Synthesis of some anilides of 2-alkyl-4-pyridinecarboxylic acids and their photosynthesis-inhibiting activity

Article abstract of DOI:10.1135/cccc19970672

Homolytic alkylation of 4-pyridinecarbonitrile with radicals generated by silver nitrate catalyzed oxidative decarboxylation of alkanoic acids and subsequent alkaline hydrolysis afforded 2-alkyl-4-pyridinecarboxylic acids 2a-2e. These were converted into

Full text of DOI:10.1135/cccc19970672

672
Miletin, Hartl, Machacek:
SYNTHESIS OF SOME ANILIDES OF 2-ALKYL-4-PYRIDINECARBOXYLIC
ACIDS AND THEIR PHOTOSYNTHESIS-INHIBITING ACTIVITY
a1
b
Miroslav MILETIN , Jiri HARTLa2 and Milos MACHACEK
a Department of Medicinal Chemistry and Drug Control, Faculty of Pharmacy, Charles University,
500 05 Hradec Kralove, Czech Republic; e-mail: 1 miletin@faf.cuni.cz, 2 hartl@faf.cuni.cz
b Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University,
500 05 Hradec Kralove, Czech Republic; e-mail: machacek@faf.cuni.cz
Received August 15, 1996
Accepted December 27, 1996
Homolytic alkylation of 4-pyridinecarbonitrile with radicals generated by silver nitrate catalyzed oxi-
dative decarboxylation of alkanoic acids and subsequent alkaline hydrolysis afforded 2-alkyl-4-pyri-
dinecarboxylic acids 2a–2e. These were converted into the halogeno-hydroxy substituted anilides
3a–3r via acyl chlorides by reaction with the respective aminophenols at 10 °C. Of the compounds
tested, 5-chloro-2-hydroxyphenylamide of 2-butyl-4-pyridinecarboxylic acid 3h showed the highest
effect upon oxygen evolution rate in spinach chloroplasts system. The structure–photosynthesis-inhi-
biting activity relationships are briefly discussed.
Key words: Homolytic alkylation; Pyridinecarboxylic acids; Photosynthesis inhibition.
Studies of relationships between chemical structure and biological activity have shown
that many herbicides acting as photosynthesis inhibitors possess in their molecules an
>N–C(=X)– group (X = O or N, not S) and a hydrophobic residue in close vicinity to
it1,2. Shipman3,4 concluded that the hydrophilic part of a herbicide binds electrostati-
cally to the terminus of an α-helix at a highly charged amino acid, whereby the hydro-
phobic part of the inhibitor extends into the hydrophobic part of the membrane.
Recently, pronounced photosynthesis-inhibiting activity has been found for alkoxy substituted
phenylcarbamates5,6, as well as for the local anaesthetic of anilide type – trimecaine7–9
,
i.e., for compounds with a –CONH– group in their molecules.
The present paper deals with the study of (di)halogeno-hydroxy substituted anilides
of 2-alkyl-4-pyridinecarboxylic acids 3a–3r as photosynthesis inhibitors. Starting 2-alkyl-
4-pyridinecarbonitriles 1a–1e were synthesized by homolytic alkylation of 4-pyridine-
carbonitrile with radicals generated by oxidative decarboxylation of alkanoic acids,
catalyzed with silver nitrate, according to Wang et al.10,11. Alkaline hydrolysis of 2-alkyl-
4-pyridinecarbonitriles 1a–1e afforded 2-alkyl-4-pyridinecarboxylic acids 2a–2e in 81–86%
yield. On treatment with thionyl chloride at reflux, the acids gave acyl chlorides which
were in turn converted into the title anilides 3a–3r (Scheme 1, Table I) by reaction with
2-amino-5-methylphenol, 2-amino-4-chlorophenol, 4-amino-2-chlorophenol, 4-amino-
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673
TABLE I
Characteristic data of 2-alkylpyridine-4-carboxanilides 3a–3r
Calculated/Found
Formula
M.w.
M.p.,°C
Yield, %
~
~
Compound
ν(C–H)
ν(C=O)
% C % H % N % Cla
3a
3b
3c
3d
3e
3f
C15H15ClN2O2
290.8
191–193
35
61.94 5.20
62.08 5.27
9.67 12.19 2 980, 2 940
1 640
1 660
1 640
1 665
1 640
1 650
1 640
1 680
1 650
1 640
1 660
1 640
1 640
1 645
1 640
9.45 11.99
2 880
C15H14Cl2N2O2 181–183
55.40 4.34
55.50 4.39
8.61 21.80
8.57 21.60
2 990
325.2
58
C16H18N2O2
270.3
141–144
36
71.09 6.71 10.36
71.15 6.79 10.19
–
–
2 990
C15H15ClN2O2
290.8
169–171
39
61.94 5.20
62.07 5.25
9.67 12.19 2 990, 2 970
9.62 12.05
C15H14Cl2N2O2 182–183
325.2 60
C15H14Br2N2O2 129–133
55.40 4.34
55.14 4.32
8.61 21.80
8.57 21.65
2 995
2 980
2 975
2 990
43.51 3.41
43.65 3.48
6.77 38.59
6.81 38.32
414.1
58
3g
3h
3i
C15H14I2N2O2
508.1
180–182.5 35.46 2.78
58 35.37 2.71
129–130.5 63.06 5.62
45 63.15 5.71
158–160.5 63.06 5.62
5.51 49.95
5.42 50.15
C16H17ClN2O2
304.8
9.19 11.63
9.08 11.52
C16H17ClN2O2
304.8
9.19 11.63 2 980, 2 940
9.06 11.51
42
63.12 5.73
3j
C16H16Cl2N2O2 185–187
339.2 58
56.65 4.75
56.77 4.81
8.26 20.90
8.14 20.63
2 980
3k
3l
C16H16Br2N2O2 134–137
44.89 3.77
44.78 3.69
6.54 37.33 2 960, 2 930
6.48 37.45
428.1
56
C16H16I2N2O2
522.1
185–186.5 36.81 3.09
5.37 48.61
5.22 48.82
2 960
2 980
2 970
2 970
62
36.72 2.94
3m
3n
3o
C16H17ClN2O2
304.8
156–158
40
63.06 5.62
63.12 5.67
9.19 11.63
9.25 11.47
C16H16Cl2N2O2 182–184.5 56.65 4.75
8.26 20.90
8.19 20.71
339.2
57
56.61 4.85
C16H17BrN2O2
340.2
150–152
42
55.03 4.91
55.15 4.97
8.02 22.88
7.89 22.70
Collect. Czech. Chem. Commun. (Vol. 62) (1997)

