Seeking Polymeric Prodrugs of Norfloxacin. Part 2. Synthesis and Structural Analysis of Polyurethane Conjugates

Oligo(ε-caprolactone) and oligolactide were synthesized via ring-opening polymerization of cyclic esters in the presence of creatinine as initiators. Thus obtained oligomers were successfully used in the synthesis of novel polyurethane conjugates of norfloxacin. The structures of the polymers and conjugates were elucidated by means of MALDI-TOF MS, NMR and IR studies.


Introduction
Pharmacy is one of the most important areas of applications of polymers. They are used as active macromolecular pharmaceutical substances, blood substitutes, auxiliary materials and excipients, reagents for synthesis of macromolecular prodrugs, in polymeric drug delivery systems, therapeutic systems, etc. [1][2][3][4][5][6][7][8][9][10][11]. Aliphatic polyurethanes have good biodegradability and biocompatibility properties. These attributes make them extremely useful in medical and pharmaceutical industries, for example to produce implants containing controlled drug delivery systems [12,13].
Aliphatic polyurethanes are usually prepared by reaction of aliphatic diisocyanate with compounds having two reactive hydrogen atoms (oligomers) reaction. Alternatively, aliphatic polyurethanes can OPEN ACCESS be prepared by non-isocyanate methods, if five-membered cyclic carbonate groups are reacted with diamines. Aliphatic polyurethanes can be synthesized by ring-opening polymerization of cyclic urethane, too [14][15][16][17][18]. Polyesters, polyethers and polycarbonates are usually used as oligomers. They were successfully synthesized by ring opening polymerization of cyclic monomers in the presence of cationic or anionic initiators, as well as coordinating and enzymatic catalysts [19][20][21][22][23][24][25][26][27][28][29][30][31]. Recently, Wang has found that creatinine, a non-toxic metabolite in the human body, shows rather satisfactory catalytic properties for polymerization of L-lactide [32]. Creatinine is a break-down product of creatine phosphate in the muscle tissue, and is usually produced in the body at a fairly constant rate (depending on the muscle mass). Chemically, creatinine is a spontaneously formed, cyclic derivative of creatine. Creatinine is chiefly filtered out of the blood by the kidneys.
Fluoroquinolones comprise an important new class of synthetic oral antibacterial agents used against various infections. They efficiently kill bacteria and/or prevent their growth. The first discovered and clinically effective quinolone was norfloxacin, that is 1-ethyl-6-fluoro-1,4-dihydro-4oxo-7-(1-piperazinyl)-3-quinolinecarboxylic acid [33]. Recently, the interaction of norfloxacin with DNA has been considered as useful in the anticancer drug design [33]. Preliminary efforts to prepare nanoparticles of poly(D,L-lactide-co-glycolide) loaded with adsorbed norfloxacin has already been reported [34]. The anticancer action of norfloxacin is supposed to be improved by anchoring the drug, using a chemical linkage, to biodegradable oligomers. Such delivery systems can transport the drug molecules more efficiently and more specifically. We would like to follow the latter scientific direction.
In this paper, we describe the synthesis of a series of linear polyurethane conjugates of norfloxacin. Chemical structures of the synthesized polymers have been confirmed using 1 H-and 13 C-solution NMR and FT-IR spectroscopy. Molecular weights of polymers have been determined using gel permeation chromatography (GPC) and a viscosity method. The present paper is the continuation of our previous work [35]. We believe that the obtained polyurethanes can find practical applications as effective drug delivery systems transporting active substances to specific body locations at the required rate.

