Synthesis of Some Amides of Substituted Pyrazine-2-carboxylic Acids and Their Photosynthesis-inhibiting Activity

¹ Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy, Charles University, 500 05 Hradec Kralove, Czech Republic

^a e-mail: dolezalm@faf.cuni.cz, 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.

Received: 15 August 2001 / Uploaded 22 August 2001

Introduction

Various compounds possessing -NHCO- group, e. g. acyl and thioacyl amides, benzamides, phenyl carbamates, etc., were found to inhibit photosynthetic electron transport [1--4]. Amides of 2-alkylpyridine-4-carboxylic [5, 6], 2-alkylsulfanyl-4-pyridinecarboxylic [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]. The inhibitory efficiency of APA concerning chlorophyll production in Chlorella vulgaris depended on the lipophilicity of the alkoxy substituent and also on its position on the aromatic ring [8--10]. The antialgal activity of APA correlated with the antifungal activity of these compounds against Candida albicans [10]. We have recently reported the synthesis of a series of amides prepared from some or 5-alkylpyrazine-2-carboxylic acids and some aminophenols [11], halogenated and alkylated anilines [12, 13]. All these amides of pyrazinoic acid possess antialgal, antifungal, and antimycobacterial properties [12, 14].

The presented study is concerned in the synthesis of another series of amides prepared from substituted pyrazin-2-carboxylic acids and alkylated or halogenated anilines. The aim of this work is to search for the structure--activity relationships and to determine the importance of increased hydrophobic properties for photosynthesis-inhibiting evaluation of newly prepared pyrazine-2-carboxylic acid amides.

Results and Discussion

The synthesis of amides is shown in Scheme 1. Condensation of chlorides of 3-chloro-pyrazine-2-carboxylic acid (1) [15] with ring substituted anilines yielded a series of 3 amides of 3-chloropyrazine-2-carboxylic acids 2a-c. Condensation of chlorides of 6-chloropyrazine-2-carboxylic (3a) [16], 5-(1,1-dimethylethyl)pyrazine-2-carboxylic (3b) [11] or 6-chloro-5-(1,1-dimethylethyl)pyrazine-2-carboxylic (3c) [11] acids with ring substituted anilines yielded a series of 9 amides of mentioned substituted pyrazine-2-carboxylic acids 4a-i.

Scheme 1: Preparation of amides of substituted pyrazine-2-carboxylic acids 2a-c and 4a-i.

The melting points, yields, and elemental analyses for the all compounds prepared are given in Table 1, and the IR and ¹H NMR spectral data for the amides 4a-i in Table 2. Calculated log P values of all derivatives studied are shown in Table 3.

Biological activity of prepared amides 2a-c and 4a-i concerning inhibition of oxygen evolution rate in spinach chloroplasts was investigated. The inhibitory activity of the compounds has been expressed as IC₅₀ values (see Table 3). However, there are no results for the compounds 4a, 4b, 4c, 4g and 4i due their low solubility in dimethyl sulfoxide.

The studied compounds inhibited photosynthetic electron transport in spinach chloroplasts what was reflected in the inhibition of oxygen evolution rate. The IC₅₀ values varied in the range from 0.065 (2a) to 1.210 mmol dm^-3 (2c). In general, the studied compounds exert very moderate photosynthesis-inhibiting activity.

Table 1. Analytical data of the amides 2a-c and 4a-i.

Compd.	X	R	Y	Formula M. w.	% Calculated / % Found						M.p./°C Yield/%
					C	H	N	F	Cl	Br
2a	-	-	H	C11H8ClN3O 233.7	56.55 56.52	3.45 3.34	17.98 17.86	- -	15.17 -	- -	269--270 75
2b	-	-	3-F	C11H7ClFN3O 251,7	52.50 52.48	2.80 3.02	16.70 16.67	7.55 -	14.09 -	- -	265--266 44
2c	-	-	3-Br	C11H7BrClN3O 312.6	42.27 42.15	2.26 2.32	13.44 13.46	- -	11.34 -	25.56 -	225--226 58
4a	Cl	H	4-Br	C11H7BrClN3O 312.6	42.27 42.19	2.26 2.23	13.44 13.36	- -	11.34 -	25.56 -	166--167 89
4b	H	(CH3)3C	4-Br	C15H16BrN3O 334,2	53.91 54.06	4.83 4.82	12.57 12.67	- -	- -	23.91 -	191--192 86
4c	Cl	(CH3)3C	4-Br	C15H15BrClN3O 368.7	48.87 49.04	4.10 4.08	11.40 11.31	- -	9.62 -	21.67 -	204--205 84
4d	Cl	H	2,4,6-CH3	C14H14ClN3O 275.7	60.98 60.87	5.12 4.98	15.24 15.01	- -	12.86 -	- -	133--134 83
4e	H	(CH3)3C	2,4,6-CH3	C18H23N3O 297.4	72.70 72.63	7.80 7.82	14.13 13.94	- -	- -	- -	120--121 88
4f	Cl	(CH3)3C	2,4,6-CH3	C18H22ClN3O 331.9	65.15 65.16	6.68 6.59	12.66 12.53	- -	10.68 -	- -	108--109 82
4g	Cl	H	4-COCH3	C13H10ClN3O2 275.7	56.64 56.48	3.66 3.47	15.24 15.01	- -	12.86 -	- -	215--216 63
4h	H	(CH3)3C	4-COCH3	C17H19N3O2 297.4	68.67 68.51	6.44 6.58	14.13 14.20	- -	- -	- -	205--206 75
4i	Cl	(CH3)3C	4-COCH3	C17H18ClN3O2 331.8	61.54 61.77	5.47 5.50	12.66 12.84	- -	10.68 -	- -	201--202 82

