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Current Organic Chemistry , 2008, 12, 667-674 667

1385-2728/08 $55.00+.00 2008 Bentham Science Publishers Ltd.

Study of Stability of New Mutual Prodrugs with AntimycobacterialActivity

Josef Jampilek* 1,2, Zuzana Reckova 1, Ales Imramovsky 3,4, Ivan Raich 5, Jarmila Vinsova 3 andJiri Dohnal 1,2

1 Zentiva a.s., U kabelovny 130, 10237 Prague 10, Czech Republic; 2 Department of Chemical Drugs, Faculty of Phar-macy, University of Veterinary and Pharmaceutical Sciences, Palackeho 1/3, 61242 Brno, Czech Republic; 3 Departmentof Inorganic and Organic Chemistry, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Hey-rovskeho 1203, 50005 Hradec Kralove, Czech Republic; 4 Departm ent of Chemistry, Faculty of Education, University of

Hradec Kralove, Namesti Svobody 301, 50003 Hradec Kralove, Czech Republic; 5 Department of Chemistry of NaturalCompounds, Faculty of Food and Biochemical Technology, Institute of Chemical Technology, Technicka 5, 166 28 Pra-gue 6, Czech Republic

Abstract: New mutual prodrugs of pyrazinamide, isoniazid, p-aminosalicylic acid and ciprofloxacine were designed andsynthesised. All the prepared compounds were tested for their antimycobacterial activities. Several discussed compoundsshowed excellent biological effects against both Mycobacterium tuberculosis and some atypical strains. These activitieswere comparable with or higher than those of the standards (pyrazinamide, isoniazid, rifampicin). These interesting com-

pounds are presented here, and their physico-chemical properties are discussed. Stabilities of all th e studied compoundswere measured by means of RP-HPLC method simulating conditions of biological testing. Decomposition half-times ofindividual 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 ini-tio /DFT calculations of electronic deficiency ( +) in methine spacer connecting active parts of the molecule. The relation-ships between the chemical structure ( -electron density in the connecting CH linker) and the stability of the studied com-

pounds are discussed.

Keywords: Antimycobacterials; Mutual prodrugs; Pyrazinamide; Isoniazid; Stability study; RP-HPLC; Mathematical model-ling; Ab init io /DFT calculations.

1. INTRODUCTION

Tuberculosis (TB), the worlds leading infectious dis-ease, caused especially by Mycobacter ium tuberculosis , isone of the most devastating diseases primarily due to severaldecades of neglect, and presents a global health threat ofescalating proportions. TB remains a deadly disease and con-tinues 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 numberof people are carriers of the latent form that creates a dan-gerous source of the illness for the future. TB is afterHIV/AIDS the second leading infectious cause of mortalitytoday. The HIV pandemic has led to a rapid growth of theTB epidemic, and increased the probability of dying of TB.The rise in TB incidence can be attributed also to the devel-opment of resistance of M. tuberculosis to commonly usedantituberculous drugs [1,2]. There is an intensified need forthe development of new antituberculotic drugs due to the

*Address correspondence to this author at the Zentiva a.s., U kabelovny130, 10237 Prague 10, Czech Republic; Department of Chemical Drugs,Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sci-ences, Palackeho 1/3, 61242 Brno, Czech Republic; Tel: +420 267 243 695;Fax: +420 272 701 331; E-mail: [email protected]

rapid growth of the immuno-compromised patient popula-

tion, 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 combina-tion with other drugs. Modifying either of these moleculeshas been a challenge taken up by several research groups [7-11]. As INH is a prodrug, its therapeutic activity requiresactivation by mycobacterial catalase-peroxidase . INH pre-vents the mycolic acid biosynthesis by inhibiting a 2- transenoyl-acyl carrier protein reductase (InhA) that belongs tothe FAS-II ( fatty acid synthetase II ) system [12].

Pyrazinamide (PZA), an analogue of nicotinamide, is afrontline tuberculosis-specific-drug that is used in combina-tion with other drugs and contributed to shortening the TBtherapy. PZA is a prodrug that requires activation or conver-sion under acidic pH condition into its active form, pyrazi-noic acid (POA), by the pyraz inamidase/nicotinamidase en-zyme encoded by pncA gene of susceptible M. tuberculosisThe target of PZA/POA appears to be the membrane, i.e.

fatty acid synthase-I [13,14]. Research efforts are required todevelop new compounds as potential antituberculous agents.Research on potential antituberculosis drugs, especiallythose based on PZA derivatives, has been performed at theFaculty of Pharmacy, Charles University for many years [15-27].

