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The Scientific World Journal

Volume 2014 (2014), Article ID 803789, 6 pages

http://dx.doi.org/10.1155/2014/803789

## The Influence of pH and Temperature on the Stability of *N*-[(Piperidine)methylene]daunorubicin Hydrochloride and a Comparison of the Stability of Daunorubicin and Its Four New Amidine Derivatives in Aqueous Solutions

^{1}Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Poznan University of Medical Sciences, 6 Grunwaldzka Street, 60-780 Poznań, Poland^{2}Department of Oncological Surgery II, Greater Poland Cancer Centre, 15 Garbary Street, 61-688 Poznań, Poland^{3}Department of Modified Antibiotics, Institute of Biotechnology and Antibiotics, 5 Starościńska Street, 02-515 Warsaw, Poland

Received 29 August 2013; Accepted 24 December 2013; Published 6 February 2014

Academic Editors: A. Nokhodchi, M. Ozyazici, and C. Pichon

Copyright © 2014 Mikołaj Piekarski et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### Abstract

The influence of pH and temperature on the stability of *N*-[(piperidine)methylene]daunorubicin hydrochloride (PPD) was investigated. Degradation was studied using an HPLC method. Specific acid-base catalysis of PPD involves hydrolysis of protonated molecules of PPD catalyzed by hydrogen ions and spontaneous hydrolysis under the influence of water zwitterions, unprotonated molecules, and monoanions of PPD. The thermodynamic parameters of these reactions, energy, enthalpy, and entropy, were calculated. Also, the stability of daunorubicin and its new amidine derivatives (piperidine, morpholine, pyrrolidine, and hexahydroazepin-1-yl) in aqueous solutions was compared and discussed.

#### 1. Introduction

Anthracyclines are one of the most widely used groups of anticancer agents. The mechanism of action of anthracyclines, in addition to their multidirectional activity, involves direct intercalation of DNA, resulting in the biosynthesis of macromolecular stoppage, induction of oxidative stress inside cells by generating free radicals, combining with DNA and its alkylation, cross-linking of DNA strands, interrupting the DNA-helicase activity, direct impact on cell membranes and interruption of their activity, and induction of DNA damage and apoptosis with topoisomerase II activity stoppage. Each of these processes is closely connected with the structure of anthracyclines. Their application is limited by their dose-dependent cardiotoxicity, which was reported shortly after anthracyclines were introduced into clinical use. Another problem noted during anthracycline therapy is the growing drug resistance of cancer cells [1–4]. Anthracyclines are compounds of a glycoside structure, containing a sugar and an aglycone moiety. The sugar is usually daunosamine whereas the aglycone moiety consists of four six-carbon rings. The complex structure of anthracyclines allows many modifications of both the aglycone and the sugar moiety [5–10]. Many novel anthracycline derivatives modified in the sugar moiety were synthesized in the Institute of Biotechnology and Antibiotics, Warsaw, Poland [11]. It was proved that a group of 3′-formamide-substituted daunorubicin derivatives shows a much lower tendency to produce free radicals. Consequently, their antitumor activity, similar to that of daunorubicin, is combined with lower toxicity to the healthy cells of the body. Derivatives modified in the daunosamine moiety, in addition to anticancer activity, are able to break the resistance barrier. As those compounds do not target topoisomerase II, the cells containing the mutated enzyme are still sensitive to those compounds [12].

The new derivatives of daunorubicin containing in the amidine group a piperidine, morpholine, pyrrolidine, or hexahydroazepine moiety (Figure 1) demonstrate antiproliferative activity similar to daunorubicin [5, 13, 14]. It was found that daunorubicin was degraded in aqueous solutions and in the solid state [15, 16]. Its degradation in aqueous solution was studied at 50°C and pH 0–14. The photodegradation of daunorubicin [17] and stability of daunorubicin in infusion fluids have also been studied [18–23]. Daunorubicin stored in polypropylene syringes at 4°C after diluting with water for injections was stable at least 43 days [18]. Studies of the stability and compatibility of a mixture of etoposide, cytarabine, and daunorubicin have shown that all of them were stable in 5% glucose solution, both when separate and mixed. The greatest stability was obtained by storing the mixture in the dark at room temperature [19].

