ES2421056A1 - Haptens, conjugates and antibodies for pyrimethanil fungicide - Google Patents

Abstract

The invention relates to haptens, conjugates and antibodies for pyrimethanil fungicide. In addition, the invention relates to the use of pyrimethanil conjugates as assay antigens or immunogens in order to obtain antibodies of the aforementioned fungicide, and to the use of the labelled derivatives of pyrimethanil as assay antigens. The invention also relates to a pyrimethanil analysis method using the antibodies obtained, at times together with assay antigens which are conjugates or labelled derivatives. The invention also provides a kit for analysing pyrimethanil, which includes antibodies of said fungicide, at times together with assay antigens which are conjugates or labelled derivatives.

Description

Haptenes, conjugates and antibodies to the fungicide pyrimethanil

The present invention relates to haptens, conjugates, labeled derivatives and antibodies to pyrimethanil. In addition, the present invention

5 also refers to a method of analysis of pyrimethanil using the antibodies obtained, sometimes together with test antigens that are conjugated or labeled derivatives.

STATE OF THE TECHNIQUE

Fungicides are the group of pesticides that most frequently

10 appears in surveillance and control programs. This is so not only because of its high use, but mainly because these plant protection products are used to fight fungal infections at times near harvest or even later (postharvest fungicides). This fact considerably increases the probability of

15 that residues of these treatments remain when the food reaches the consumer, which forces the regulatory and control bodies to be more vigilant and ideally to increase controls. Fungal attacks on stored fruits are one of the main reasons for economic losses in agriculture. The intensive use of conventional fungicides,

20 such as thiabendazole or imazalil, has led in recent years to the emergence of resistant strains and therefore to a less effective treatment with these products. Given this circumstance, the agrochemical companies launched R&D programs aimed at developing new products that presented innovative mechanisms of action and that allowed us to combat

25 Fungal infections effectively, while being safe and compatible with integrated pest management programs. These products are beginning to gradually replace older products, which are less acceptable from a toxicological and environmental point of view.

Among the most relevant fungicides developed in the last decade with postharvest applications, pyrimethanil stands out [H.J. Rosslenbroich and D. Stuebler, Crop Prot. 2000, 19, 557-561]. This pesticide belongs to the family of anilinopyrimidines whose biochemical mode of action is common to this

5 family and different from the rest of fungicides. Pyrimethanil (4,6-dimethyl-N-phenylpyrimidin-2-amine) is a product developed by AgrEvo and marketed worldwide by companies such as Bayer CropScience, BASF

or Janssen Pharmaceutical. Currently, the most common commercial names are Scala and Penbotec, also in the form of 10 formulations combined with a wide variety of different fungicides, among which Philabuster (with imazalil) and FlintStar (with trifloxistrobin). Its use as a postharvest fungicide is authorized both in citrus fruits and in stone fruits and seeds. Pyrimethanil was included in Annex I of the EU in 2006. Among the crops for which the EU has established limits

Maximum 15 residues explicitly include citrus fruits (10 ppm), stone fruits (3–10 ppm), pip fruits (5 ppm), grapes and strawberries (5 ppm), in addition to a variety of vegetables and vegetables.

The analytical methodologies used for the analysis of these fungicides are fundamentally of the instrumental type, especially 20 gas chromatography (GC) and high performance liquid chromatography (HPLC), coupled to different detectors according to the type of compound to be analyzed and the required sensitivity . These techniques are characterized by their ability to simultaneously analyze several residues with high precision and accuracy. However, although they are essential in many circumstances, they often involve the use of laborious and high-cost methodologies, which should be carried out by highly qualified personnel in well-equipped centralized laboratories and usually away from production areas. These limitations condition the suitability of these techniques to undertake the analysis of large numbers of samples and to obtain 30 results in the short term, two aspects that would contribute to guarantee the safety of the food sold and to carry out further studies.

exhaustive on the exposure of consumers to these fungicides through food.

Immunoassays are bioanalytical techniques based on the interaction of an antigen (the analyte) with an antibody that specifically recognizes it. However, a pesticide is a small organic molecule that constitutes a single antibody binding site, so in this type of analytical techniques the interaction between the analyte and the antibody is carried out by displacing the binding between the antibody and a labeled analyte analog. Thus, in the presence of the analyte a competition is established between it and the analog 10 marked by antibody binding. Usually, the labeling is carried out with an enzymatic activity, thus giving rise to enzyme immunoassays. When one of the participants in the reaction is also immobilized on a solid support, the technique is called ELISA (Enzyme-Linked Immunosorbent Assay). The first enzyme immunoassays for 15 pesticides were developed during the first half of the 80s. Since then until now the number of pesticides for which immunoassays have been developed has increased dramatically, assuming several tens and covering the main groups of compounds: insecticides , herbicides and fungicides. In fact, a large number of these tests are commercially available in the form of kits with different formats. The growing acceptance of immunoassays as complementary techniques to chromatography for the analysis of small organic molecules is due to the fact that it is a simple, fast and low-cost methodology, exhibiting at the same time high sensitivity and specificity. The immunoassays 25 allow the target analyte to be specifically detected in very complex mixtures, considerably simplifying the laborious sample preparation procedures, which in turn results in an increase in the sampling capacity. In addition, immunoassays can be performed in portable formats, which makes them independent of centralized laboratories and makes them suitable for analysis at production points. The analytical excellencies attributed to immunoassays have already been

