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Study of Stability of New Mutual Prodrugs with Antimycobacterial Activity

Josef Jampilek*1,2, Zuzana Reckova1, Ales Imramovsky3,4, Ivan Raich5, Jarmila Vinsova3 and Jiri Dohnal1,2
Zentiva a.s., U kabelovny 130, 10237 Prague 10, Czech Republic; 2Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackeho 1/3, 61242 Brno, Czech Republic; 3Department of Inorganic and Organic Chemistry, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 50005 Hradec Kralove, Czech Republic; 4Department of Chemistry, Faculty of Education, University of Hradec Kralove, Namesti Svobody 301, 50003 Hradec Kralove, Czech Republic; 5Department of Chemistry of Natural Compounds, Faculty of Food and Biochemical Technology, Institute of Chemical Technology, Technicka 5, 166 28 Prague 6, Czech Republic
Abstract: New mutual prodrugs of pyrazinamide, isoniazid, p-aminosalicylic acid and ciprofloxacine were designed and synthesised. All the prepared compounds were tested for their antimycobacterial activities. Several discussed compounds showed excellent biological effects against both Mycobacterium tuberculosis and some atypical strains. These activities were comparable with or higher than those of the standards (pyrazinamide, isoniazid, rifampicin). These interesting compounds are presented here, and their physico-chemical properties are discussed. Stabilities of all the studied compounds were measured by means of RP-HPLC method simulating conditions of biological testing. Decomposition half-times of individual mutual prodrugs were determined. Experimentally found data were processed using the program MATLAB, reaction orders and rate constants were calculated. The experimental results were subsequently correlated with ab initio/DFT calculations of electronic deficiency (+) in methine spacer connecting active parts of the molecule. The relationships between the chemical structure ( -electron density in the connecting CH linker) and the stability of the studied compounds are discussed.
1

Keywords: Antimycobacterials; Mutual prodrugs; Pyrazinamide; Isoniazid; Stability study; RP-HPLC; Mathematical modelling; Ab initio/DFT calculations. 1. INTRODUCTION Tuberculosis (TB), the worlds leading infectious disease, caused especially by Mycobacterium tuberculosis, is one of the most devastating diseases primarily due to several decades of neglect, and presents a global health threat of escalating proportions. TB remains a deadly disease and continues to reach approximately 2 million deaths annually. One-third of the worlds population is currently infected; more than 5000 people die of TB every day. A great number of people are carriers of the latent form that creates a dangerous source of the illness for the future. TB is after HIV/AIDS the second leading infectious cause of mortality today. The HIV pandemic has led to a rapid growth of the TB epidemic, and increased the probability of dying of TB. The rise in TB incidence can be attributed also to the development of resistance of M. tuberculosis to commonly used antituberculous drugs [1,2]. There is an intensified need for the development of new antituberculotic drugs due to the rapid growth of the immuno-compromised patient population, and growth of multi-drug resistant (MDR) strains [3-6]. Isoniazid (INH) has been widely applied as the first-line drug for the treatment of tuberculosis, usually in combination with other drugs. Modifying either of these molecules has been a challenge taken up by several research groups [711]. As INH is a prodrug, its therapeutic activity requires activation by mycobacterial catalase-peroxidase. INH prevents the mycolic acid biosynthesis by inhibiting a 2-transenoyl-acyl carrier protein reductase (InhA) that belongs to the FAS-II (fatty acid synthetase II) system [12]. Pyrazinamide (PZA), an analogue of nicotinamide, is a frontline tuberculosis-specific-drug that is used in combination with other drugs and contributed to shortening the TB therapy. PZA is a prodrug that requires activation or conversion under acidic pH condition into its active form, pyrazinoic acid (POA), by the pyrazinamidase/nicotinamidase enzyme encoded by pncA gene of susceptible M. tuberculosis. The target of PZA/POA appears to be the membrane, i.e. fatty acid synthase-I [13,14]. Research efforts are required to develop new compounds as potential antituberculous agents. Research on potential antituberculosis drugs, especially those based on PZA derivatives, has been performed at the Faculty of Pharmacy, Charles University for many years [1527].
2008 Bentham Science Publishers Ltd.

*Address correspondence to this author at the Zentiva a.s., U kabelovny 130, 10237 Prague 10, Czech Republic; Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackeho 1/3, 61242 Brno, Czech Republic; Tel: +420 267 243 695; Fax: +420 272 701 331; E-mail: josef.jampilek@zentiva.cz

1385-2728/08 $55.00+.00

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COOH

OH

F O 1

COOH

N 2

N 3

N H N O N N N N O N H N N N H H N O O N N H N N N H COOH

OH

F O 4

COOH

Fig. (1). The studied amides as prodrugs of pyrazine-2-carboxamide and isoniazid.

