Synthesis of 3-substituted Isoxazolecarboxamides as ...

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Apr 12, 2008 - high fungicidal activities against Alternaria alternate, Botrytis cinerea, Rhizoctonia solani, Fusarium culmorum, and Phytophthora cactorum.
Synthesis of 3-substituted Isoxazolecarboxamides as Potential Fungicides Mirosław Gucma1, W. Marek Gołębiewski1*, Bolesław Morytz1, Hayley Charville2 and Andrew Whiting2 1 Institute of Industrial Organic Chemistry, Annopol 6, 03-236 Warsaw, Poland Phone: 48 22 811 1231, fax: 48-22 811 0799, email: [email protected]; [email protected] 2 Department of Chemistry, Durham University, Science laboratories, South Road, Durham DH1 3LE, United Kingdom Phone: 44 (0) 191 334 2081, fax: 44 (0)191 386 1127; email: [email protected]; [email protected] Abstract A series of new 3-substituted isoxazolecarboxamides have been prepared from aldehydes. A key step was a 1,3-dipolar cycloaddition of nitrile oxides to -unsaturated esters. Some of these compounds exhibited high fungicidal activities against Alternaria alternate, Botrytis cinerea, Rhizoctonia solani, Fusarium culmorum, and Phytophthora cactorum. Keywords: Chlorination, cycloaddition, fungicides, isoxazolecarboxamides, regioselectivity, synthesis INTRODUCTION Many carboxamides listed in the Pesticide Manual show biological activity mostly as herbicides and fungicides [1]. Herbicidal activity is presumably due to either: 1) the inhibition of fatty acid biosynthesis (particularly by chloroacetamides); 2) the inhibition of phytoenone desaturase (the enzyme involved in the biosynthesis of the carotenoids [2]); 3) the inhibition of cell elongation and division (flufenacet); 4) the inhibition of cell wall biosynthesis (isoxaben); or 5) the inhibition of photosynthetic electron transport at the photosystem II receptorsite (pentanochlor). In addition, fungicidal potency of some amides results from inhibition of mitochondrial functions by disrupting the succinate dehydrogenase complex in the respiratory electron transport chain (flutolanil) [3]. However, fungicidal activity of some phenyl amides, such as ofurace, also results from inhibition of rybosomic RNA synthesis and fenhexamid disrupts sterol biosynthesis. The biological activity of simple isoxazole derivatives [4] prompted us to synthesize a number of 3-arylisoxazolecarboxamides in order to evaluate their biological activity and several amides showed high biological activity against Alternaria alternata, Botrytis cinerea, Rhizoctonia solani, Fusarium culmorum, Phytophthora cactorum and Blumeria graminis [5,6]. Therefore, in continuation of our search for new potent compounds, we have prepared a series of new 3-alkyland 3-aryisoxazolecarboxamides and studied their fungicidal activity and we report herein, the results of these studies. RESULTS AND DISCUSSION The isoxazolecarboxamides listed in Tables 1 and 2 were prepared in a few steps starting from aldehydes which were oximated with hydroxylamine and chlorinated with NCS in DMF. The corresponding isoxazole system was obtained by a 1,3-dipolar cycloaddition reaction of , -unsaturated esters and nitrile oxides generated in situ in the presence of triethylamine in most cases (Huisgen method) [7] or on a basic (Amberlyst A-21) column in the case of the synthesis amide 10 [8] (Scheme 1). Some 3-aryl-4,5-dihydroisoxazolinecarboxylates were dehydrated to give the corresponding isoxazoles using an NBS bromination, followed by potassium acetate induced dehydrobromination [9]. The title amides were synthesized by reaction of the acid chlorides prepared by saponification of the corresponding esters, followed by reaction of the acids with oxalyl chloride, followed by acylation of the aromatic or heterocyclic amines in the presence of tertiary amines (Method A). In the case of weakly nucleophilic aromatic amines, the amidation process was carried out by activation of amines with n-butyllithium in diethyl ether (Method B) or by formation of lithium amides with t-butyllithium (Method C) to avoid formation of side products due to degradation of the acid chlorides [10] and to increase the yield of the amides.

2 OH OH CH2=CHCO2Et NCS Cl N NH2OH H N 3 2 Base R 69-93% R DMF R 63-91% 74-95% R = Aryl (R1), t -Bu

H 1

O

NaOH

O

N

1. (COCl)2

CO2H

71-91% R

2.