Ring-opening polymerization of cyclic esters
The first test of L-lactide (L-LA) polymerization in the presence of creatinine (CE) was described by Wang [32]. Thus obtained poly(L-lactide) (PLLA), terminated by hydroxyl and carboxyl groups, had M n = 6,700-14,000 Da and narrow polydispersity (PD = 1.20-1.57). We have decided to carry out more detailed investigation of that reaction by extending the range of monomers to ε-caprolactone (CL) and rac-lactide (D,L-LA). The aim was to obtain a low-molecular weight polyesters, terminated at both sides by hydroxyl groups, which can be subsequently used as segments in polyurethane conjugates.  The polymerization reactions of CL, D,L-LA and L-LA in the presence of CE and ethylene glycol (EG) were carried out at 160 ºC for 96 h. Under these conditions the cyclic monomers underwent ring-opening polymerizations to give low-molecular weight polyesters terminated by hydroxyl chainend groups (Scheme 1, Table 1).
The chemical structures of the obtained polymers were confirmed by 13 C-, 1 H-NMR and IR studies. Figures 1 and 2 show typical MALDI-TOF spectra of the obtained products.   (Table 1, run no. 9).
The MALDI-TOF spectrum of PCL comprises two series of peaks. The most prominent series of peaks is characterized by a mass increment of 114 Da, which is equal to the mass of the repeating unit in the PCL polymer ( Figure 1). This series is assigned to PCL terminated with a hydroxyl group and detected as the Na + adduct (residual mass: RM = 41 Da). The second series of the peaks is also from PCL terminated with a hydroxyl group, but corresponds to the K + adduct (RM = 57 Da).
The MALDI-TOF spectrum of PLA comprises two series of peaks, too ( Figure 2). The main series comes from PLA terminated with a hydroxyl group and corresponds to the Na + adduct (RM = 42 Da), while the second series of smaller peaks is also from PLA terminated with a hydroxyl group, but corresponds to the K + adduct (RM = 57 Da). In the MALDI-TOF spectrum of PLA both populations of chains of even and odd number of lactic acid m.u. can be observed. The odd number of acid m.u. shows that under the process conditions the polymer chain undergoes intermolecular transesterification (leading to an exchange of segments), which is a typical phenomenon for the polymerization of lactides [20]. Formation of PCL and PLA macrocycles was not observed.
The number-average molecular weights determined from GPC for CL oligomers lie in the 3,100-4,900 Daltons range, and the polydispersity indexes in the 1. The mechanism of the ring-opening polymerization of L-LA in the presence CE was proposed by Wang [32]. The coordination-insertion mechanism was postulated. The kinetic and mechanistic studies of the polymerization of cyclic monomers in the presence of CE are underway in our laboratory, too. The results are to be presented in next papers.
As polyurethane segments, we used PCL diols of M n = 2,400 and 2,600 Da (Table 1), PLA diols of M n = 1,800 and 2,000 Da, and commercially available OEAD of M n = 1,000 Da and OCL of M n = 2,000 Da. The HDI:oligodiol:NOR molar ratio was always 2:1:1, which corresponds to a so called isocyanate index equal to 1. Stannous octoate (SnOct 2 ) or dibutyltin dilaurate (DLDBSn) were applied as the polyaddition catalysts. The reaction conditions and molecular weight of the obtained products are listed in Table 2. We found that the synthesized by us and commercial polyesters diols underwent polyaddition giving polyurethanes of M v = in the range of 14,200-42,800 Da, as determined using the viscosity method. The degree of polyaddition was dependent on the kind of catalyst used. Then, in all the systems studied, the polyurethanes obtained in the presence of DLDBSn were characterized by higher molecular weights than those prepared using SnOct 2 . Scheme 2. The synthesis scheme of polyurethane conjugates (two-step process). The chemical structures of the prepared polymers were confirmed by 13 C, 1 H-NMR and IR studies. Figure 3 presents typical 1 H-NMR spectrum of the obtained polyurethane conjugate.
Preliminary studies on the release of the norfloxacin from polyurethane conjugates have made. It has turned out that the drug is gradually released from the obtained polyurethane conjugates (Figure 4).  Reaction conditions: temp. 65 °C, time -3h (for the one-step process) or 6h (for the two-step process), molar ratio HDI: macrodiol: NOR: catalyst = 2:1:1:0.01; PCL1 (M n = 2,600 Da), PCL2 (M n = 2,400 Da), PLA1 (M n = 2,000 Da), PLA2 (M n = 1,800 Da); * I -one-step process, II -twostep process; M v -average molecular weight from the viscosity measurements; ** -calculated by the weight method.     (Table 3, Run no. 3, degradation in phosphate buffer, after 21 days). Table 3 shows the degree of norfloxacin released from of the selected polyurethane conjugates as function of time under mild conditions in HCl buffer (pH=1) and phosphate buffer (pH=7,4), respectively. Preliminary results show the rate of norfloxacin release from polyurethane conjugates depends on the structure of the polymer and the order of hydrolysis is as follows: poly(lactide-polyurethane) > poly(ethylene adipate-polyurethane) > poly(ε-caprolactone-polyurethane). The results suggest a high stability of obtained polyurethane conjugates to chemical hydrolysis at pH 7,4 than 1.
Kinetics of the NOR release from polyurethane conjugates are still under study and will be presented in the next paper. We shall also discuss correlation between the structure of the polyurethane conjugates and the drug release rate.

