Field of the invention

The invention relates to uses of compounds in the treatment of plants. It particularly concerns compounds that can be used in a treatment of a plant to promote plant growth. The invention also relates to compounds that can be used to prevent or reduce stomatal closure or to cause stomatal opening in a plant. The invention further provides compounds for use in a herbicidal treatment of a plant. New compounds and a method for their manufacture are also described.

Background to the invention

It is known that reactive oxygen species (ROS), such as hydrogen peroxide, and reactive nitrogen species, such as nitric oxide (NO), are involved in the control of cellular events in plants. Such compounds are known to effect plant physiology. For example, both ROS and NO can cause stomatal closure (L. Lamattina et al, Plant Physiol., 126 (2001), pages 1 196 to 1204; and X. Zhang et al, Plant Physiol., 126 (2001), pages 1438 to 1448).

However, hydrogen sulfide (H ₂ S) is often thought to be a phytotoxin, which is harmful to the growth and development of plants. H ₂ S has been found to inhibit oxygen release from young seedlings of six rice cultivars and phosphorus uptake was inhibited in this plant species (M.M. Joshi et al, Phytophatology, 65 (1975), pages 1 175 to 1 170).

Also, fumigation of a variety of plants, namely Medicago, grapes, lettuce, sugar beets, pine and fir, with 3000 parts per billion (ppb) of H ₂ S caused lesions on leaves, defoliation and reduced growth of the plants (C.R. Thompson et al, Environ. Sci. Technol, 12 (1978), pages 550 to 553). These results provide support for the role of H ₂ S as a phytotoxin.

It has also been reported that transpiration rates of several species of plants, including maize, pumpkins and spinach, were unaffected by short-term exposure to atmospheric H ₂ S (L.J. De Kok et al, New Phytol, 112 (1989), page 533 to 542).

Summary of the invention

The invention is based on the finding that the treatment of plants with a H ₂ S releasing compound can prevent guard cells from closing the stomata found on the plants' leaves. In particular, the treatment prevented stomata from closing in low light conditions, and even in the dark. Stomata are pores that are used in the photosynthesis and respiration processes of a plant. During photosynthesis, carbon dioxide in air enters through the stomata openings and is converted into organic compounds, such as sugar, which is used as a source of chemical energy. Water vapour is released through the stomata into the atmosphere by transpiration.

It is known that the rate of transpiration and photosynthesis of a plant is related to the size of its stomata openings. Generally, plants control the size of their stomata openings to balance the amount of water lost through transpiration with the amount of carbon dioxide that can pass through the pores to allow photosynthesis to occur. The invention allows a plant to continue photosynthesising and generating energy, particularly in low light conditions, thereby enhancing the overall growth of the plant.

An aspect of the invention relates to the use of a compound in a treatment of a plant to promote plant growth wherein the compound is a salt comprising an anion of formula (1 ):

or a conjugate acid thereof, wherein:

Ri , R ₂ , R ₃ , R4 and R ₅ are each independently selected from H, halogen, optionally substituted Ci -C| ₀ alkyl, optionally substituted C ₂ -Ci ₀ alkenyl, optionally substituted C ₂ - Cio alkynyl, optionally substituted C ₃ -Cio cycloalkyl, optionally substituted C ₃ -Ci ₀ cycloalkenyl, optionally substituted C3-C10 cycloalkynyl, hydroxy, optionally substituted C1-C10 alkoxy, amino, mono-(Ci-C ₁₀ alkyl)amino and di-(C Cio alkyl)amino groups; and

Y is a nitrogen containing heterocyclyl group having from 3 to 14 ring atoms, a nitrogen containing heteroaryl group having from 3 to 14 ring atoms, an optionally substituted mono-(Ci-Cio alkyl)amino group, an optionally substituted di-(Ci-Ci ₀

alkyl)amino group or an optionally substituted tri-(Ci -Ci ₀ alkyl)amino group.

It has been shown that a compound of formula (1 ) can release H ₂ S slowly (Ling Li et al, Circulation, 1 17 (2008), pages 2351 to 2360). It has been reported that this compound is able to mimic the slow release of endogenous ¾S from cells in animals, thereby avoiding a sudden, concentrated dose of H ₂ S. Concentrated doses of H ₂ S would be obtained by the direct use of H ₂ S gas or by administering compounds such as NaSH.

It is believed that the slow release of H ₂ S is not only important for preventing stomatal closure in plants, but that it is also of benefit in keeping the stomata open for a prolonged period of time.

A further aspect of the invention relates to the use of a compound to prevent or reduce stomatal closure or to cause stomatal opening in a plant, wherein the compound is a salt comprising an anion of formula (1) or a conjugate acid thereof, wherein:

Ri, R ₂ , R ₃ , R4 and R ₅ are each independently selected from H, halogen, optionally substituted C1 -C10 alkyl, optionally substituted C ₂ -Cio alkenyl, optionally substituted C ₂ - C10 alkynyl, optionally substituted C ₃ -Cio cycloalkyl, optionally substituted C3-Ci ₀ cycloalkenyl, optionally substituted C ₃ -Ci ₀ cycloalkynyl, hydroxy, optionally substituted Ci-Ci ₀ alkoxy, amino, mono-(C _r Ci ₀ alkyl)amino and di-(Ci-Ci ₀ alkyl)amino groups; and

Y is a nitrogen containing heterocyclyl group having from 3 to 14 ring atoms, a nitrogen containing heteroaryl group having from 3 to 14 ring atoms, an optionally substituted mono-(Ci-Ci ₀ alkyl)amino group, an optionally substituted di-(Ci-C ₁₀ alkyl)amino group, or an optionally substituted tri-(Ci-Cio alkyl)amino group.

In hot and dry conditions or environments, where water is scarce, some plants close their stomata during the day to prevent them from drying out and dying. Also, some herbicides (and pathogens) may be able to enter a plant through its stomata thereby causing the death of the plant.

Another aspect of the invention is the use of a compound in a herbicidal treatment of a plant, wherein the compound is a salt comprising an anion of formula (1) or a conjugate acid thereof, wherein:

Ri, R ₂ , R3, R4 and R ₅ are each independently selected from H, halogen, optionally substituted C|-Cio alkyl, optionally substituted C ₂ -Cio alkenyl, optionally substituted C ₂ - C10 alkynyl, optionally substituted C ₃ -Ci ₀ cycloalkyl, optionally substituted C3-C10 cycloalkenyl, optionally substituted C ₃ -C _]0 cycloalkynyl, hydroxy, optionally substituted C1 -C10 alkoxy, amino, mono-(Ci-Cio alkyl)amino and di-(Ci-C] ₀ alkyl)amino groups; and Y is a nitrogen containing heterocyclyl group having from 3 to 14 ring atoms, a nitrogen containing heteroaryl group having from 3 to 14 ring atoms, an optionally substituted mono-(Ci-Cio alkyl)amino group, an optionally substituted di-(Ci-Cio alkyl)amino group or an optionally substituted tri-(Ci-Ci ₀ alkyl)amino group. The uses of the invention relate to a method, which comprises the step of treating a plant with the compound as defined above or a combination as defined below. The method may be for treating a plant to promote plant growth; for preventing or reducing stomatal closure or for causing stomatal opening in a plant; or for herbicidally treating a plant.

The invention also provides a combination comprising:

(a) a compound, which is either a salt comprising an anion of formula (1 ) or a conjugate acid thereof, wherein:

Ri, R ₂ , R ₃ , R4 and R ₅ are each independently selected from H, halogen, optionally substituted C1-C10 alkyl, optionally substituted C ₂ -C) ₀ alkenyl, optionally substituted C ₂ -Cio alkynyl, optionally substituted C3-C10 cycloalkyl, optionally substituted C3-C10 cycloalkenyl, optionally substituted C3-C 10 cycloalkynyl, hydroxy, optionally substituted C|-Ci ₀ alkoxy, amino, mono-(Ci-Cio alkyl)amino and di-(Ci-Cio alkyl)amino groups; and

Y is a nitrogen containing heterocyclyl group having from 3 to 14 ring atoms, a nitrogen containing heteroaryl group having from 3 to 14 ring atoms, an optionally substituted mono-(Ci-C ₁₀ alkyl)amino group, an optionally substituted di-(C]-Cio alkyl)amino group or an optionally substituted tri-(Ci-Cio alkyl)amino group; and

(b) a herbicide or one or more plant nutrients.