5

H2N

N R2

6

N

O

CO2Et 4

R

O

O

N H

R2

7

R

Scheme 1. Synthesis of the amides Four types of amides were prepared. The first group of amides were the 3-aryl (alkyl)-2-isoxazoline (isoxazole)5-anilides 7; The second group of compounds included the 3-aryl (alkyl)-2-isoxazoline (isoxazole)-5-pyridinyl amides 8; The third group contained the 3-aryl (alkyl)-2-isoxazoline-5-piperazinylamides 9; And the fourth group contained the 3,4,5-trisubstited 2-isoxazolines 10 and 11 (Fig. 1).

N

O

O

N

N H

N

R2

R

O

R

O N R2

N 9

8

N

O

Et Cl H N

CF3

O I

N H 11

O

Cl

10

F3C

N

O

i-Pr

CF3

Fig 1. The other types of prepared amides Several attempts to perform direct amide bond formation from the corresponding carboxylic acids and amines with boronic acid-based catalysts were unsuccessful, even with the most forcing known direct amide conditions [11]. Indeed, even 10 times normal catalyst loadings were ineffective at effecting the conversion shown in Scheme 2, presumably due to lack of reactivity of both the carboxylic acid and hindrance of the aniline function.

N

O

CO2H +

12 Et

Br NH2

Br Br

A (10%) or B (10%) no reaction

13

B(OH)2 A:

N(i -Pr)2, fluorobenzene, 85oC, 6 days

B: B(OH)3 , toluene, 120o C , 7 days Scheme 2. Attempted direct amide synthesis The physical properties and elemental analyses (or HRMS) of amides 7-11 are presented in Table 1 and their NMR spectra are shown in Table 2.

3 Table 1. Physical properties and Elemental Analyses (HRMS) of Amides 7-11 Comp. b

R

7aa 7bb 7cb 7db

R2

C6H5 H 4-CF3C6H4 H 4-CF3C6H4 4-OCH3 2,3,64-Br Cl3C6H2 2,3,64-Br 7e Cl3C6H2 2,3,64-Br,3-CF3 7f Cl3C6H2 4-EtC6H4 2,5-Cl2 7g 4-EtC6H4 4-sec-Bu 7h t-Bu 2-CN 7i t-Bu 2,6-Cl2,4-NO2 7j t-Bu 2,3,5,6-Cl4 7k C6H5 2,3,5,6-F4 8a 4-EtC6H4 2,3,6-F3,5-Cl 8b 8cb 2-CF3C6H4 2,3,6-F3,5-Cl 8db 3,52,6-F2,3,5-Cl2 (CF3)2C6H3 t-Bu 2,6-F2,3,5-Cl2 8e t-Bu 2,3,5,6-F4 8f 2,3,6-F3C6H2 2,3,6-F3,5-Cl 8g 2,4,5-F3C6H2 2,3,5,6-F4 8h 9ab 4-CF3C6H4 Et 2,3,6Me 9b Cl3C6H2 t-Bu Et 9c 10 11 a [12], b Isoxazole derivative, c [M+Na].

Yield (%) 92 63 95 39

Method of prep. A A A A

Mp (oC)

31

A

wax

61

A

wax

536.8763 (536.8769)

79 57 27 57 52 72 28 33 24

A A A B B C B C C

glass wax 124-127 glass glass 124-126 wax 168-170 165-166

59.38 (59.51) 4.38 (4.44) 75.10 (75.39) 7.61 (7.48) 66.20 (66.40) 6.62 (6.33) 46.74 (46.68) 4.29 (4.20) 43.86 (43.78) 3.52 (3.67) 355.0683 (355.0676) 53.49 (53.20) 3.28 (3.41) 45.55 (45.57) 1.39 (1.44) 40.39 (40.34) 1.06 (1.00)

71 64 59 51 66 86

C C C C A A

109–114 104-106 111-113 116-119 100-102 79-81

44.14 (44.33) 3.81 (3.72) 49.14 (48.91) 3.84 (4.11) 431.9978 (431.9950)c 45.96 (45.81) 1.69 (1.54) 57.59 (57.78) 5.01 (5.13) 398.0175 (398.0206)

60 27 39

A B A

64-66 wax 238-240

62.79 (62.89) 48.32 (48.11) 54.02 (53.99)

