Synthesis of 2-Hydroxy-5-( 1-( 2-( pyrazine-2-carbonyl ) hydrazono ) hexyl ) Benzoic Acid , A Pyrazinamide Analog of Salicylic Acid

The occurrence of resistant strains of Mycobacterium tuberculosis has driven current research on combining current anti-tuberculosis drugs and other bioactive molecules to enhance their efficacy against susceptible and resistant strains of the bacteria. In this study, a salicylic acid derivative of pyrazinamide, 2-hydroxy-5-(1-(2-(pyrazine-2carbonyl)hydrazono)hexyl) benzoic acid was synthesized and characterized. The compound was prepared by coupling a pyrazinamide moiety, one of the first line drugs used to treat tuberculosis and a salicylic acid derivative with a 6-carbon alkyl chain. The salicylic acid derivative was generated via Friedel-Crafts acylation of methyl salicylate followed by base hydrolysis of the acylated product. This was coupled with the pyrazinamide moiety via imine formation. The product, 2-hydroxy-5-(1-(2-(pyrazine-2carbonyl)hydrazono)hexyl) benzoic acid was obtained as an off-white powder in 62% yield.


INTRODUCTION
Tuberculosis (TB) is one of the leading causes of death in the world from an infection of acid-fast bacilli Mycobacterium tuberculosis (Todar, 2012).
This bacteria commonly infects the lungs but may also attack other organs such as the kidneys, spine and the brain.In the treatment of the disease, the first line of drugs prescribed includes isoniazid, rifampin, ethambutol and pyrazinamide.A second line of drugs that includes kanamycin, capreomycin and amikacin are administered upon the development of multi-drug resistant strains of tuberculosis (MDR-TB) which is resistance of the bacteria against the first line of drugs (WHO, 2012;CDC, 2012).further worsened by the increase in the number of cases which are infected with MDR-TB, many cases of which are also found in Eastern Europe and Central Asia (WHO, 2012).
The increase in the development of multidrug resistant strains of tuberculosis (MDR-TB) and extensively-drug resistant strains (XDR-TB) which is resistance to the fist line and second-line of drugs used to treat the disease respectively has resulted to a vast amount of research on the modification of the first-line of drugs used to treat TB.
One of the first-line of drugs used to treat the disease, pyrazinamide (PZA) plays a unique role in shortening the treatment period from nine months to six months (Zhang et al., 2003).This is due to its sterilizing activity, which kills a population of persistent tubercle bacilli that are not affected by other drugs (Vergara et al., 2009).Unlike isoniazid and rifampin, PZA has an activity against multidrug resistant strains that favors its usage over other drugs.However, several studies have shown that this drug is inactive against Mycobacterium bovis and other resistant strains of mycobacterium that has a mutation in their pncA gene.This gene codes for the pyrazinamidase enzyme, which converts PZA to pyrazinoic acid (POA) as it enters the cell of the bacteria as shown in Figure 1 ( Mitchison and Zhang, 2011).Occurrence of resistance towards the PZA drug, stirred up a huge amount of research work on the modification of the drug to enhance its effectivity against resistant strains of M. tuberculosis.Vast arrays of modifications were already done on the PZA nucleus, most of which resulted to an increased potency of the drug compared to the base molecule against resistant strains of M. tuberculosis and also towards other non-tuberculous mycobacterial strains (Vergara et al., 2009;Sriram et al., 2006;Imramovsky et al., 2007;Zitko et al., 2012).
Most of the modifications done on the PZA drug (Table 1) were made by fusing it with other potential molecules which may improve its activity.These PZA derivatives mostly retained the original structure of the PZA nucleus (Vergara et al., 2009;Sriram et al., 2006;Imramovsky et al., 2007).
The creation of PZA derivatives of Mannich bases has generated analogs with an increased lipophilic character.The synthesis of these Mannich base derivatives of PZA were done via microwave assisted reaction of the PZA molecule with formaldehyde and a secondary amino function of substituted piperazines.This enables the attachment of the PZA core via the methylene group of the Mannich base.One of the synthesized analogs, 1cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-(3-methyl-4-((pyrazine-2-carboxamido)methyl)piperazin-1-yl)-4-oxoquinoline-3carboxylic acid (A), was the most active compound in vitro against MDR-TB with an minimum inhibitory concentration (MIC) of 0.20 µg/ml.This is 125 times greater than the potency of the parent PZA drug against MDR-TB (MIC of PZA = 25.0 µg/ml).The increased anti-TB activity is attributed to the increased lipophilic character of the PZA analog.The calculated logP values of these compounds showed higher values compared to the PZA drug (-1.31).(Sriram et al., 2006).
In another related study, PZA was linked with known anti-TB drugs such as isoniazid, paminosalicylic acid and ciprofloxacin by the CH fragment.This was done by the reaction of pyrazinamide and N,N-dimethylformamide dimethyl acetal, to create an activated pyrazine derivative, N-(dimethylaminomethylene) pyrazine-2-carboxamide.This allows for the substitution reaction of nucleophilic amino moieties to the activated PZA molecule to generate the derivatives.The higher lipophilic character of 4-(2-Pyrazinecarbonyliminomethyl)aminosalicylic acid (B), one of the

