Third International Electronic Conference on Synthetic Organic Chemistry (ECSOC-3), www.mdpi.org/ecsoc-3.htm, September 1-30, 1999

[C0004] 

Synthesis of N,N'-diarylalkanediamides and their antimycobacterial and antialgal activity

L. Kubicova¹*, K. Waisser¹, J. Kunes¹, K. Kralova², Z. Odlerova³, M. Slosarek⁴, J. Janota⁴, Z. Svoboda⁵

Department of Inorganic and Organic Chemistry¹, Faculty of Pharmacy, Charles University, Heyrovskeho 1205, 500 05 Hradec Kralove, Czech Republic. Tel. +420 49 5067339, Fax +420 49 5210002, *e-mail: [email protected]
Institute of Chemistry², Faculty of Natural Sciences, Comenius University, Mlynska dolina CH-2, 842 15 Bratislava, Slovak Republic
Institute of Preventive and Clinic Medicine³, Limbova 14, 835 01 Bratislava, Slovak Republic
National Institute of Public Health⁴, Srobarova 48, 100 42 Praha 10, Czech Republic
Institute of Experimental Biopharmaceutics⁵, Heyrovskeho 1207, 500 02 Hradec Kralove, Czech Republic

Received: 16 August 1999 / Uploaded: 21 August 1999

Abstract: A set of N,N'-diarylalkanediamides was synthesised. 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 caused by their low aqueous solubility, and hence by a restricted passage of the inhibitor through the hydrophilic regions of thylakoid membranes.

Keywords: N,N'-diarylalkanediamides, antimycobacterial activity, antialgal activity.

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], 2,3-diarylpentanediamides display activity against Gram-positive bacteria [3], and N,N'-substituted 2-halobutanediamides 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 one [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 compounds 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 OER-inhibiting 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 antimycobaterial 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 acyl chlorides 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 acyl chlorides were prepared from the corresponding acids by the reaction with phosphorus pentachloride (succinyl chloride) or thionyl chloride (all other acyl chlorides). 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 characteristic data of compounds 1--46 are given in Table 1 and Table 2.

Scheme 1

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 the 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 above 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(4-methoxyphenyl)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-CH₃, 4-OCH₃ and 3,4-Cl₂, a quasi-parabolic dependence of the inhibitory activity on m was found, with maximum inhibition for N,N'-diaryloctanediamides (m = 6; R = 4-CH₃ (39) and 4-OCH₃ (40)) and N,N'-bis(3,4-dichlorophenyl)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].