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Miletin, Hartl, Machacek:
TABLE I
(Continued)
Calculated/Found
%H
%N %Cla
Formula
M.w.
M.p.,°C
Yield, %
~
~
Compound
ν(C–H)
ν(C=O)
%C
3p
3q
3r
C16H16Br2N2O2
428.1
133–136 44.89 3.77
63 44.82 3.85
135–137 64.05 6.01
38 63.97 5.98
184–186 57.80 5.14
61 58.00 5.26
6.54 37.33
6.51 37.19
2 980
1 640
1 645
1 660
C17H19ClN2O2
318.8
8.79 11.12
8.77 11.00
2 985, 2 970
2 990, 2 940
C17H18Cl2N2O2
353.2
7.93 20.07
8.19 19.89
a
% Br for 3f, 3k, 3o, 3p; % I for 3g, 3l.
2,6-dichlorophenol, 4-amino-2-bromophenol, 4-amino-2,6-dibromophenol, and 4-amino-
2,6-diiodophenol at 10 °C. Keeping the low temperature was essential in order to avoid
the partial esterification of acyl chlorides with aminophenols. Structure of the products
1
1
was proven by IR and H NMR spectra. In the H NMR spectra (Table II) the protons
of alkyl groups as well as the protons of aromatic rings were observed at the expected
values with expected splitting patterns and intensities.
2
N
N
N
R
NH
1
R
CN
R
COOH
R
C
O
1
2
3
R
3
1
2
3
1
2
3
R
R
R
R
R
R
3
3
R
1, 2
C H
C H
3-Cl
4-OH
C H
4 9
3,5-Cl 4-OH
2
3,5-Br 4-OH
2
a
b
c
d
e
a
b
c
d
e
f
j
3
7
3
7
i-C H
3
C H
3,5-Cl 4-OH
C H
4
k
l
7
3
7
2
9
9
C H
i-C H 4-CH 2-OH
3
C H
4
3,5-I
2
4-OH
4-OH
4
9
7
3
t-C H
4
i-C H 5-Cl
3
2-OH
t-C H 3-Cl
4 9
m
n
o
p
q
r
9
7
C H
i-C H 3,5-Cl 4-OH
3
t-C H 3,5-Cl 4-OH
4 9 2
5
11
7
2
i-C H 3,5-Br 4-OH
3
t-C H 3-Br
4-OH
7
2
4 9
i-C H 3,5-I
3
4-OH
2-OH
4-OH
t-C H 3,5-Br 4-OH
g
h
i
7
2
4
9
2
C H
5-Cl
3-Cl
C H 3-Cl
5 11
4-OH
3,5-Cl 4-OH
4
9
C H
C H
5
4
9
11
2
SCHEME 1
Collect. Czech. Chem. Commun. (Vol. 62) (1997)

