic compounds and such complexes are successfully used to increase the chemical stability, solubility and bioavailability of many compounds [9].
M.V. Ol’khovich et al. / Fluid Phase Equilibria 384 (2014) 68–72
69
Table 1 Characteristics of chemicals used in this study. Chemical name
CAS number
Source
1H-indole-3-methanol, 1-pentyl-a,a-bis(1-pentyl1H-indol-3-yl)Methylium, tris(1-pentyl-1H-indol-3-yl)-, chloride (1:1) 2-Hydroxypropyl-b-cyclodextrin
1225221-24-8
Synthesis –
Recrystallization 0.99
HPLCa 1H NMRb
1362426-61-6
Synthesis –
Recrystallization 0.99
HPLC 1H NMR
128446-35-5
Aldrich
–
–
a b
Initial mass fraction purity
0.97
Purification method
Final mass fraction purity
–
Analysis method
High performance liquid chromatography. Hydrogen-1 nuclear magnetic resonance.
Native CDs are known to interact with human tissues and to extract cholesterol and other cell membrane components, particularly if renal tubules are accumulated in the cells, thus having a nephrotoxic effect [10]. Chemical modification of original CDs allowed us to obtain safer derivatives with the same or better complexing ability. Among the numerous cyclodextrin derivatives 2-hydroxypropyl-b-cyclodextrin (2HPb-CD) has the highest solubility in water and bioavailability [11]. In works [12–16], the authors used 2HP-b-CD to increase the solubility of triclosan, glibenclamide, ketoconazole, ganciclovir ether acyl prodrugs. Besides, some toxicological studies have revealed that 2HP-b-CD is well tolerated by people when taken orally and intravenously [17]. The goal of the present study is to obtain stable inclusion complexes of 1H-indole-3-methanol, 1-pentyl-a,a-bis(1-pentyl1H-indol-3-yl) (I) and methylium, tris(1-pentyl-1H-indol-3-yl)-, chloride with 2-hydroxypropyl-b-cyclodextrin with improved solubility and to investigate thermodynamics of the complexation process. 2. Experimental 2.1. Materials The compounds 1H-indole-3-methanol, 1-pentyl-a,a-bis(1pentyl-1H-indol-3-yl) (I) and methylium, tris(1-pentyl-1H-indol3-yl)-, chloride (1:1) (II) were synthesized by the method described in [6]. 2-Hydroxypropyl-b-cyclodextrin was received from Aldrich. The major information of the used chemicals is given in Table 1. As the water phase we used a phosphate buffer pH 7.4 modeling a vascular system medium. Phosphate buffer (0.06 M,
N
pH 7.4) was prepared combining the K2HPO4 (9.1 g in 1 L) and NaH2PO412H2O (23.6 g in 1 L) salts [18]. Salts K2HPO4 and NaH2PO412H2O were supplied by Merck (99% purity). Ionic strength was adjusted by adding potassium chloride. All chemicals were of AR grade. The pH values were measured by using Electroanalytical Analyser, Type OP-300, Radelkis, Budapest standardized with pH 1.68, 6.86 and 9.22 solutions. 2.2. Solubility measurements The study of inclusion complex solubility consisted in quantitative determination of the “guest” compound solubility (the substance under consideration) at different cyclodextrin concentrations. Based on the obtained data we plotted “guest” phase solubility diagrams of different cyclodextrin concentrations [19]. An excessive amount of the compound under study was added to the phosphate buffer solution with 2HP-b-CD. The 2HPb-CD concentration varied from 0 to 0.2 mol/L. The prepared solutions were placed into glass test tubes and mixed in a temperature-controlled air bath within the temperature range of 293–315 K for 72 h. After the balance was reached, the solutions were centrifuged and filtered out through a 0.20 mm Millex1 HA filter (Ireland). The concentration of the dissolved compounds was determined by the spectrophotometer Cary 50 (Varian) using a 1 cm quartz cell. The complexes stoichiometry was measured by the isomolar series method (Job’s method) [20]. The measurements were conducted based on a series of solutions in which the total amount of molar concentration of cyclodextrin and the compounds under study was constant, and their ratio was changing continuously.
OH N
C N
N
N
N
Cl
I
II Fig. 1. Structural formulas of the compounds I and II.
O
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M.V. Ol’khovich et al. / Fluid Phase Equilibria 384 (2014) 68–72
Fig. 2. UV–vis absorption spectra for compounds I and II in buffer solution at different concentrations 2HP-b-CD.