Tables

Table 1. Experimental data of the compounds

Compd.	Formula (M. w.)	m	R	M. p. (°C) Yield (%)	IR nu(C=O) (cm-1)
1	C16H16N2O2 (268.32)	0	2-CH3	212a) 65.2	1668
2	C16H16N2O2 (268.32)	0	3-CH3	135--137a) 59.7	1666
3	C14H10N4O6 (330.26)	0	4-NO2	357a) 61.4	1706
4	C14H10Br2N2O2 (398.05)	0	4-Br	329--331a) 57.6	1666
5	C16H14Br2N2O2 (426.11)	2	4-Br	281a) 92.4	1652
6	C16H12Cl4N2O2 (406.10)	2	3,4-Cl2	258--259b) 93.1b)	1659b)
7	C16H16N2O2 (268.32)	2	H	231--233a) 94.6	1663
8	C18H20N2O4 (328.37)	2	4-OCH3	256--257a) 89.5	1648
9	C16H14Cl2N2O2 (337.21)	2	4-Cl	288--290 90.3	1652
10	C18H20N2O2 (296.37)	2	4-CH3	273--275 91.4	1655
11	C16H14F2N2O2 (304.30)	2	3-F	206--207 88.5	1663
12	C16H14F2N2O2 (304.30)	2	4-F	244--245 92.2	1651
13	C16H14N4O6 (358.31)	2	3-NO2	228--230 87.5	1675
14	C20H24N2O2 (324.42)	2	3,4-(CH3)2	231--232 85.2	1651
15	C16H14Cl2N2O2 (337.21)	2	3-Cl	233--235a) 92.3	1668
16	C24H32N2O2 (380.53)	2	4-C4H9	232--234 81.0	1659
17	C22H28N2O2 (352.48)	2	4-isoC3H7	234--236 82.5	1656
18	C24H32N2O2 (380.53)	2	4-secC4H9	178--179 80.7	1655
19	C20H26N4O2 (354.45)	2	4-N(CH3)2	282.5--283.5a) 88.4	1645
20	C18H14N4O2S2 (382.45)	2	c)	282--285 91.5	1694
21	C17H16Cl2N2O2 (351,23)	3	4-Cl	243 69.5	1664
22	C19H22N2O4 (342.39)	3	4-OCH3	224 62.2	1659
23	C17H18N2O2 (282.34)	3	H	225a) 84.3	1673
24	C19H22N2O2 (310.40)	3	4-CH3	221a) 54.4	1664
25	C17H14Cl4N2O2 (420.12)	3	3,4-Cl2	267b) 63.0b)	1679b)
26	C17H16Br2N2O2 (440.13)	3	4-Br	257a) 57.5	1664
27	C20H24N2O2 (324.42)	4	4-CH3	252 67.4	1659
28	C18H20N2O2 (296.37)	4	H	238a) 75.0	1660
29	C18H18N4O6 (386.36)	4	3-NO2	239 58.3	1667
30	C18H18Cl2N2O2 (365.26)	4	3-Cl	197 59.8	1663
31	C18H16Cl4N2O2 (434.15)	4	3,4-Cl2	277b) 58.8b)	1670b)
32	C20H24N2O4 (356.42)	4	2-OCH3	152 40.3	1659
33	C20H24N2O4 (356.42)	4	4-OCH3	234 64.5	1648
34	C18H18Cl2N2O2 (356.26)	4	4-Cl	239 66.7	1656
35	C24H28N2O6 (440.50)	4	4-COOC2H5	217 54.0	1708, 1693
36	C20H20Cl4N2O2 (462.20)	6	3,4-Cl2	167--168b) 34.6b)	1671b)
37	C20H22Cl2N2O2 (393.31)	6	4-Cl	197--198 81.3	1659
38	C20H24N2O2 (324.42)	6	H	186--188a) 57.2	1659
39	C22H28N2O2 (352.48)	6	4-CH3	224--226a) 54.7	1656
40	C22H28N2O4 (384.48)	6	4-OCH3	220--221 63.2	1652
41	C20H22Br2N2O2 (482.21)	6	4-Br	253a) 53.6	1664
42	C21H22Cl4N2O2 (476.23)	7	3,4-Cl2	170--171b) 52.2b)	1680b)
43	C21H24Cl2N2O2 (407.34)	7	4-Cl	196--197 61.4	1660
44	C21H26N2O2 (338.45)	7	H	180--181a) 62.7	1671
45	C23H30N2O2 (366.50)	7	4-CH3	197--198a) 61.4	1662
46	C23H30N2O4 (398.50)	7	4-OCH3	194--196 43.9	1655
a) M. p. values from literature: Compound, value (°C) [ref.]: 1, 209 [8]; 2, 133 [8]; 3, 359 [9]; 4, 321--322 [10]; 5, 284 [11]; 7, 230,5 [12]; 8, 256 [13]; 15, 232 [14]; 19, 277-280 [15]; 23, 223 [11]; 24, 218 [11]; 26, 256 [11]; 28, 235 [11]; 38, 186--7 [16]; 39, 219 [11]; 41, 248 [11]; 44, 186--7 [16]; 45, 198 [11]. b) The data of compounds 6, 25, 31, 36 and 42 were taken from [7]. c) The compounds is N,N'-bis(2-benzothiazolyl)butanediamide.

Table 2. ¹H NMR spectroscopic data and elemental analyses of selected compounds

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)

a) not tested

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)

Table 5. Inhibition of chlorophyll production in Chlorella vulgaris by N,N'-diarylbutanediamides (m = 2; concentrations of compounds were constant, 75 µmol dm^-3)

Experimental

General

The melting points were determined on a Kofler block and are uncorrected. The samples for elemental analysis and biological tests were dried over P₂O₅ 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. TLC was performed using petroleum ether : ethyl acetate (1:1), and chloroform : aceton (9:1) as the mobile phases. ¹H NMR spectra of new compounds were recorded for DMSO-d₆ solutions at ambient temperature on a Varian Mercury-Vx BB 300 spectrometer (operating at 300 MHz). Chemical shifts were recorded as delta values in parts per million (ppm), and were indirectly referenced to tetramethylsilane via the solvent signal (2.49 for ¹H).

Synthesis of acyl chlorides

Succinyl choride
Phosphorus pentachloride (328 mmol) was added to a finely powdered succinic acid (400 mmol), and the mixture was heated at 120°C for 5 hours. Phosphorus oxychloride was then distilled off, and the crude product was purified by vacuum distillation (yield 74.5 %, b. p. 104-105 °C / 3.33 kPa, lit. [17] b. p. 103--104 °C / 3.33 kPa).

Suberyl choride
Suberic acid (287 mmol) was heated with thionyl chloride (1260 mmol) at 120 °C for 4 hours. After the removal of the excess reagent by distillation, the crude product was purified by distillation at reduced pressure (yield 69.20 %, b. p. 145°C / 1.33 kPa, lit. b. p. [18] 149--150 °C / 1.60 kPa). The same protocol was used for the preparation of the remaining acyl chlorides (yield; b. p.; b. p. [ref]): glutaryl chloride (90.7 %; 104 °C / 2.53 kPa; 100 °C / 2.00 kPa [19]); adipoyl chloride (81.2 %; 130--132 °C / 2.40 kPa; 130--132 °C / 2.40 kPa [20]); azelayl chloride (71.4 %; 162 °C / 1.20 kPa; 165 °C / 1.73 kPa [20]).

