Synthesis and Chemistry of Agrochemicals III - American Chemical

Chapter 18

Cyanoacrylate Inhibitors of Photosynthetic Electron Transport

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Structural Requirements for Inhibitor Potency and Herbicidal Activity John L. Huppatz, Helen G. McFadden, Marie-Luise Huber, and Leslie F. McCaffery Commonwealth Scientific and Industrial Research Organisation, Division of Plant Industry, Canberra 2601, Australia Readily synthesized derivatives of 2-cyanoacrylic acid are potent inhibitors of photosynthetic electron transport. The structural features associated with optimum activity in this class of inhibitor are described with particular emphasis on the steric factors involved. Generally, inhibition of the Hill reaction correlates well with post-emergence herbicidal activity, though a series of highly active inhibitors devoid of herbicidal activity was discovered.

Photosynthesis is a particularly attractive target for the design of potential commercial herbicides. It has the obvious advantage of being a metabolic function unique to higher plants and certain bacteria, with no counterpart in mammalian physiology. Compounds specifically blocking the photosynthetic process might therefore be expected to present minimal toxicological problems. Furthermore, the complexities of photosynthesis have been largely unraveled and the structure of the reaction center at the molecular level is now known in considerable detail. Several sites for interference with the photosynthetic process have been identified but by far the largest group of photosynthetic herbicides act by inhibiting photosynthetic electron transport at photosystem II (PSII). These compounds, which include the urea, triazinone and uracil herbicides (7), owe their phytotoxicity to an ability to bind to a 32 kD polypeptide (the D l , or Q protein) in the PSII reaction center in chloroplasts. This type of PSII inhibitor displaces the native plastoquinone, Q , from its binding site, thereby blocking electron flow and initiating a chain of events which ultimately results in plant death (2). The PSII herbicide binding site has shown an intriguing affinity for a wide range of chemically diverse structures, a property which makes rationalization of the structure/activity relationships between the different herbicide classes difficult (3,4)- The susceptibility of the PSII site to a wide range of chemical types has resulted in a steady flow of new structures with herbicidal properties in recent years. Paradoxically, no new PSII inhibitor herbicide has made a significant impact on world agriculture for many B

B

0097-6156/92/0504-0186$06.00/0 © 1992 American Chemical Society In Synthesis and Chemistry of Agrochemicals III; Baker, Don R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.


18.

Cyanoacrylate Inhibitors of Photosynthesis 187

HUPPATZ ET AL.

years. The reasons for this are complex but include the continued effectiveness and low cost of established PSII herbicides, particularly the urea and triazine families, and the inability of most PSII inhibitors to match the field performances of other modem herbicides, particularly the sulfonylureas. Theoretically, there is no known reason why PSII inhibitor herbicides could not approach the low dose rates characteristic of the sulfonylureas (5). This is particularly true for highly potent inhibitors of photosynthetic electron transport, provided properties of foliar penetration and transport within plant tissue were favorable. Compounds based on 2-cyanoacrylic acid esters were first reported as herbicides in 1969-71 in a series of patents (6-8), but little information was given on the spectrum of activity and the mode of action was not discussed. A detailed study of compounds of general structure 1 (9) revealed that herbicidal activity was associated with Nmethylanilino cyanoacrylates 1, [R^phenyl, R =H, and R =simple alkyl (2-4 carbon atoms)] and that phytotoxicity apparently resulted from inhibition of photosynthetic electron transport (9). Subsequently, structure 1 was modified to produce vinylogous ureas of general structure 2 (10). These compounds had similar structural requirements to the original series on the "left-hand side" of the molecule (a p-hydrogen atom and an N-methylanilino function) and the same stereochemistry. They were also inhibitors of the Hill reaction; a property accounting for the potent phytotoxicity of some members of the series (10). The most potent compounds in the 2-cyanoacrylate series were found to be compounds with quite different structural features from the N-methylanilino analogues 1 and 2. Significantly, they are characterized by a cis orientation of the amino and carbonyl functions as shown in general structure 3. Moreover, in contrast to the N-methyl-N-aryl analogues 1 and 2, they incorporate a hydrogen atom attached to the enamine nitrogen and a ^-substituent R that is other than hydrogen. With favorable substitution at R , R and R , 2-cyanoacrylates 3 are extraordinarily potent inhibitors of photosynthetic electron transport. Moreover, the activity of these molecules was found to be highly sensitive to minor structural variation thereby providing significant insight into the topography of the binding site occupied by the inhibitors (11-16). This review will summarize the structural requirements of 2-cyanoacrylates 3 associated with potent inhibition of photosynthetic electron transport and highlight features which provide some understanding of the topographical limits to the binding of this type of molecule. 2

