Sixth International Electronic Conference on Synthetic Organic Chemistry (ECSOC-6), http://www.mdpi.org/ecsoc-6, 1-30 September 2002

[C004]

Substituted pyrazine-2-carboxamides, their synthesis and photosynthesis-inhibiting activity

Martin Dolezal1a*, Helena Kutilova1, Katarina Kralova2, Jiri Kunes3

1 Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy, Charles University, 500 05 Hradec Kralove, Czech Republic
a e-mail: [email protected], tel. +420 49 5067272, fax +420 49 5512423
2 Institute of Chemistry, Faculty of Natural Sciences, Comenius University, 842 15 Bratislava, Slovak Republic
3 Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University, 500 05 Hradec Kralove, Czech Republic
* Author to whom correspondence should be addressed.

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Abstract: Condensation of chlorides of substituted pyrazine-2-carboxylic acids with ring-substituted anilines yielded a series of amides of 6-chloropyrazine-2-carboxylic, 5-tert-butylpyrazine-2-carboxylic or 5-tert-butyl-6-chloro-pyrazine-2-carboxylic acids. Products were tested for their photosynthesis-inhibiting activity. The most active inhibitor of oxygen evolution rate in spinach chloroplasts was 5-tert-butyl-6-chloro-pyrazine-2-carboxylic acid (5-bromo-2-hydroxy-phenyl)-amide (IC50 = 0.0419 mmol dm-3). The presence of phenolic group in the benzene moiety determined to an increased photosynthesis-inhibitory activity.

Keywords: Amides of 5-alkyl-6-chloro-pyrazine-2-carboxylic acids; Photosynthesis inhibition; Spinach chloroplasts

Introduction

Various compounds possessing -NHCO- group were found to inhibit photosynthetic electron transport [1—4]. Amides of 2-alkylpyridine-4-carboxylic [5,6], 2-alkylsulfanylpyridine-4-carboxylic [6,7] acids inhibited oxygen evolution rate in Chlorella vulgaris and their inhibitory activity depended on the lipophilicity of the compounds. Several esters of alkoxy substituted phenylcarbamic acids showed the antialgal activity against Chlorella vulgaris [8—10]. We have recently reported the synthesis of a series of amides prepared from the substituted pyrazine-2-carboxylic acids and some aminophenols [11], halogenated and alkylated anilines [12—15]. All these amides possess some antialgal, antifungal, and antimycobacterial properties [12,14,16].

The presented study is concerned in the synthesis of another series of amides prepared via anilinolysis of substituted pyrazin-2-carboxylic acid chlorides with alkoxylated, hydroxylated and/or halogenated anilines. The aim of this work is to search for the structure—activity relationships in the mentioned series, i.e. to continue in studying of the substituent variability influence on the biological activity, and to determine the importance of increased hydrophobic properties for photosynthesis-inhibiting evaluation of newly prepared substituted pyrazine-2-carboxamides.

Results and Discussion

The synthesis of amides is shown in Scheme 1. Condensation of chlorides of 6-chloropyrazine-2-carboxylic (1a) [17], 5-(1,1-dimethylethyl)pyrazine-2-carboxylic (1b) [11] or 6-chloro-5-(1,1-dimethylethyl)pyrazine-2-carboxylic (1c) [11] acids with ring-substituted anilines yielded a series of 12 amides of mentioned pyrazine-2-carboxylic acids 2a—l. The melting points, yields, elemental analyses, and the IR, 1H and 13C NMR spectral data for the all compounds prepared are given in Experimental. Calculated log P values of all derivatives studied are shown in Table 1.

Twelve studied compounds inhibited photosynthetic electron transport in spinach chloroplasts (see Table 1). The inhibitory activity of the studied compounds was relatively low, the most efficient inhibitors were compounds 2h (IC50 = 80.3 µmol dm-3) and mainly 2i (IC50 = 41.9 µmol dm-3), i.e. compounds with the hydroxylic group on the benzene ring (R2 = 2-OH-5-Br). These results are in the good correspondence with our previous observation about the importance of phenolic moiety for the photosynthesis-inhibiting activity in the studied compound type [11]. The increased hydrophobic parameters of studied compounds possess only secondary influence on the biological effect.