Type of Modification: Increased Lipophilicity
MIC/logP values: 3.13 µg/ml vs 6-60 µg/ml (PZA) c , 0.11 vs -1.31 (PZA) d (B) N-acylhydrazone derivatives of PZA (Vergara et al., 2009).synthesized compounds in the study showed higher activity against M.tuberculosis, with an MIC value of 3.13 µg/ml.The higher lipophilicities of these compounds enables easy transport through cellular membrane of the mycobacteria, resulting to lower MIC values compared to the parent PZA drug (Imramovsky et al., 2007).

Type of Modification
Another way of attaching the PZA nucleus to other molecules to improve its effectivity is via imine bond formation.N-acylhydrazones containing the PZA nucleus were synthesized by reactions involving monosubstituted benzaldehyde moieties and a derivatized PZA core.This generated compounds with ewithdrawing groups (e.g.Cl, F, CN and NO2) attached in the ortho and meta position of the benzaldehyde ring to exhibit better activity (MIC = 50-100µg/mL) compared with PZA (MIC > 100 µg/mL) (Vergara et al., 2009).Some bioactive molecules have been studied upon its co-administration with the PZA drug.Weak acids such as benzoic acid, fatty acids and salicylic acid were co-administered alongside PZA and showed lower colony forming units (CFU) of M. tuberculosis in both normal and nutrient starved incubation conditions (Chen et al., 2007).Non-steroidal anti-inflammatory drugs, specifically ibuprofen and aspirin, upon co-administration with PZA have also shown an increase in potency of the drug in a mouse model infected with Mycobacterium tuberculosis H37Rv (Byrne et al., 2007).
In view of these results, we envisioned the synthesis of a pyrazinamide analog with a salicylic acid derivative.The attachment of the PZA nucleus via imine formation afforded an easy pathway of fusing the PZA moiety with an acylated salicylic acid derivative.This allows for the retention of the original PZA nucleus.The addition of the alkyl chain may increase the lipophilicity of the compound and aide in its diffusion through the mycobacterial cell wall (Sriram et al., 2006;Imramovsky et al., 2007).The evidence on the enhanced effectivity of the PZA drug in the presence of weak acids may be further investigated with this lead compound.This compound may show an increase in the efficacy of the pyrazinamide drug towards susceptible and resistant strains of Mycobacterium tuberculosis.