144-146 226-228 glass wax

Elem. Anal. (%, calc) C H 72.41 (72.16) 5.42 (5.30) 362.0518 (362.0528) 59.79 (59.67) 3.55 (3.62) 42.75 (43.04) 1.64 (1.81) 43.07 (42.84)

2.39 (2.25)

9.49 (9.44) 3.01 (2.83) 3.87 (3.83)

Table 2. 1H NMR and 13C NMR Spectral Data of Amides 7-11 Compd. 7a

7b 7c

7d 7e

7f

7g

7h

1

H NMR / 13C NMR ( ppm) (200MHz, CDCl3,): 8.53 (bs, 1H, NH), 7.69 (m, 2H, H-2’,-6’), 7.58 (m, J = 7.6 Hz, 2H, H-2”,-6”), 7.48–7.22 (m, 5H, H-3’,-4’,-5’,-3”,-5”), 5,26 (dd, J = 10.7; 6.3 Hz, 1H, H-5), 3.81 (d, J = 10.7 Hz, 1H, H-4), 3.78 (d, J = 6.3 Hz, 1H, H-4) (200MHz, CDCl3,): 9.35 (bs, 1H, NH), 7.99 (d, J = 8.1 Hz, 1H, H-2”), 7.78 (d, J = 8.1 Hz, 1H, H6”), 7.68 (d, J = 7.6 Hz, 2H, H-3’,-5’), 7.42 (s, 1H, H-4), 7.22 (m, 3H, H-2’,-6’,-4”) (200MHz, CDCl3,): 7.98 (d, J = 8.2 Hz, 2H, H-3’,-5’), 7.77 (d, J = 8.2 Hz, 2H, H-2’,-6’), 7.60 (dm, J = 8.4 Hz, 2H, H-2'',-6''), 7.38 (s, 1H, H-4), 6.94 (dm, J = 8.4 Hz, 2H, H-3'',-5''), 3.84 (s, 3H, OCH3) (200MHz, CDCl3,): 8.38 (s, 1H, NH), 7.59 (d, J = 8.6 Hz, 2H, H-2",-6"), 7.51 (d, J = 8.7 Hz, 1H, H-4'), 7.45 (d, J = 8.7 Hz, 1H, H-5'), 7.40 (d, J = 8.6 Hz, 2H, H-3",-5"), 7.12 (s, 1H, H-4) (500MHz, CDCl3,): 8.54 (s, 1H, NH), 7.62 (d, J = 8.7 Hz, 2H, H-2",-6"), 7.50 (d, J = 8.7 Hz, 1H, H-4'), 7.40 (d, J = 8.7 Hz, 2H, H-3",-5"), 7.34 (d, J = 8.7 Hz, 1H, H-5'), 5.33 (dd, J = 11.5; 5.0 Hz, 1H, H-5), 3.76 (dd, J = 18.0; 11.5 Hz, 1H, H-4a), 3.67 (dd, J = 18.0; 5.0 Hz, 1H, H-4b) (200MHz, CDCl3,): 8.70 (bs, 1H, NH), 7.97 (s, 1H, H-2”), 7.69 (s, 1H, H-4’), 7.68 (s, 1H, H-5’), 7.51 (d, J = 8.7 Hz, 1H, H-5”), 7.34 (d, J = 8.7 Hz, 1H, H-6”), 5.35 (dd, J = 10.9; 5.3 Hz, 1H, H-5), 3.74 (d, J = 10.9 Hz, 1H, H-4a), 3.69 (d, J = 5.3 Hz, 1H, H-4b) (200MHz, CDCl3,): 9.2 (s, 1H, NH), 8.48 (d, J = 2 Hz, 1H, H-6"), 7.61 (d, J = 8.0 Hz, 2H, H-6',2'), 7.31 (d, J = 8.5 Hz, 1H, H-3"), 7.26 (d, J = 8.0 Hz, 2H, H-5',-3'), 7.05 (dd, J = 8.0; 2.5 Hz, 1H, H-4"), 5.27 (dd, J = 10.5; 5.5 Hz, 1H, H-5), 3.78 (d, J = 10.5 Hz, 1H, H-4a), 3.77 (d, J = 5.5 Hz, 1H, H-4b), 2.68 (q, J = 7.5 Hz, 2H, CH2), 1.24 (t, J = 7.5 Hz, 3H, CH3) (200MHz, CDCl3,): 8.49 (s, 1H, NH), 7.60 (d, J = 8.2 Hz, 2H, H-6',-2'), 7.48 (d, J = 8.4 Hz, 2H, H-