The invention further provides a compound, which is a salt comprising an anion of formula (1) or a conjugate acid thereof, wherein:

Ri, R ₂ , R3, _t and R ₅ are each independently selected from H, halogen, optionally substituted C1-C10 alkyl, optionally substituted C ₂ -Ci ₀ alkenyl, optionally substituted C ₂ - Cio alkynyl, optionally substituted C ₃ -C ₁₀ cycloalkyl, optionally substituted C ₃ -Ci ₀ cycloalkenyl, optionally substituted C3-Ci ₀ cycloalkynyl, hydroxy, optionally substituted Ci-Cio alkoxy, amino, mono-(Ci-Cio alkyl)amino and di-(Ci-Cio alkyl)amino groups; and

Y is a nitrogen containing heterocyclyl group having from 3 to 14 ring atoms, a nitrogen containing heteroaryl group having from 3 to 14 ring atoms, an optionally substituted mono-(Ci-Ci ₀ alkyl)amino group, an optionally substituted di-(Ci-C| ₀ alkyl)amino group or an optionally substituted tri-(Ci-Cio alkyl)amino group;

with the proviso that the compound is not morpholinium 4- methoxyphenyl(morpholino) phosphinodithioate. Preferably, the compound is not phenyl(piperidin-l -yl)phosphinodithioic acid, o-bromophenyl(piperidin-l - yl)phosphinodithioic acid, w-aminophenyl(piperidin-l-yl)phosphinodithioic acid, ?- ethylphenyl(piperidin-l-yl)phosphinodithioic acid, sodium phenyl(piperidin-l- yl)phosphinodithioate, potassium o-methylphenyl(piperidin- 1 -yl)phosphinodithioate, calcium ?-fluorophenyl(piperidin-l -yl)phosphinodithioate, ammonium p- methoxyphenyl(piperidin- 1 -yl)phosphinodithioate or hydrazinium m- trifluoromethylphenylphosphinopiperidinodithioate.

A further aspect of the invention relates to a method of preparing a compound as defined above, whi of formula (2):

(2)

with either:

(i) a compound of formula (3), or a conjugate base thereof:

Y-H

(3)

or

(ii) a compound of formula (3a):

B:

(3a)

wherein:

Ri , R ₂ , R ₃ , R4 and R ₅ are each independently selected from H, halogen, optionally substituted C1-C 10 alkyl, optionally substituted C ₂ -C| ₀ alkenyl, optionally substituted C ₂ - Cio alkynyl, optionally substituted C3-Ci ₀ cycloalkyl, optionally substituted C3-C10 cycloalkenyl, optionally substituted C3-Ci ₀ cycloalkynyl, hydroxy, optionally substituted C| -Cio alkoxy, amino, mono-(C|-C[ ₀ alkyl)amino and di-(Ci -Cio alkyl)amino groups; and Y is a nitrogen containing heterocyclyl group having from 3 to 14 ring atoms, a nitrogen containing heteroaryl group having from 3 to 14 ring atoms, an optionally substituted mono-(Ci-Ci ₀ alkyl)amino group or an optionally substituted di-(Ci-Cio alkyl)amino group; B is a nitrogen containing heterocyclyl group having from 3 to 14 ring atoms, a nitrogen containing heteroaryl group having from 3 to 14 ring atoms or an optionally substituted tri-(Ci-Cio alkyl)amino group;

with the proviso that when Ri, R ₂ , R4 and R ₅ are H, and R ₃ is a methoxy group, then the compound of formula (3) is not morpholine. Preferably, (i) when Ri, R ₂ , R4 and R ₅ are H, and R ₃ is a methoxy group, then the compound of formula (3) is not morpholine or piperidine; (ii) when Ri, R ₂ , R ₃ , R4 and R ₅ are H, then the compound of formula (3) is not piperidine; (iii) when Ri is -Br and R ₂ , R3, R4 and R ₅ are H, then the compound of formula (3) is not piperidine; (iv) when R ₂ is -NH ₂ and Ri, R ₃ , R4 and R ₅ are H, then the compound of formula (3) is not piperidine; (v) when R ₃ is ethyl and R|, R ₂ , 4 and R ₅ are H, then the compound of formula (3) is not piperidine; (vi) when Ri is methyl and R ₂ , R ₃ , R4 and R ₅ are H, then the compound of formula (3) is not piperidine; (vii) when R ₃ is -F and Ri, R ₂ , R» and R ₅ are H, then the compound of formula (3) is not piperidine, and/or (viii) when R ₂ is -CF ₃ and Rj, R ₃ , R ₄ and R ₅ are H, then the compound of formula (3) is not piperidine.

Brief description of drawings

Figure 1 is a reaction scheme showing the synthesis of morpholinium 4- methoxyphenyl(morpholino) phosphinodithioate (GYY4137) from Lawesson's reagent (2,4-bis(4-methoxyphenyl)-l ,3,2,4-dithiadiphosphetane 2,4-disulfide compound).

Figure 2 is a histogram showing the effect on the aperture size of stomata of leaf samples of Arabidopsis thaliana ecotype Columbia that were exposed to direct lighting for 2.5 hours and treated with NaSH.

Figure 3 is a histogram showing the effect on the aperture size of stomata of leaf samples of Arabidopsis thaliana ecotype Columbia that were exposed to direct lighting for 2.5 hours and treated with the compound GYY4137.

Figure 4 is a histogram showing the effect on the aperture size of stomata of leaf samples of Arabidopsis thaliana ecotype Landsberg erecta, which were not exposed to high lighting and were treated directly with abscisic acid (ABA) or the compound

GYY4137.

Figure 5 is a histogram showing the effect on aperture size of stomata in leaf samples, which have been treated with NaSH or GYY4137 in either the light (indicated on the horizontal axis by "L") or in the dark (indicated on the horizontal axis by "D"). Figure 6 shows images of fluorescence caused by the use of NO specific dye 4,5- diaminofluorescein diacetate (DAF2-DA) after treating epidermal fragments as follows: A: Control with no treatment; B: ABA (50 μΜ) treatment; C: GYY4137 (100 μΜ) treatment alone; D: ABA treatment in the presence of GYY4137; E: NaSH (100 μΜ) treatment alone; F: ABA treatment in the presence of NaSH.

Figure 7 is a histogram showing the effect on aperture size of stomata in leaves of Capsicum annum, which have been treated with NaSH or GYY4137.

Figure 8 shows images of fluorescence caused by the use of NO specific dye 4,5- diaminofluorescein diacetate (DAF2-DA) after treating epidermal fragments of leaves of Capsicum annum.

Figure 9 is a graph showing the effect of GYY4137 on the growth of Arabidopsis thaliana.

Figure 10 is a histogram showing the effect of GYY4137 on the growth of shoots and roots of Arabidopsis thaliana.

Detailed description of the invention

Generally, the invention relates to a compound in a salt form, where the anion of the salt is an anion of formula (1) and is able to slowly release H ₂ S. Generally, the salt will also possess a counter cation.

Typically, the cation is an organic cation of the formula XH ⁺ , where X is a base, such as ammonia (e.g. XH ⁺ is NH ₄ ⁺ ), a mono-(Ci-Ci ₀ alkyl)amine, (e.g. XH ⁺ is MeNH ₃ ⁺ , EtNH ₃ ⁺ ), di-(C]-Cio alkyl)amine (e.g. XH ⁺ is Me ₂ NH ₂ ⁺ , Et ₂ NH ₂ ⁺ ), a nitrogen containing heterocycle having from 3 to 14 ring atoms (e.g. XH ⁺ is N-morpholinium, N-piperidinium, N-pyrrolidinum) or a nitrogen containing heteroaryl compound having from 3 to 14 ring atoms (e.g. XH ⁺ is pyridinium). Preferably, X is a di-(Ci-Cio alkyl)amine, a nitrogen containing heterocycle having from 3 to 14 ring atoms or a nitrogen containing heteroaryl compound having from 3 to 14 ring atoms. More preferably X is a CI-CH nitrogen containing heterocycle, particularly morpholine, piperidine or pyrrolidine.