demonstrated in many practical applications, where they have competed favorably with chromatographic techniques [M.C. Hennion, Analysis 1998, 26,149-155; A. Abad et al., J. Chromatogr. A 1999, 833, 3–12; A. Abad et al., J. Agric. Food Chem. 2001, 49, 1707-1712; N.A. Lee and I.R. Kennedy, J. AOAC

5 Int. 2001, 84, 1393-1406; FA. Esteve-Turrillas et al., Anal. Chim. Minutes 2010, 682, 93-103].

However, to date the use of immunoassay technology to analyze the content of the fungicide pyrimethanil in a sample has not been described.

10 Therefore, there is a need, particularly in the food and / or agricultural industry, to develop an analytical method that allows the determination or detection by means of pyrimethanil immunoassay technology, a fungicide widely used today, preferably by use of a kit comprising at least one pyrimethanil antibody.

15 DESCRIPTION OF THE INVENTION

The present invention provides pyrimethanil haptens, compounds of formula (I), which have adequate functionalization, in different positions of the molecule, for obtaining pyrimethanil conjugates or pyrimethanil labeled derivatives.

A first aspect of the present invention describes a compound of general formula (I):

T − L − Y

(I)

characterized in that 25 T is selected from the group consisting of R-I, R-II, R-III, R-IV and R-V;

HH N N NN

N

N R-I

R-II

H H NN

NN N

N R-III

R-IV

NN N

R-V

where RI is selected from the group consisting of [2 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl], [3 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl] and [4- ((4,6dimethylpyrimidin-2-yl) amino) phenyl];

R-II is [4,6-dimethyl-2- (phenylamino) pyrimidin-5-yl]; R-III is selected from the group consisting of [2 - ((4-methylpyrimidin-2yl) amino) phenyl], [3 - ((4-methylpyrimidin-2-yl) amino) phenyl] and [4 - ((4 -methylpyrimidin-2-yl) amino) phenyl]; R-IV is selected from the group consisting of [6-methyl-2- (phenylamino) pyrimidin-4

20 yl] and [6-methyl-2- (phenylamino) pyrimidin-5-yl]; and R-V is [N- (4,6-dimethylpyrimidin-2-yl) -N- (phenyl) amino]; L is a hydrocarbon chain of 2 to 40 carbon atoms, where the chain is linear or branched, saturated or unsaturated, and said hydrocarbon chain comprises between 0 and 10 heteroatoms that are selected from the

25 group consisting of S, O and N; Y is a functional group selected from the group consisting of −COOH, −NH2, −N3, −CH2Br, −CH2I, −CHO, −SH, −SO3H, −OSO2Ph, −NH − NH2, and −OSO2Ar; with the condition of:

a) when T is [4 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl], -L-Y is different from -CH2CH2-NH2; b) when T is [4 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl], -L-Y is different from −CONHCH2-CO2H; 5 c) when T is [4 - ((4-methylpyrimidin-2-yl) amino) phenyl], -L-Y is different from -CHCH3-CO2H; d) when T is [6-methyl-2- (phenylamino) pyrimidin-4-yl], -L-Y is different from −OCH2-CO2H; e) when T is [N- (4,6-dimethylpyrimidin-2-yl) -N- (phenyl) amino], L is a

10 hydrocarbon chain of 2 to 40 carbon atoms, linear or branched, saturated or unsaturated, with the proviso that said hydrocarbon chain does not comprise any heteroatom; f) when T is [N- (4,6-dimethylpyrimidin-2-yl) -N- (phenyl) amino], -L-Y is different from - (CH2) 3-NH2.

In the present patent application, aryl (-Ar) is understood as an aromatic carbocyclic group with a single ring, for example phenyl, multiple rings, for example biphenyl, or multiple condensed rings where at least one of them is aromatic, for example 1,2,3,4-tetrahydronaphthyl, 1-naphthyl, 2-naphthyl. The aryl group may be substituted or not. Preferably, the aryl group is 4

20 methylphenyl.