Synthesis of N,N'-diarylalkanediamides

N,N'-diarylethanediamides (1--4)
Sodium (1 g) was dissolved in absolute ethanol (100 ml), 3-methylaniline (300 mmol) followed by diethyl oxalate (150 mmol) were added to the solution, and the reaction mixture was heated at reflux for 1 hour. The crude product was filtered off, washed with water, and crystallized from ethanol. The yields, melting points, and IR spectral data of compounds 1--4 are given in Table 1.

Other N,N'-diarylalkanediamides
Succinyl chloride (16 mmol) was added dropwise to a stirred solution of 4-methylaniline (32 mmol) 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 remaining amides were prepared in an analogous fashion. The yields, melting points, IR, and NMR spectral data as well as elemental analyses are summarised in Table 1 and Table 2.

Biological assays

Antimycobacterial activity
Antimycobacterial evaluation of N,N'-diarylalkanediamides (m = 0, 3, 4, 6, 7) was carried out in a semisynthetic liquid protein-containing Sula medium (IMUNA, Sarisske Michalany), buffered to pH 7.2. The following mycobacterial strains were used: Mycobacterium tuberculosis H₃₇Rv, M. kansasii PKG8, M. avium No. 80/72 and M. fortuitum 1021. The MICs were determined after 14 days of incubation at 37 °C. The compounds were added to the medium in a dimethyl sulfoxide solution. The final concentrations were 1000; 333; 111; 37; 12.3; and 4.1 µmol dm^-3.

Antimycobacterial activity of butanediamides (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 dimethyl sulfoxide solutions. The final concentrations 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.

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 dimethyl sulfoxide (DMSO) as their solubility in water was insufficient. The antialgal activity was expressed as the percentage of inhibition of the untreated control.

Acknowledgements: This study was supported by the Ministry of Education of the Czech Republic (CEZ J 13/98: 11600001), and by the Scientific Grant Agency of the Ministry of Education of the Slovak Republic (grant No. 1/4013/97).

References

[1] Waisser K., Odlerova Z., Grunert R.: Pharmazie 1989, 44, 162.

[2] Dave M. P., Patel J. M., Langalia N. A., Shah S. R., Thaker K. A.: J. Indian Chem. Soc. 1985, 62, 386.

[3] Al-Arab Mohammad M., Na'was Tarek E., Abu-Yousef Imad A.: Indian J. Chem. 1990, 29B, 694.

[4] Stauffer Chemical Co.: Ger. Offen. 24 51 512 (1975).

[5] Raynes K., Galatis D., Cowman A. F., Tilley L., Deady L. W.: J. Med. Chem. 1995, 38, 204.

[6] Kubicova L., Kadlcikova J., Waisser K., Kralova K., Sersen F., Slosarek M., Janota J., Svoboda Z.: Folia Pharm. Univ. Carol. 1998, 23 Suppl., 148.

[7] Kubicova L., Kralova K., Waisser K.: Chem. Papers 1999, 53, in press.

[8] Lapkin I. I., Andrejchikev Ju. S.: Zh. Org. Khim. 1965, 1, 1212.

[9] Price Ch. C., Velzen B. H.: J. Org. Chem. 1947, 12, 386.

[10] CIBA-GEIGY: U. S. 3906033 (1975); Chem. Abstr. 1976, 84, 4633.

[11] Barnicoat C. R.: J. Chem. Soc. 1927, 2926.

[12] Kirshanov, Egorova: Zh. Obsch. Khim. 1953, 23, 1920.

[13] Ueda Mitsuru, Morishima Makoto, Kakuta Urayumi, Sugiyama Yunichi: Macromolecules 1992, 25, 6580.

[14] Sanna A., Repetto G.: Gazz. Chim. Ital. 1927, 57, 777.

[15] Chen Ku Lin, Chang Shih Tai, Chi Chieh Chang: Yao Hsuch Pao 1957, 5, 333; ref. Chem. Abstr. 1961, 55, 25862i.

[16] Hill J. W., Wallace H. C.: J. Amer. Chem. Soc. 1933, 55, 5023.

[17] Vorlaender D.: Justus Liebigs Ann. Chem. 1894, 280, 167.

[18] V. Auwers, Schmidt: Chem. Ber. 1913, 46, 479.

[19] Hoffmann H. M. R., Haase K., Geschwinder P. M.: Synthesis 1982, 3, 237.

[20] Etaix: Ann. Chim. (Paris) 1896, 9, 369.

[21] Kralova K., Sersen F., Melnik M.: J. Trace Microprobe Techn. 1998, 16, 491.

[22] Inskeep W. P., Bloom P. R.: Plant. Physiol. 1985, 77, 483.

All comments on this poster should be sent by e-mail to (mailto:[email protected] ona.edu) [email protected] with C0004 as the message subject of your e-mail.