4

7i

7j 7k

8a

8b

8c

8d 8e 8f

8g

8h 9ab

9b

9c

10

11

6",-2"), 7.25 (d, J = 8.2 Hz, 2H, H-5',-3'), 7.14 (d, J = 8.2 Hz, 2H, H-5",-3"), 5.23 (dd, J = 10.6; 6.5 Hz, 1H, H-5), 3.77 (d, J = 6.5 Hz, 1H, H-4a),3.76 (d,J = 10.6 Hz, 1H, H4b), 2.68 (q, J = 7.5 Hz, 2H, CH2Ph), 2.57 (m, 1H, CHPh), 1.56 (q, J = 6.8 Hz, 2H, CH2 [CH]CH3), 1.24 (q, J = 7.5 Hz, 3H, CH3CH2Ph), 1.21 (q, J = 7.0 Hz, 3H, CH3CH), 0.79 (t, J = 7.2 Hz, 3H, CH3CH2CH) (200MHz, CDCl3,): 9.00 (s, 1H, NH), 8.22 (d, J = 7.8 Hz, 1H, H-6’), 7.63 (d, J = 7.8 Hz, 1H, H3’), 7.61 (td, J = 7.6; 1.5 Hz, 1H, H-5’), 7.25 (td, J = 7.6; 1.5 Hz, 1H, H-4’), 5.12 (dd, J = 8.4; 7.8 Hz, 1H, H-5), 3.41 (d, J = 8.4 Hz, 1H, H-4a), 3.41 (d, J = 7.8 Hz, 1H, H-4b), 1.24 (s, 9H, C(CH3)3); (50.3MHz, CDCl3,): 170.5, 167.3, 139.2, 134.1, 132.8, 125.4, 122.3, 115.9, 104.5, 39.5, 33.4, 28.3 (3C) (200MHz, CDCl3,): 8.60 (s, 1H, NH), 8.26 (s, 2H, H-3’, H-5’), 5.18 (dd, J = 8.2; 7.5 Hz, 1H, H-5), 3.43 (d, J = 8.2 Hz, 1H, H-4), 3.42 (d, J = 7.5 Hz, 1H, H-4), 1.24 (s, 9H, CH3)3C) (200MHz, CDCl3,): 8.47 (s, 1H, NH), 7.60 (s, 1H, H-4’), 5.16 (t, J = 7.8 Hz, 1H, H-5), 3.42 (d, J = 7.8 Hz, 2H, H-4), 1.24 (s, 9H, C(CH3)3); (50.3MHz, CDCl3,): 170.1, 167.4, 133.9, 132.3, 131.3, 130.0, 39.8, 33.4, 28.2 (200MHz, CDCl3,): 8.61 (bs, 1H, NH), 7.70 (d, J = 7.0 Hz, 1H, H–6’), 7.69 (d, J = 8.2 Hz, 2H, H– 2’), 7.50–7.39 (m, 3H, H-3,-4’, 5’), 5.38 (dd, J = 10.4; 8.5 Hz, 1H, H-5), 3.83 (d, J = 10.4 Hz, 1H, H-4a), 3.84 (d, J = 8.5 Hz, 1H, H-4b) (200MHz, CDCl3,): (bs, 1H, NH), 7.73 (d, J = 8.3 Hz, 2H, H-2’,-6’), 7.30 (d, J = 8.3 Hz, 2H, H-3’,5’), 4.99 (dd, J = 9.0; 3.0 Hz, 1H, H-5), 4.69 (dd, J = 9.6; 9.0 Hz, 1H, H-4a), 4.56 (dd, J = 9.6; 3.0 Hz, 1H, H-4b), 2.68 (q, J = 7.5 Hz, 2H, CH2CH3), 1.27 (t, J = 7.5 Hz, 3H) (200MHz, CDCl3,): 8.46 (s, 1H, NH), 7.86 (m, 1H, H-3’), 7.68 (m, 3H, H-4’, -5’,-6’), 7.34 (d, J = 0.8 Hz, 1H, H-4); (50.3MHz, acetone-d6,): 163.4, 162. 7, 154.1, 152.1 (ddd, J = 214.6; 12.7; 3.3 Hz), 148.2 (dt, J = 242.4; 15.8 Hz), 139.9 (ddd, J = 261.1; 27.8; 6.9 Hz), 133.6 (q, J = 0.8 Hz), 133.0, 131.7, 129.2 (q, J = 30.9 Hz), 127.9, 127.6 (q, J = 5.5 Hz, 2C), 110.6 (q, J = 2.4 Hz) (200MHz, CDCl3,): 8.32 (s, 2H, H-6’,-2’), 8.30 (s, 1H, NH), 8.05 (s, 1H, H-4'), 7.55 (s, 1H, H-4) (200MHz, CDCl3,): 8.68 (s, 1H, NH), 5.17 (dd, J = 8.8; 6.7 Hz, 1H, H-5), 3.43 (d, J = 8.8 Hz, 1H, H-4a), 3.42 (d, J = 6.7 Hz, 1H, H-4b), 1.24 (s, 9H, (CH3)3C) (200MHz, CDCl3,): 8.60 (s, 