The addition of diphenylcarbazide (an artificial electron donor acting in the intermediate Z+/D+ on the donor side of photo system II [18]) to spinach chloroplasts inhibited by the studied compounds caused complete restoration of the photosynthetic electron transport. This indicates that the primary donor of PS II (P680) was not be damaged by the studied compounds. Previous EPR experiments with some amides of pyrazin-2-carboxylic acid showed that the site of action of these compounds in the photosynthetic apparatus of spinach chloroplasts is intermediate D2 on the donor side of photo system II [19].

Scheme 1: Synthesis of some substituted pyrazine-2-carboxamides 2a—l


Experimental

General

Melting points were determined on a Kofler apparatus and are uncorrected. Elemental analyses were obtained using an EA 1110 CE instrument (Fisons Instruments S.p.A., Milan). The IR spectra were recorded on a Nicolet Impact 400 spectrometer in KBr pellets. The 1H NMR spectra were measured for solutions in CDCl3 with a Varian Mercury - Vx BB 300 spectrometer operating at 300 MHz. Chemical shifts were recorded as δ values in parts per million (ppm), and were indirectly referenced to tetramethylsilane via the solvent signal (7.26 for 1H). Multiplicities are given together with the coupling constants (in Hz). Log P values were computed using a program ACD/LogP ver. 1.0 (Advanced Chemistry Development Inc., Toronto).

Synthesis of amides 2a—l

A mixture of acid (i.e. 6-chloropyrazine-2-carboxylic [17], 5-tert-butylpyrazine-2-carboxylic [11] or 5-tert-butyl-6-chloro-pyrazine-2-carboxylic [11] acids, 0.05 mol) and thionyl chloride (5.5 cm3, 75 mmol) in 20 cm3 of dry benzene was refluxed for about 1 h. Excess of thionyl chloride was removed by repeated evaporation with dry benzene in vacuo. The crude acyl chloride dissolved in 50 cm3 of dry acetone was added drop wise to a stirred solution of the corresponding substituted aniline (50 mmol) in 50 cm3 of dry pyridine keeping at the room temperature. After the addition was complete, stirring continued for another 30 min. The reaction mixture was then poured into 100 cm3 of cold water and the crude amide was collected and recrystallized from aqueous ethanol.

6-Chloro-pyrazine-2-carboxylic acid (3-methoxy-phenyl)-amide (2a). Appropriate aniline afforded 2a in 74% yield, m.p. 139-140 °C. For C12H10ClN3O2 (263.7) calculated: 54.66% C, 3.62% H, 13.45% Cl, 15.94% N; found: 54.72% C, 3.59% H, 16.09% N. IR spectrum: 3355 (NH), 2838 (OCH3), 1681 (CO). 1H NMR (300 MHz, CDCl3) δ 9.39-9.37 (m, 2H, H3, NH), 8.80 (d, 1H, J=0.55 Hz, H5), 7.50 (t, 1H, J=2.20 Hz, H2'), 7.30 (d, 1H, J=7.96 Hz, H4'), 7.24-7.19 (m, 1H, H5'), 6.74 (ddd, 1H, J=7.96 Hz, J=2.47 Hz, J=1.10 Hz, H6'), 3.84 (s, 3H, OCH3). 13C NMR (75 MHz, CDCl3) δ 160.2, 159.3, 147.5, 147.4, 143.9, 142.2, 138.0, 129.9, 112.2, 111.1, 105.5, 55.4.