3

2

CH R

1

CH

3

_ N R

2 / r

1

CN ~^C00R

1

R

3

3

1 _ Ny _ / H

/

2

C

R

N

^

C

O

N

H

—

1

— N R

COOR^ CN

2

3

In Synthesis and Chemistry of Agrochemicals III; Baker, Don R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

2

188

SYNTHESIS AND CHEMISTRY OF AGROCHEMICALS III

Chemistry. Synthetically, cyanoacrylates are readily accessible with a range of substituents possible for each of R , R and R . Initially compounds were prepared by a two-step procedure involving the formation of an ethoxymethylene compound 4 from the appropriate cyanoacetic ester and a diethyl (or trimethyl) ortho ester followed by reaction with an appropriate amine to give compounds of general structure 3 [Figure 1, route (a)]. Two important limitations restrict the usefulness of this reaction pathway as a completely general synthetic method. Firstly, the range of ortho esters commercially available or conveniently prepared is limited, thereby restricting the versatility of the reaction with respect to R . Secondly, the reaction either failed or gave very modest yields when applied to cyanoacetamides, thereby severely restricting the range of possible analogues. The most obvious alternative procedure involved acylating a cyanoester or amide, followed by methylation of the enol form of the acyl derivative. The Rathke and Cowan procedure (77) for the acylation of acetoacetic and malonic esters was successfully extended to cyanoacetic esters and amides. This method involved condensation of a cyanoacetic ester or amide and an acid chloride in the presence of triethylamine and anhydrous magnesium chloride [Figure 1, route (b)] resulting in excellent yields of the


1

2

3

RCOCI/ Et N/MgCI 3

3

CNCHjCOOR

2

> I H (b)

COOR

3

5 (b) CH N 2

2

T H R

1 _

I i N ^yCOOFT R

2

/

r

R NH 1

2

9

^CN

4

R0 Y_y 4

R

2

/

„ C

M N

^C00R

3

4 ( R = Et) 4

3

6

( R = Me) 4

Figure 1


18. HUPPATZETAL.

Cyanoocrylate Inhibitors of Photosynthesis 189

acylated product 5. The methoxymethylene intermediates 6 were obtained by facile methylation of the acyl derivatives with ethereal diazomethane. Reaction with amines, either directly or in refluxing acetonitrile, completed this alternative synthesis of cyanoacrylates 3. The stereochemistry of the 2-cyanoacrylates 3 is an important determinant of biological activity. Compounds in this series have a Z-configuration (i.e. a cis orientation of the amino and ester functions), although compounds unsubstituted in the p-position 3 (R =H) can exist as either geometric isomer (13). The latter compounds are at best weak inhibitors of the Hill reaction and have little or no herbicidal activity. Stereochemistry was assigned from spectral (PMR and infrared) data (13,18) and supported by an x-ray structure determination of one of the more potent inhibitors 3 (R =4-chlorobenzyl; R =wopropyl; R =ethoxyethyl) (18). The x-ray data confirmed the Z-stereochemistry and demonstrated the presence of a planar core stabilized by a strong intramolecular hydrogen bond between the ester carbonyl oxygen and the hydrogen atom attached to the enamine nitrogen (18).


2

2

3

Structural Requirements for Hill Reaction Activity. Optimization of structure 3 to achieve maximum potency in inhibiting photosynthetic electron transport was achieved by systematically varying the substituents R , R and R . The intrinsic activity of each analogue was rapidly and reliably assessed using the Hill reaction in isolated pea chloroplasts. Compounds were assayed for Hill inhibition activity using chloroplast fragments isolated from the leaves of Pisum sativum, the electron acceptor being the blue dye, 2,3\6-trichlorophenolindophenol. The activity was expressed as p/ , i.e. -log /5o, where 7 was the molar concentration required to decrease the level of dye reduction to 50% of that obtained in the absence of the compound. While Hill reaction data do not necessarily correlate well with whole plant activity, a significant in vitro inhibition is a prerequisite for in vivo phytotoxicity. Other factors, such as leaf penetration, translocation and detoxification in plant tissue, ultimately determine whole plant performance and species selectivity. However, from the viewpoint of establishing the structural features necessary to maximize affinity for the binding site, the Hill reaction provides valuable insights and, when combined with glasshouse screening data, enables useful design rules to be formulated. For inhibition of photosynthetic electron transport the nitrile function is mandatory; replacement by hydrogen, acyl, amide or ester (9) groups eliminates activity. The ester group may be replaced by an amide or keto function though these analogues are not as effective, either as Hill inhibitors or as herbicides, and will not be considered further. 1(