1H, NH), 5.18 (dd, J = 9.2; 6.3 Hz, 1H, H-5), 3.43 (d, J = 9.2 Hz, 1H, H-4a), 3.42 (d, J = 6.3 Hz, 1H, H-4b), 1.23 (s, 9H, C(CH3)3); (50.3MHz, CDCl3,): 169.8, 167.8, 39.8, 33.5, 28.1 (200MHz, CDCl3,): 8.64 (bs, 1H, NH), 7.29 (tdd, J = 9.2; 5.2; 3.8 Hz, 1H, H-4’), 6.98 (tdd, J = 9.2; 3.8; 2.2 Hz, 1H, H-5’), 5.41 (dd, J = 10.7; 5.8 Hz, 1H, H-5), 3.90 (dt, J = 10.8; 1.3 Hz, 1H, H-4a), 3.87 (dd, J = 5.8; 1.3 Hz, 1H, H-4b) 8.59 (bs, 1H, NH), 7.72 (sym. m, 1H, H-6’), 7.05 (td, J = 10.2; 6.4 Hz, 1H, H-3’), 5.39 (dd, J = 8.8; 8.4 Hz, 1H, H-5), 3.88 (d, J = 8.4 Hz, 1H, H-4a), 3.86 (d, J = 8.8 Hz, 1H, H-4b) (200MHz, CDCl3,): 7.95 (d, J = 8.1 Hz, 2H, H-5’, H-3’), 7.76 (d, J = 8.1 Hz, 2H, H-6’,-2’), 7.13 (s, 1H, H-4), 3.90 (t, J = 4.8 Hz, 4H, H-2",-6"), 2.65 (t, J = 4.8 Hz, 4H, H-3",-5"), 2.55 (q, J = 7.2 Hz, 2H, CH2CH3), 1.18 (t, J = 7.2 Hz, 3H, CH2CH3) (200MHz, CDCl3,): 7.55 (d, J = 2.4 Hz, 1H, H-4'), 7.51 (d, J = 2.4 Hz, 1H, H-6’), 5.43 (dd, J = 11.2; 7.6 Hz, 1H, H-5), 4.26 (dd, J = 17.3; 7.6 Hz, 1H, H-4a), 3.91-3.78 (m, 2H, H-2’’eq,-6’’eq), 3.66-3.51 (m, 2H, H-2’’ax,-6’’ax), 3.56 (dd, J = 17.3; 11.2 Hz, 2H, H-4), 2.55-2.33 (m, 4H, H-3’’,5’’), 2.34 (s, 3H, NCH3); (50.3MHz, CDCl3,): 165.2, 156.3, 135.0, 133.0, 131.6, 131.4, 129.9, 128.9, 78.8, 55.2, 54.5, 46.0, 45.8, 42.6, 38.8 (200MHz, CDCl3,): 5.15 (dd, J = 11.0; 7.5 Hz, 1H, H-5), 3.86-3.79 (m, 2H, H-2’eq, H-6’eq), 3.82 (dd, J = 17.0; 7.5 Hz, 1H, H-4a), 3.57-3.47 (m, 2H, H-2’ax,-6’ax), 3.00 (dd, J = 17.0; 11.0 Hz, 1H, H-4b), 2.54 (m, 2H, H-3’ax,-5’ax), 2.50 (m, 1H, H-14ax or H-16ax), 2.45 (q, J = 7.3 Hz, 2H, OCH2), 2.37 (ddd, J = 11.3; 8.2; 3.5 Hz, 1H, H-16ax or H-14ax ), 1.23 (s, 9H, C(CH3)3), 1.10 (t, J = 7.3 Hz, 3H, CH3CH2); (50.3MHz, CDCl3,): 167.2, 166.3, 77.5, 53.1, 52.5, 52.4, 46.0, 42.7, 36.3, 33.3, 28.3 (3C), 12.1 (200MHz, CDCl3,): 7.95 (d, J = 8.1 Hz, 2H, H-3’,-5’), 7.70 (d, J = 8.1 Hz, 2H, H-2’,-6’), 7.60 (s, 2H, H-3”,-5”), 7.22 (s, 1H, NH), 5.12 (m, 1H, H-5), 4.20 (d, J = 4.2 Hz, 1H, H-4), 1.81 (m, 2H, CH2CH3), 1.59 (t, J = 7.2 Hz, 3H, CH3CH2) (200MHz, CDCl3,): 8.53 (s, 1H, NH), 7.65 (d, J = 8.9 Hz, 2H, H-5”,-3”), 7.63 (d, J = 8.2 Hz, 2H, H-3’”,-5’”), 7,54 (d, J = 8.2 Hz, 2H, H-3’, H-5’), 7.46 (d, J = 8.2 Hz, 2H, H-2’”,-6’”), 7.37 (d, J = 8.9 Hz, 2H, H-2”,-6”), 7.19 (d, J = 8.2 Hz, 2H, H-2’,-6’), 5.26 (d, J = 3.1 Hz, 1H, H-5), 4.94 (d, J = 3.1 Hz, 1H, H-4), 2.87 (septuplet, J = 6.9 Hz, 1H, CH[CH3]2), 1.22 (d, J = 6.9 Hz, 6H, CH[CH3]2)