EXPERIMENTAL
General Analytical Procedure.All reagents (ZnCl2, methyl salicylate, pyrazinoic acid, hydrazine hydrate, and hexanoyl chloride) used were analytical grade with ≥99% purity.All chemicals and solvents used were purchased from Sigma-Aldrich Chemicals, Singapore and Merck Chemicals, Philippines except for the pyrazine-2-hydrazide which was prepared in a previous study (Lagua, 2011).
The purity of the product in each reaction was determined by thin layer chromatography.The TLC plates used were 4x2 cm in dimension and pre-coated with silica gel (Fluka).Visualizing agents for TLC were ultraviolet light and iodine (I2) powder chamber.
For the characterization of the target compounds, functional groups were determined using Nicolet-500 FT-IR Spectrometer.The mass spectra of the compounds were obtained using Bruker Mass Spectrometer either in the positive or negative ion mode.The 1 H-NMR spectra were obtained using Jeol 400 MHz Nuclear Magnetic Resonance Spectrometer at the National Chemistry Instrumentation Center (NCIC) Ateneo de Manila University.The melting points of the target compound and all intermediate products were obtained using the Fischer-Johns Mel-Temp Apparatus.

Synthesis of Methyl-5-n-hexanoylsalicylate
(2).Zinc chloride (2.1872 g, 16.04 mmol), methyl salicylate (1.70 mL, 13.12 mmol) and dichloromethane (6.00 mL) were mixed in a 100 mL round bottom flask.To this mixture, hexanoyl chloride (2.00 mL, 14.31 mmol) was added drop-wise forming a cloudy white mixture.Stirring of the reaction mixture for 48 hours produced a dark brown colored solution.The reaction mixture was extracted with CH2Cl2 (20 mL) followed by washing with distilled water (10 mL x 2).The organic layer was dried with anh.Na2SO4, filtered and concentrated in vacuo producing a viscous dark brown liquid.Column chromatography separation using 100% n-C6H14 was carried out to remove the starting material.This was followed by using 60% n-C6H14/CH2Cl2 as solvent system to isolate the target compound.