The aim of our work was to evaluate the stability of *N*-[(piperidine)methylene]daunorubicin hydrochloride (PPD) in a wide range of pH and to determine the pH range in which it is the most stable. Also, the stability of daunorubicin and its four amidine derivatives was compared.

In order to establish the observed rate constants an isocratic HPLC method was used.

#### 2. Materials and Methods

##### 2.1. Samples

*N*-[(Piperidine)methylene]daunorubicin hydrochloride (PPD) was synthesized in the Department of Modified Antibiotics, Institute of Biotechnology, Warsaw, Poland [11]. PPD is a reddish powder, freely soluble in water and methanol. Quinine hydrochloride (Sigma-Aldrich Logistik GmbH, Germany) was used as an internal standard. Sodium laurisulfate (A.C. reagent Sigma-Aldrich Logistik GmbH, Germany) and all other chemicals and solvents were obtained from Merck KGaA, Germany, and were of analytical or high-performance liquid chromatographic grade.

##### 2.2. Chromatographic Conditions

Chromatographic separation and quantitative analysis were performed by using an HPLC method [24]. The analytical system consisted of a Shimadzu SPD-20A Prominence UV/VIS detector and a Rheodyne with a 50 *μ*L loop. An LiChrospher RP-18 column (125 mm × 4 mm, 5 *μ*m particle size, Merck, Germany) was used as the stationary phase. The mobile phase consisted of a mixture of 9 volumes of acetonitrile, 1 volume of methanol, and 10 volumes of a solution containing 2.88 g L^{−1} of sodium laurisulfate and 1.6 mL L^{−1} of phosphoric acid. The flow rate was 1.5 mL min^{−1} and UV detection was performed at 254 nm. Although the method was evaluated and validated for the determination of four derivatives of daunorubicin, the selectivity was examined during a stability study of PPD.

##### 2.3. Kinetic Procedures

The degradation of PPD in aqueous solutions was studied at 313, 325, 333, and 343 K in the pH range 0.43–5, at 303, 308, and 313 K at pH above 5.5, and in sodium hydroxide solutions at 298, 303, 308, and 313 K. The pH values of the reaction solutions and those of the buffer standards were measured at reaction temperatures. The pH values of the reaction solutions in HCl and NaOH were calculated from the equations or . The activity coefficients and were obtained or calculated from the literature data [25]. The ionic strength of all the solutions was adjusted to 0.50 mol L^{−1} with a solution of sodium chloride (4 mol L^{−1}). Solutions of the desired pH and ionic strength of 0.50 mol L^{−1} were heated to the required temperatures and then a sample of PPD was added. The initial concentration of PPD was 0.2 mg mL^{−1}. At selected times, determined by the rate of degradation, samples of the solutions (0.5 mL) were collected and instantly cooled with a mixture of ice and water. Samples with pH above 7.5 were neutralized by using HCl solutions at concentrations ensuring that their pH was approximately 2. To such samples, 0.5 mL of the internal standard solution (quinine hydrochloride solution) was added. 50 *μ*L of samples of the solutions was injected in the column.

#### 3. Results and Discussion

##### 3.1. Observed Rate Constants

The observed rate constants of PPD degradation were determined in a pH range of 0.43–13.54 and were described by the equation of a pseudo-first-order reaction: where and are the time-dependent concentration and the initial concentration of PPD at time and , respectively, is the observed rate constant of the pseudo-first-order reaction of PPD degradation. The number of measurements of for each series ranged from 8 to 12.

##### 3.2. Buffer Catalysis

Under the conditions of this study, the rate constants () did not depend on the total concentrations of the phosphate, acetate, and borate buffers, which indicated that the components of the buffers did not catalyze the degradation of PPD. To verify that the differences between determined at different buffer concentrations were not statistically significant, the parallelism test was used. Since in the reaction solutions of PPD in HCl, phosphate, acetate, and borate buffers as well as in NaOH, general acid-base catalysis was not observed, in the whole range of pH the values of .

##### 3.3. pH-Rate Profiles

The rate constants determined in hydrochloric acid, sodium hydroxide, phosphate, borate, and acetate buffers were used to calculate the relationship log (Figure 2).