In quest for biologically more potent antimycobacterials a series of new PZA and INH derivatives were prepared as mutual prodrugs. Some discussed compounds showed comparable activity with PZA/INH not only against M. tuberculosis, but they exhibited excellent activities also against some non-tuberculosis strains [15]. Prodrugs are pharmacologically inactive derivatives of active drugs. They are designed for various purposes, e.g to maximize the amount of the active drug that reaches its site of action, through manipulation of the physicochemical, biopharmaceutical or pharmacokinetic properties of the drug. Prodrugs are converted into the active drug within the cell/the body through enzymatic or non-enzymatic reactions. In mutual prodrugs two active drugs are combined in one molecule, and synergistic affects of both components can be expected. In the present study, PZA and INH were linked by a CH group with one another or with another appropriate antibacterial component -p-aminosalicylic acid (PAS) or ciprofloxacine (CPF); see Fig. (1). These new molecules are known as mutual prodrugs and can exhibit a prolonged release, synergistic action of the components, and higher hydrophobicity, e.g. easier transport through the mycobacterial membrane [14,28]. Antimycobacterial MIC values of the studied compounds were determined after the incubation at 37 C for 7, 14 and 21 days [15]. The stability testing should indicate whether the intact molecule is responsible for their excellent potency or whether they are first decomposed to original drugs which then exhibit synergistic effects. Decomposition of the studied compounds was measured by means of a RP-HPLC method simulating conditions of biological testing. Decomposition half-times were determined. Experimentally found data were subsequently processed using the program MATLAB, rate constants and the reaction half-times were determined by evaluation of error array of the kinetic model describing degradation process.

2. EXPERIMENTAL Synthesis and full characteristics of the discussed derivatives of pyrazine-2-carboxamide and isoniazid are described in ref. [15]. 2.1. Chromatographic Conditions Working standards of pyrazinamide p.a., isoniazid p.a., p-aminosalicylic acid p.a. and ciprofloxacine p.a. were purchased from Sigma-Aldrich. The HPLC separation module Waters Alliance 2695 XE and Waters Photodiode Array Detector 2996 (Waters Corp., Milford, MA, U.S.A.) were employed. The HPLC separation process was monitored by Millennium32 Chromatography Manager Software, Waters 2004 (Waters Corp., Milford, MA, U.S.A.). For compounds 1-3 the chromatographic column Phenomenex Synergi Polar-RP 4 m, 4.6250 mm, (Phenomenex, Torrance, CA, U.S.A.) was used. The mixture of phosphate buffer 0.05 M (pH = 6.0) and MeCN p.a. (55:45) was applied as a mobile phase and 20% MeCN was chosen as a sample solvent. The detection wavelength was 265 nm. The flow was 1.0 ml/min, injection volume 20 l, column temperature 30 C and sample temperature 37 C. Time of analysis was 10 min. For compounds 4-6 the chromatographic column Discovery HS F5 5 m, 4.6150 mm, (Supelco, Bellefonte, PA, U.S.A.) was used. The mixture of phosphate buffer 0.05 M (pH = 2.0) and MeCN p.a. (65:35) was applied as a mobile phase and 20% MeCN was chosen as a sample solvent. Other chromatographic conditions were the same as for compounds 1-3. The initial concentrations of all compounds 1-6 were about 1.2 mg/ml. The stability testing was performed in triplicate for each compound.

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Fig. (2). Decomposition of the compound 1.

2.2. Mathematical Modelling Reaction orders and rate constants were calculated using the program MATLAB ver. R2006a (The Math Works, Natick, Massachusets, U.S.A.). 2.3. Ab initio/DFT Calculations Ab initio/DFT calculations (geometry optimizations, charge calculations) were performed in Gaussian 03W [29] at various levels of theory. For the calculation of the charges and polarizable conductor calculation (CPCM) solvation model [30], as implemented in Gaussian 03W, Merz, Singh and Kollman procedure [31,32] was used to simulate the H2O or MeCN medium. During geometry optimizations, all structures 1-6 were pre-optimized at the HF/3-21G level, then refined at the HF/6-31G(d) level, both in a gas phase. Final optimization as well as charge calculations were performed at the B3LYP/631G(d,p) level [33] for both H2O and MeCN medium. Atomic charges were calculated as a 4:1 weighted average for values obtained in water and MeCN, respectively. 3. RESULTS AND DISCUSSION The decomposition of the studied compounds was measured by means of RP-HPLC methods simulating conditions of biological testing. The methods were validated and the selected parameters met the criteria. Validations were performed according to the ICH Guidelines. System suitability for PZA: i) repeatability of 5 injections: RSD = 0.02%, ii) USP tailing 1.05. Validation (validated parameters): i) precision repeatability (independent analyses of 6 sample solutions): RSD = 0.29%, ii) linearity (LOQ PZA 100% concentration): correlation coefficient r = 0.9998, n = 11, iii) accuracy recovery: = 100.9%, RSD = 0.62%.