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675
The photosynthesis-inhibiting activity (Table III) for the chosen anilides was deter-
mined by measuring their inhibitory effect upon oxygen evolution rate in spinach chlo-
roplasts system5. It was impossible to determine the corresponding IC50 values for
3′,5′-dihalogeno-4′-hydroxy derivatives 3b, 3e, 3f, 3g, 3j, 3k, and 3l due to their too
low water solubility. In addition, as seen in comparison of the results obtained with
2-tert-butyl and 2-pentyl derivatives 3n, 3p, and 3r with those of 3′-monohalogeno-
4′-hydroxy analogues 3m, 3o, and 3q, the introduction of a further halogen into the 5′
position of an aniline moiety was detrimental to the activity in all cases examined. The
much lower activity shown by dihalogeno derivatives 3n, 3p, and 3r could be ascribed
also to their lower solubility and to their decreased ability to pass through the hydro-
philic regions of the thylakoid membranes. In contrast to the 3′-halogeno-4′-hydroxy
series, the 5′-chloro-2′-hydroxy derivatives displayed higher photosynthesis-inhibiting
activity. Of the anilides tested, the most active compound was the anilide 3h with an
IC50 of 31.4 µmol dm–3.
TABLE II
1H NMR chemical shifts of 2-alkylpyridine-4-carboxanilides 3a–3f, 3h–3r
Arom.
Alkyl group(s)
other CH2
Compound
H-3
H-5
H-6 H-2′ H-5′ H-6′
CH2 or CH
CH3
0.93 t
3a
3b
3c
3d
3e
3f
7.70 7.65 8.66 7.83 6.98 7.51
2.82 t
2.83 t
3.13 m
3.13 m
3.13 m
3.13 m
2.87 t
2.84 t
2.84 t
2.87 t
2.84 t
–
1.75 m
1.75 m
7.72 7.67 8.68 7.82
–
7.82
0.93 t
a
7.76 7.67 8.66
7.75 7.66 8.68
–
–
6.66 7.44
–
–
–
–
1.29 d, 2.25 s
1.29 d
b
–
–
–
–
7.76
7.82
8.02
7.79c
7.71 7.65 8.69 7.82
1.29 d
7.72 7.65 8.69 8.02
1.29 d
d
3h
3i
7.79c 7.72 8.69
–
1.72 m, 1.35 m 0.92 t
1.71 m, 1.35 m 0.92 t
1.71 m, 1.35 m 0.92 t
1.71 m, 1.35 m 0.92 t
1.71 m, 1.35 m 0.92 t
7.69 7.65 8.66 7.84 6.99 7.51
3j
7.71 7.66 8.68 7.83
7.78 7.70 8.71 8.02
7.71 7.65 8.67 8.22
–
–
–
7.83
8.02
8.22
3k
3l
3m
3n
3o
3p
3q
3r
7.85c 7.65 8.70 7.83c 6.99 7.51
7.82c 7.66 8.72 7.82c 7.82c
7.84c 7.65 8.70 7.84c 6.98 7.56
7.85 7.66 8.72 8.01 8.01
7.69 7.64 8.66 7.83 6.98 7.51
–
–
–
–
1.37 s
1.37 s
1.37 s
1.37 s
–
–
–
–
–
2.82 t
2.83 t
1.73 m, 1.33 me 0.88 t
1.72 m, 1.32 me 0.87 t
7.70 7.66 8.67 7.82
–
7.82
b
c
d
e
a 6.77 (H-3′); 6.95 (H-3′), 7.13 (H-4′); overlapping signals; 6.97 (H-3′), 7.14 (H-4′); 4 H.
Collect. Czech. Chem. Commun. (Vol. 62) (1997)