5-tert-Butyl-pyrazine-2-carboxylic acid (3-methoxy-phenyl)-amide (2b). Appropriate aniline afforded 2b in 81% yield, m.p. 79-80 °C. For C16H19N3O2 (285.4) calculated: 67.35% C, 6.71% H, 14.73% N; found: 67.48% C, 6.69% H, 14.95% N. IR spectrum: 3360 (NH), 2841 (OCH3), 1677 (CO). 1H NMR (300 MHz, CDCl3) δ 9.65 (bs, 1H, NH), 9.39 (d, 1H, J=1.37 Hz, H3), 8.62 (d, 1H, J=1.37 Hz, H6), 7.55 (t, 1H, J=2.20 Hz, H2'), 7.28 (t, 1H, J=7.97 Hz, H5'), 7.22-7.17 (m, 1H, H4'), 6.72 (ddd, 1H, J=7.97 Hz, J=2.47 Hz, J=1.10 Hz, H6'), 3.85 (s, 3H, OCH3), 1.45 (s, 9H, CH3). 13C NMR (75 MHz, CDCl3) δ 167.8, 161.1, 160.2, 142.9, 141.3, 1389.0, 138.6, 129.8, 111.9, 110.7, 105.2, 55.3, 37.0, 29.7.

5-tert-Butyl-6-chloro-pyrazine-2-carboxylic acid (3-methoxy-phenyl)-amide (2c). Appropriate aniline afforded 2c in 78% yield, m.p. 128-129 °C. For C16H18ClN3O2 (319.8) calculated: 60.09% C, 5.67% H, 11.09% Cl, 13.14% N; found: 59.88% C, 5.62% H, 13.18% N. IR spectrum: 3380 (NH), 2840 (OCH3), 1686 (CO). 1H NMR and 13C NMR spectral data were not obtained.

6-Chloro-pyrazine-2-carboxylic acid (3,5-dimethoxy-phenyl)-amide (2d). Appropriate aniline afforded 2d in 64% yield, m.p. 211-212 °C. For C13H12ClN3O3 (293.7) calculated: 53.16% C, 4.12% H, 12.07% Cl, 14.31% N; found: 52.81% C, 4.29% H, 14.02% N. IR spectrum: 3370 (NH), 2964, 2838 (OCH3), 1685 (CO). 1H NMR (300 MHz, CDCl3) δ 9.38 (s, 1H, H3), 9.34 (bs, 1H, NH), 8.81 (s, 1H, H5), 6.98 (d, 2H, J=2.19 Hz, H2', H6'), 6.33-6.30 (m, 1H, H4'), 3.82 (s, 6H, OCH3). 13C NMR (75 MHz, CDCl3) δ 161.2, 159.3, 147.6, 147.4, 143.9, 142.2, 138.5, 98.2, 97.7, 55.5

5-tert-Butyl-pyrazine-2-carboxylic acid (3,5-dimethoxy-phenyl)-amide (2e). Appropriate aniline afforded 2d in 82% yield, m.p. 135-136 °C. For C17H21N3O3 (315.4) calculated: 64.74% C, 6.71% H, 13.32% N; found: 63.85% C, 6.71% H, 13.23% N. IR spectrum: 3360 (NH), 2961, 2838 (OCH3), 1690 (CO). 1H NMR (300 MHz, CDCl3) δ 9.62 (bs, 1H, NH), 9.38 (d, 1H, J=1.37 Hz, H3), 8.62 (d, 1H, J=1.38 Hz, H6), 7.00 (d, 2H, J=2.20 Hz, H2', H6'), 6.29 (t, 1H, J=2.20 Hz, H4'), 3.82 (s, 6H, OCH3), 1.44 (s, 9H, CH3). 13C NMR (75 MHz, CDCl3) δ 167.8, 161.1, 161.1, 142.9, 141.3, 139.1, 139.0, 97.9, 97.2, 55.4, 37.1, 29.7

5-tert-Butyl-6-chloropyrazine-2-carboxylic acid (3,5-dimethoxy-phenyl)-amide (2f). Appropriate aniline afforded 2f in 49% yield, m.p. 123-124 °C. For C17H20ClN3O3 (349.8) calculated: 58.37% C, 5.76% H, 10.13% Cl, 12.01% N; found: 58.57% C, 5.91% H, 12.05% N. IR spectrum: 3376 (NH), 2960, 2839 (OCH3), 1698 (CO). 1H NMR (300 MHz, CDCl3) δ 9.31 (bs, 1H, NH), 9.25 (s, 1H, H3), 6.99 (d, 2H, J=2.20 Hz, H2', H6'), 6.30 (t, 1H, J=2.20 Hz, H4'), 3.82 (s, 6H, OCH3), 1.55 (s, 9H, CH3). 13C NMR (75 MHz, CDCl3) δ 164.6, 161.1, 159.8, 145.7, 141.0, 140.2, 138.7, 98.1, 97.5, 55.4, 39.0, 28.3