The semilogarithmic relationship indicated that in water solutions at pH 0.43–13.54, the following reactions occurred.(i)Degradation of protonated molecules of PPD catalyzed by hydrogen ions (),(ii)Spontaneous hydrolysis of zwitterions (), unprotonated molecules (), and monoanions () of PPD under the influence of water.

The total reaction rate was equal to the sum of partial reaction rates: where is the hydrogen ion activity and are the fractions of the molecules of PPD.

The catalytic rate constants were calculated from the plots = , which were linear with a positive slope that was equal to (Figure 3). The catalytic rate constants of the spontaneous hydrolysis of PPD monoanions were calculated as the mean values of at pH above 12. The catalytic rate constants and were calculated from the equation = using the values at pH 5.5–9 where the concentrations of forms = (Figure 4). The plots were linear and the value for = 1 was equal to the catalytic rate constant , whereas the value of for corresponded to the catalytic rate constant .

The values of , , and were calculated at 303, 308, and 313 K and next extrapolated from the Arrhenius relationship to 323, 333, and 343 K. The calculated theoretical profile of = (pH) and that obtained from the experimental results were nearly identical, indicating that the choice of the equation describing the total rate of PPD degradation was correct (Figure 2).

##### 3.4. Influence of Temperature

Based on the Arrhenius relationship ln , linear plots of ln were used to calculate the energy of activation (), the entropy () and enthalpy (), and the preexponential coefficient () for the partial reactions (Table 1). The lowest energy of activation was observed in the reaction of spontaneous hydrolysis of PPD zwitter ions. The entropy of all reactions under the influence of water (spontaneous hydrolysis) was negative, which suggested the bimolecular character of these reactions. The positive values of entropy for the reactions of protonated molecules of PPD catalyzed by hydrogen ions indicated a positive participation of entropy of protonation reaction.

The linear relationships of and (Figure 5) were obtained for the degradation of protonated molecules of PPD catalyzed by hydrogen ions and spontaneous hydrolysis of , PPD, and molecules under the influence of water, which suggested that all reactions occurred according to the same mechanism of a bimolecular reaction.

##### 3.5. Influence of Ionic Strength

The influence of ionic strength was studied in hydrochloric acid (0.10 mol L^{−1}, 343 K) and sodium hydroxide solution (0.10 mol L^{−1}, 308 K) and interpreted according to the Brönsted-Bjerrum equation. It was found that in the solutions of hydrochloric acid a positive salt effect was observed (the slope () of the relationship log was 1.29 ± 0.63, whereas in the sodium hydroxide solutions no influence of the ionic strength was observed (). These results confirmed that in hydrochloric acid the degradation of PPD occurred as a reaction of protonated molecules of PPD catalyzed by hydrogen ions and in the sodium hydroxide solutions as a spontaneous hydrolysis of PPD under the influence of water.

#### 4. A Comparison of the Stability of Daunorubicin and Its Four New Amidine Derivatives in Aqueous Solutions

Specific acid-base catalysis of daunorubicin involves reactions catalyzed by hydrogen and hydroxyl ions and spontaneous hydrolysis under the influence of water depending on the DAU charge [15] whereas specific acid-base catalysis of the new amidine derivatives of DAU comprises hydrolysis of protonated molecules and spontaneous hydrolysis under the influence of water depending on the charge of the derivatives [26–28]. In the case of amidine, DAU derivatives hydrolysis catalyzed by hydroxide ions is not observed.

By comparing the relationship log for daunorubicin (DAU) and for its four amidine derivatives, pyrrolidine (PMD), morpholine (MMD), piperidine (PPD), and hexahydroazepin-1-yl (HMD) [15, 26–28] (Figure 6), it is possible to conclude the following:(i)daunorubicin demonstrates the greatest stability in the pH range 4–6;(ii)in the entire pH range daunorubicin is the most stable compound;(iii)the smallest differences in the rate of degradation of DAU and its four derivatives are observed in an acidic environment, where the dominant reaction is the degradation of protonated molecules catalyzed by hydrogen ions;(iv)significant differences in the degradation rate are observed in spontaneous hydrolysis under the influence of water;(v)in the pH range 4–6, in which DAU demonstrates the greatest stability, its derivatives undergo degradation at a greater rate from 3 (PMD) to 5 orders (MMD) of magnitude.(vi)MMD demonstrates the greatest stability at pH about 2.5 whereas PPD, PMD, and HMD at about 3.5;(vii)in the pH range 6–10 the stability of DAU and its four derivatives compares as follows: DAU ≫ PMD > PPD > HMD > MMD.

#### Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

#### References

- M. Piekarski and A. Jelińska, “Anthracyclines still prove effective in anticancer therapy,”
*Mini-Reviews in Medicinal Chemistry*, vol. 13, no. 5, pp. 627–634, 2013. View at Publisher · View at Google Scholar - G. N. Hortobágyi, “Anthracyclines in the treatment of cancer. An overview,”
*Drugs*, vol. 54, no. 4, pp. 1–7, 1997. - G. Minotti, P. Menna, E. Salvatorelli, G. Cairo, and L. Gianni, “Anthracyclines: molecular advances and pharmacologie developments in antitumor activity and cardiotoxicity,”
*Pharmacological Reviews*, vol. 56, no. 2, pp. 185–229, 2004. View at Publisher · View at Google Scholar · View at Scopus - R. B. Weiss, “The anthracyclines: will we ever find a better doxorubicin?”
*Seminars in Oncology*, vol. 19, no. 6, pp. 670–686, 1992. View at Scopus - M. Wasowska, J. Wietrzyk, A. Opolski, J. Oszczapowicz, and I. Oszczapowicz, “Effect of structural modifications of anthracyclines on the ability to overcome drug resistance of cancer cells,”
*Anticancer Research*, vol. 26, no. 3, pp. 2009–2012, 2006. View at Scopus - J. Madej, S. Dzimira, and M. Wąsowska, “New derivatives of anthracycline antibiotics with lower cardiotoxicity,”
*Journal Chemotherapy*, vol. 16, pp. 154–158, 2004. - M. Ripamonti, L. Capolongo, G. Melegaro et al., “Morpholinylanthracyclines: cytotoxicity and antitumor activity of differently modified derivatives,”
*Investigational New Drugs*, vol. 14, no. 2, pp. 139–146, 1996. View at Scopus - E. Fan, W. Shi, and T. L. Lowary, “Synthesis of daunorubicin analogues containing truncated aromatic cores and unnatural monosaccharide residues,”
*Journal of Organic Chemistry*, vol. 72, no. 8, pp. 2917–2928, 2007. View at Publisher · View at Google Scholar · View at Scopus - Y.-G. Gao, W. Priebe, and A. H. Wang, “Substitutions at C2′ of daunosamine in the anticancer drug daunorubicin alter its DNA-binding sequence specificity,”
*European Journal of Biochemistry*, vol. 240, no. 2, pp. 331–335, 1996. View at Scopus - J. Pawłowska, J. Tarasiuk, E. Borowski, M. W̧sowska, I. Oszczapowicz, and C. R. Wolf, “The ability of new formamidine sugar-modified derivatives of daunorubicin to stimulate free radical formation in three enzymatic systems: NADH dehydrogenase, NADPH cytochrome P450 reductase and xanthine oxidase,”
*Acta Biochimica Polonica*, vol. 47, no. 1, pp. 141–147, 2000. View at Scopus - M. Wąsowska, J. Oszczapowicz, I. Oszczapowicz, and A. Owoc, Patent C07H 15/252, 2006.
- F. Arcamone, F. Animati, G. Capranico et al., “New developments in antitumor anthracyclines,”
*Pharmacology and Therapeutics*, vol. 76, no. 1–3, pp. 117–124, 1997. View at Publisher · View at Google Scholar · View at Scopus - M. Wasowska, I. Oszczapowicz, J. Wietrzyk et al., “Influence of the structure of new anthracycline antibiotics on their biological properties,”
*Anticancer Research*, vol. 25, no. 3, pp. 2043–2048, 2005. View at Scopus - E. Ciesielska, K. Studzian, M. W̧sowska, I. Oszczapowicz, and L. Szmigiero, “Cytotoxicity, cellular uptake and DNA damage by daunorubicin and its new analogues with modified daunosamine moiety,”
*Cell Biology and Toxicology*, vol. 21, no. 3-4, pp. 139–147, 2005. View at Publisher · View at Google Scholar · View at Scopus - J. H. Beijnen, O. A. G. J. Van der Houwen, M. C. H. Voskuilen, and W. J. M. Underberg, “Aspects of the degradation kinetics of daunorubicin in aqueous solution,”