The products synthesized were tested for herbicidal, insecticidal, acaricidal and fungicidal activity. The most promising biological potency was found against fungal isolates (see Table 3).

5 Table 3. Fungicidal inhibitory activity of compounds 7-11 at 200 g/ml a Comp.

R

R2

Botrytis Fusarium Phytophtora Rhizoctonia Blumeria cinerea culmorum cactorum solani graminis C6H5 H 0 0 0 0 1 7a 4-CF3C6H4 H 0 0 0 0 0 7bb 7cb 4-CF3C6H4 4-OCH3 1 1 0 2 7db 2,3,6-Cl3C6H2 4-Br 1 2 1 1 2,3,6-Cl3C6H2 4-Br 2 2 2 2 2 7e 2,3,6-Cl3C6H2 4-Br 0 0 2 1 2 7f 4-EtC6H4 2,5-Cl2 1 0 0 0 1 7g 4-EtC6H4 4-sec-Bu 2 1 0 1 0 7h t-Bu 2-CN 1 7i t-Bu 2,6-Cl2-4-NO2 2 1 2 2 0 7j t-Bu 2,3,5,6-Cl4 1 0 2 2 0 7k C6H5 2,3,5,6-F4 1 2 2 1 8a 4-EtC6H4 2,3,6-F3-5-Cl 3 2 2 2 8b 2-CF3C6H4 2,3,6-F3-5-Cl 0 1 1 0 8cb 3,5-(CF3)2C6H3 2,6-F2,3-5-Cl2 1 2 1 2 8db t-Bu 2,6-F2-3,5-Cl2 3 1 3 3 1 8e t-Bu 2,3,5,6-F4 2 1 2 2 8f 2,3,6-F3C6H2 2,3,6-F3,5-Cl 2 2 2 2 8g 2,4,5-F3C6H2 2,3,5,6-F4 3 2 3 3 8h 4-CF3C6H4 Et 1 2 2 2 9ab 2,3,6-Cl3C6H2 Me 1 1 2 2 1 9b t-Bu Et 0 0 0 0 0 9c 0 1 1 10 0 0 0 0 11 2 2 2 3 Chlorothalonil a At 0-3 scale: 0 = 0-20% growth inhibition, 1 = 21-50% growth inhibition, 2 = 51-80% growth inhibition, 3 = 81-100% growth inhibition; b Isoxazole derivative. In the first group of amides (7a-7k), no activity was observed for the unsubstituted anilides 7a and 7b. Moderate activity was found for isoxazolineamides substituted at both phenyl rings, as well as the 3-t-butyl compound, which was particularly promising for compounds with electron-withdrawing groups (EWG) on aromatic rings (such as 7e). The corresponding isoxazole derivative 7d showed lower activity. From the second group of amides (8a-8g), the best results were obtained for pyridyl amides containing EWG substituents on both aromatic rings (e.g. 8h) and for the t-butyl derivative (8e). These compounds were more potent than the reference pesticide chlorothalonil The substitution pattern was also important for activity; low activity was observed for amides with an ortho-substituted and an aldehyde-derived phenyl ring (8c). Similarly, the di-ortho-substituted pyridyl derivative 8g was less active than the ortho,meta-analogue 8h. Piperazinyl compounds, from the third group of amides, were characterized by moderate fungicidal potency, however, the 3-t-butyl derivative was devoid of activity. Introduction of the third isoxazoline substituent, whether alkyl or aryl cases, such as compounds 10 and 11 led to lower biological activity. The amide 10 was prepared also in optically active form from the known chiral compound, methyl 3-(4-trifluoromethylphenyl)-5-ethyl-4,5-dihydroisoxazole-4-carboxylate [12] by saponification, conversion to the acid chloride (with oxalyl chloride) and amidation by Method B. Unfortunately, no increase in fungicidal activity was observed compared to the racemate. In conclusion, the biological activity of the amides reported herein depends on a combination of electronic and steric effects. High biological potency can be correlated with low electron density of the ring systems. Effective conjugation of the phenyl and isoxazole rings is essential, whereas, steric congestion in both rings is detrimental to biological activity and presumably results from the loss of coplanarity of the ring systems. In this context, good activity of 3-alkylisoxazolineamides was unexpected and deserves further study.