In some circumstances, the anion of formula (1) may be in equilibrium with its conjugate acid. Both the anion of formula (1) and its conjugate acid (formula (4) below) may be present.

(1) (4)

In one embodiment of the invention, the cation is an inorganic cation, which is an alkali metal ion, such as Na ⁺ or K ⁺ , an alkaline earth cation, such as Ca ²⁺ or Mg ²⁺ , or a metal cation of an element selected from Fe, Mo, Cu, Mn, Zn and Ni. The inorganic cation may be selected to provide a source of a plant nutrient.

Ri, R ₂ , R ₃ , R4 and R ₅ can each be independently selected from optionally substituted CpCio alkyl, optionally substituted C ₂ -Cio alkenyl, optionally substituted C ₂ - C 10 alkynyl, optionally substituted C ₃ -Cio cycloalkyl, optionally substituted C3-C10 cycloalkenyl, optionally substituted C3-C10 cycloalkynyl, and optionally substituted C1-C10 alkoxy. Preferably, each of R|, R ₂ , R ₃ , R4 and R ₅ is unsubstituted.

When R|, R ₂ , R ₃ , R4 or R ₅ is substituted, then preferably each of R _\ , R ₂ , R ₃ , R4 and R ₅ is independently substituted with one or more substituents selected from hydroxy, C|- Cio alkoxy, halogen, C ₃ -C ₇ cycloalkyl and C ₆ -Ci4 aryl.

Generally, R _\ , R ₂ , R ₃ , R4 and R5 are each independently selected from H, halogen,

C 1-C10 alkyl, hydroxy, Ci-Cio alkoxy, amino, mono-(Ci-C ₁₀ alkyl)amino and di-(Ci-Ci ₀ alkyl)amino groups, and Y is a nitrogen containing heterocyclyl group having from 3 to 14 ring atoms, a nitrogen containing heteroaryl group having from 3 to 14 ring atoms, an optionally substituted mono-(Ci-Ci ₀ alkyl)amino group or an optionally substituted di-(Ci- Cio aIkyl)amino group.

Typically, Rj, R ₂ , R ₃ , R» and R ₅ are each independently selected from H, Ci-Ci ₀ alkyl and C1 -C10 alkoxy groups. In one embodiment, the invention relates to compounds where at least one of Ri and R ₅ is H. Preferably, both Ri and R ₅ are H.

Y comprises a nitrogen atom. Generally, Y is bonded to the phosphorus atom in formula (1) above by a nitrogen atom.

The compounds of the invention may be obtained by reacting a nucleophilic compound with a 1 ,3,2,4-dithiadiphosphetane 2,4-disulfide compound of formula (2).

A readily available source of a 1,3,2,4-dithiadiphosphetane 2,4-disulfide compound of formula (2) is Lawesson's reagent. It is preferred that Rj, R ₂ , R4 and R ₅ are H, and R ₃ is a C1 -C10 alkoxy group. More preferably Ri, R ₂ , R4 and R ₅ are H and R ₃ is a methoxy group.

The nucleophilic compound reacts with the phosphorus atom in the

dithiadiphosphetane unit in the compounds of formula (2) and becomes incorporated in the resulting product. The nucleophilic compound may be represented by formula (3):

Y-H

(3)

or may be a conjugate base thereof (e.g. Y ^" ), or the nucleophilic compound may be represented by formula (3a):

B:

(3a).

Preferably the method of the invention comprises the step of reacting a compound of formula (2) with a compound of formula (3).

As represented in formula (3a), B is a compound that comprises a group or atom having a lone pair of electrons, which are preferably on a nitrogen atom.

Y in formula (1) above is a nitrogen containing heterocyclyl group having from 3 to 14 ring atoms, a nitrogen containing heteroaryl group having from 3 to 14 ring atoms, an optionally substituted mono-(Ci-C|o alkyl)amino group, an optionally substituted di-(Ci- C|o alkyl)amino group or an optionally substituted tri-(Ci-Ci ₀ alkyl)amino group.

Preferably, Y is a nitrogen containing heterocyclyl group having from 3 to 14 ring atoms, more preferably Y is a nitrogen containing heterocyclyl group having from 3 to 14 ring atoms in which the nitrogen atom is directly bonded to the phosphorus atom in the anion of formula (1 ). More preferably, Y is selected from:

In another embodiment, Y is preferably a benzylamino group (PhCH ₂ NH-).

Typically, Y-H is a nitrogen containing heterocycle having from 3 to 14 ring atoms, a nitrogen containing having from 3 to 14 ring atoms, an optionally substituted mono-(C|- C| ₀ alkyl)amine or an optionally substituted di-(Ci-C]o alkyl)amine.

It is preferred that the nucleophilic compound Y-H or Y ^" has a nucleophilic nitrogen atom, such that the phosphorus atom in formula (1) becomes directly bonded to the nitrogen atom in Y.

Y in formula (1) above may be positively charged. When the nucleophilic compound is represented by formula (3a), then Y in formula (1) above will have a positive charge. Y in formula (1) may then be represented by B ⁺ . Y is positively charged because the lone pair on B, which is preferably present on a nitrogen atom, forms a covalent bond with the phosphorus atom. If Y is positively charged, then the compound of the invention may be a zwitteron. A counter cation, such as an organic cation (XH ⁺ ) or an inorganic cation as described above, would not then be present.

When Y in formula (1) is B ⁺ , then preferably Y is a nitrogen containing

heterocyclyl group having from 3 to 14 ring atoms, a nitrogen containing heteroaryl group having from 3 to 14 ring atoms or an optionally substituted tri-(Ci-C| ₀ alkyl)amino group. Preferably Y is a nitrogen containing heteroaryl group having from 3 to 14 ring atoms.

Generally, the nitrogen atom of the heterocyclyl group, the heteroaryl group tri-(alkyl)amino group is bonded to the phosphorus atom (i.e. the phosphorus atom shown in formula (1) above), when Y is B ⁺ .

When Y in formula (1) is B ⁺ , then Y is preferably a heterocyclyl group or a heteroaryl group selected from 1 -(2H-pyrrolyl), 3-oxazolyl, 2-isoxazolyl, 3-thiazolyl, 2- isothiazolyl, 1-pyridinyl, 1-pyridazinyl, 1-pyrimidinyl, 1-pyrazinyl, l-(l,3,5-triazinyl), 1- indolizinyl, l -(3H-indolyl), 2-(lH-indolizinyl), 3-benzthiazolyl, 5-(4H-quinolizinyl), 1 - quinolinyl, 2-isoquinolinyl, 1 -cinnolinyl, 2-cinnolinyl, 2-phthalazinyl, 1 -quinazolinyl, 3- quinazolinyl, 1-quinoxalinyl, l -(l ,8-naphthyridinyl), 1-pteridinyl, 3-pteridinyl, 5- pteridinyl, 1 -quinuclidinyl, 10-acridinyl and 5-phenazinyl. More preferably, Y is selected from 1 -(2H-pyrrolyl), 1-pyridinyl, 1-pyridazinyl, 1-pyrimidinyl, 1-pyrazinyl, 1 -( 1 ,3,5- triazinyl), l -(3H-indolyl), 1 -quinolinyl and 2-isoquinolinyl. Even more preferably, Y is 1 - pyridinyl, 1 -pyridazinyl or 1 -pyrimidinyl, especially 1-pyridinyl.