6-Chloro-pyrazine-2-carboxylic acid (5-bromo-2-hydroxy-phenyl)-amide (2g). Appropriate aniline afforded 2g in 71% yield, m.p. 154-155 °C. For C11H7BrClN3O2 (328.6) calculated: 40.21% C, 2.15% H, 24.32% Br, 10.79% Cl, 12.79% N; found: 40.51% C, 1.93% H, 13.05% N. IR spectrum: 3370 (NH), 1682 (CO). 1H NMR and 13C NMR spectral data were not obtained.

5-tert-Butyl-pyrazine-2-carboxylic acid (5-bromo-2-hydroxy-phenyl)-amide (2h). Appropriate aniline afforded 2h in 86% yield, m.p. 184-185 °C. For C15H16BrN3O2 (350.2) calculated: 51.44% C, 4.60% H, 22.82% Br, 12.00% N; found: 51.39% C, 5.61% H, 11.94% N. IR spectrum: 3368 (NH), 1685 (CO). 1H NMR (300 MHz, CDCl3) δ 9.55 (bs, 1H, NH), 9.37 (d, 1H, J=1.1 Hz, H3), 8.60 (d, 1H, J=1.1 Hz, H6), 8.08 (d, 1H, J=2.47 Hz, H3'), 7.47 (dd, 1H, J=8.79 Hz, J=2.47 Hz, H5'), 7.02 (d, 1H, J=8.79 Hz, H6'), 5.66 (bs, 1H, OH), 1.44 (s, 9H, CH3). 13C NMR (75 MHz, CDCl3) δ 167.9, 161.0, 149.4, 142.9, 141.1, 139.0, 131.2, 123.6, 120.9, 116.1, 110.1, 37.1, 29.7

5-tert-Butyl-6-chloro-pyrazine-2-carboxylic acid (5-bromo-2-hydroxy-phenyl)-amide (2i). Appropriate aniline afforded 2i in 77% yield, m.p. 160-161 °C. For C15H15BrClN3O2 (384.7) calculated: 46.84% C, 3.93% H, 20.77% Br, 9.22% Cl, 10.92% N; found: 47.09% C, 4.12% H, 11.13% N. IR spectrum: 3373 (NH), 1691 (CO). 1H NMR (300 MHz, CDCl3) δ 9.28 (bs, 1H, NH), 9.25 (s, 1H, H3), 8.06 (d, 1H, J=2.47 Hz, H3'), 7.49 (dd, 1H, J=8.79 Hz, J=2.47 Hz, H5'), 7.03 (d, 1H, J=8.79 Hz, H6'), 5.65 (bs, 1H, OH), 1.55 (s, 9H, CH3). 13C NMR (75 MHz, CDCl3) δ 164.7, 159.7, 149.7, 145.8, 140.8, 140.2, 130.8, 123.8, 121.2, 116.1, 110.1, 39.0, 28.3

6-Chloro-pyrazine-2-carboxylic acid (3,4-dichloro-phenyl)-amide (2j). Appropriate aniline afforded 2i in 83% yield, m.p. 132-133 °C. For C11H6Cl2N3O (302.6) calculated: 43.67% C, 2.00% H, 35.15% Cl, 13.89% N; found: 43.51% C, 1.78% H, 14.11% N. IR spectrum: 3370 (NH), 1690 (CO). 1H NMR (300 MHz, CDCl3) δ 9.41 (bs, 1H, NH), 9.38 (s, 1H, H3), 8.83 (s, 1H, H5), 8.00 (d, 1H, J=2.47 Hz, H2'), 7.59 (dd, 1H, J=8.79 Hz, J=2.47 Hz, H6'), 7.45 (d, 1H, J=8.79 Hz, H5'). 13C NMR (75 MHz, CDCl3) δ 159.3, 147.8, 147.4, 143.2, 142.1, 136.1, 132.9, 130.7, 130.6, 128.3, 121.5, 119.0