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668 Current Organic Chemistry, 2008, Vol. 12, No. 8 Jampilek et al.

In quest for biologically more potent antimycobacterialsa series of new PZA and INH derivatives were prepared asmutual prodrugs. Some discussed compounds showed com-

parable activity with PZA/INH not only against M. tubercu-losis , but they exhibited excellent activities also againstsome non-tuberculosis strains [15].

Prodrugs are pharmacologically inactive derivatives ofactive drugs. They are designed for various purposes, e.g tomaximize the amount of the active drug that reaches its siteof action, through manipulation of the physicochemical, bio-

pharmaceutical 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 onemolecule, and synergistic affects of both components can beexpected.

In the present study, PZA and INH were linked by a CHgroup with one another or with another appropriate antibac-terial component - p-aminosalicylic acid (PAS) or ciproflox-acine (CPF); see Fig. ( 1). These new molecules are known asmutual prodrugs and can exhibit a prolonged release, syner-gistic action of the components, and higher hydrophobicity,e.g. easier transport through the mycobacterial membrane[14,28].

Antimycobacterial MIC values of the studied compoundswere determined after the incubation at 37 C for 7, 14 and21 days [15]. The stability testing should indicate whetherthe intact molecule is responsible for their excellent potencyor whether they are first decomposed to original drugs whichthen 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 deter-mined. Experimentally found data were subsequently proc-essed using the program MATLAB, rate constants and thereaction half-times were determined by evaluation of errorarray of the kinetic model describing degradation process.

2. EXPERIMENTAL

Synthesis and full characteristics of the discussed deriva-tives of pyrazine-2-carboxamide and isoniazid are describedin 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 pur-chased 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 ChromatographyManager Software, Waters 2004 (Waters Corp., Milford,MA, U.S.A.).

For compounds 1-3 the chromatographic column Phe-nomenex Synergi Polar-RP 4 m, 4.6 250 mm, (Phe-nomenex, 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 chosenas a sample solvent. The detection wavelength was 265 nm.The flow was 1.0 ml/min, injection volume 20 l, columntemperature 30 C and sample temperature 37 C. Time of

analysis was 10 min.For compounds 4-6 the chromatographic column Discov-ery HS F5 5 m, 4.6 150 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 forcompounds 1-3.

The initial concentrations of all compounds 1-6 wereabout 1.2 mg/ml. The stability testing was performed in trip-licate for each compound.

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

N

N N N

N

O

N

F COOH

O

N

N N N

HO

H N

O N

N

N N N

H

O

OH

COOH

O

H N

N N

N N

F COOH

O

N

O

H N

N NH

O

H N

N N

O

H N

N NH

OH

COOH N

1 2 3

4 5 6

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2.2. Mathematical Modelling

Reaction orders and rate constants were calculated usingthe 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 chargesand polarizable conductor calculation (CPCM) solvation

model [30], as implemented in Gaussian 03W, Merz, Singhand Kollman procedure [31,32] was used to simulate theH2O or MeCN medium.

During geometry optimizations, all structures 1-6 were pre-optimized at the HF/3-21G level, then refined at theHF/6-31G(d) level, both in a gas phase. Final optimization aswell as charge calculations were performed at the B3LYP/6-31G(d,p) level [33] for both H 2O and MeCN medium.Atomic charges were calculated as a 4:1 weighted averagefor values obtained in water and MeCN, respectively.

3. RESULTS AND DISCUSSION

The decomposition of the studied compounds was meas-ured by means of RP-HPLC methods simulating conditionsof biological testing. The methods were validated and theselected parameters met the criteria. Validations were per-formed according to the ICH Guidelines.

System suitability for PZA: i) repeatability of 5 injec-tions: RSD = 0.02%, ii ) USP tailing 1.05. Validation (vali-dated parameters): i) precision repeatability (independentanalyses 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 injec-tions: RSD = 0.08%, ii ) USP tailing 1.03. Validation (vali-dated parameters): i) precision repeatability (independentanalyses 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 been0.02% (i.e. 50 ng/ml) under the above mentioned conditions,regarding the concentration of sample solution. The quantita-tion limit (LOQ) is triple of this value, i.e. 0.06%.