*International Journal of Pharmaceutics*, vol. 31, no. 1-2, pp. 75–82, 1986. View at Scopus - J. Cielecka-Piontek, A. Jelińska, M. Zajac, M. Sobczak, A. Bartold, and I. Oszczapowicz, “A comparison of the stability of doxorubicin and daunorubicin in solid state,”
*Journal of Pharmaceutical and Biomedical Analysis*, vol. 50, no. 4, pp. 576–579, 2009. View at Publisher · View at Google Scholar · View at Scopus - M. J. Wood, W. J. Irwin, and D. K. Scott, “Photodegradation of doxorubicin, daunorubicin and epirubicin measured by high-performance liquid chromatography,”
*Journal of Clinical Pharmacy and Therapeutics*, vol. 15, no. 4, pp. 291–300, 1990. View at Scopus - M. J. Wood, W. J. Irwin, and D. K. Scott, “Stability of doxorubicin, daunorubicin and epirubicin in plastic syringes and minibags,”
*Journal of Clinical Pharmacy and Therapeutics*, vol. 15, no. 4, pp. 279–289, 1990. View at Scopus - R. Chevrier, V. Sautou, V. Pinon, F. Demeocq, and J. Chopineau, “Stability and compatibility of a mixture of the anti-cancer drugs etoposide, cytarabine and daunorubicine for infusion,”
*Pharmaceutica Acta Helvetiae*, vol. 70, no. 2, pp. 141–148, 1995. View at Publisher · View at Google Scholar · View at Scopus - J. H. Beijnen, H. Rosing, A. De Vries Ph., and W. J. M. Underberg, “Stability of anthracycline antitumour agents in infusion fluids,”
*Journal of Parenteral Science and Technology*, vol. 39, no. 6, pp. 220–222, 1985. View at Scopus - G. K. Poochikian, J. C. Cradock, and K. P. Flora, “Stability of anthracycline antitumor agents in four infusion fluids,”
*American Journal of Hospital Pharmacy*, vol. 38, no. 4, pp. 483–486, 1981. View at Scopus - J. Karlsen, H. Thonnensen, O. Resberg, S. Horne, and T. J. Skobba, “Stability of cytotoxic intravenous solutions subjected to freeze-thaw treatment,”
*Norvegica Pharmaceutica Acta*, vol. 45, no. 2, pp. 61–67, 1983. View at Scopus - J. Cielecka-Piontek, A. Jelińska, A. Dołhań et al., “Stability of new anticancer agents in intravenous solutions,”
*Asian Journal of Chemistry*, vol. 24, no. 2, pp. 769–772, 2012. - M. Lłukawska, I. Oszczapowicz, A. Jelińska et al., “Development and validation of RP-HPLC method for determination of novel derivatives of daunorubicin,”
*Chemia Analityczna*, vol. 54, no. 5, pp. 907–917, 2009. View at Scopus - E. Pawełczyk and T. Hermann,
*The Fundamentals of Stability of Drugs*, PZWL, Warsaw, Poland, 1982 (Polish). - A. Krause, A. Jelińska, J. Cielecka-Piontek et al., “Acid-base catalysis of
*N*-[(morpholine)methylene]-daunorubicin,”*Drug Development and Industrial Pharmacy*, vol. 38, no. 8, pp. 1024–1028, 2012. View at Publisher · View at Google Scholar - P. Zalewski, M. Zajac, A. Jelinska, J. Cielecka-Piontek, and I. Oszczapowicz, “Stability study of anticancer agent
*N*-[(Hexahydroazepin-1-yl)methyl] daunorubicin in Aqueous Solutions Using HPLC method,”*Asian Journal of Chemistry*, vol. 23, no. 2, pp. 835–838, 2011. View at Scopus - A. Jelińska, J. Uszak, J. Cielecka-Piontek et al., “Stability of [(
*N*-pyrrolidine)metylene]daunorubicin in aqueous solutions,”*Reaction Kinetics and Catalysis Letters*, vol. 98, no. 1, pp. 69–75, 2009. View at Publisher · View at Google Scholar · View at Scopus