According to a preferred embodiment, the compound of formula (I) is characterized in that L is a linear hydrocarbon chain of 2 to 20 carbon atoms. Preferably, said linear hydrocarbon chain comprises between 2 and 8 carbon atoms.

According to another additional preferred embodiment, the compound of formula (I) of the present invention is characterized in that Y is selected from the group consisting of −COOH, −CHO, −NH2 and −SH. Preferably, the compound of formula (I) object of the present invention is characterized in that Y is -COOH.

According to another preferred embodiment, the compound of formula (I) as described in this patent application is characterized in that T is [3 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl] or [4 - ((4 , 6-dimethylpyrimidin-2-yl) amino) phenyl]; L is a linear hydrocarbon chain of 2 to 20 carbon atoms; and I know

5 select from the group consisting of −COOH, −CHO, −NH2 and −SH. Preferably, the compound of formula (I) of the present invention is characterized in that L is a linear hydrocarbon chain of 2 to 8 carbon atoms.

According to an even more preferred embodiment, the compound of formula (I) of this invention is selected from the group consisting of

H H

NN

(CH2) 5CO2H N N N

N

(CH2) 5CO2H

Y

According to another preferred embodiment, the compound of formula (I) as it is

15 described in this patent application is characterized in that T is [6-methyl-2 (phenylamino) pyrimidin-4-yl]; L is a linear hydrocarbon chain of 2 to 20 carbon atoms; and Y is selected from the group consisting of −COOH, −CHO, −NH2 and −SH. Preferably, the compound of formula (I) of the present invention is characterized in that L is a hydrocarbon chain

20 linear from 2 to 8 carbon atoms.

According to an even more preferred embodiment, the compound of formula (I) of this invention is

H

HO2C (H2C) 4 N N N

According to another preferred embodiment, the compound of formula (I) as described in this patent application is characterized in that T is [N- (4,6-dimethylpyrimidin-2-yl) -N- (phenyl) amino]; L is a linear hydrocarbon chain of 2 to 20 carbon atoms; and Y is selected from the group consisting of

5 −COOH, −CHO, NH2 and −SH. Preferably, the compound of formula (I) of the present invention is characterized in that L is a linear hydrocarbon chain of 2 to 8 carbon atoms.

According to an even more preferred embodiment, the compound of formula (I) of this invention is (CH2) 4CO2H

NN N

A second aspect of the present invention describes a compound of formula (II):

15 [T − L − Z] n − P

(II)

characterized in that T is selected from the group consisting of R-I, R-II, R-III, R-IV and R-V; where

H H

NN

NN

N

N R-III

R-IV HH NN

NN

N

N

R-I

R-II

NN N

R-V

RI is selected from the group consisting of [2 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl], [3 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl] and [4- ( (4,6-dimethylpyrimidin-2-yl) amino) phenyl];

R-II is [4,6-dimethyl-2- (phenylamino) pyrimidin-5-yl]; R-III is selected from the group consisting of [2 - ((4-methylpyrimidin-2yl) amino) phenyl], [3 - ((4-methylpyrimidin-2-yl) amino) phenyl] and [4 - ((4 -methylpyrimidin-2-yl) amino) phenyl]; R-IV is selected from the group consisting of [6-methyl-2- (phenylamino) pyrimidin-4

10 yl] and [6-methyl-2- (phenylamino) pyrimidin-5-yl]; and R-V is [N- (4,6-dimethylpyrimidin-2-yl) -N- (phenyl) amino]; L is a hydrocarbon chain of 2 to 40 carbon atoms, where the chain is linear or branched, saturated or unsaturated, and said hydrocarbon chain comprises between 0 and 10 heteroatoms that are selected from the

15 group consisting of S, O and N; Z is a functional group selected from the group consisting of −CONH−, −NHCO−, −NHCONH−, −NHCSNH−, −OCONH−, −NHOCO−, −OCSNH−, −SCONH−, −S−, −S− S−, −NH (C = NH) -, −OCO−, −CO−, −CHOH−, −N = N−, −NH−, −NR−,

Y

 ;

P is a natural or synthetic polypeptide of molecular weight greater than 2000 daltons; Y

25 n is a number with a value between 1 and 500.

The value of n indicates the degree of conjugation, that is, the molar ratio between the fraction derived from the compound of formula (I) and P, the natural or synthetic polypeptide of molecular weight greater than 2000 daltons in the compound of formula

(II) resulting.

The compound of formula (II) of the present invention can be used for the production of antibodies or together with a pyrimethanil antibody, to determine or detect this fungicide in a sample using the technology of

competitive immunoassay.