EXPERIMENTAL Reagent grade chemicals were used without further purification, unless otherwise noted. Elemental analyses were performed at Microanalysis Laboratory of Institute of Organic Chemistry, Polish Academy of Sciences,

6 Warsaw. Melting points were determined in capillary tubes and are uncorrected. Spectra were obtained as follows: IR spectra on JASCO FTIR-420 spectrometer, 1H NMR spectra on Varian 500 UNITY plus-500 and Varian 200 UNITY plus 200 spectrometers in deuterated chloroform using TMS as internal standard, ESI mass spectra on Micromass LCT. Flash-chromatography was carried out using silica gel S 230-400 mesh (Merck). Hydroximinoyl acid chlorides were prepared from the corresponding aryl aldehyde oximes and NCS in DMF [10]. General procedure for the cycloaddition reactions A solution of chloro oxime (13 mmol) in anhydrous toluene (15 mL) was added dropwise over 30 min. to a stirred mixture of anhydrous toluene, anhydrous NEt3 (6 mL), MgSO4 (2 g) and ethyl acrylate (8 mL, 80.0 mmol). The reaction mixture was stirred overnight at room temperature, diluted with toluene (50 mL), washed with water (5 x 100 mL) and evaporated in vacuo. General procedure for amide synthesis with tertiary amines (Method A) A solution of an aniline derivative (1.2 mmol) in anhydrous toluene (10 ml) was added with stirring to an acid chloride followed by dry triethyl amine (4 ml, 30.0 mmol). The solution was stirred under reflux for 1 h and overnight at room temperature. Water (10 ml) was added, the organic layer was washed with 3% hydrochloric acid solution and water, and was dried over magnesium sulfate. A crude amide obtained after evaporation of the solvent was purified by crystallization. General procedure for amide synthesis with n-butyl lithium (Method B) 2.5 M solution of n-BuLi in hexanes (0.2 ml, 0.5 mmol) was added dropwise at -78 oC to a stirred solution of 4aminopyridine derivative (0.4 mmol) in dry diethyl ether. Stirring was continued for 1 h and a solution of acid chloride (0.3 mmol) in dry diethyl ether (or HMPA) was added dropwise. The mixture was stirred for 5h at 0 oC and overnight at room temperature. The reaction was quenched with ammonium chloride solution, product was extracted with methylene chloride and purified by flash chromatography. General procedure for amide synthesis with t-butyl lithium (Method C) 1.7 M solution of t-BuLi in hexanes (1 ml, 1.7 mmol) was added dropwise at -78 oC to a stirred solution of an aniline derivative (1.2 mmol) in dry diethyl ether. Stirring was continued for 1 h and a solution of acid chloride (0.4 mmol) in dry HMPA was added dropwise. The mixture was stirred for 4 h at 0 oC and overnight at room temperature. The reaction was quenched with ammonium chloride solution, product was extracted with diethyl ether, washed with water and purified by flash chromatography. (+)-3-(4-Trifluoromethylphenyl)-4,5-dihydroisoxazole-5-ethyl-4-carboxylic acid 4-trifluoromethyl-2,6dichlorophenylamide (10a) It was prepared by Method B. The enantiomeric excess of separated regioisomers was determined by HPLC analysis (Astec Cyclobond I 2000 RN); [α]D= + 37.0 (c 0.2, acetone), ee = 82 %; IR (KBr) 3433, 3240, 2926, 2850, 1668, 1525, 1394, 1327, 1172, 1132, 1073, 1020, 930, 890, 850, 810 cm -1. Acknowledgements This work was supported in part by the Polish Ministry of Science and Higher Education Research (Grant 429E142/S/2009)

