Synthesis and Pharmacological Effects of Diosgenin–Betulinic Acid Conjugates

The target diosgenin–betulinic acid conjugates are reported to investigate their ability to enhance and modify the pharmacological effects of their components. The detailed synthetic procedure that includes copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition (click reaction), and palladium-catalyzed debenzylation by hydrogenolysis is described together with the results of cytotoxicity screening tests. Palladium-catalyzed debenzylation reaction of benzyl ester intermediates was the key step in this synthetic procedure due to the simultaneous presence of a 1,4-disubstituted 1,2,3-triazole ring in the molecule that was a competing coordination site for the palladium catalyst. High pressure (130 kPa) palladium-catalyzed procedure represented a successful synthetic step yielding the required products. The conjugate 7 showed selective cytotoxicity in human T-lymphoblastic leukemia (CEM) cancer cells (IC50 = 6.5 ± 1.1 µM), in contrast to the conjugate 8 showing no cytotoxicity, and diosgenin (1), an adaptogen, for which a potential to be active on central nervous system was calculated in silico. In addition, 5 showed medium multifarious cytotoxicity in human T-lymphoblastic leukemia (CEM), human cervical cancer (HeLa), and human colon cancer (HCT 116). Betulinic acid (2) and the intermediates 3 and 4 showed no cytotoxicity in the tested cancer cell lines. The experimental data obtained are supplemented by and compared with the in silico calculated physico-chemical and absorption, distribution, metabolism, and excretion (ADME) parameters of these compounds.

2 1.1. Applied methods of conversion of 7 to 8, graphical image Scheme S1. Applied methods of conversion of 7 to 8.

Physico-chemical and ADME characteristics
The partition coefficient (log P) and the distribution coefficient (log D) are the most important molecular descriptors [S1]. In chemical and pharmaceutical sciences, both, log P and log D are the measures of hydrophilicity or hydrophobicity of the studied compound, and are useful for estimating distribution of a drug within the body. The parameter log D shows the dependence on the pH of the matrix. Hydrophobic drugs are then preferentially distributed to hydrophobic compartments (e.g., lipid bilayer of cells), while hydrophilic drugs are preferentially distributed to hydrophilic compartments (e.g., blood serum). The distribution coefficient is a pH dependent value, and, therefore, the value at pH = 7.4 (the physiological 3 pH value of blood serum) is of particular importance (see also Table 2). Thus, log P expresses a ratio of concentrations of non-ionized compound between two phases, non-polar (octanol) and polar (water), while log D expresses the ratio of the sum of the concentrations of all forms of the compound (ionized and non-ionized) in each of the two phases. In pharmacology, log P and log D indicate how easily the drug can reach its intended target in the body, how strong its effect will be once it reaches its target and how long it will remain in the body in an active form. The log P values calculated for 1, 2 and 5-8 exceed the values given by the Lipinski  (Table 4). Another supportive parameter is the predicted aqueous solubility, log S. The parameter S (in mol dm -3 ) is the concentration of the solute in a saturated solution that is in equilibrium with the crystalline solid, and it is a pH dependent parameter.
Standard range for log S at pH 7.4 is -6.5/+0.5. Among the studied compounds, only 1, 2, 5 and 6 show values for log S in this range (Table 4).
The importance of some of the ADME parameters for evaluation of pharmacokinetic properties of the prepared compounds is also summarized in Table 2. The blood brain barrier (BBB) and plasma protein binding are two of the important factors affecting distribution of the compound in the human body. Several parameters assist in evaluation of each potential drug for its BBB transport [S2]. The rate of brain penetration, log PS, is a logarithm of the permeability-surface area coefficient that measures the ability of a drug to cross the BBB and to move into brain tissue over time. It is one of the relevant parameters for evaluation of the rate of BBB penetration. The extent of brain penetration parameter, log PB, indicates if the drug might be active or inactive on the central nervous system (CNS). The typical values for log PB are -1.5/+1.5. Most of the current drugs show the log PB value up to +2 (active on CNS) or down to -2 (inactive on CNS). The log PB values calculated for 1, 2 and 5-8 indicate that 1 is slightly active on CNS, while 2 and 5-8 are inactive on CNS (Table 2; Fig.   S1 and Fig. S2). However, the complex of the BBB parameters is completed by the brain/plasma equilibration rate, the parameter expressed as log PS * fu,brain that is a mathematical modeling parameter based on time required for reaching brain equilibrium. It is dependent on the brain unbound fraction (fu,brain). This value indicates if the drug may potentially be active on CNS together with the log BB parameter, the predicted brain/blood partition coefficient. The parameter log BB is a hybrid parameter determined by permeability, plasma and brain tissue binding, and active transport mechanism, standard range -3.0/+1.2.
The log BB values calculated for 1 and 5-8 appear in the required range of this parameter.
Nevertheless, there are numerous exceptions already known.
Bioavailability represents another important ADME parameter. Among the studied compounds, the calculated bioavailability for 5, 7 and 8 is lower than that for 1, 2 and 6.
However, the experimental data show that only 5 and 7 showed cytotoxicity to the tested cancer cell lines.
Plasma protein binding (PPB) gives calculated quantity of a drug bound to a protein.
The Lipinski [28] and Ghose [29] rules describe molecular properties important for a small molecule drug pharmacokinetics in the human body, including their absorption, distribution, metabolism and excretion (ADME parameters). The importance of several ADME parameters, namely blood-brain barrier and plasma protein binding, reflects the distribution of the compound in the human body [S3]. A comparison of the calculated physico-chemical properties with the measured cytotoxicity shows that every time a new class of compound is being investigated, no available experimental screening may be intentionally omitted (Tables   3 and 4).

Calculated activity on central nervous system (CNS)
Fig. S1 shows the calculated activity of diosgenin (1) and betulinic acid (2) on central nervous system. Blue dots represent currently used drugs that are CNS active, while orange dots 5 represent currently used drugs that are CNS inactive. The green dot shows the calculated position of diosgenin (1). The calculation was made through the ACD/Labs software [27]. In the below shown graph, log PS * fu,brain (the brain/plasma equilibration rate) appears on the xaxis, and log BB (a hybrid parameter determined by permeability, plasma and brain tissue binding, and active transport mechanism) appears on the y-axis. Based on the position of the green dot, diosgenin (1) displays certain potential to become active on central nervous system, which result of the calculation is in good agreement that 1 was determined as adaptogen.
In turn, the compounds 5 and 7 appeared to have no CNS activity at all, as shown in the Fig.   S2.