5-tert-Butyl-pyrazine-2-carboxylic acid (3,4-dichloro-phenyl)-amide (2k). Appropriate aniline afforded 2k in 76% yield, m.p. 143-144 °C. For C15H15Cl2N3O (324.2) calculated: 55.57% C, 4.66% H, 21.87% Cl, 12.96% N; found: 55.63% C, 4.71% H, 13.08% N. IR spectrum: 3365 (NH), 1685 (CO). 1H NMR (300 MHz, CDCl3) δ 9.67 (bs, 1H, NH), 9.37 (d, 1H, J=1.37 Hz, H3), 8.61 (d, 1H, J=1.37 Hz, H6), 8.01 (d, 1H, J=2.48 Hz, H2'), 7.58 (dd, 1H, J=8.79 Hz, J=2.47 Hz, H6'), 7.43 (d, 1H, J=8.79 Hz, H5'), 1.45 (s, 9H, CH3). 13C NMR (75 MHz, CDCl3) δ 168.2, 161.2, 143.0, 140.7, 139.0, 136.9, 133.0, 130.6, 127.7, 121.3, 118.9, 37.1, 29.7

5-tert-Butyl-6-Chloro-pyrazine-2-carboxylic acid (3,4-dichloro-phenyl)-amide (2l). Appropriate aniline afforded 2l in 83% yield, m.p. 113-114 °C. For C15H14Cl3N3O (358.7) calculated: 50.23% C, 3.93% H, 29.65% Cl, 11.72% N; found: 55.63% C, 4.71% H, 13.08% N. IR spectrum: 3390 (NH), 1685 (CO). 1H NMR (300 MHz, CDCl3) δ 9.38 (bs, 1H, NH), 9.25 (s, 1H, H3), 8.01 (d, 1H, J=2.47 Hz, H2'), 7.59 (dd, 1H, J=8.79 Hz, J=2.48 Hz, H6'), 7.44 (d, 1H, J=8.79 Hz, H5'), 1.55 (s, 9H, CH3). 13C NMR (75 MHz, CDCl3) δ 165.1, 159.9, 145.8, 140.5, 140.3, 136.5, 133.0, 130.7, 128.2, 121.6, 119.1, 39.1, 28.2

Study of inhibition of oxygen evolution rate in spinach chloroplasts

The inhibition of oxygen evolution rate (OER) in spinach chloroplasts by the studied compounds was investigated spectrophotometrically (Specord UV VIS, Zeiss, Jena) in the presence of an electron acceptor 2,6-dichlorophenol—indophenol, using method described in Ref. [20]. The compounds were dissolved in dimethyl sulfoxide (DMSO) because of their low water solubility. The used DMSO volume fractions (up to 5 vol. %) did not affect the oxygen evolution. The inhibitory efficiency of the studied compounds has been expressed by IC50 values, i.e. by molar concentration of the compounds causing 50 % decrease in the oxygen evolution relative to the untreated control. Comparable IC50 value for a selective herbicide atrazine [21] is about 1.0 µmol dm-3.

Table 1: OER inhibition in spinach chloroplasts (IC50) and calculated lipophilicity (log P) of compounds 2a—l.

