Synthesis of New Fluorine Substituted Heterocyclic Nitrogen Systems Derived from p-Aminosalicylic Acid as Antimycobacterial Agents

Some new fluorine substituted heterocyclic nitrogen systems 2–17 have been synthesized from ring closure reactions of substituted p-amino salicylic acids (PAS). The Schiffs base of PAS was cyclized with chloroacetyl chloride and mercaptoacetic acid to give azetidinone 2, thiazolidinone 3, and spiro-fluoroindolothiazoline-dione 10. However, PAS when reacted directly with 4fluorobenzoyl chloride and 5-oxazolinone yielded derivatives 4, 5, and 7. Aminomethylation of PAS using formaldehyde and piperidine or piperazine formed N-alkyl and N,N-dialkyl derivatives (11 and 12 respectively) upon fluorinated benzoylation gave compounds 13 and 14. Similarly, treatment of PASwith thiosemicarbazide 15 and subsequent cyclizationwith diethyl oxalate yielded the fluorinated heterocycle 17.The structures of the fluorinated heterocyclic systems have been established on the basis of elemental analysis, H NMR, C NMR, and MS spectral data. Some of the targets exhibited a high inhibition towardsMycobacterium strain with favorable log P values.


Introduction
Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis.After AIDS, tuberculosis is the second leading cause of death from an infectious disease worldwide [1][2][3][4][5].The frequent coinfection of TB in HIV patients further complicates the selection of an appropriate treatment regimen.During, recent years, Mycobacterium tuberculosis has developed increased resistance against drugs.The multidrug-resistant (MDR-TB) and extensively drugresistant (XDR-TB) strains of tuberculosis are considered as some of the most challenging threats to global health [6,7].Medicinal researchers are continuing all over the world in order to have a safe and effective therapeutic strategy against these resistant strains.
The treatment involves the administration of multiple drugs because it is clear that monotherapy leads to the development of resistance.Aminosalicylic acid (PAS) which was introduced as an antitubercular medicine in 1948 is being used in combination with the second line therapeutic regimen against multidrug-resistant and extensively drugresistant strains [8].In a recent study a salicylic acid analog, benzofuran salicylic acid (1-A09; Figure 1), has been found to show Mycobacterium protein tyrosine phosphatase B inhibiting activity [9].This analog of salicylic acid has provided an innovative therapeutic starting point for the treatment of TB, including MDR and XDR forms, that is not only complementary, but also synergistic with current drugs.
Fluorine is a well-known bioisostere in various organofluorine compounds as antimycobacterial agents [10].The introduction of fluorine has already shown to modulate the stereoelectronic parameters of organic molecules [11,12].Substitution of fluorine into a potential drug molecule not only alters the electronic environment, but also influences the p avalue of neighboring Bronsted acid/base centers, polarity, and the influence on lipophilicity as expressed by the distribution coefficient.The introduction of fluorine substituent in bioactive molecules can often improve their pharmacological properties as increased membrane permeability, enhanced hydrophobic binding, and stability against metabolic transformation.Furthermore, it has also been shown that selective organofluorine interactions with protein residues can be used to substantially enhance protein-ligand binding affinity and selectivity [13].In an extension of our previous study, in the area of synthesis of bioactive compounds for the treatment of infectious diseases [14][15][16][17][18][19][20], the present work aims at the synthesis of some new fluorinated heterocyclic systems incorporating PAS as anti-Mycobacterium agents.

Experimental
Melting points were determined in an electrothermal Bibby Stuart Scientific Melting Point SMP (US).The IR spectra were recorded using KBr discs on a Perkin Elmer Spectrum RXI FT-IR systems number 53529.