3. Results and discussion As works [21–23] show, the substitution of OH-groups surrounding the CD cavity leads to a significant increase in the stability of the forming inclusion complexes. The introduced substituents increase the size of the macrocyclic cavity, change its hydrophobicity and affect the flexibility of the CD molecule allowing it to get involved in additional interactions with the guest molecule and thus retaining it inside the complex. Such results were obtained by studying 2HPb-CD complexes with diclofenac [21], nabumetone [22], nitrophenol [23]. Therefore, it is of interest to investigate 2HP-b-CD interactions with 1H-indole-3-methanol, 1-pentyl-a,a-bis(1-pentyl-1H-indol3-yl) (I) and its derivative chloride (II), the structural formulas of which are presented in Fig. 1. The ability of CDs to form inclusion complexes with hydrophobic compounds is the basis for their application as solubilizing agents [24]. Permeation of the molecules of biologically active substances into a macrocyclic cavity and their arrangement within it are provided by nonvalent interactions (van der Waals, Table 2 The solubilities of compounds studied in HP-b-CD buffer solutions (pH 7.4) at T = 298.15 K and pressure p = 0.1 MPa. SCD (mol/L)
SDrug (104 mol/L)
I 0.000 0.005 0.029 0.046 0.051 0.075 0.087 0.098 0.113 0.179
0.06 0.12 0.50 0.76 0.83 0.12 0.14 0.16 0.18 0.27
II 0.000 0.001 0.005 0.007 0.010 0.017 0.022 0.025 0.031 0.042 0.051
0.21 0.43 2.13 3.51 5.11 8.21 11.03 13.20 16.01 21.93 29.01
Standard uncertainties are u(T) = 0.05 K, ur(S) = 0.04, u(p) = 0.05.
Fig. 3. Phase-solubility diagram of compounds I and II in the presence of 2HP-b-CD in buffer at 298 K.
hydrophobic, electrostatic, hydrogen bonding and steric effects). Besides, if the “guest” molecule has hydrophobic and hydrophilic fragments of a similar size, CDs prefer bonding with the former. The hydrophobic fragments in the compounds under study are alkyl chains and aromatic rings. UV–vis absorption spectra for the pure compounds and at the addition different concentrations of cyclodextrin have been studied in buffer pH 7.4 (Fig. 2). As it can be seen from Fig. 2, the results of UV–vis spectra how the absorption spectrum of inclusion complexes aqueous solutions coincides with the pure compounds, the maximum peaks appear at 476 nm. A solution increases with the addition of 2HP-b-CD. The similarity of the changes reveals that the inclusion complexes have been formed between b-CD and compounds I–II [25]. To study the dissolution processes of compounds I and II with 2HP-b-CD, we measured solubility of these substances in buffer solutions with different concentrations of cyclodextrin (Table 2). Fig. 3 shows the phase solubility diagrams of the compounds under consideration at the temperature of 298 K. It was determined that the maximum solubility of compound I – 2.74 104 mol/L is reached in the solution with the concentration of 2HP-b-CD 0.179 mol/L. The resulting value of solubility is greater than the initial concentration in buffer 6.32106 mol/L in 43 times. The solubility of compound II in buffer 2.12105 mol/L by adding the 2HP-b-CD solution (0.051 mol/L) increased 137 time and reached
Fig. 4. Job’s curves for complex formation of 2HP-b-CD with compounds I and II at in buffer at 298 K.
M.V. Ol’khovich et al. / Fluid Phase Equilibria 384 (2014) 68–72
Fig. 5. Kinetic dependences of solubility for compounds I and II in the presence of various concentrations of 2HP-b-CD. 3
2.91 10 mol/L. The considerable solubility increase proves that the 2HP-b-CD and the drugs interact with each other and form complexes. For all of the obtained inclusion complexes the solubility increased linearly as the 2HP-b-CD concentration grew, therefore, the plotted phase diagrams can be viewed as AL-type charts [19]. The linearity of these dependences indicates that 1:1 component ratio complexes are formed between the compounds under study and 2HP-b-CD. The stoichiometric parameters of the obtained 1:1 inclusion complexes were confirmed by the Job’s method. To do this, we plotted a diagram showing the dependence of the measured solution optical density (D), proportional to the complex formation, on the mole fractions of both components. Fig. 4 represents Job’s curves of the studied systems with the minimum solubility values observed if the mole ration is equal to 0.5 which corresponds to 1:1 complex formation. The stability constant of the 1:1 inclusion complexes were calculated based on the solubility diagram by equation: K¼
slope S0 ð1 slopeÞ
(1)
where K is stability constant; S0 is solubility of compound studied in buffer. The inclusion complex stability constants at 298 K for compounds I and II were equal to 252.94 2.78 L/mol and 2404.56 5.40 L/mol, respectively. To check the stability of the obtained complexes we studied the solubility kinetics of compounds I and II. The solubility kinetic dependences are represented in Fig. 5. As Fig. 5 shows, the inclusion complex of compound I with 2HP-b-CD is remained stable for a long time. The solubility of compound II is decreased in time in the presence of 2HP-b-CD. It was estimated that in buffer solution pH 7.4 individual compound II is hydrolyzing during 60 h Table 3 The values of the parameters of Eq. (1) and the stability constants of the complex 2HP-b-CD with a compound I in buffer pH 7.4 at pressure p = 0.1 MPa.