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Miletin, Hartl, Machacek:
Turning now to the effect of pyridine nucleus modification, comparison of IC50
values of 3′-chloro-4′-hydroxyanilides of 2-alkyl-4-pyridinecarboxylic acids with
straight-alkyl chain 3a, 3i, and 3q revealed that increased alkyl chain length caused an
enhancement of the activity. This result is expected for good correlation between lipo-
philicity and photosynthesis-inhibiting activity and has been found previously5,6,12,13
.
The introduction of 2-tert-butyl group, as represented by the anilide 3m, increased activity
by factor of 4 relative to 3i. Neglecting the contribution of branching at the α-carbon, the
effect of lipophilicity on photosynthesis inhibition can be illustrated also in 5′-chloro-
2′-hydroxy series (compare 3d with 3h).
Because the halogeno-hydroxy substituted anilides of 2-alkyl-4-pyridinecarboxylic
acids represent a novel structural type of photosynthesis-inhibiting agents, additional
work on the structure–activity relationships and the determination of their site of action
in photosystem is in progress.
TABLE III
Photosynthesis-inhibiting activity of some 2-alkylpyridine-4-carboxanilides (IC50 values from oxygen
evolution rate measurement in spinach chloroplasts system)
Compound
3a
3d
3h
3i
3m
3n
3o
3p
3q
3r
IC50, µmol dm–3 266.0 78.3
31.4 195.7 50.8 413.5 85.8 207.0 78.1 239.1
EXPERIMENTAL
Melting points were determined on a Boëtius apparatus and are uncorrected. Purity of intermediates
and products was checked by TLC on Silufol UV254 plates (Kavalier Votice, Czech Republic). Column
chromatography was performed on the Silpearl (Kavalier Votice, Czech Republic). Samples for
elemental analysis were dried at about 100 Pa over phosphorus pentoxide at room temperature for 6 h.
~
IR spectra (ν, cm–1) were recorded on a Perkin–Elmer model 577 spectrometer in KBr pellets.
1H NMR spectra (δ, ppm; J, Hz) were determined for solutions in hexadeuteriodimethyl sulfoxide
with tetramethylsilane as the internal standard with a BS 587 (80 MHz) or BS 494 (100 MHz) apparatus
(Tesla Brno, Czech Republic).
2-Alkyl-4-pyridinecarbonitriles 1a–1e
2-Propyl-, 2-isopropyl- and 2-butyl-4-pyridinecarbonitriles (1a–1c) were prepared according to Wang
et al.10,11. Crude products were purified by column chromatography using petroleum ether–ethyl ace-
tate (80 : 20) as the eluent. The observed boiling points agreed with those previously described10,11
.
By the same procedure 2-tert-butyl-4-pyridinecarbonitrile (1d, m.p. 51–53 °C, ref.14 gives 50–52 °C)
and 2-pentyl-4-pyridinecarbonitrile (1e, b.p. 134–136 °C/1.06 kPa, ref.14 gives 116–126 °C/0.93 kPa)
were also prepared in 43% and 41% yield, respectively.
Collect. Czech. Chem. Commun. (Vol. 62) (1997)