2-Hydroxy-4-[(piperidin-1-ylmethyl)amino]benzoic Acid (11).
To a solution of PAS (1.53 g, 0.01 mol) in MeOH (20 mL), piperidine (0.85 g, 0.01 mol) and formaldehyde (37%, 2 mL) were added.The reaction mixture was stirred at room temperature for 5 h.To this mixture an excess amount of distilled water was added and the mixture was left overnight.All the new compounds obtained were tested for in vitro anti-tuberculosis activity against M. tuberculosis H37Rv using the BACTEC 12 medium using a broth microdilution assay, the Microplate Alamar Blue Assay (MABA) [21,22].Rifampicin was used as the standard (Table 1).Of these compounds, the ones which exhibited >90% inhibition in the primary screen (MIC < 6.25 g/mL) were considered at lower concentrations against M. tuberculosis H37Rv in order to determine the actual MIC, using MABA in the level 2 of the screening (Table 2).Rifampin (RMP) was used as the reference compound (RMP MIC = 0.015-0.125mg/mL).

Results and Discussion
3.1.Chemistry.The condensation of p-aminosalicylic acid (PAS) with p-fluorobenzaldehyde in methanol produced the  Schiff base 1.The IR spectra showed two absorption bands at 3383 cm −1 and 1668 cm −1 for the OH and CO groups, respectively along with a characteristic C=N absorption at 1602 cm −1 .Their 1 H NMR spectra exhibited beside the aromatic protons a singlet of one proton intensity at  8.30 for the CH=N as well as two exchangeable singlets at  11.02 and  5.45 for the COOH and the phenolic OH, respectively.The structure of the above compound was further confirmed from its 13 C NMR and MS data (experimental section).Similarly, cycloaddition [23] of compound 1 with chloroacetyl chloride in dry benzene afforded the azetidinone 2, while with thioglycolic acid in dry dioxane it afforded the 4-[2-(4-fluorophenyl)-4-oxo-thiazolidin-3-yl]-2-hydroxybenzoic acid 3.The IR spectra of 2 and 3 showed two carbonyl absorptions at 1700-1777 cm −1 and 1669-1672 cm −1 for the azetidinone and COOH groups, respectively, as well as OH bands in the regions 3268-3383 cm −1 .The 1 H NMR spectra of 2 exhibited beside the aromatic protons at  7.08-7.96two doublets at  5.23 and 5.51 (J = 9.0 Hz) for H-2 and H-3 protons, respectively.On the other hand the thiazolidine derivative 3 showed beside the seven aromatic protons at ).The IR spectra of 4 and 5 showed two carbonyl absorption at 1700-1777 cm −1 and 1668-1673 cm −1 for the azetidinone and COOH carbonyl groups, respectively, as well as OH bands in the regions 3268-3383 cm −1 .Compound 4 also exhibited an absorption band at 3440 cm −1 which is attributed to a free OH group.However, compound 5 exhibited a third carbonyl at 1762 cm −1 for the ester group.The structures of the above compounds were further confirmed by their 1 H NMR, 13 C NMR and MS data.
The treatment of PAS with oxazolone 6 in refluxing dry pyridine afforded the imidazolone 7. Its IR spectra showed two carbonyl absorptions at 1690 cm −1 (C=O of imidazolone) and 1665 cm −1 (C=O, COOH).The 1 H NMR spectra of 2 exhibited beside the aromatic protons at  7.12-8.06two exchangeable singlets at  11.20 and  5.48 for the COOH and the phenolic OH groups, respectively.The structure of 7 was further supported by the 13 C NMR spectral data which showed the expected number of aliphatic and aromatic carbons.
Similarly, condensation of 5-fluoroisatin 8 with PAS in methanol yielded 5-fluoroisatin Schiff base 9 which upon cycloaddition with thioglycolic acid in dry dioxane afforded the spirothiazolidine derivative 10.Compound 10 can be also obtained directly from refluxing of compound 8 and PAS with thioglycolic acid in dry dioxan in one step (Scheme 1).The 13 C NMR spectra of Schiff base 9 showed beside the aromatic carbons two carbonyl carbons at  161.9 and  172.3 as well as a C=N signal at  164.2.The spiroderivative 10 exhibited three carbonyl signals at  168.4, 171.2, and 171.9 in addition to a methylene carbon signal at  30.1.However, the 1 H NMR spectrum of compound 10 showed very characteristic two doublets of C-5 proton at  3.84 and 3.95 showing geminal coupling (J = 12.4 Hz).The structures of compounds 9 and 10 were further confirmed by their MS spectra which showed the molecular ion peak M + + 2 at m/z 376.
The aminomethylation of PAS using formaldehyde and piperidine or piperazine in methanol produced the N-alkyl 11 and N,N  -dialkyl 12 derivatives, respectively.Benzoylation of compounds 11 and 12 on warming it with 4-fluorobenzoyl chloride in DMF led to the formation of the benzoyl-or dibenzoyl derivatives 13 or 14, respectively (Scheme 2).The structures of the above compounds 11-14 were confirmed by showed beside the two carbonyl absorptions at 1640 cm −1 and 1663 cm −1 a C=S band at 1317 cm −1 .The structure of the above compound was further confirmed from its 13 C NMR which showed the expected number of aliphatic and aromatic carbons as well as a thiocarbonyl signal at  182.3 in addition to three carbonyl signals at  155.8, 157.5, and 171.8 (carboxyl) (Scheme 3).Further confirmation of the structure of 17 was done by its MS spectral data.