System suitability for INH: i) repeatability of 5 injections: RSD = 0.08%, ii) USP tailing 1.03. Validation (validated parameters): i) precision repeatability (independent analyses of 6 sample solutions): RSD = 0.14%, ii) linearity (LOQ INH 100% concentration): correlation coefficient r = 0.9998, n = 11, iii) accuracy recovery: = 100.3%, RSD = 0.54%. The detection limits (LOD) of PZA and INH have been 0.02% (i.e. 50 ng/ml) under the above mentioned conditions, regarding the concentration of sample solution. The quantitation limit (LOQ) is triple of this value, i.e. 0.06%. The applied methods were developed with respect to sufficient resolution among the peaks of the compounds in the chromatogram. Commercially available drugs (PZA, INH, PAS, CPF) and evaluated mutual prodrugs were used for the development of the methods. The discussed prodrugs 1-6 were divided into two groups PZA and INH derivatives according to the main component in the compounds. PZA and INH were used for validation of both methods, because an increase in concentration of PZA or INH as the main products of decomposition was observed, though an increase in concentration of PAS or CPF could also be observed. The mixture of DMSO/water was used as a solvent for antimycobacterial evaluation. Physico-chemical properties of DMSO are not appropriate to RP-HPLC due to their high UV absorbance, high level of noise and destruction of endcapped RP columns. Based on these facts, MeCN was chosen as a convenient solvent and DMSO replacement. Experimental data determined by means of HPLC method were subsequently processed using commercially available software MATLAB. The decomposition as well as decomposition half-time (1/2) for each of the discussed compounds are shown in Figs. (2-7). The half-time of the reaction shows 50% decrease of compound content [%]. Reaction orders and rate constants are calculated from decomposition of the studied prodrugs [34-38], therefore

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Fig. (3). Decomposition of the compound 2.

Fig. (4). Decomposition of the compound 3.

only figures illustrating prodrug decomposition are shown. The reaction order of the decomposition of the compounds investigated in an 80% aqueous solution followed pseudofirst-order degradation kinetics [34-38]. The reaction rate constants were calculated from the slopes of the plots of the natural logarithm of the residual reactant fraction versus time by the statistical regression analysis method. The rate constants and half-times of the decomposition were subsequently calculated for the compounds 1-6, see Table 1. The courses and half-times of the hydrolysis are graphically illustrated, see Figs. (2-7). The calculated half-times were in good agreement with the ones estimated from experimental data.

The compounds 1, 2 and 6 exhibited significant degradation under the chosen experimental conditions. In fact they are decomposed completely within 7 days. The compounds 3, 4 and 5 show about 10%, 30% and 19% of their initial concentration after 7 days, about 1%, 10% and 4% of their initial concentration after 14 days and about 0.1%, 3% and 1% of their initial concentration after 21 days, respectively. It can be assumed that the determined antimycobacterial activities [15] of the combinations of PZA-CPF (1), PZAINH (2) and INH-PAS (6) summarized individual effects of original drugs (PZA, INH, PAS, CPF). In contrast, the remaining prodrugs PZA-PAS (3), INH-CPF (4) and INH-INH (5) decompose more slowly, and their antimycobacterial

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Fig. (5). Decomposition of the compound 4.

Fig. (6). Decomposition of the compound 5.

potencies determined after 7 days of incubation reflect the combined effects of these new chemical/molecular entities and their components. According to ref. [15], the PZA-prodrugs 1-3 are more potent than the PZA itself. However, the INH-prodrugs 4-6 do not exhibit higher potency than INH. The same applies for assays against atypical strains in which only the prodrug INH-CPF (4) is significantly more potent than the INH itself. Due to these facts it may be concluded, that the effects of the most active compounds 1, 2, 6 are probably caused by the activity of the original drugs and/or their synergism. Only an excellent activity of the mutual prodrug 4 (INH-CPF) against atypical strains is the result of this compound itself. Its

higher potency is due to the presence of CPF and also probably due to higher hydrophobicity of INH combined with CPF. Pyridine and especially pyrazine are very specific molecules. The nitrogen atoms have an increased -electron density, compared with carbon atoms, which influences physicochemical properties and reactivity of both compounds [23,39-45]. Pyrazine is a more electron-deficient molecule than pyridine due to the presence of two atoms of nitrogen. It can be assumed, according to these presented facts, that also PZA is a more electron-deficient molecule than INH. It means that -electron density in connecting CH linker in the compounds 1-3 (PZA-prodrugs) is lower than the one in the