The nucleophilic compound may also form the counter cation to the anion of formula (1 ), if it is suitably basic. Thus, the counter cation to the anion of formula (1 ) may be an organic cation that is a conjugate acid of the nucleophilic compound. It is preferred that the nucleophilic compound of formula (3), YH, or the conjugate base thereof, Y ^" , is the same as X (in the cation of formula XH ⁺ ). When the nucleophilic compound has the formula YH, then the cation XH ⁺ may also be represented as YH ₂ ⁺ . Similarly, when the nucleophilic compound has the formula Y ^' , then the cation XH ⁺ may be represented as YH.

Typically, when X is a di-(C]-Ci ₀ alkyl)amine, a nitrogen containing heterocycle having from 3 to 14 ring atoms or a nitrogen containing heteroaryl compound having from 3 to 14 ring atoms, then preferably YH is the same as X.

In the method of preparing a compound of the invention, the step of reacting a compound of formula (2) with a compound of formula (3) typically involves adding a solution of the compound of formula (3) dropwise to the compound of formula (3) to form a reaction mixture. Preferably, the solution of the compound of formula (3) is a solution in a chlorinated solvent, such as dichloromethane (CH ₂ C1 ₂ ) or chloroform (CHC1 ₃ ).

After the compound of formula (2) has been added to the compound of formula (3), the reaction mixture is stirred for 0.5 to 12 hours, preferably 1 to 5 hours, more preferably 1.5 to 3 hours. Typically, the reaction mixture is stirred at 15 °C to 35 °C.

The invention preferably relates to uses or methods involving the compounds morpholinium 4-methoxyphenyl(morpholino) phosphinodithioate, piperidinium 4- methoxyphenyl(piperidino) phosphinodithioate or pyrrolidinium 4-methoxyphenyl (pyrrolidino) phosphinodithioate, or compositions comprising such compounds.

In one aspect, the compound morpholinium 4-methoxyphenyl(N-morpholino) phosphinodithioate is excluded from the compounds of the invention or a method of preparing such a compound. Preferably, the compound is not morpholinium 4- methoxyphenyl(N-rnorpholino) phosphinodithioate, phenyl (piperidin-1 - yl)phosphinodithioic acid, o-bromophenyl(piperidin-l -yl)phosphinodithioic acid, m- aminophenyl(piperidin- 1 -yl)phosphinodithioic acid, -ethylphenyl(piperidin- 1 - yl)phosphinodithioic acid, sodium phenyl(piperidin-l -yl)phosphinodithioate, potassium o- methylphenyl(piperidin- l-yl)phosphinodithioate, calcium -fluorophenyl(piperidin-l- yl)phosphinodithioate, ammonium -methoxyphenyl(piperidin-l-yl)phosphinodithioate or hydrazinium n-trifluoromethylphenylphosphinopiperidinodithioate, and optionally the compound is not thiomorpholinium 4-methoxyphenyl(thiomorpholin-4-yl)

phosphinodithioate, piperidinium 4-methoxyphenyl(piperidin-l-yl) phosphinodithioate, benzylammonium 4-methoxyphenyl(benzylamino) phosphinodithioate, pyrazolium 4- methoxyphenyl(pyrazolin- 1 -yl) phosphinodithioate or piperazinium 4-methoxyphenyl(N- piperazinyl) phosphinodithioate.

In another embodiment of the invention, the anion of a compound of the invention is not henyl(piperidin-l -yl) phosphinodithioate, o-brornophenyl(piperidin-l-yl) phosphinodithioate, m-arninophenyl(piperidin-l-yl) phosphinodithioate, p- ethylphenyl(piperidin- 1 -yl) phosphinodithioate, o-methylphenyl(piperidin- 1 -yl) phosphinodithioate, -fluorophenyl(piperidin-l -yl) phosphinodithioate, p- methoxyphenyl(piperidin- 1 -yl) phosphinodithioate, wj-trifluoromethylphenyl

phosphinopiperidinodithioate, 4-methoxyphenyl(thiomorpholin-4-yl) phosphinodithioate, 4-methoxyphenyl(benzylamino) phosphinodithioate, 4-methoxyphenyl(pyrazolin- 1 -yl) phosphinodithioate or 4-rnethoxyphenyl(N-piperazinyl) phosphinodithioate.

The method of preparing a compound of the invention preferably also excludes the preparation of compounds where the anion is phenyl (piperidin-l-yl)phosphinodithioate, o- bromophenyl(piperidin- 1 -yl)phosphinodithioate, m-aminophenyl(piperidin- 1 - yl)phosphinodithioate, -ethylphenyl(piperidin-l -yl)phosphinodithioate, o- methylphenyl(piperidin-l -yl)phosphinodithioate, -fluorophenyl(piperidin-l- yl)phosphinodithioate, /?-methoxyphenyl(piperidin- 1 -yl)phosphinodithioate, m- trifluoromethylphenylphosphinopiperidinodithioate, 4-methoxyphenyl(thiomo holin-4-yl) phosphinodithioate, 4-methoxyphenyl(benzylamino) phosphinodithioate, 4- methoxyphenyl(pyrazolin-l-yl) phosphinodithioate or 4-methoxyphenyl(N-piperazinyl) phosphinodithioate.

The invention is generally concerned with a plant or plants that has/have stomata. Stomata are present in the sporophyte generation of all land plant groups, except liverworts.

Typically, the plant is a sporophyte generation of plant with the proviso that the plant is not a liverwort.

It is preferred that the plant is a dicotyledon or a monocotyledon.

In one embodiment, the plant is a monocotyledon. Examples of suitable monocotyledon plants include grains (e.g. rice, wheat, maize, etc.), pasture grasses, sugar cane, bamboo, members of the palm family (Arecaceae), members of the banana family (Musaceae), members of the ginger family {Zingiber aceae), members of the onion family (Alliaceae), lilies, daffodils, irises, amaryllis, orchids, carinas, bluebells and tulips.

In one embodiment, the plant is a root crop (i.e. a plant that has an edible underground plant structure). Examples of suitable plants that are root crops include cassava, sweet potato, beet, carrot, rutabaga, turnip, parsnip, radish, yam, horseradish, sassafras, angelica, sarsaparilla and licorice.

In one embodiment, the plant is a member of the genus Arabidopsis.

In another embodiment, the plant is a member of the genus Capsicum.

Typically, the plant is treated with the compound in an aqueous solution at a concentration of 0.5 μΜ to 250 μΜ, preferably a concentration of 1 μΜ to 200 μΜ, more preferably 10 μΜ to 200 μΜ.

In another embodiment, the plant is treated with the compound in an aqueous solution at a concentration of 0.01 μΜ to 1500 μΜ, preferably a concentration of 0.1 μΜ to 1000 μΜ.

It is preferred that the plant is treated with the compound simultaneously, concurrently, separately or sequentially with either (i) one or more plant nutrients, or (ii) one or more herbicides.

An aspect of the invention concerns treating a plant to promote plant growth. It may be necessary for the growth environment of the plant to be controlled to promote plant growth. For example, the growth environment may be controlled in a greenhouse or in a field. Generally, this will involve monitoring the environmental condition of the plant and supplying it with one or more plant nutrients as needed.

Generally, it is preferred that the plant is grown indoors.

Typically, the temperature, air humidity, moisture level of the soil, light intensity, light duration, the amount of C0 ₂ and/or the amount of plant nutrients fed to the plant are controlled when the growth environment of a plant is controlled.

It is known that plants have different photoperiods and respond differently to varying strengths of light intensity. The exact conditions of the growth environment and the amount of compound of the invention that are used to promote growth will depend on the specific plant that is to be treated. However, these may easily be determined by a person skilled in the art using commonly available techniques. Typically, the relative humidity of the growth environment is at least 60%, preferably at least 75%, more preferably at least 80%.

The average temperature of the growth environment is preferably from 15 to 30 °C, more preferably 18 to 25 °C.

Typically, the plant is treated with a compound of the invention under conditions where the amount of C0 ₂ is from 400 ppm to 1200 ppm, preferably 450 ppm to 1000 ppm, more preferably 500 ppm to 800 ppm, even more preferably 550 ppm to 600 ppm. The amount of C0 ₂ in ppm generally refers to the amount of C0 ₂ in the air of the growth environment of the plant.