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The pesticide Manual XIII ed., Tomlin, C. D. S., Ed.;British Crop Protection Council: Alton, 2003. White, H. Conference of British Crop Protection Council, Brighton, 1999. Hewitt, H.G. Fungicides in crop protection, Cab International, Cambridge, 1998. Wielkopolski, W.; Śmieszek, R. ; Witek, R.S. Synthesis and herbicidal activity of isoxazole-substituted 1-aminoethylphosphonates and 1-hydroxyethylphosphonates. Pest. Sci. 1994, 40, 107-112. Gołębiewski, W.M.; Gucma, M; Krawczyk, M. „Pochodne 3fenylo(alkilo)izoksazolo-5-karboksamidów i sposób ich wytwarzania”, Patent application P-386683 April 12, 2008.

7 Gołębiewski, W.M.; Gucma, M. Synthesis and biological activity of 3-substituted isoxazolecarboxamides. Monatsh. Chem. 2010, 141, 461-469. Bast, K.; Christl, M.; Huisgen, R.; Mack, W.; Sustman, R. Additionen des Benzonitriloxids an olefinische und acetylenische Dipolarophile. Chem. Ber. 1973, 106, 3258-3274. Sibi, M.P.; Itoh, K.; Jasperse, C.P. Chiral Lewis acid catalysis in nitrile oxide cycloaddition. J. Am. Chem. Soc. 2004, 126, 5366-5367. Bianchi, G.; Grünanger, P. Conversion of 2-isoxazolines to isoxazoles. Tetrahedron 1965, 21, 817-822. Gołębiewski, W.M.; Gucma, M. New fragmentation of 2-isoxazoline-5-carboxylic acid chlorides. J. Heterocycl. Chem. 2006, 43, 509-513. Charville, H.; Jackson, D.; Hodges, G.; and Whiting, A. The thermal and boron-catalysed direct amide formation reactions: mechanistically understudied yet important processes. Chem. Commun., 2010, 46, 1813-1823. (a) Quilico, A.; Stagno D’Alcontres, G.; Grünager, P. Nitrile oxides. II. Nitrile oxides and compounds containing double bonds. Gazz. Chim. Ital. 1950, 80, 479-495; (b) Chistokletov, V.N.; Troshchenko, A.T. Addition of benzonitrile oxide to unsaturated compounds. II. Addition of benzonitrile oxide to dienes, and to halogen-substituted unsaturated hydrocarbons. Izv. Sibirsk. Otd. Akad. Nauk SSSr, Ser. Khim. Nauk, 1963, 147-151 (Chem. Abstr. 1963, 59, 13960). Gołębiewski, W. M.; Gucma, M. Enantioselective 1,3-dipolar cycloaddition reactions using chiral lanthanides catalysts. J. Heterocycl. Chem. 2008, 45, 1687-1693.

[6] [7] [8] [9] [10] [11]

[12]

[13]

Synthesis of 3-substituted Isoxazolecarboxamides as Potential Fungicides Mirosław Gucma1, W. Marek Gołębiewski1*, Bolesław Morytz1, Hayley Charville2 and Andrew Whiting2 Graphical abstract

OH CH2=CHCO2Et N Base

Cl

N

O

CO2Et

N

O

O N H

R2 R Isoxazolecarboxamides, prepared from aldehydes, show biological activity as fungicides R

R