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 -1.5 1.6 Q is the total heat Data: ManVar013_NDH obstacle is their apparent low solubility. Workers have 0.4 1.4

Model: OneSites 1.4 ΔH is the enthalpy of binding -2.0 pKa = 9.15 1.2 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 1.2 1.0 K is the binding constant N 1.11 ±0.0328 Neutral kcal/mole injectant of boronic acid containing analog (4). It was also noted that solubility could be increased by adding 1 0.0 0.8 n is the number of binding sites -2.5 K2.43E3±158 Anion ΔH -5269 ±202.9 0.8 0.6 V is the volume of the reaction cell monosaccharides to the aqueous solutions (4). Further, the formulators of Velcade® observed an increase 2468101214 ΔS -2.18 0.6 0.4 -3.0 Absorbance (AU) pH [L] is the ligand concentration in the solubility of the drug when it was lyophilized in the presence of mannitol (5). They attributed this to 0.4 Titrant Vol./1 Equiv. Vol. 0.2 [M] is the receptor concentration 0.2 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 0 77.588.599.51010.511 is fit to a modified Henderson-Hasselbach equation. soluble trimeric boroxine species shown in orange (Figure 2). A further explanation of this increased 190 210 230 250 270 290 pH -4.0 Figure 9 0123456 Wavelength (nm) 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 HO 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 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. As effect of glucose and fructose on the apparent pKa of 4-MBBA. The UV spectrophotometric 6mg/mLsolution units or greater for the highest binding polyols, when alkyl or aryl boronic acids form pKa lowering studies. Further, the binding constants observed are on the the number of hydroxyls on the polyol increases, the effect on 4-MBBA increases. The technique could not be used due to spectral interference, most likely the carbonyl group. The pKa: 9-10 pKa: ~6 mannitol experiment confirms that both the UV spectrophotometric and potentiometric titration boronate esters. Second, the rank order of the polyols’ effect is maintained. same order as those predicted from the pKa lowering studies. greatest effect comes from mannitol and xylitol, as the pKa lowering effect plateaus at techniques yield the same results, when accounting for the different concentrations used in the around pKa 6.2, almost a full 3 units below the original pKa of ~9.2. experiments. Fructose decreased the apparent pKa of 4-MBBA slightly greater than did mannitol, and much greater than did glucose. Acknowledgements References Future Work Conclusions 1. W. Yang, X. Gao, and B. Wang. Boronic acid compounds as potential pharmaceutical agents. Med Res Rev 23: The authors would like to acknowledge; Alana Toro-Ramos for her assistance in 346-68 (2003). developing the methodology for pKa determination by UV spectrophotometry, Kaisa 2. Millennium Pharmaceuticals. VELCADE® (bortezomib) for Injection PRESCRIBING INFORMATION, Kinnari for her assistance in the UV spectrophotometry studies, and Dr. Richard Cambridge, MA, 2004. •pKa determination by UV spectroscopic and potentiometric titrations have identified that some high binding Prankerd for his guidance and training in the ITC experiments. •The solid state of boronic acids and boronate esters will be studied, 3. T. Mc Cormack, W. Baumeister, L. Grenier, C. Moomaw, L. Plamondon, B. Pramanik, C. Slaughter, F. Saucy, This research was financially supported by: R. Stein, F. Zuhl, and L. Dick. Active site-directed inhibitors of Rhodococcus 20 S proteasome. Kinetics and with a particular emphasis on how to exploit the formulation of boronic polyols decrease the pKa of a boronate by over three units. The high binding polyols include some common mechanism. J Biol Chem 272: 26103-9 (1997). •The Takeru Higuchi Predoctoral Fellowship acid containing drugs with polyols in order to maximize the binding pharmaceutical excipients. •The NCI Predoctoral Training Grant 4. K. Yoshino, A. Suzuki, Y. Mori, H. Kakihana, C. Honda, Y. Mishima, T. Kobayashi, and K. Kanda. •The Takeru Higuchi–Nigel Manning Intersearch Program Improvement of solubility of p-boronophenylalanine by complex formation with monosaccharides. phenomena. Strahlentherapie und Onkologie: Organ der Deutschen Rontgengesellschaft. [et al] 165: 127-9 (1989). •This effect occurs in both aryl and alkyl boronic acids, and is maintained regardless of the starting pKa of the •The Department of Pharmaceutical Chemistry, University of Kansas 5. L. Plamondon, L. Grenier, J. Adams, and S.L. Gupta. Formulation of Boronic Acid Compounds. United States unbound boronic acid. Further, the rank order of polyol effect is maintained from alkyl to aryl boronic acids. Patent. Patent No. US 6,699,835 B2, March 2, 2004. •The solubility and dissolution of boronates will be explored in greater 6. A. Albert & E.P. Serjeant. The Determination of Ionization Constants: A Laboratory Manual. London, UK: detail to further elucidate the mechanism of boronate ester and boroxine •Isothermal titration calorimetry confirms that a boronic acid-polyol binding correlates with this pKa lowering Chapman and Hall Ltd.; 1971. dissolution to yield free boronic acids. effect. •Solubility studies show that pKa lowering of boronic acids by polyols has a large effect on the solubility of these molecules, especially near physiologic pH.