In the northern hemisphere, during the late autumn, winter and early spring, plants are generally exposed to relatively low levels of sunlight. It can be difficult to grow plants under such conditions without the aid of an artificial source of light, such as a grow lamp. The invention may allow plants to perform photosynthesis at relatively low levels of light and thereby to continue to grow, without the assistance of an artificial light source.

In one embodiment, the plant is treated with a compound of the invention under conditions where the light intensity is from 1 lux to 1000 lux, preferably 5 lux to 500 lux, more preferably 50 lux to 250 lux.

The invention may promote plant growth by promoting plant transpiration, which usually occurs in the dark or under low levels of light.

In another embodiment, the plant is treated with a compound of the invention under conditions where the light intensity is from 10 ^"4 lux to 0.5 lux, preferably 10 ^"3 lux to 0.25 lux, more preferably 0.01 to 0.1 lux.

The invention may also improve the growth of a plant during the daytime under conditions where there is plenty of light and the plant is adequately supplied with nutrients.

In another embodiment, the plant is treated with a compound of the invention under conditions where the light intensity is from 2000 lux to 5000 lux, preferably 2500 lux to 4000 lux, more preferably 3000 lux to 3500 lux.

Generally, the plant is exposed to 8 to 16 hours of light a day.

A compound of the invention may be used with one or more plant nutrients, which assist in promoting plant growth. Suitable plant nutrients are minerals or compounds that provide sources of nitrogen (e.g. nitrate), phosphorus (e.g. phosphate), potassium, calcium (e.g. lime), magnesium (e.g. lime), sulphur, iron, manganese, zinc, copper, boron, chlorine, and/or molybdenum. A fertilizer may include one or more of the plant nutrients. Another aspect of the invention concerns a herbicidal treatment of a plant. When a plant is treated with a compound of the invention, its stomata will be prevented from closing. In hot and dry conditions, the plant will continue to transpire, such that it will dry out and die. The treatment may also allow pathogens to enter the plant through its stomata and cause disease, which may kill the plant.

Typically, the plant is treated with a compound of the invention under conditions where the daytime temperature is an average of at 25 °C, preferably is at least 30 °C.

It is preferred that the relative humidity of the plant environment is less than 50%, preferably less than 40%, more preferably less than 30%, even more preferably less than 20%, especially less than 10%.

It is preferred that the plant is treated with a compound of the invention under conditions where the light intensity is at least 10,000 lux, preferably at least 25,000 lux, more preferably at least 50,000, even more preferably at least 100,000, especially at least 120,000 lux.

Typically, the plant is a weed, such as Ailanthus altissima, Bermuda grass, bindweed, broadleaf plantain, burdock, clover, creeping Charlie, dandelion, goldenrod, Japanese knotweed, kudzu, leafy spurge, milk thistle, poison ivy, ragweed, sorrel, St John's wort, sumac, wild carrot, wood sorrel, common ragwort, spear thistle, creeping or field thistle, curled dock, broad leaved dock, chickweed, or barnyard grass.

A compound of the invention may be used with one or more herbicides. The compound of the invention may increase the efficacy of the herbicide by preventing a plant from closing its stomata.

Typically, the herbicide is a post-emergent herbicide. Post-emergent herbicides are generally applied after a plant or crop has emerged.

Examples of suitable herbicides include acetochlor, acifluorfen, aclonifen, acrolein, alachlor, alloxydim, ametryn, amicarbazone, amidosulfuron, aminopyralid, amitrole, anilofos, asulam, atrazine, azimsulfuron, beflubtiamid, benazolin, benefin, bensulfuron, bensulide, bentazon, benzofenap, bifenox, bispyribac, bromacil, bromoxynil, butachlor, butafenacil, butroxydim, butylate, cacodylic acid, carbetamide, carfentrazone,

chlorsulfuron, chlortoluron, cinmethylin, clethodim, clodinfop, clomazone, clopyralid, cloransulam-mezthyl, cyanazine, cycloate, cyclosulfamuron, cycloxydim, cyhalofop, DCPA, 2,4-D, dazomet, desmedipham, desmetryn, dicamba, dichlobenil, dichlorprop, diclofop, diclosulam, difenzoquat, diflufenican, diflufenzopyr, dimethenamid, diquat, dithiopyr, diuron, DSMA, endothall, EPTC, ethalfluralin, ethametsulfuron, ethofumesate, ethoxysulfuron, fenoxaprop, fentrazamide, flazasulfuron, florasulam, fluazifop-P, flucarbazone-sodium, flufenacet, flumetsulam, flumiclorac, flumioxazin, fluometuron, flupyrsulfuron, flurchloridone, fluridone, fluroxypyr, fluthiacet, foramsulfuron, fosamine, glufosinate, glyphosate, halosulfuron, haloxyfop, hexazinone, imazamethabenz, imazamox, imazapic, imazapyr, imazaquin, imazethapyr, iodosulfuron, isoxaben, isoxaflutole, lactofen, linuron, mecoprop, mefluidide, mesotrione, metham, metolachlor, metribuzin, metsulruron, MSMA, napropamide, naptalam, nicosulfuron, norflurazon, oryzalin, oxadiazon, oxasulfuron, oxyfluorfen, paraquat, pebulate, pelargonic acid, pendimethalin, phenmedipham, picloram, picolinafen, pinoxaden, primisulfuron, prodiamine, prometon, prometryn, pronamide, propanil, propaquizafop, propoxycarbazone, prosulfocarb, propazine, prosulfuron, pyrazon, pyrasulfuron-ethyl, pyridate, pyrithiobac, pyrosulam, quinclorac, quinmerac, quizalofop, rimsulfuron, sethoxydim, siduron, simazine, sulcotrione, sulfentrazone, sulfometuron, sulfosulfiiron, tebuthiuron, tepaloxydim, terbacil, terbutryn, thiazopyr, thifensulfuron, thiobencarb, topramezone, tralkoxydim, triallate, triasulfuron, tribenuron, triclopyr, trifloxysulfuron, trifluralin, triflusulfuron and vernolate.

The amount of the compound of the invention that is used to treat a plant will depend upon on the activity of the particular compound in question, the nature of the plant being treated and the desired effect. Further factors include the environmental conditions of the plant being treated. For example, high doses of the compound may be expected to have a herbicidal effect, particularly if the environmental conditions of the plant under treatment will not support enhanced plant growth. A person skilled in the art would readily be able to determine the amount of compound of the invention for achieving an effect in accordance with the use of the invention using rudimentary tests or standard tests that are well known in the art.

Definitions

Any reference to "promote plant growth" as used herein refers to an increase in the dry weight of an individual plant or an increase in crop yield compared to that which would be obtained without using a compound of the invention under otherwise identical conditions. The term "treatment" as used herein refers to the application of a compound or composition of the invention to a plant, such as by spraying a solution or composition of the invention over or around the plant.

Any reference to an "increase in efficacy of a herbicide" as used herein refers to an increase in the herbicidal effectiveness of the herbicide. Thus, a larger number of plants may be killed when the herbicide is used with a compound of the invention compared to that which would be obtained without using a compound of the invention under otherwise identical conditions. Alternatively, the amount or concentration of the herbicide that is used to kill a specific number or quantity of plants will be lower when used with a compound of the invention, compared to the amount or concentration of herbicide that would be used without using a compound of the invention under otherwise identical conditions.

The term "halogen" as used herein refers to a -F, -CI, -Br or -I moiety.

The term "hydroxy" as used herein refers to a -OH moiety.

The term "alkyl" as used herein refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 10 carbon atoms (unless otherwise specified), which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated or fully unsaturated). Thus, the term "alkyl" includes the sub-classes alkenyl, alkynyl, cycloalkyl, cycloalkenyl and cycloalkynyl below.