Instrumentation
The polymerization products were characterized by means of 1 H-and 13 C-NMR (Varian 300 MHz), and FTIR spectroscopy (Spectrum 1000, Perkin Elmer). The NMR spectra were recorded in CDCl 3 or DMSO-d 6 . The IR spectra were recorded from KBr pellets. Relative molecular mass and molecular mass distributions were determined using MALDI-TOF MS, GPC and viscosity techniques. The MALDI-TOF spectra were measured in the linear mode on a Kompact MALDI 4 Kratos analytical spectrometer using a nitrogen gas laser with 2-[(4-hydroxyphenyl)diazenyl] benzoic acid (HABA) as a matrix [the samples were dissolved in CHCl 3 (10 mg/mL) and THF (20 mg/mL)]. The average molecular mass and the molecular mass distribution of the macromolecular conjugates were measured by means of the GPC technique (LabAlliance) at 25 °C (the samples were dissolved in chloroform). The device was calibrated with polystyrene standards.
The hydroxyl number of the obtained polycarbonate diols was determined according to the conventional method, based on the reaction with acetic acetate [15].
The amount of released norfloxacin was determined by a UV-Vis spectrophotometry (UV-1202 Shimadzu) at the adsorption maximum of the free drug in aqueous buffered solutions (λ max = 279 nm) using a 1 cm quartz cell.
HPLC-analysis of Norfloxacin was performed on a Phenomenex-RPC 18 column (250 × 4.6 mm, 5 µm) (Dionex P-580 chromatograph). The eluent was a mixture of water/acetonitrile/trifluoroacetic acid (80/20/0.1/%). The norfloxacin was spectrophotometrically detected at 279 nm.  (Table 1). When the reaction time was completed, the reaction product was dissolved in CH 2 Cl 2 , then precipitated from cold methanol using diluted hydrochloric acid (5% aqueous solution) and finally dried under vacuum for 7 days. The precipitation was repeated three times.

Macromolecular conjugates synthesis
Polymeric conjugates of NOR were synthesised using both the one-step and two-step methods. In the one-step method, at the beginning NOR was added to the mixture of oligomers, HDI and the catalyst. Then, this reaction mixture was stirred for 3 hours at 65 °C. In the two-step method, oligoestrodiols were mixed with HDI (25 mmol) in a molar ratio 1:2 and dissolved in DMSO. This solution was placed in a three-necked flask equipped with a stirrer and a thermometer. Afterwards, a catalyst was added to the flask and the reaction mixture was left with stirring for 3 hours at 65 °C. Then, the solution of NOR in DMSO was added dropwise into the reactor with the prepolymer under vigorous stirring (in the molar ratio of NOR to prepolymer equal 1:1). After the addition procedure was completed, the reaction mixture was left with stirring for additional 3 hours at 65 °C. Then, it was washed with diluted hydrochloric acid (5% aqueous solution) and water. The precipitation was repeated three times. The conjugates isolated from the solution's organic phase were kept under vacuum at room temperature for no more than one week.

Conclusions
It has been proved that the ring-opening polymerization of L-LA, D,L-LA and ECL in the presence of CE is a very efficient method of the synthesis of low-molecular weight polyesters, terminated by hydroxyl groups. Polymerization at 160 °C in bulk produced polymers with a high yield (even ca. 90% in some cases). It has been then shown that oligoesters prepared in this way may be applied for the synthesis of polyurethane conjugates of NOR. The synthesis of those conjugates was done in three steps. First, the ring-opening homo-or copolymerization of L-LA, D,L-LA and ECL in the presence of CE and EG was carried out. In the second step, the prepolymer was obtained. In the final step, the reaction of the prepolymer with NOR was performed. The possibility of using the obtained conjugates as prodrugs of norfloxacin is currently in progress. The latter application requires yet careful, subsequent in vitro and in vivo examinations. We believe that the obtained polyurethane conjugates of norfloxacin are good potential candidates for carriers in drug delivery systems.