Synthesis of N,N’-Diarylalkanediamides and Their Antimycobacterial and Antialgal Activity

A set of N,N'-diarylalkanediamides was synthesized. The compounds were tested for their antimycobacterial and antialgal activity. The antimycobacterial activity of N,N'-diarylalkanediamides depends on the lipophilicity of the respective acid. Antimycobacterially active substances were found only in the series of N,N'-diarylethanediamides and N,N'-diarylbutanediamides. Other compounds (derivatives of pentane-, hexane-, octane- and nonanediamide) were inactive against various strains of mycobacteria. The compounds inhibited growth and chlorophyll production in Chlorella vulgaris. Their relatively low antialgal activity is probably connected with their lowered aqueous solubility, and hence by a restricted passage of the inhibitor through the hydrophilic regions of thylakoid membranes.


Introduction
As the end of the 20 th century witnesses a sharp rise in the incidence of mycobacterial infections, the development of new antimycobacterial drugs is presently of utmost importance and should proceed at a rapid pace.When exploring a possible link between antituberculous activity and the ability to form chelates with heavy metals, we prepared a set of N,N'-diarylethane-and -propanediamides, which were evaluated in vitro against Mycobacterium tuberculosis, and some of them showed significant activity [1].Other authors have described various kinds of biological activity of alkanediamide derivatives as well.For example, some 2-methylcarbonylbutanediamides are active against M. tuberculosis [2], 2,3diarylpentanediamides display activity against Gram-positive bacteria [3], and N,N'-substituted 2halobutanediamides act as herbicides [4].Raynes et al. studied the influence of the length of the connecting chain on the antimalarial activity of bisquinolines; the derivative of butanediamide was the most efficient [5].In our previous study [6] we found that N,N'-bis (3,4-dichlorophenyl)butanediamide effectively inhibited oxygen evolution rate (OER) in spinach chloroplasts and that this compound interacted with the pigment-protein complexes in photosystem 2. The increase of the length of the connecting chain in the series of N,N'-bis (3,4-dichlorophenyl)alkanediamides led to the decrease of OERinhibiting activity in spinach chloroplast [7].The decrease in biological activity with increasing lipophilicity of the compounds is probably linked to their lowered aqueous solubility, and hence to a restricted passage of the inhibitor through the hydrophilic regions of thylakoid membranes.
This study is focused on the synthesis of a large set of N,N'-diarylalkanediamides and on the study of antimycobacterial and antialgal activity of these compounds.

Results and Discussion
N,N'-Diarylalkanediamides, with the exception of N,N'-diarylethanediamides, were prepared from the corresponding anilines by treatment with the appropriate alkanedioyl dichlorides in pyridine at 0°C.The reaction mixtures were allowed to stand at room temperature and after 24 hours they were poured into water.The products were filtered off, washed with water and crystallized from ethanol.The alkanedioyl dichlorides were prepared from the corresponding acids by the reaction with phosphorus pentachloride (butanedioyl dichloride) or thionyl chloride (all other alkanedioyl dichlorides).N,N'-Diarylethanediamides were prepared from diethyl oxalate by the reaction with the corresponding aniline in the presence of sodium ethanolate.All syntheses are outlined in Scheme 1.The yields, physical properties and analytical data for compounds 1-46 are given in Table 1 and Table 2. Compounds 1-46 were tested for their in vitro antimycobacterial activity against Mycobacterium tuberculosis, M. avium, M. fortuitum, and M. kansasii.We found that the antimycobacterial activity of N,N'-diarylalkanediamides depends on the lipophilicity of the respective acid.Active substances were found only in the series of N,N'-diarylethanediamides and N,N'-diarylbutanediamides, but their antimycobacterial activity was mostly low.The MIC values of the active compounds are given in Table 3.All derivatives of pentane-, hexane-, octane-and nonanediamide were inactive against the studied mycobacteria strains.
The inhibition of chlorophyll production in statically cultivated algae Chlorella vulgaris by selected derivatives was investigated at a constant inhibitor concentration of 75 µmol dm -3 .The antialgal activity of the compounds was generally low, and the observed inhibition of algal chlorophyll production varied in the range of 14.2 (10) to 57.9% (40) (Table 4 and Table 5).The antialgal activity of N,N'diarylbutanediamides was relatively low, varying in the range of 14.2 (10) to 43.6% (15) (Table 5).The most effective inhibitor from the series of N,N'-diarylalkanediamides was N,N'-bis(4methoxyphenyl)octanediamide (40), causing 57.9% inhibition of chlorophyll production (Table 4).Antialgal activity of substituted N,N'-diarylalkanediamides with the same substituent R was proportional to the number of methylene groups in the connecting chain of the molecule (m = 2-4, 6, 7) for derivatives with R = H and 4-Cl, respectively.For derivatives with R = 4-CH 3 , 4-OCH 3 and 3,4-Cl 2 , a quasi-parabolic dependence of the inhibitory activity on m was found, with maximum inhibition for N,N'-diaryloctanediamides (m = 6; R = 4-CH 3 (39) and 4-OCH 3 (40)) and N,N'-bis(3,4dichlorophenyl)hexanediamide (m = 4; 31).The relatively low biological activity of the compounds is probably a consequence of their low aqueous solubility, and hence a restricted passage of the inhibitor through the hydrophilic regions of thylakoid membranes.An efficient inhibition of photosynthetic electron transport in spinach chloroplasts by N,N'-bis(3,4-dichlorophenyl)butanediamide has been observed previously [6,7].b The data of the compound were taken from [7].