G (K)
S0 106 (mol/L)
Slope 103
K (L/mol)
Correlation coefficient
293.15 298.15 303.15 311.15 315.15
2.50 6.32 11.30 23.68 39.47
1.533 0.018 1.596 0.017 1.637 0.018 1.601 0.015 1.748 0.014
614.13 7.36 252.94 2.78 145.12 1.59 67.72 0.63 44.28 0.35
0.9996 0.9998 0.9994 0.9991 0.9987
Standard uncertainties are u(T) = 0.05 K, ur(S0) = 0.04, u(p) = 0.05.
71
Fig. 6. Logarithmic dependence of the stability constants of the inclusion complex 2HP-b-CD–compound I on the reciprocal temperature (R = 0.9972).
and reveals the solubility decrease from 2.12 105 mol/L to 6.32 106 mol/L. The enhanced solubility of compound II in the presence of 2HP-b-CD remains constant during a long period and does not decrease lower than that of the individual one that can be explained by the CD solubilizing action. Diagrams of phase solubility at different temperatures were plotted for the stable inclusion complex of compound I. It was determined that the compound solubility in the presence of 2HPb-CD increases as the temperature rises. This may be caused by the release of water molecules bound in the cyclodextrin cavity [26]. The values of parameters of Eq. (1) and stability constants of the obtained inclusion complexes at different temperatures are presented in Table 3. It has been determined that the stability constants decrease as the temperatures grow which is typical of the exothermic process of complexation [27,28]. The thermodynamic and structural characteristics of complexation allow us to evaluate the solubilizing and stabilizing action of cyclodextrin and the degree of changing of the physico-chemical properties of guest-molecules introduced into the cyclodextrin cavity. The thermodynamic parameters of forming the inclusion complex 2HP-b-CD – compound I were obtained from the logarithmic dependence of the stability constant on the reciprocal temperature (Fig. 6). To calculate the complexing enthalpy and entropy values, we used the integral form of the van Hoff’s equation [29]:
DH 0
lnK ¼
RT
þ
DS 0
(2)
R
where DH and DS are the enthalpy and entropy of complex formation, respectively, T is the temperature, R is the gas constant. The Gibbs energy change was calculated by equation:
DG0 ¼ RTlnK
(3)
The obtained thermodynamic parameters are shown in Table 4. The negative values of the changes in the Gibbs energy and the enthalpy indicate that the interactions of compound I and 2HPTable 4 Thermodynamic parameters of the formation complex 2HP-b-CD with a compound I in buffer pH 7.4 at 298 K.
DG (kJ mol1) DH (kJ mol1) TDS (kJ mol1)
Complex
K (L/mol)
2HP-b-CD– I
252.94 2.78 13.70 0.15
90.7 5.2
76.9 4.4
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b-CD are spontaneous and exothermic. The negative DH values indicate of quite strong molecular interactions caused by both van der Waals forces and hydrogen bond formation [30]. As the authors state [28] these interactions are thought to result from the hydrophobic guest permeation into the cyclodextrin cavity as well as the dehydration of the guest molecule. The negative changes of the complexation entropy can be explained by the reduction of the translational and rotational degrees of freedom of the guest molecule when the latter is introduced into the cyclodextrin cavity and by the fact that a more ordered system is created. The excess (in absolute magnitude) of the enthalpy term over the entropic one indicates that the inclusion process is enthalpy-determined. 4. Conclusion By using UV-spectroscopy we have studied the behavior of pharmacologically active substances in a buffer solution containing different 2HP-b-CD concentrations. The phase solubility diagrams of the studied compounds showed that drug solubility rises linearly as the 2HP-b-CD concentration increases. The linear solubility growth can be explained to the formation of inclusion complexes between compounds I, II and 2HP-b-CD with a 1:1 stoichiometric ratio. It has been found that the obtained 2HP-b-CD–compound II complex is unstable in a pH 7.4 buffer solution. The stability constants of the inclusion complex of compound I and the main thermodynamic parameters DG , DH and DS are calculated. The conclusion is made that the formation of a supramolecular 2HP-b-CD–compound I complex is an exothermal enthalpy-determined process driven by hydrogen bond formation and van der Waals interactions, particularly the dispersion attraction of the aromatic ring and alkyl chains of the molecule to the internal hydrophobic cavity of the 2HP-b-CD molecule. The research results have shown that it is possible to use 2HP-b-CD to significantly increase the solubility of the studied drugs in aqueous solutions that would reduce the necessary dosage, and hence toxicity of the proposed drugs.
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