RESULTS AND DISCUSSION
The relevance of the pyrazinamide drug in the treatment of tuberculosis engages current research in the derivatization of the drug to be effective against resistant strains of mycobacteria.The retention of the pyrazinamide nucleus and its coupling with a vast set of compounds which may enhance its effectivity has been the study of a significant amount of research (Vergara et al., 2009;Sriram et al., 2006;Imramovsky et al., 2007).Specifically the attachment of the pyrazinamide core through the formation of an N-acylhydrazone group has proven to be a simple method used in a previous study to effectively fuse the PZA core with potential compounds that may improve its efficacy against M. tuberculosis (Vergara et al., 2009).This method has been adopted by the group to create a pyrazinamide analog with a salicylic acid moiety, which is done by creating a salicylic acid derivative with an acyl chain, to which the amine group of the pyrazinamide molecule may be fused via imine formation (Scheme 1).Previous study shows that the coadministration of weak acids lowered the number of CFU in normal and nutrientstarved media of mycobacteria (Chen et al., 2007).This has prompted the group to fuse the pyrazinamide core to a salicylic acid molecule by the attachment of an acyl chain which contains a ketone group.This may be used as the point of attachment to the derivatized pyrazinamide nucleus in the hope that the resulting compound would increase the effectivity of the PZA drug against susceptible and resistant strains of mycobacteria.The attachment of an alkyl chain to the salicylic acid moiety was done to increase the lipophilicity of the salicylic acid derivative to be attached to the PZA ring.This was done via Friedel-Crafts acylation of methyl salicylate with hexanoyl chloride in the presence of zinc chloride as catalyst.Friedel-Crafts acylation reactions are useful alternative routes to direct alkylation reactions since it provides a less reactive aromatic compound which limits the generation of over alkylated products.This was then deemed to be the best pathway to attach an alkyl chain to the methyl salicylate precursor.The reaction proceeds via the generation of an acylium ion, followed by the bond formation using two pi-electrons from the methyl salicylate ring generating a carbocation intermediate.The removal of a proton adjacent to the carbocation regenerates the aromatic ring (Scheme 2).This afforded methyl-5-n-hexanoylsalicylate (2) in 20.01%yield.The low yield may be attributed to the substantial amount of unreacted starting material and the presence of some by-products generated after the reaction.The low reactivity of the zinc chloride catalyst may have contributed to the relatively low yield (Heany, 1991).
Methyl-5-n-hexanoylsalicylate (2) was generated as yellow needle like crystals with a sharp melting point (41-43 °C).The TLC pure product (Rf = 0.48; 2:3 Hexane-CH2Cl2) gave an Rf value that is lower than that of methyl salicylate (Rf value = 0.61; 2:3 Hexane-CH2Cl2) which confirms that the addition of an acyl chain has increased the polarity of the molecule.The IR spectrum of the compound showed a signal at 1677 cm -1 which signifies the attachment of the ketone group.The mass spectrum of the compound showed a pseudomolecular ion peak at 273.11236 [M+Na] and 251.12995 [M+H] which are consistent with the expected mass of compound corresponding to a molecular formula of C14H18O4 (m/z = 250.32).
To generate the salicylic acid group, the methyl salicylate derivative (2) was reacted with NaOH to allow hydrolysis of the ester group to carboxylic acid functionality.This generated 5-n-hexanoylsalicylic acid (3) as orange crystals in high yield (87.29 %).This structural change was confirmed by the broad signal at 3466 -2508 cm -1 in the IR spectrum of the compound which corresponds to the carboxylic acid OH.The one-spot TLC profile with an Rf value of 0.52 (25:1:1 ethyl acetate-EtOH-acetic acid) and sharp melting point of the salicylic acid derivative further confirms the purity of the compound.The expected mass of m/z = 236.29 corresponding to the molecular formula of the compound (C13H16O4) was confirmed by the presence of a pseudomolecular ion peak at 235.09884[M-H].This salicylic acid derivative was then attached to the pyrazinamide nucleus via an imine formation reaction.The PZA molecule was first converted to pyrazine-2-hydrazide to allow for the reaction between the amino group of the hydrazide and the ketone group of the acyl chain in the salicylic acid moiety (Scheme 3) as adopted from the procedure used by Vergara and co-workers in the synthesis of N-acylhydrazones (Vergara et al., 2009).
This route afforded the target compound (Figure 2) as an off-white powder in 62.43 % yield.The crude product generated after the reaction only required washing with water to remove the unreacted pyrazine-2-hydrazide and salicylic acid derivative.The structure of compound (4) was confirmed by 1 H-NMR.The singlet at 11.04 ppm confirms the presence of the NH proton of the amide group attached to the pyrazinamide moiety which is similar to the data generated in previous studies (Swamy, et al., 2007).The absence of any doublet in the same region signifies the attachment of the amino group to the ketone functionality.
The relevant signals (Table 2) attributed to the pyrazinamide ring are given by the peaks at 8.81 -8.82 ppm (dd, J = 1.5, 2.5 Hz) due to the H5 proton.This shows ortho and meta coupling with the signals at 9.28 and 8.96 ppm which corresponds to H6 (J = 2.4 Hz) and H3 (J = 1.5 Hz) protons respectively.The attachment of the acyl chain at the para position relative to the OH group in the salicylic acid ring was further confirmed by the signal of the H22 proton at 8.04 ppm (dd, J = 2.4, 8.8 Hz).This confirms the ortho coupling of this proton with the with the H21 proton at 7.07 ppm (d, J = 8.8 Hz) and its meta coupling with the H18 proton at 8.30 ppm (d, J = 2.3 Hz).
The protons at the alkyl chain were also confirmed with the multiplet signal at 1.34 ppm corresponding to the methylene protons in the chain.The triplet at 2.87 ppm (J = 7.8 Hz) correspond to the methylene protons nearest the imine bond, as shown in the table below.
Scheme 2. Mechanism of Friedel-Crafts Acylation for the synthesis of compound (2).
Scheme 3. Mechanism of Imine formation for the synthesis of compound (4).