In the context of alkyl groups, the prefix Ci-Cio denotes the number of carbon atoms, or range of number of carbon atoms present in that group. Thus, the term "CpCio alkyl" refers to an alkyl group having from 1 to 10 carbon atoms. The first prefix may vary according to the nature of the alkyl group. Thus, if the alkyl group is an alkenyl or alkynyl group, then the first prefix must be at least 2 (e.g. C ₂ -Ci ₀ ). For cyclic (e.g. cycloalkyl, cycloalkenyl, cycloalkynyl) or branched alkyl groups, the first prefix must be at least 3 (e.g. C ₃ -C,o).

Examples of saturated alkyl groups include methyl (CO, ethyl (C ₂ ), propyl (C ₃ ), butyl (C ₄ ), pentyl (C ₅ ), hexyl (C ₆ ), heptyl (C ₇ ), octyl (C _g ), nonyl (C ₉ ) and decyl (Cio).

Examples of saturated linear alkyl groups include, but are not limited to, methyl

(Ci), ethyl (C ₂ ), n-propyl (C ₃ ), n-butyl (C ₄ ), n-pentyl (amyl) (C ₅ ), n-hexyl (C ₆ ), and n- heptyl (C ₇ ). Examples of saturated branched alkyl groups include iso-propyl (C ₃ ), iso-butyl (C ₄ ), sec-butyl (C ₄ ), tert-butyl (C ₄ ), iso-pentyl (C ₅ ), and neo-pentyl (C ₅ ).

The term "alkenyl" refers to an alkyl group having one or more carbon-carbon double bonds. Examples of unsaturated alkenyl groups include ethenyl (vinyl, -CH=CH ₂ ), 1 -propenyl (-CH=CH-CH ₃ ) and 2-propenyl (allyl, -CH-CH=CH ₂ ).

The term "alkynyl" refers to an alkyl group having one or more carbon-carbon triple bonds. Examples of unsaturated alkynyl groups include, but are not limited to, ethynyl (ethinyl, -C≡CH) and 2-propynyl (propargyl, -CH ₂ -C≡CH).

The term "cycloalkyl" refers an alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a carbocyclic compound (i.e. a compound where all of the ring atoms are carbon atoms). The ring may be saturated or unsaturated (e.g. partially unsaturated or fully unsaturated), which moiety has from 3 to 10 carbon atoms (unless otherwise specified). Thus, the term "cycloalkyl" includes the sub-classes cycloalkenyl and cycloalkynyl. Preferably, each ring has from 3 to 7 ring carbon atoms. Examples of cycloalkyl groups include those derived from (i) saturated monocyclic hydrocarbon compounds: cyclopropane (C ₃ ), cyclobutane (C ₄ ), cyclopentane (C ₅ ), cyclohexane (C ₆ ), cycloheptane (C ₇ ) and methylcyclopropane (C ₄ ); (ii) unsaturated monocyclic hydrocarbon compounds: cyclopropene (C ₃ ), cyclobutene (C ₄ ), cyclopentene (C ₅ ), cyclohexene (C ₆ ), methylcyclopropene (C ₄ ) and

dimethylcyclopropene (C ₅ ); (iii) saturated polycyclic hydrocarbon compounds: thujane

(do), carane (Cio), pinane (Cio), bornane (C ₎₀ ), norcarane (C ₇ ), norpinane (C ₇ ), norbornane (C ₇ ), adamantane (Ci ₀ ), decalin (C] ₀ ); (iv) unsaturated polycyclic hydrocarbon compounds: camphene (Cio), limonene (Cio), pinene (Ci ₀ ); and (v) polycyclic hydrocarbon compounds having an aromatic ring: indene (C9), indane (C9) and tetraline (Cio).

Typically, any reference to an alkyl group described herein is a C1-C10 alkyl group, preferably a Ci-C ₈ alkyl group, more preferably a Ci-C alkyl group, and is especially a Ci-C ₄ alkyl group. The alkyl groups in the invention are preferably saturated alkyl groups or saturated cycloalkyl groups, more preferably saturated, unbranched alkyl groups.

The phrase "optionally substituted" as used herein refers to a parent group which may be unsubstituted or which may be substituted with a substituent. In the context of a mono-(alkyl)amino group, di-(alkyl)amino group or their respective amines, or a tri- (alkyl)amino group the term refers to the substitution of each alkyl moiety in the amino or amino group. Thus, for example, in a di-(alkyl)amino group, each alkyl moiety may be substituted with substituents that are the same or different. The term "substituents" is used herein in the conventional sense and refers to a chemical moiety, which is covalently attached to, or if appropriate, fused to, a parent group.

Typically, a group or moiety may be "optionally substituted" with one or more "substituents" selected from hydroxy, Ci-Ci ₀ alkoxy, halogen, C ₃ -C ₇ cycloalkyl and C ₆ -Ci ₄ aryl. It is preferred that the optionally substituted group is substituted with one or more C6-Ci4 aryl groups, more preferably a single C ₆ -Ci ₄ aryl group.

The term "aryl" as used herein refers to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 6 to 14 ring carbon atoms (unless otherwise specified). Preferably, each ring has from 6 to 10 ring carbon atoms. More preferably, the aryl group is a phenyl group.

The term "alkoxy" used herein refers to an alkyl-oxy group, where the alkyl group is as defined above and has from 1 to 10 carbon atoms (unless otherwise specified). The alkyl moiety in an alkoxy group is preferably a saturated alkyl group or a saturated cycloalkyl group. More preferably the alkyl moiety is a saturated, unbranched alkyl group. Examples of CpCio alkoxy groups include -OMe (methoxy), -OEt (ethoxy), -0( ⁿ Pr) (n- propoxy), -Ο('ΡΓ) (isopropoxy), -0( ⁿ Bu) (n-butoxy), -0( ^s Bu) (sec-butoxy), -0(¾u) (isobutoxy), and -O('Bu) (tert-butoxy).

The term "amino" as used herein refers to a -NH ₂ moiety.

The term "mono-(alkyl)amino" as used herein refers to a monovalent moiety obtained by removing a hydrogen atom from a nitrogen atom of a primary alkyl amine compound. The alkyl moiety is as defined above and has from 1 to 10 carbon atoms (unless otherwise specified). It is preferred that the alkyl moiety in a mono-(alkyl)amino group is a saturated alkyl group or a saturated cycloalkyl group. More preferably the alkyl moiety is a saturated, unbranched alkyl group. Examples of mono-(Ci-Ci ₀ alkyl)amino groups include -NHCH ₃ , -NHCH ₂ CH ₃ , -NHCH(CH ₃ ) ₂ and -NHC(CH ₃ ) ₃ .

The term "di-(alkyl)amino" as used herein refers to a monovalent moiety obtained by removing a hydrogen atom from a nitrogen atom of a secondary alkyl amine compound. The alkyl moieties are as defined above and each alkyl moiety independently has from 1 to 10 carbon atoms (unless otherwise specified). It is preferred that each alkyl moiety is a saturated alkyl group or a saturated cycloalkyl group. More preferably each alkyl moiety is a saturated, unbranched alkyl group. Examples of di-(Ci-Ci ₀ alkyl)amino groups include -N(CH ₃ ), -N(CH ₃ )CH ₂ CH ₃ , -N(CH ₂ CH ₃ )(CH(CH ₃ ) ₂ ) and -N(CH ₃ )C(CH ₃ ) ₃ . The term "tri-(alkyl)amino" as used herein refers to a tertiary alkyl amine, which typically is able to covalently bond or coordinate to another atom or moiety by the lone pair of electrons on the nitrogen atom. The alkyl moieties are as defined above and each alkyl moiety independently has from 1 to 10 carbon atoms (unless otherwise specified). It is preferred that each alkyl moiety is a saturated alkyl group or a saturated cycloalkyl group. More preferably each alkyl moiety is a saturated, unbranched alkyl group.

Examples of tri-(Ci-Ci ₀ alkyl)amino groups include N(CH ₃ ) _3> N(CH ) ₂ CH ₂ CH ₃ ,

N(CH ₂ CH ₃ ) ₂ (CH(CH ₃ ) ₂ ) and N(CH ₃ ) ₂ C(CH ₃ ) ₃ .