General
The melting points were determined on a Kofler block and are uncorrected.The samples for elemental analyses and biological tests were dried over P 2 O 5 at 61°C and 66 Pa for 24 h.Elemental analyses were performed on a C,H,N,S analyzer (FISONS AE 1110, Milano).The IR spectra were measured in KBr on a Nicolet Impact 400 apparatus.The purity of the compounds was checked by TLC, using petroleum ether:ethyl acetate (1:1) and chloroform:acetone (9:1) as the mobile phases. 1 H NMR spectra of new compounds were recorded for DMSO-d 6 solutions at ambient temperature on a Varian Mercury-Vx BB 300 spectrometer (operating at 300 MHz).Chemical shifts were recorded as δ values in ppm, and were indirectly referenced to tetramethylsilane via the solvent signal (2.49 for 1 H).
Other N,N'-diarylalkanediamides  Butanedioyl dichloride (0.016 mol) was added dropwise to a stirred solution of an appropriate aniline (0.032 mol) in pyridine (20 ml) at 0°C.The reaction mixture was allowed to stand at ambient temperature for 24 hours, and then poured into water (100 ml).The product was filtered off and crystallized from ethanol.The yields, melting points, IR and NMR spectral data as well as elemental analyses are summarized in Table 1 and Table 2.
Antimycobacterial activity of N,N'-diarylbutanediamides (m = 2) was determined in Sula semisynthetic medium (SEVAC, Prague).For evaluation of their in vitro antimycobacterial activity, the following strains were used: M. tuberculosis CNCTC My 1/47, M. kansasii CNCTC My 235/80, M. avium CNCTC My 80/72 and M. fortuitum CNCTC My 187/73 from the National Institute of Public Health, Prague.The compounds were added to the medium in DMSO solutions.The final concentra-tions were 1000; 500; 250; 125; 62; 31; 16; 8; 4 µmol dm -3 .The minimum inhibitory concentrations were determined after incubation at 37°C for 21 days.MIC was the lowest concentration of a substance (on the above-stated concentration scale), at which inhibition of the growth of mycobacteria occurred.The compound is considered as active, when its MIC is lower than 1000 µmol dm -3 .DMSO concentration in the medium was maximum for concentration 1000 µmol dm -3 of the tested compound and the control samples contained the same DMSO amount (10 v/v %).The applied DMSO content did not affect the antimycobacterial activity.

Antialgal activity
The inhibitory effect of selected N,N'-diarylalkanediamides on algal chlorophyll (Chl) production has been investigated in statically cultivated Chlorella vulgaris (96 hours; photoperiod 16 h light / 8 h dark; illumination: 5 000 lx; pH = 7.2; Chl content at the beginning of cultivation: 0.5 mg dm -3 ) at room temperature and a constant inhibitor concentration 75 µmol dm -3 according to Kralova et al. [21].Chl content of algal suspensions was determined spectrophotometrically following its extraction into N,N-dimethylformamide according to Inskeep and Bloom [22].The compounds were dissolved in DMSO as their solubility in water was insufficient.DMSO concentration in the algal suspensions did not exceed 0.75 v/v % and the control samples contained the same DMSO amount as the suspensions treated with the tested compounds.The antialgal activity was expressed as the percentage of inhibition of the untreated control.

Table 1 .
Physical properties and analytical data of compounds 1-46.

Table 3 .
Antimycobacterial activity of the active compounds expressed as MIC (µmol dm-3).(m= number of methylene groups in the connecting chain of the compounds).

Table 4 .
Inhibition of chlorophyll production in Chlorella vulgaris by N,N'-diarylalkanediamides (m = number of methylene groups in the connecting chain of the compounds; concentrations of compounds were constant, 75 µmol dm -3 ).