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Fig. (7). Decomposition of the compound 6. Table 1. The Calculated Rate Constants, Half-Times of the Decompositions and Calculated +-Values of the Charges of the Electronic Deficiencies on Methine Carbon in the Connecting Linker in 20% MeCN Medium
Compound 1 2 3 4 5 6 k [h-1] 0.200 0.040 0.014 0.007 0.010 0.500 1/2 [h] 3.5 17.3 49.5 99.0 69.3 1.4 +-values [20% MeCN] 0.564 0.608 0.675 0.322 0.442 0.580

compounds 4-6 (INH-prodrugs). The optimum value of electron density in CH linker between PZA/INH and the second part of prodrug molecule is important for the stability of the discussed compounds, as described elsewhere [11]. The half-times of the studied compounds 1-6 were compared to the calculated values of electronic deficiency (+) in methine linker between both connected molecules. Results of DFT point charges calculated in Gaussian 03W [29] are presented in Table 1. Electronic densities of methine moiety both in water and in acetonitrile were calculated. From these results methine carbon electronic densities of all studied compounds in 20% MeCN solution were derived. These results are practically the same as the charges in water. As expected, the methine carbon in PZA-prodrugs 1-3 shows higher electronic deficiencies (higher +-values) than in INH-prodrugs 4-6. From the half-times of INH-derivatives 4-6 when compared to their calculated methine electronic deficiency (+) it can be concluded, that they correspond to each other. Decrease of charge + (increase of electron density) on the methine carbon causes the increase of the halftime (stability increase) of the individual INH-prodrugs (6 < 5 < 4), see Fig. (8). Absolutely different properties can be

observed with PZA-derivatives 1-3. According to the data presented above, it can be stated, that the increase in electronic deficiency (+) causes the increase in stability PZAprodrugs (1 < 2 < 3), see Fig. (8). A hypothesis of probable decomposition/hydrolysis of the discussed mutual prodrugs 1-6 leading either to liberation of the original antituberculosis drugs or to generation of a drug and a formylamino derivative of INH, PZA, PAS or CPF is shown in Scheme 1. Mechanism of decomposition probably starts with a nucleophilic attack of water to electron-deficient methine. Successive proton transfer generates a neutral molecule. Another molecule of water breaks CN bond and INH/PZA molecule is then liberated. The formylamino derivative of the second component is generated at the same time. Formyl moiety is unstable, formylamino derivatives are decomposed, and the molecule of the second drug is formed, see Scheme 1. The value of + charge is very important for reactivity (stability) of the compounds. Generally, it could be assumed that the values of electronic deficiency + in CH linker ranging from 0.564 to 0.608 (prodrugs 1, 2, 6) is convenient for water nucleophilicity, see Fig. (8). Nonetheless other struc-

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Fig. (8). Dependence of calculated half-times [h] on DFT calculated methine carbon charge +-values of the discussed PZA/INH-prodrugs 16.

+ PZA / INH N H C R N H2O PZA / INH N

OH2 CH R N

proton transfer

OH PZA / INH N H CH R N

H2O

OH PZA / INH NH2 + HO CH R N -H2O O

H C R N HN R

R = fragment of INH, PZA, PAS or CPF

Scheme 1. General scheme of a possible decomposition of the discussed mutual prodrugs 1-6.

tural factors must be also taken in account. As discussed above and shown in Fig. (8), INH-prodrugs decompose better with higher electron deficiency (+) in methine group. On the contrary, decomposition of PZA-prodrugs decreases with an increase in +. A more detailed study of stability and metabolism of the studied compounds dealing with elucidating the mechanisms underlying their decomposition is under intensive investigation. 4. CONCLUSIONS Six new antituberculosis efficient compounds were presented here. Their synthesis and characterization was described by Imramovsky et al. [15]. Physico-chemical properties of these mutual prodrugs were discussed. Stabilities of all the prodrugs were simulated. The experimental results were correlated with ab initio/DFT calculations of electronic

deficiency (+) in the methine carbon in the spacer connecting both active parts of the molecule. According to all the obtained results it can be postulated, that the stability of all the discussed prodrugs is strongly dependent on -electron density in the connecting CH linker. The stability of PZAprodrugs 1-3 increases with the increase in electronic deficiency (+) in the methine moiety. On the contrary, the stability of INH-prodrugs 4-6, increases with the decrease in electronic deficiency ( +) in the methine linker. This experience will be used in the design of new antimycobacterial potential compounds. ACKNOWLEDGEMENTS This study was supported by MSM 0021620822 and MSM 6046137305.

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