The term "nitrogen containing heterocyclyl" used herein refers to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a non-aromatic, nitrogen containing heterocyclic compound, which moiety has from 3 to 14 ring atoms (unless otherwise specified), where at least one of the ring atoms is a nitrogen atom. The ring of heterocyclic compound may be fully saturated or unsaturated.

Examples of nitrogen containing heterocyclyl groups include those derived from aziridine, azetidine, pyrrolidine, pyrroline, 2H-pyrrole or 3H-pyrrole, piperidine, dihydropyridine, tetrahydropyridine, azepine, imidazolidine, pyrazolidine, imidazoline, pyrazolone, piperazine, tetrahydrooxazole, dihydrooxazole, tetrahydroisoxazole, dihydroisoxazole, morpholine, tetrahydrooxazine, dihydrooxazine, oxazine, thiazoline, thiazolidine, thiomorpholine, oxadiazine, and oxathiazine.

Typically, any reference to a nitrogen containing heterocyclyl group refers to a group having from 3 to 14 ring atoms, preferably 4 to 12 ring atoms, more preferably 5 to 10 ring atoms, even more preferably 6 to 8 ring atoms. It is also preferred that the heterocyclyl group is saturated.

It is preferred that any reference to a heterocyclyl group refers to a group having no more than 3 ring heteroatoms, where at least one of which is nitrogen. More preferably, the heterocyclyl group has no more than 2 ring heteroatoms, where one of which is nitrogen and the other is either oxygen or nitrogen.

The term "nitrogen containing heteroaryl group" used herein refers to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a nitrogen containing heteroaromatic compound, which moiety has from 3 to 14 ring atoms (unless otherwise specified), where at least one of the ring atoms is a nitrogen atom.

Examples of nitrogen containing heteroaryl groups include those derived from pyrrole, pyridine, oxazole, isoxazole, isoxazine, oxadiazole, oxatriazole, thiazole, isothiazole, imidazole, pyrazole, pyridazine, pyrimidine, pyrazine, triazole, triazine, tetrazole, indole, isoindole, indolizine, indoline, isoindoline, purine, benzimidazole, indazole, benzoxazole, benzisoxazole, benzodioxole, benzofurazan, benzotriazole, quinoline, isoquinoline, quinolizine, benzoxazine and benzodiazine.

Typically, any reference to a nitrogen containing heteroaryl group refers to a group having from 3 to 14 ring atoms, preferably 4 to 12 ring atoms, more preferably 5 to 10 ring atoms, even more preferably 6 to 8 ring atoms.

It is preferred that any reference to a heteroaryl group refers to a group having no more than 3 ring heteroatoms, where at least one of which is nitrogen. More preferably, the heteroaryl group has no more than 2 ring heteroatoms, where one of which is nitrogen and the other is either oxygen or nitrogen.

The invention will now be illustrated by the following, non-limiting examples.

Examples

Synthesis of morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate

(GYY4137

Morpholine (20 mmol) in methyl chloride (CH ₂ C1 ₂ , 6 mL) was added dropwise (room temperature) to a CH ₂ C1 ₂ solution (6 mL) of 2,4-bis(4-methoxyphenyl)-2,4- dithioxo-l,3,2,4-dithiadiphosphetane (4.0 mmol). The reaction mixture was stirred at room temperature for 2 hours. The precipitate was filtered and washed several times with CH ₂ C1 ₂ . The product was a white solid (67% yield) and was pure as determined byΉ nuclear magnetic resonance. The compound (GYY4137), which has a melting point of 159.8 °C to 164.0 °C, is soluble in water up to 30 mg/mL (pH 7.4). The nuclear magnetic resonance characteristics of the compound (GYY4137) are as follows: Ή nuclear magnetic resonance (300 MHz, acetone-D6, 300 K): δ = 8.03 to 8.1 1 (m, 2H, aromatic CH), 6.88 to 6.90 (m, 2H, aromatic CH), 3.94 (m, 4H, CH), 3.82 (s, CH ₃ ), 3.50 to 3.53 (m, 4H, CH),

3.36 to 3.40 (m, 4H, CH), 2.87 to 2.92 (dd, J = 9.7, 5.4 Hz, CH), 2.04 to 2.09 (m, 4H, CH); ¹³ C nuclear magnetic resonance (75 MHz, acetone-D6, 300K): δ = 132.7 (aromatic CH), 132.5 (aromatic CH), 1 12.2 (aromatic CH), 1 12.0 (aromatic CH), 66.8 (CH ₂ ), 63.6 (CH ₃ ), 54.6 (CH ₂ ), 54.0 (CH ₂ ), 44.9 (CH ₂ ), 43.3 (CH ₂ ). The infrared (film) values were 3019 cm ^"1 , 1215 cm ^"1 and 756 cm ^"1 . The H ₂ S release from the compound (GYY4137) in vitro has been assessed with the use of 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and by amperometry as described in Ling Li et al (Circulation, 1 17 (2008), pages 2351 to 2360).

Example 1 Plant growth

Wild-type, both Landsberg erecta and Columbia ecotypes, Arabidopsis thaliana were sown in Levington's F2 compost (Avoncrop, Bristol, UK) and grown under a 12 hour photo-period in plant growth chambers (Sanyo Gallenkamp,Loughborough, UK). Fully expanded wild-type leaves were harvested at 4 to 5 weeks and used immediately. Stomatal assays

Stomatal bioassays were carried out as described previously by Desikan et al (Proceedings of the National Academy of Science, USA, 99 (2002), pages 16319 to 16324). Whole leaves were incubated in MES-KCl buffer [10 mM 2-morpholino ethane sulfonic acid (MES), 5 mM KC1, 50 μΜ CaCl ₂ , pH 6.15] for 2.5 hours under light conditions (e.g. 20 μΕ m ^'2 s ^"1 ) before the addition of various compounds, or left in the dark. The whole leaves were used and placed in the Petri dishes (lower epidermis downwards) containing 1, 10 or 100 μΜ of NaSH or GYY4137 in 3 mL MES, pH 6.15. Stomatal apertures were observed in epidermal fragments after a further 2 hour incubation. Apertures were measured using a light microscope (40x) and imaging camera with LEICA QWIN image processing and analysis software (Leica Microsystems and Imaging Solutions, Cambridge, UK).

Measurement of nitric oxide using DAF2-DA

Nitric oxide accumulation was estimated using the specific NO dye DAF2-DA (Calbiochem, Nottingham, UK), using the method described previously by Desikan et al (Proceedings of the National Academy of Science, USA, 99 (2002), pages 16319 to 16324). Epidermal fragments in MES-KCl buffer (10 mM MES, 5 mM KC1, 50 μΜ CaCl ₂ , pH 6.15) were loaded with 15 μΜ DAF2-DA for 15 min before washing with MES-KCl buffer for 20 min. Fragments were subsequently incubated for a further 25 min in the presence of various compounds before images were visualized. Images were visualized using CLSM (excitation 488 nm, emission 515 ran; Nikon PCM2000, Kingston-upon-Thames, UK). Images acquired were analysed using SCION IMAGE software (Scion, Frederick, MD, USA).

Results

To assess whether H ₂ S could cause stomatal closure, leaves were exposed to light to open the stomata, and then treated with the plant hormone abscisic acid (ABA) or the H ₂ S donor NaSH.

As can be seen in Figure 2, ABA caused a significant closure of the stomata following a 2.5 hr treatment. However, no closure was seen on NaSH treatment, and in fact the stomatal opened more in a dose-dependent manner. Even with 1 μΜ NaSH treatment a significant amount of opening compared to the control was seen.

The compound GYY4137 is able to release H ₂ S slowly and its ability to affect stomatal movements was also assessed. Once again, no closure was seen and the stomata were more open than the control, albeit to a lesser extent than seen with NaSH (compared Figure 2 with Figure 3). Maximal opening was seen with 10 μΜ of GYY4137. As the concentration was increased further the effect was reduced, with the stomata opening becoming more comparable to that of the controls.