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677
2-Alkyl-4-pyridinecarboxylic Acids 2a–2e
2-Alkyl-4-pyridinecarbonitrile 1a–1e (60 mmol) in ethanol (10 ml) was mixed with 25% aqueous solution
of sodium hydroxide (15 ml, 120 mmol) and refluxed until the evolution of ammonia ceased. The
reaction mixture was then diluted with twofold volume of water and acidified with 10% hydrochloric
acid to pH 4–5. The crude product was collected, washed with water, and recrystallized from aqueous
ethanol. TLC was performed using petroleum ether–ethyl acetate–acetic acid (50 : 45 : 5) as the
mobile phase.
2-Propyl-4-pyridinecarboxylic acid (2a), m.p. 187–189 °C. Yield 81%. For C9H11NO2 (165.2) cal-
culated: 65.44% C, 6.71% H, 8.48% N; found: 65.29% C, 6.82% H, 8.27% N. IR spectrum: 2 985,
1
2 960, 2 895 (C–H); 2 435 (COOH); 1 705 (C=O). H NMR spectrum: 0.91 t, 3 H (CH3); 1.73 m, 2 H
(CH2); 2.83 t, 2 H (CH2Ar); 7.66 d, 1 H, J = 4.9 (H-5); 7.71 s, 1 H (H-3); 8.69 d, 1 H, J = 4.9 (H-6).
2-Isopropyl-4-pyridinecarboxylic acid (2b), m.p. 183–186 °C. Yield 82%. For C9H11NO2 (165.2)
calculated: 65.44% C, 6.71% H, 8.48% N; found: 65.31% C, 6.79% H, 8.26% N. IR spectrum:
1
2 980, 2 955, 2 890 (C–H); 2 440 (COOH); 1 710 (C=O). H NMR spectrum: 1.26 d, 6 H [(CH3)2];
3.13 m, 1 H (CH); 7.66 d, 1 H, J = 4.4 (H-5); 7.68 s, 1 H (H-3); 8.67 d, 1 H, J = 4.4 (H-6).
2-Butyl-4-pyridinecarboxylic acid (2c), m.p. 179–181 °C (ref.15 gives m.p. 181 °C). Yield 82%. 1H NMR
spectrum: 0.91 t, 3 H (CH3); 1.31 m and 1.72 m, 2 H (CH2) each; 2.83 t, 2 H (CH2Ar); 7.63 d, 1 H,
J = 4.9 (H-5); 7.67 s, 1 H (H-3); 8.67 d, 1 H, J = 4.9 (H-6).
2-tert-Butyl-4-pyridinecarboxylic acid (2d), m.p. 174–176 °C. Yield 86%. For C10H13NO2 (179.2)
calculated: 67.02% C, 7.31% H, 7.82% N; found: 67.15% C, 7.47% H, 7.68% N. IR spectrum:
1
2 975, 2 890 (C–H); 2 440 (COOH); 1 705 (C=O). H NMR spectrum: 1.35 s, 9 H [(CH3)3]; 7.63 d,
1 H, J = 4.9 (H-5); 7.84 s, 1 H (H-3); 8.71 d, 1 H, J = 4.9 (H-6).
2-Pentyl-4-pyridinecarboxylic acid (2e), m.p. 167–169 °C. Yield 86%. For C11H15NO2 (193.2) cal-
culated: 68.37% C, 7.82% H, 7.25% N; found: 68.55% C, 7.91% H, 7.06% N. IR spectrum: 2 980,
1
2 950, 2 880 (C–H); 2 450 (COOH); 1 750 (C=O). H NMR spectrum: 0.93 t, 3 H (CH3); 1.31 m, 4 H
[(CH2)2]; 1.72 m, 2 H (CH2); 2.81 t, 2 H (CH2Ar); 7.63 d, 1 H, J = 4.6 (H-5); 7.66 s, 1 H (H-3); 8.67 d,
1 H, J = 4.6 (H-6).
2-Alkylpyridine-4-carboxanilides 3a–3r
A mixture of 2-alkyl-4-pyridinecarboxylic acid 2a–2e (50 mmol) and thionyl chloride (5.5 ml, 75 mmol)
in dry benzene (10 ml) was refluxed for about 1 h. Excess of thionyl chloride was removed by re-
peated evaporation with dry benzene in vacuo. The crude acyl chloride dissolved in 50 ml of dry
acetone was added dropwise to a stirred solution of the corresponding aminophenol (50 mmol) in dry
pyridine (50 ml) keeping the temperature at 10 °C. After the addition of aminophenol was complete,
stirring at 10 °C continued for another 30 min. The reaction mixture was then poured into cold water
(200 ml). Crude anilide was collected and recrystallized from aqueous ethanol. TLC was performed
in petroleum ether–ethyl acetate (50 : 50) as the mobile phase. The yields, melting points, elemental
analyses, and IR spectral data of 2-alkylpyridine-4-carboxanilides 3a–3r are given in Table I and
1
their H NMR chemical shifts in Table II.
Measurement of Oxygen Evolution Rate
The oxygen evolution rate in spinach chloroplasts was investigated spectrophotometrically (Specord
UV-VIS, Zeiss Jena, Germany) in the presence of electron acceptor 2,6-dichlorophenolindophenol ac-
cording to method described in ref.5. Due to low water solubility the studied compounds were dis-
solved in dimethyl sulfoxide (DMSO) so that the applied DMSO concentration (up to 5% v/v) did
not affect oxygen evolution. The inhibitory efficiency of the studied compounds has been expressed
Collect. Czech. Chem. Commun. (Vol. 62) (1997)

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Miletin, Hartl, Machacek:
by IC50 values, i.e., by molar concentration of the compounds causing 50% decrease in the activity
with respect to the untreated control. The results are summarized in Table III.
This study was supported by the Grant No. 204/96 from the Grant Agency of Charles University. The
authors thank Dr K. Kralova, Institute of Chemistry, Comenius University, Bratislava, for providing
data of the photosynthesis inhibition. They also thank Mrs D. Karlickova (Department of Medicinal
Chemistry and Drug Control, Faculty of Pharmacy, Charles University) and Mrs J. Zizkova (Depart-
ment of Inorganic and Organic Chemistry of the same Faculty) for performing the elemental analyses
and recording the IR spectra.
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