Physical and Chemical Properties of Boronic Acids: Formulation Implications William A. Marinaro and Valentino J. Stella Department of Pharmaceutical Chemistry, The University of Kansas Isothermal Titration Purpose Solubility Calorimetry The objective of the following research is to determine the effect of various polyols on the physical and Figure 13 chemical properties of two model boronic acids; 4-methoxybenzeneboronic acid (4-MBBA) shown in Figure 4 Solubility of 4-MBBA Vs. pH Figure 5 Figure 4 demonstrates the Figure 6 ITC Instrumental Diagram Figure 14 Solubility Vs. 1/[H+] large effect mannitol has on the structure 1, and isobutylboronic acid (IBA) illustrated in structure 2. The research will quantify the ability of Solubility Vs. 1/[H+] Isothermal titration calorimetry 30mM Mannitol Titrated into 35 No Mannitol (solubility solubility of 4-MBBA. Highlighted 1mM 4-MBBA at pH 11.6 the polyols, first, to form boronate esters with the model pH mg/mL) 35 500mM Mannitol or ITC, is a calorimetric 30 (solubility mg/mL) in green is the effect at physiologic analytical techniques which Time (min) Structure 1 Structure 2 0.00 2.00 4.00 6.00 8.00 10.00 12.00 30 compounds. Second, to determine the effect that this pH, a 10-fold increase in solubility. -10 0 10 20 30 40 50 60 70 80 90100110120130140150160170180 HO 100 25 measures the heat evolved or OH formation has on the pKa and solubility of the boronate Equation 1 25 pKa’: 5.87 Figures 5 and 6 illustrate the direct absorbed upon titrating a 0 B O 20 Ka[HA]0 B 20 compound. This information should provide crucial 10 S = [HA]0 + + effect on the pKa as determined solution of ligand into a solution -5 HO HO 15 [H ] 15 from the solubility pH profile, as of receptor. The instrumental -10 insights to the formulation chemist when developing 4-Methoxybenzeneboronic acid Isobutylboronic acid 1 Solubility (mg/mL) 10 setup is shown in figure 13. As Solubility (mg/mL)10 described by equation 1. Through (4-MBBA) µcal/sec (IBA) boronic acid containing pharmaceuticals. 5 pKa’: 6.39 the mole ratio increases during -15 5 figure 6 one can see that the ratio of Solubility (mg/mL) Solubility 0.1 pKa’: 8.69 the titration and there is less free 0 -20 0 mannitol to boronic acid has an 0.0E+00 5.0E+09 1.0E+10 1.5E+10 receptor, the total heat per No Mannitol (solubility mg/mL) 500mM Mannitol (solubility mg/mL) 0.0E+00 2.0E+07 4.0E+07 6.0E+07 8.0E+07 effect on solubility through pKa. 1/[H+] (M-1) 1/[H+] (M-1) injection decreases, as shown in figure 14. This profile is 0 described, for a molecule with a Background single set of receptors, by -2 equation 2. Thus, the data may be fit, as in figure 15, and one may solve for the variables: N, kcal/mole of injectant Boronic acid compounds are the focus of increasing interest as therapeutic agents due to their ability -4 K, ΔH, and ΔS 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 to inhibit enzyme activity, among other characteristics. Many peptide boronic acid compounds have been Effect of Polyols on Boronic Acid pKa Values Figure from User’s Manual Revision 12705, Molar Ratio Calorimetry Sciences Corp., Lindon, UT investigated as enzyme inhibitors with implications in pathologies as varied as Alzheimer’s disease, cervical cancer, blood clotting disorders, hepatitis C, etc. (1). In 2003 the drug Velcade® (bortezomib), figure 1, Figure 15 30mM Mannitol Titrated into was approved by the FDA as a second-line treatment for multiple myeloma, it acts via inhibition of the 26S 1mM 4-MBBA at pH 11.6 Potentiometric titrations were also performed using a Mettler-Toledo proteasome in mammalian cells (2). As shown in scheme 1, the role of the boronic acid moiety in this A large change in the UV spectra of 4-MBBA is observed at 1.4 235nm, this was utilized in pKa determination by UV DL-53 autotitrator and the pKa values were calculated as described Equation 2 0.0 1.2 inhibition is to interact with the hydroxyl of the N-terminal threonine of the β-subunit of the proteasome (3). spectrophotometric titrations (Figure 7). Figure 8 by Albert and Serjeant (6). Figure 9 shows the type of data ⎡ 2 ⎤ 1+ []M ⋅n⋅ K − ()1+ []M ⋅n⋅ K − []L ⋅ K + 4⋅ K ⋅[]L -0.5 While the pharmacologic properties of these 1.0 generated, in this case 4-MBBA alone yields a pKa of 9.31±0.01. Q = V ⋅ΔH ⋅ ⎢[]L + ⎥ 2⋅ K OH OH ⎢ ⎥ Pharmacophore 0.8 ⎣ ⎦ B B Pharmacophore compounds appears to be impressive, there are obstacles -1.0 Peptide HO Peptide OH O Figure 7 UV Spectra of 4-MBBA, Neutral and Anion to their formulation into suitable dosage forms. One 0.6 Where: Scheme 1 1.4 -1.5 1.6 Q is the total heat Data: ManVar013_NDH 0.4 obstacle is their apparent low solubility. Workers have 1.2 1.4 Model: OneSites ΔH is the enthalpy of binding -2.0 1.2 pKa = 9.15 1.0 Chi^2/DoF = 1527 observed solubility values for boronic acid containing compounds well below that of the respective non- Absorbance at 235nm (AU) 0.2 K is the binding constant 1 N 1.11 ±0.0328 Neutral 0.8 kcal/mole injectant of boronic acid containing analog (4). It was also noted that solubility could be increased by adding 0.8 0.0 n is the number of binding sites -2.5 K2.43E3±158 0.6 0.6 Anion V is the volume of the reaction cell ΔH -5269 ±202.9 monosaccharides to the aqueous solutions (4). Further, the formulators of Velcade® observed an increase 2468101214 0.4 ΔS -2.18 0.4 -3.0 Absorbance (AU) pH [L] is the ligand concentration 0.2 0.2 in the solubility of the drug when it was lyophilized in the presence of mannitol (5). They attributed this to Titrant Vol./1 Equiv. Vol. 0 [M] is the receptor concentration The titration of 4-MBBA is shown (Figure 8). The curve 0.0 -3.5 the formation of boronic acid esters, shown in green, and the simultaneous avoidance of forming the less 190 210 230 250 270 290 77.588.599.51010.511 Wavelength (nm) is fit to a modified Henderson-Hasselbach equation. soluble trimeric boroxine species shown in orange (Figure 2). A further explanation of this increased pH -4.0 Figure 9 0123456 solubility is a pKa lowering effect that polyols impart to boronic acids (Figure 3). The mechanism by Molar Ratio which polyols help solubilize boronic acid containing compounds is not completely understood and Figure 15 deserves further study. Interaction of various polyols with 4-methoxybenzene boronic Figure 10Apparent pKa of 4-MBBA Vs. Polyol Concentration Figure 11 Figure 12 Apparent pKa of 4-MBBA Vs. Polyol Concentration by Apparent pKa of IBA Vs. Polyol Concentration by Potentiometric acid as the receptor in ITC experiments UV Spectrophotometric Titration Potentiometric Titration Titration Figure 2 9.4 Figure 1 9.50 2,3- O OH OH 8.9 12.00 9.00 H 11.50 N N B R 8.4 Glycerin 8.50 Ligand: mannitol sorbitol erythritol glycerol butanediol N OH OH 11.00 H B 7.9 Erythritol 8.00 Glucose 10.50 Glucose O O O 7.50 Mannitol 10.00 Mannitol N O OH 7.4 Xy li t ol 7.00 Fructose 9.50 Fructose B B Mannitol 6.9 pKa Apparent -1 R O R B 6.50 9.00 Sorbitol K (M ) ±SD 2767±39 6254±605 118±NA 23.7±0.7 13.2±0.8 Apparent pKa Apparent R O OH 8.50 solid 6.4 6.00 Structure of Velcade® (bortezomib) 4-MBBA Apparent pKa 8.00 solid 5.50 5.9 7.50 0 0.1 0.2 0.3 0.4 0.5 0.6 HO OH 0 0.1 0.2 0.3 0.4 0.5 0.6 0 0.1 0.2 0.3 0.4 0.5 0.6 OH Polyol Concentration (M) Polyol Concentration (M) HO n ±SD 1.05±0.00 1.07±0.08 0.84±NA NA NA Polyol Concentration (M) HO OH Figure 3 HO HO Sorbitol HO OHHO O OHHO HO H O OH CH2 CH2 OH O OH HO O H H OH ΔH (kJ/mol) O OH H HO HO OH HO OH H OH HO OH OH H HO OH OH ±SD -21.5±1.9 -26.7±0.6 -24.3±NA NA NA R B + R R B R HO OH OH H OH OH OH HO H OH HO OH HO Fructose Figure 12 applies the potentiometric technique to the study of IBA an alkyl boronic HO OH OH HO OH Glucose OH HO O B B Glycerin Erythritol Xylitol Mannitol acid. It is critical to demonstrate that this phenomenon occurs with alkyl boronic R OH R OH acids, because most boronic acid containing drugs are alkyl, not aryl boronic acids. The ITC data confirms the rank order of polyol effects observed during Figure 11 illustrates the utility of the potentiometric technique, described above, in studying the First, this series of experiments shows that a similar drop in pKa occurs, roughly 3 boronic acid boronate ester < 1mg/mL solution Figure 10 demonstrates the correlation between polyol size and pKa lowering effect.