As H ₂ S appeared to cause the opening, the experiment was repeated without the previous exposure to light to cause opening prior to treatment with the H ₂ S releasing compound GYY4137. In this experiment, the control aperture size was smaller and ABA caused closure. Once again treatment with the compound GYY4137 resulted in stomatal opening, with the effect being dose dependent (see Figure 4). High concentrations had a larger effect than seen with the pre-light treatment with opening increasing up to 200 μΜ GYY4137 (see Figure 4).

The effects of both NaSH and GYY4137 were assessed following dark treatment, a condition which would cause physiological closure of the stomata. After 2 hours in the dark control stomata were seen to close (see "Control D" in Figure 5; "L" = light, "D" = dark), but if treated simultaneously with either NaSH or GYY4137 the stomata did not close as much. Treatment with ΙΟΟμΜ of GYY4137 in the dark resulted in stomata that were open to the same extent as the samples which had been treated with light (see the result for "100 μΜ GYY D 2hr" in Figure 5). A similar trend was seen if the treatments were carried out over 4 hours, although the effects of both GYY4137 and NaSH were less. The treatment of leaves with either ABA or darkness will cause the production of NO, and therefore DAF2-DA mediated fluorescence was used in conjunction with confocal microscopy to assess the accumulation of NO in samples which had been treated with H ₂ S, either administered as NaSH or GYY4137. To assess the ability of the compounds to interfere with the DAF-based assays, the levels of DAF2-based fluorescence, which was caused by NO release from SNP was assessed in a fluorimeter in the presence and absence of NaSH or GYY4137. No reduction in DAF2 fluorescence was seen, suggesting that these compounds do no interfere. Therefore in the presence of either NaSH or GYY4137, DAF2 would be a representative measure of NO accumulation, as reported by C. Garcia-Mata et al (Plant Physiol. , 128 (2002), pages 790 to 792).

As can be seen in Figure 6 untreated control samples showed no increase in DAF2- mediated fluorescence, whereas ABA treatment caused a large increase in the NO- mediated signal seen in the guard cells. Treatment with either NaSH or GYY4137 on their own did cause a small rise in fluorescence, suggesting that these compounds may stimulate NO production, or reduce NO scavenging in the stomata. However, more significantly, when GYY4137 or NaSH were added at the same time as ABA, the fluorescence seen was considerably less than when ABA was added on it own, suggesting that the GYY4137 or NaSH had reduced the NO accumulation which usually results from ABA treatment (see Figure 6).

ABA caused a significant accumulation of NO as estimated by DAF2-DA fluorescence, but both NaSH and GYY4137 reduced this accumulation to a large extent (see Figure 6). Neither compounds used completely removed the NO, but the presence of NO was considerably less.

Example 2

The effects of both NaSH and GYY4137 on the plant species Capsicum anuum were studied. The leaves were from Capsicum anuum plants that were between 6 and 7 weeks old were analysed.

Stomatal assays

Stomatal bioassays were carried out as described by Desikan et al (Proceedings of the National Academy of Science, USA, 99 (2002), pages 16319 to 16324). Epidermal peels were incubated in MES-KC1 buffer [10 mM 2-morpholino ethane sulfonic acid (MES), 5 mM KC1, 50 μΜ CaCl ₂ , pH 6.15] for 2.5 hours under direct lighting (in 60 to 100 IE m ^"2 s ^"1 ) before the addition of the compounds.

In experiment (A), samples were sheltered from direct lighting and treated with ABA or NaHS. In experiment (B), samples were sheltered from direct lighting and treated with ABA or GYY 4137 for the next 2 h and left under day light conditions before stomata apertures were analyzed. Apertures were measured using a light microscope and imaging camera with LEICA QWIN image processing and analysis software (Leica Microsystems and Imaging Solutions, Cambridge, UK).

Measurement of nitric oxide using DAF2-DA

Nitric oxide accumulation was estimated using the specific NO dye DAF2-DA

(Calbiochem, Nottingham, UK), using the method described previously by Desikan et al (Proceedings of the National Academy of Science, USA, 99 (2002), pages 16319 to 16324). Epidermal fragments in MES-KC1 buffer (10 mM MES, 5 mM KC1, 50 μΜ CaCl ₂ , pH 6.15) were exposed to the direct lightning for 2 h. After 2 h samples were loaded with 30 μΜ DAF2-DA for 15 min before washing with MES-KC1 buffer; three times for 10 min. Fragments were subsequently incubated for a further 30 min in the presence of the compounds before images were visualized using CLSM (excitation 488 nm, emission 515 ran; Nikon PCM2000, Kingston-upon-Thames, UK). Images acquired were analyzed using SCION IMA GE software (Scion, Frederick, MD, USA).

Experiment (A) was a control sample with no treatment. In experiment (B), the epidermal fragments were treated with ABA. In experiment (C), the fragments were treated with 100 μηι of NaHS. Experiment (D) relates to ABA treatment in the presence of NaHS. Experiment (E) is treatment with 100 μπι of GYY4137. Experiment (F) relates to ABA treatment in the presence of NaHS. Results

As can be seen in Figure 7A, NaSH caused stomata to open further, even though the leaf tissue had been exposed to the light. Stomata were able to close, as ABA treatment demonstrated, therefore showing that the stomata were not defective. When the

experiments were repeated with GYY4137 (see Figure 7B) a smaller but similar effect of the addition of the H ₂ S donor was seen. The NO experiment was performed to investigate if NO accumulation is effected in Capsicum when treated with H ₂ S donors. The presence of a H ₂ S donor dramatically reduced the amount of NO that was measured following ABA treatment. This suggests that H ₂ S is having an effect on NO metabolism, which may account for the stomata aperture measurements.

Example 3

The effect of GYY4137 on growth of soil-grown Arabidopsis thaliana

Arabidopsis thaliana (Col-0) were grown in plastic pots (5 cm diameter) in

Levington F2 compost plus vermiculite (4: 1 , F2 compost:vermiculite) in a controlled environment room (20°C, 65% RH, 16 h light period, light intensity 200 μπιοΐ photons m ^"2 s ^"1 ) for 2 weeks after sowing. After 2 weeks the plants were watered twice weekly to runoff with GYY4137 dissolved in water for 4 weeks. The shoots (rosettes) were then cut off at soil surface, freeze dried and weighed.

Results

The data shown in Figure 9 are fresh weight per rosette ± standard error (n = 8).

There was a significant effect (one way ANOVA, p<0.05) of GYY4137 on the biomass of the rosettes with maximum growth at 0.01 mM. The highest concentration of GYY4137 decreased growth.

Example 4 The effect of GYY4137 on growth of Arabidopsis thaliana

Arabidopsis thaliana seed were surface-sterilized for 4 min in 10% (v/v) household bleach and for 4 min in ethanol: water :bleach mixture (7:2: 1 by vol.), and rinsed twice for 2 min in sterile water and stratified at 4°C for 2 days. Square Petri dishes (12 cm x 12 cm) were half filled with sterile half-strength Murashige-Skoog medium (Sigma-Aldrich) gelled with 0.5% (w/v) Phytagel (Sigma-Aldrich). Sterile seeds (20 per plate) were sown onto the surface of the gel. Lids were sealed with gas permeable tape and the dishes placed in a controlled environment room (20°C, 65% RH, 1 h light period, light intensity 200 μπιοΐ photons m ^"2 s ^"1 ) for 2 weeks. After this period, roots and shoots from each plate were blotted dry and weighed. Results

The data shown in Figure 10 are mean fresh weight/plant ± standard error (n = 4 separate dishes).

There was a significant effect (one way ANOVA, pO.001) of GYY4137 on the biomass of the leaves with maximum growth at 10 μΜ (Figure 10). Higher concentrations of GYY4137 decreased shoot growth. Root biomass followed the same pattern (one way ANOVA, pO.01).