50

50

1

The substituent R . With compounds of general formula 4, significant activity can be achieved with diverse lipophilic groups (Table I). Studies with straight-chain alkyl substituents (11,12) suggested that the alkyl group was interacting with a large, unconstrained hydrophobic area in the thylakoid membrane and increased activity was associated with the increased lipophilic nature of R . With alkylamino-2-cyanoacrylates 3 (R^QHan+i, R =H, R =CH CH20CH CH3), the p/ increased by 0.5 unit with each additional methylene group, suggesting that inhibitors of this type partition into the membrane as they would into octanol (72). 1

2

3

2

2

50


190

SYNTHESIS AND CHEMISTRY OF AGROCHEMICALS III

Table I. Activity of Compounds of General Formula (4) as Inhibitors of the Hill Reaction R NH ^ 1

CH CH


3

Compound No

2

^C00CH CH 0CH CH 2

2

CN

2

3

(4)

R

n-C H 8

plg^

1

1 7

-

7-15

6 7 8

X = H X = 4CI X = 2CI

4-80 5-75 4-45

9 10 11

X = H X = 4CI X = 2CI

6-20 8-70 5-85

CH

12 13 14

3

racemate S - ( + ) isomer R - ( - ) isomer

6-65 7-10 4-90



18,

Cyanoacrylate Inhibitors of Photosynthesis 191

HUPPATZETAL.

However, with compounds in which a phenyl ring was incorporated into R , lipophilic interactions were modified by steric effects (14). Phenylamino derivatives (e.g. compounds 6-8, Table I) were relatively weak inhibitors, though activity could be increased markedly by 3- or 4-substitution (e.g. compound 7). Inclusion of a methylene group between the phenyl and amino functions (compound 9) resulted in an activity increase much greater than would have been expected on the basis of increased lipophilicity alone. Substitution in the phenyl ring can significantly affect Hill activity, with an increase in potency of up to 300-fold observed with a favorable substituent (e.g. compound 10). Simple QSAR analyses of substituted phenylamino- and benzylamino 2-cyanoacrylates showed a high degree of correlation between lipophilicity of the substituent and activity (79). Moreover, there was strong evidence that the phenyl rings of the two series interact differently with the hydrophobic domain of the receptor site, with the increased flexibility of the benzylamino series enabling these compounds to bind with greater affinity. Previous studies with phenylurea inhibitors indicated an adverse effect on activity of orr/io-substitution in the phenyl ring (20). In the case of the benzylamino-2-cyanoacrylates, activity can be reduced almost 1000-fold when an 2-chloro substituent replaces 4-chloro (compare 10 and 11). The orr/io-substituent must exert sufficient steric influence to prevent the phenyl ring achieving the conformation required for optimal interaction with the binding site. The importance of steric interactions within the hydrophobic domain occupied by R was reinforced by differences in potency observed between the enantiomers 13 and 14 (75). The S(-i-) isomer 13 is 200-fold more active than the corresponding R(-) isomer 14. Thus, the topographical characteristics of the binding site discriminate between the different spatial orientation of the groups around the chiral center in 13 and 14 (75). 1

2

1

The P-substituent, R . In 2-cyanoacrylates 3 where R was alkyl-, aryl- and aralkylamino, inhibition of photosynthetic electron transport was enhanced by alkyl substitution at the P-carbon. With straight-chain alkyl groups, activity reached a maximum when a P-ethyl substituent was present in the molecule (13,14). Further increasing the alkyl chain length decreased activity indicating that the R region of the receptor site also has strict spatial requirements. The data presented in Table II clearly indicate that steric arrangement of the carbon atoms is a primary determinant of binding affinity. Maximum activity appears to be associated with a branch at the a-carbon. The compound 15 (R=/s

Synthesis and Chemistry of Agrochemicals III - American Chemical

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