Table 2. IR and ¹H NMR spectral data of the amides 4a-i.

Compd.

IR (cm-1)

1H-NMR (delta, ppm; J in Hz)

(C=O)

NH

H3

H5

H6

H2'

H3'

H4'

H5'

H6'

CH3

C(CH3)3

4a

1685

9.40bs

9.38d overlapped

8.82s

-

7.70-7.63m AA', BB'

7.55-7.48m AA', BB'

-

7.55-7.48m AA', BB'

7.70-7.63m AA', BB'

-

1.45s

4b

1685

9.66bs

9.38d J=1.51

-

8.61d J=1.51

7.69-7.63m AA', BB'

7.52-7.46m AA', BB'

-

7.52-7.46m AA', BB'

7.69-7.63m AA', BB'

-

-

4c

1690

9.37s

9.26s

-

-

7.69-7.64m AA', BB'

7.53-7.47m AA', BB'

-

7.53-7.47m AA', BB'

7.69-7.64m AA', BB'

-

1.55s

4d

1680

8.87bs

9.39bs

8.82s

-

-

6.95s

-

6.95s

-

2.30s 3H 2.23s 6H

-

4e

1685

9.09bs

9.39d J=1.65

-

8.64d J=1.65

-

6.95s

-

6.95s

-

2.30s 3H 2.23s 6H

1.46s

4f

1690

8.85bs

9.26s

-

-

-

6.94s

-

6.94s

-

2.30s 3H 2.23s 6H

1.56s

4g

1680

a

a

a

a

a

a

a

a

a

a

a

4h

1685

9.85bs

9.39d J=1.37

-

8.63d J=1.37

7.90-7.83m AA', BB'

8.04-7.97m AA', BB'

-

8.04-7.97m AA', BB'

7.90-7.83m AA', BB'

2.60s

1.45s

4i

1690

9.55bs

9.27s

-

-

7.90-7.83m AA', BB'

8.04-7.96m AA', BB'

-

8.04-7.96m AA', BB'

7.90-7.83m AA', BB'

2.60s

1.55s

^a not measured

Table 3. IC₅₀ values concerning inhibition of oxygen evolution rate in spinach chloroplasts by the tested amides 2a-c and 4a-i and calculated log P values of the compounds in comparison with standard (atrazine).

Compd.	IC50 [mmol dm-3]	log P
2a	0.065	1.63 +- 0.41
2b	0.168	2.12 +- 0.49
2c	1.210	2.84 +- 0.49
4a	a	3.42 +- 0.48
4b	a	3.99 +- 0.47
4c	a	5.11 +- 0.49
4d	0.495	3.64 +- 0.41
4e	0.434	4.21 +- 0.41
4f	0.195	5.33 +- 0.42
4g	a	2.14 +- 0.43
4h	0.664	2.70 +- 0.42
4i	a	3.83 +- 0.44
Atrazine	0.001	1.03 +- 0.62

^a not measured

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 delta 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/Log P ver. 1.0 (Advanced Chemistry Development Inc., Toronto).

Synthesis of amides 2a-c and 4a-i

A mixture of acid (i. e. 3-chloropyrazine-2-carboxylic [15], 6-chloropyrazine-2-carboxylic [16], 5-(1,1-dimethylethyl)pyrazine-2-carboxylic [11] or 6-chloro-5-(1,1-dimethylethyl)-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 dropwise 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 200 cm³ of cold water and the crude amide was collected and recrystallized from aqueous ethanol.

Study of inhibition of oxygen evolution rate in spinach chloroplasts

The oxygen evolution rate in spinach chloroplasts was investigated spectrophotometrically (Specord UV VIS, Zeiss, Jena) in the presence of an electron acceptor 2,6-dichlorophenol--indophenol, by method described in Ref. [17]. 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 [18] is about 0.001 mmol dm^-3.

Acknowledgements. This study was supported by the Ministry of Education of the Czech Republic (No. 11160001 and No. FRVS 1676/G4/2001) 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 for their skillful technical assistance and Dr. D. Mikulasova from the Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Bratislava, for her assistance in the preparation of chloroplasts.

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