Comp. IC50 [mmol dm-3] Log P
2a 0.4998 2.42 ± 0.42
2b 0.7995 2.98 ± 0.41
2c 0.6440 4.10 ± 0.43
2d 0.5330 2.46 ± 0.43
2e 0.3172 3.02 ± 0.42
2f 0.4351 4.14 ± 0.45
2g 0.1462 3.34 ± 0.50
2h 0.0803 3.91 ± 0.49
2i 0.0419 5.03 ± 0.51
2j 0.1048 4.15 ± 0.44
2k 1.5251 4.72 ± 0.43
2l 0.1301 5.84 ± 0.45
Atrazine 0.0010 1.03 ± 0.62

Acknowledgements. This study was supported by the Ministry of Education of the Czech Republic (No. 11160001) and by the Scientific Grant Agency of the Ministry of Education of the Slovak Republic and the Slovak Academy of Sciences (Grant No. 1/7262/20). We also thank D. Karlickova, J. Zizkova, and T. Vojtisek from the Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Czech Republic, for their skillful technical assistance and Dr. D. Mikulasova from the Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Bratislava, Slovak Republic, for her assistance in the preparation of chloroplasts.

References

  1. Good, N. E.: Plant. Physiol. 1961, 36, 788.
  2. Kralova, K., Sersen, F., Cizmarik, J.: Chem. Pap. 1992, 46, 266.
  3. Kralova, K., Sersen, F., Miletin, M., Hartl, J.: Chem. Pap. 1998, 52, 52.
  4. Kralova, K., Sersen, F., Kubicova, L., Waisser, K.: Chem. Pap. 1999, 53, 328.
  5. Miletin, M., Hartl, J., Machacek, M.: Collect. Czech. Chem. Commun. 1997, 62, 672.
  6. Kralova K., Loos D., Miletin M., Klimesova V.: Folia Pharm. Univ. Carol. 1998, 23 Suppl., 77.
  7. Miletin M., Dolezal M., Hartl J., Kralova K., Machacek M.: The Third International Electronic Conference on Synthetic Organic Chemistry (ECSOC-3), September 1-30, 1999 (http://www.unibas.ch/mdpi/ecsoc-3/c0005/c0005.htm).
  8. Kralova K., Loos D., Cizmarik J.: Collect. Czech. Chem. Commun. 1994, 59, 2293.
  9. Kralova K., Loos D., Cizmarik J.: Photosynthetica 1994, 30, 155.
  10. Kralova K., Bujdakova H., Cizmarik J.: Pharmazie 1995, 50, 440.
  11. Dolezal, M., Hartl, J., Miletin, M., Machacek, M., Kralova K.: Chem. Pap. 1999, 53, 126.
  12. Dolezal, M., Vicik, R., Miletin, M., Kralova, K.: Chem. Pap. 2000, 54, 245.
  13. Dolezal, M., Miletin, M., Hartl, J., Kralova, K., Kunes, J.: The Fourth International Electronic Conference on Synthetic Organic Chemistry (ECSOC-4), September 1-30, 2000 (http://www.unibas.ch/mdpi/ecsoc-4/c0028/c0028.htm).
  14. Dolezal, M., Hartl, J., Miletin, M.: Folia Pharm. Univ. Carol. 2000, 25, 15 (http://www.faf.cuni.cz/folia/25/2502dolezal.htm).
  15. Dolezal M., Miletin M., Hejsky R., Kralova K., Kunes J.: The Fifth International Electronic Conference on Synthetic Organic Chemistry (ECSOC-5), September 1-30, 2001 (http://www.mdpi.net/ecsoc-5/c0009/c0009.htm).
  16. Dolezal M., Miletin M., Kunes J., Kralova K.: Molecules 2002, 7, 363 (http://www.mdpi.net/molecules/papers/70300363.pdf).
  17. Abe, Y., Shigeta, Y., Uchimaru, F., Okada, S., Ozasayama, E.: Japan. 69 12,898 (1969); Chem. Abstr. 1969, 71, 112979y.
  18. Jegerschöld C. and Styring, S., FEBS Lett. 1991, 280, 87.
  19. Dolezal M., Kralova K., Sersen F., Miletin M.: Folia Pharm. Univ. Carol. 2001, 26, 13-20 (http://www.faf.cuni.cz/folia/26/2602dolezal.htm).
  20. Kralova, K., Sersen, F., Sidoova, E.: Chem. Pap. 1992, 46, 348.
  21. Carpentier, R., Fuerst, E. P., Nakatani, H. Y., Arntzen, C. J.: Biochim. Biophys. Acta 1985, 808, 293.