The applied methods were developed with respect to suf-ficient resolution among the peaks of the compounds in thechromatogram. Commercially available drugs (PZA, INH,PAS, CPF) and evaluated mutual prodrugs were used for thedevelopment of the methods. The discussed prodrugs 1-6were divided into two groups PZA and INH derivativesaccording to the main component in the compounds. PZAand INH were used for validation of both methods, becausean increase in concentration of PZA or INH as the main

products of decomposition was observed, though an increasein concentration of PAS or CPF could also be observed.

The mixture of DMSO/water was used as a solvent forantimycobacterial evaluation. Physico-chemical properties ofDMSO are not appropriate to RP-HPLC due to their highUV absorbance, high level of noise and destruction of end-capped RP columns. Based on these facts, MeCN was cho-sen as a convenient solvent and DMSO replacement.

Experimental data determined by means of HPLCmethod were subsequently processed using commerciallyavailable software MATLAB. The decomposition as well asdecomposition half-time ( 1/2) for each of the discussed com-

pounds are shown in Figs. ( 2-7). The half-time of the reac-tion shows 50% decrease of compound content [%].

Reaction orders and rate constants are calculated fromdecomposition of the studied prodrugs [34-38], therefore

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

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only figures illustrating prodrug decomposition are shown.The reaction order of the decomposition of the compoundsinvestigated in an 80% aqueous solution followed pseudo-first-order degradation kinetics [34-38]. The reaction rateconstants were calculated from the slopes of the plots of thenatural logarithm of the residual reactant fraction versus time

by the statistical regress ion analysis method. The rate con-stants and half-times of the decomposition were subse-quently calculated for the compounds 1-6, see Table 1. Thecourses and half-times of the hydrolysis are graphically illus-trated, see Figs. ( 2-7). The calculated half-times were ingood agreement with the ones estimated from experimentaldata.

The compounds 1, 2 and 6 exhibited significant degrada-tion under the chosen experimental conditions. In fact theyare decomposed completely within 7 days. The compounds3, 4 and 5 show about 10%, 30% and 19% of their initialconcentration after 7 days, about 1%, 10% and 4% of theirinitial concentration after 14 days and about 0.1%, 3% and1% of their initial concentration after 21 days, respectively.

It can be assumed that the determined antimycobacterialactivities [15] of the combinations of PZA-CPF ( 1), PZA-INH ( 2) and INH-PAS ( 6) summarized individual effects oforiginal drugs (PZA, INH, PAS, CPF). In contrast, the re-maining prodrugs PZA-PAS ( 3), INH-CPF ( 4) and INH-INH(5) decompose more slowly, and their antimycobacterial

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


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potencies determined after 7 days of incubation reflect thecombined effects of these new chemical/molecular entitiesand 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 appliesfor assays against atypical strains in which only the prodrugINH-CPF ( 4) is significantly more potent than the INH itself.Due to these facts it may be concluded, that the effects of themost active compounds 1, 2, 6 are probably caused by theactivity of the original drugs and/or their synergism. Only anexcellent activity of the mutual prodrug 4 (INH-CPF) againstatypical 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 withCPF.

Pyridine and especially pyrazine are very specific mole-cules. The nitrogen atoms have an increased -electron den-sity, compared with carbon atoms, which influences physico-chemical properties and reactivity of both compounds[23,39-45]. Pyrazine is a more electron-deficient moleculethan pyridine due to the presence of two atoms of nitrogen. Itcan be assumed, according to these presented facts, that alsoPZA is a more electron-deficient molecule than INH. Itmeans that -electron density in connecting CH linker in thecompounds 1-3 (PZA-prodrugs) is lower than the one in the



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compounds 4-6 (INH-prodrugs). The optimum value of -electron density in CH linker between PZA/INH and thesecond part of prodrug molecule is important for the stabilityof 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 ( +) inmethine linker between both connected molecules. Results ofDFT point charges calculated in Gaussian 03W [29] are pre-sented in Table 1. Electronic densities of methine moiety

both in water and in acetonitrile were calculated. From theseresults methine carbon electronic densities of all studiedcompounds in 20% MeCN solution were derived. These re-sults 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) thanin INH-prodrugs 4-6. From the half-times of INH-derivatives4-6 when compared to their calculated methine electronicdeficiency ( +) it can be concluded, that they correspond toeach other. Decrease of charge + (increase of electron den-sity) on the methine carbon causes the increase of the half-time (stability increase) of the individual INH-prodrugs ( 6

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