-Thioamidopyridines, and Their Antimycobacterial and Photosynthesis-Inhibiting Activity

Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Bratislava, Slovak Republic*Author to whom correspondence should be addressed.Received: 8 March 1999 / Revised and Accepted: 28 February 2000 / Published: 3 March 2000Abstract: A group of 26 new 2-halogeno-6-alkylsulfanyl- and 2,6-bis-alkylsulfanyl-4-amidopyridines and corresponding thioamidopyridines was synthesised. Some of the ami-dopyridines and all thioamidopyridines were tested for their antimycobacterial activityagainst atypical mycobacterial strains. Promising photosynthesis-inhibiting activity was alsofound for some of the amidopyridines.Keywords: Thioamidopyridines, antimycobacterial activity, photosynthesis-inhibiting ac-tivity.IntroductionSome events during the past decade have dramatically changed the nature and magnitude of theproblem of tuberculosis. The HIV epidemic and increasing resistance to antituberculous drugs dictatethe need of development of new antituberculotics [1-3].


Introduction
Some events during the past decade have dramatically changed the nature and magnitude of the problem of tuberculosis.The HIV epidemic and increasing resistance to antituberculous drugs dictate the need of development of new antituberculotics [1][2][3].
In our recent study [4], we modified the structure of therapeutically used antituberculous drugs ethionamide and prothionamide.Some of the more lipophilic derivatives showed promising activity against atypical mycobacterial strains.The present study extends the scope of lipophilic derivatives of 4-thioamidopyridines. Since it has been recently reported that 2-alkylsulfanyl-4-thioamidopyridines showing antimycobacterial [5] and antifungal activity [6] inhibit photosynthetic processes in algae and plant chloroplasts [7,8], the synthesised compounds were tested for their both antimycobacterial and photosynthesis-inhibiting activity.
To better understand the structure-activity relationships, log P values were calculated (Table 3).We found that the lipophilicities of the most potent antimycobacterial compounds were different for all four strains employed.In the 4-thioamidopyridine series (14-26), the highest activities against M. tuberculosis were observed for compounds 22-24 with log P values between 5.75 and 7.34.The antimycobacterial activity of 4-thioamidopyridines against other three strains showed a sharp dependence on lipophilicity.In the case of M. kansasii and M. avium, the most active 4-thioamidopyridines 16 and 22 showed log P values ranging from 4.86 to 5.75, while the compounds with the highest activity against M. fortuitum, 23 and 24, exhibited log P values 6.81 and 7.34.
The tested compounds also inhibited photochemical activity of spinach chloroplasts.The IC 50 values, i. e., concentrations of the compounds causing 50% decrease of oxygen evolution rate in spinach chloroplasts with respect to the untreated control, are listed in Table 3. From the comparison of IC 50 values of the 2-halogeno substituted 4-amido (1-7) and 4-thioamidopyridines (14-21) it can be concluded that amidopyridines exhibit greater inhibitory activity than the corresponding thioamidopyridines.For compounds 9-12, a pronounced decrease in photosynthesis-inhibiting activity with the increasing lipophilicity of the compounds has been confirmed.This is in good agreement with the previously obtained results concerning photosynthesis-inhibiting activity of 2-alkylsulfanyl-4thioamidopyridines in spinach chloroplasts and Chlorella vulgaris [7,8].In the 4-amidopyridine series, the most active compounds 7, 9, 3, and 6 showed log P in the range of 3.12-5.0,whereas the inhibitory activity of thioamidopyridines with log P > 3.27 showed a pronounced decrease.
Using EPR spectroscopy it was found that in the suspension of spinach chloroplasts the studied 4thioamidopyridines interact with D + intermediate, i.e., with the radical of tyrosine 161 (Tyr D ) which is located in D 2 protein on the donor side of photosystem 2 [10], and due to this interaction the photosynthetic electron transport from the oxygen evolving complex to the core of photosystem 2 is impaired.The same site of action in the photosynthetic apparatus of spinach chloroplasts has also been confirmed for the structurally similar 2-alkylsulfanyl-4-thioamidopyridines [7].
Table 1.Analytical data of the prepared compounds.

General
Melting points were determined on a Kofler block, and are uncorrected.IR spectra were recorded on a Nicolet Impact 400 spectrometer in chloroform. 1 H NMR spectra were determined for solutions in CDCl 3 with TMS as the internal standard with a BS 587 ( Tesla, Brno ) 80 MHz apparatus.Column chromatography was performed on silica gel (Silpearl, Kavalier Votice).Elemental analyses were performed on a EA 1110 CHNS-O CE INSTRUMENTS elemental analyser.
Lipophilicity of the compounds was computed using a program ACD/LogP version 1.0 (Advanced Chemistry Development Inc., Toronto).

Synthesis of 2-alkylsulfanyl-6-hexylsulfanyl-4-amidopyridines 9-13
To a stirred solution of 2-chloro-6-hexylsulfanyl-4-amidopyridine (3) (10 mmol) and the appropriate thiol (10 mmol) in anhydrous N,N-dimethylformamide (10 mL) sodium methoxide (10 mmol) in methanol (5 ml) was added dropwise.The reaction mixture was heated to about 50°C, stirred and maintained at this temperature until TLC indicated a complete reaction.TLC was performed using petroleum ether : ethyl acetate (2:1) as the mobile phase.The mixture was poured into cold water.The crude product was filtered off, purified by column chromatography (petroleum ether : ethyl acetate, 2:1) and recrystallised from aqueous ethanol.The yields and melting points are given in Table 1, and the IR and NMR spectroscopic data in Table 2.

Synthesis of 2,6-disubstituted 4-thioamidopyridines 14-26
To a stirred solution of 2,6-disubstituted 4-amidopyridine (10 mmol) in anhydrous toluene (10 ml) Lawesson's reagent (5 mmol) was added and the reaction mixture was heated at reflux until TLC indi-cated a complete reaction.TLC was performed using petroleum ether : ethyl acetate (4:1) as the mobile phase.The mixture was then evaporated under reduced pressure, the crude product was purified by column chromatography (petroleum ether : ethyl acetate, 4:1), and recrystallised from aqueous ethanol.The melting points and yields are given in Table 1, and the IR and NMR spectroscopic data in Table 2.
The MIC values were determined after 14 days of incubation at 37°C.The oxygen evolution rate (OER) in spinach chloroplasts was determined spectrophotometrically (Specord UV VIS Zeiss Jena, Germany) by the Hill reaction.The measurements were carried out in phosphate buffer (20 mmol, pH = 7.2) containing sucrose (0.4 mol.dm -3 ), MgCl 2 (5 mmol.dm - ) and NaCl (15 mmol.dm - ) using 2,6-dichlorophenol-indophenol as electron acceptor.Chlorophyll content in the samples was 30 mg.dm -3 and the samples were irradiated (~100 W.m -2 ) from 10cm distance with a halogen lamp (250 W) using a water filter to prevent warming of the samples (suspension temperature 22 o C).The compounds were dissolved in dimethyl sulfoxide (DMSO) because of their limited water solubility.The applied DMSO concentration (up to 5%) did not affect OER.

Table 2 .
IR and 1 H NMR spectroscopic data of the prepared compounds.

Table 3 .
MIC of the tested compounds against used mycobacterial strains, IC 50 values concerning inhibition of oxygen evolution rate in spinach chloroplasts by the tested compounds and calculated logP values of the prepared compounds.