Antimycobacterial Activity.
The results of the in vitro evaluation of antituberculosis activity are reported in Tables 1 and 2. During the preliminary screening compounds 1-5, 7, 9-14, and 17 were tested (Table 1) for their antimycobacterial activity; one of the compounds 17 has exhibited 100% inhibition at this concentration while other compounds exhibited between 92 and 98% inhibition at the same concentration.Compounds 3-5, 10, 13, and 17 have shown inhibition between 98 and 100%.Therefore, these are selected for the second level screening to determine the actual minimum inhibitory concentration (MIC).Compounds 5 and 13 have shown a slight improvement in the antitubercular activity in the second level and were found to be the most promising candidates of PAS analogs with MIC values 4.25 M and 3.78 M, respectively (Table 2).
The IC 50 and MIC data are used to calculate the selectivity index (SI) of each compound as an estimate of a therapeutic window and a mechanism to identify candidates for efficacy studies in vivo (Table 2).Compounds 3-5, 10, 13, and 17 have shown selectivity index values 4.64, 4.32, 3.29, 4.32, 3.17, and 4.80 respectively.Furthermore, all compounds have shown log P values in the accepted range (1.93-4.2) of druglikeness.However compounds 4, 10, and 17 show medium log P value (∼2.0) and make them suitable candidates for a possible oral drug.
In our previous research work we prepared p-aminosalicylic acid analogs keeping in mind the mutual prodrug concept [24].However, this paper includes the introduction of fluorine in almost all the PAS analogs.The reason for the induction of fluorine into these analogs is due to the fact that fluorine is much more lipophilic than hydrogen, so incorporating fluorine atoms in PAS analogs make them more fat soluble.This means it partitions into membranes much more readily, and hence these analogs have a higher bioavailability and metabolic stability.

Conclusion
Fluorine substituted heterocyclic systems containing pamino salicylic acid were synthesized as antimycobacterial agents.Some derivatives selected for the second level screening have shown favorable partition coefficient values to support druglikeness of these compounds.However their selectivity index is not very high.Further optimization of these PAS analogs is recommended in order to have a compound with the optimum structure features and the required biological activity.

Table 1 :
Results of the primary antituberculosis screening of compounds 1-17.
a MIC (minimum inhibitory concentration) of Rifampicin: 0.125-0.25 g/mL versus M. tuberculosis H37Rv.b Growth inhibition of virulent H37Rv strains of M. tuberculosis.

Table 2 :
Results of second level antituberculosis assay.
a Actual minimum inhibitory concentration (MABA assay).b Calculated log .