According to a preferred embodiment, the compound of formula (II) is characterized in that L is a linear hydrocarbon chain of 2 to 20 carbon atoms. Preferably, said linear hydrocarbon chain comprises between 2 and 8

10 carbon atoms

According to another additional preferred embodiment, the compound of formula (II) of the present invention is characterized in that Z is selected from the group consisting of −CONH−, −NHCO−, −NH−, −S−,

Preferably, the compound of formula (II) object of the present invention is characterized in that Z is −CONH−.

According to another preferred embodiment of the present invention, the compound of formula (II) described in this patent application is characterized in that P is

20 selects from the group consisting of albumin, thyroglobulin, hemocyanin, βgalactosidase, peroxidase, phosphatase and oxidase. Preferably, when P is albumin, it is egg albumin or serum albumin.

According to another preferred embodiment, the compound of formula (II) of the present invention is characterized in that

T is [3 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl] or [4 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl]; L is a linear hydrocarbon chain of 2 to 20 carbon atoms;

Z is selected from the group consisting of −CONH−, −NHCO−, −NH−, −S−, and

; Y

5 P is selected from the group consisting of albumin, thyroglobulin, hemocyanin, β-galactosidase, peroxidase, phosphatase and oxidase.

According to an even more preferred embodiment, the compound of formula (II) of the present invention is characterized in that T is [3 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl] or [4 - ((4,6- dimethylpyrimidin-2-yl) amino) phenyl]; L is a chain

10 linear hydrocarbon of 5 carbon atoms; Z is −CONH−; P is selected from the group consisting of albumin and peroxidase; and n is a value selected between 1 and 50.

According to another preferred embodiment, the compound of formula (II) of the present invention is characterized in that

15 T is [6-methyl-2- (phenylamino) pyrimidin-4-yl]; L is a linear hydrocarbon chain of 2 to 20 carbon atoms; Z is selected from the group consisting of − CONH−, −NHCO−, −NH−, −S−, and; Y

P is selected from the group consisting of albumin, thyroglobulin, hemocyanin, β-galactosidase, peroxidase, phosphatase and oxidase.

According to an even more preferred embodiment, the compound of formula (II) of the present invention is characterized in that T is [6-methyl-2- (phenylamino) pyrimidin

25 4-yl]; L is a linear hydrocarbon chain of 4 carbon atoms; Z is −CONH−; P is selected from the group consisting of albumin and peroxidase; and n is a value selected between 1 and 50.

According to another preferred embodiment, the compound of formula (II) of the present invention is characterized in that T is [N- (4,6-dimethylpyrimidin-2-yl) -N- (phenyl) amino]; L is a linear hydrocarbon chain of 2 to 20 carbon atoms; Z is selected from the group consisting of −CONH−, −NHCO−, −NH−, −S−,

Y

; Y

P is selected from the group consisting of albumin, thyroglobulin, hemocyanin, β-galactosidase, peroxidase, phosphatase and oxidase.

According to an even more preferred embodiment, the compound of formula (II) of the present invention is characterized in that T is [N- (4,6-dimethylpyrimidin-2-yl) -N (phenyl) amino]; L is a linear hydrocarbon chain of 4 carbon atoms; Z is −CONH−; P is selected from the group consisting of albumin and

15 peroxidase; and n is a value selected between 1 and 50.

A third aspect of the present invention describes a compound of formula (III):

[T − L − Z] m − Q

(III)

20 characterized in that T, L and Z have the same meaning defined above for the compound of formula (II); Q is a non-isotopic marker; and m is an integer with an integer value between 1 and 1000.

In the present invention, "marker" means any molecule or fragment that gives rise to a signal measurable by any type of analytical technique. In the present invention, Q of the compound of formula (III) identifies a fragment or a chemical detector, marker or tracer molecule.

This compound of formula (III) can be used with a pyrimethanil antibody to determine or detect this fungicide in a sample using competitive immunoassay technology.

According to a preferred embodiment, the compound of formula (III) is characterized in that L is a linear hydrocarbon chain of 2 to 20 carbon atoms. Preferably, L is a linear hydrocarbon chain of 2 to 8 carbon atoms.

According to another additional preferred embodiment, the compound of formula (III) of the present invention is characterized in that Z is selected from the group consisting of −CONH−, −NHCO−, −NH−, −S−, and

.

Preferably, the compound of formula (III) is characterized in that Z is 15 −CONH−.

According to another additional preferred embodiment, the compound of formula (III) of the present invention is characterized in that Q is biotin, a luminescent compound, a fluorophore, a label coupled to an indirect detection system, micro or nanoparticles or others.

Preferably, Q is selected from the group consisting of biotin, fluorescein or any one of its derivatives, a cyanine fluorophore, a rhodamine fluorophore, a coumarin fluorophore, a ruthenium bipyryl, luciferin or any one of its derivatives, a acridinium ester and micro-or nanoparticles of colloidal gold, carbon or latex.

According to another preferred embodiment, the compound of formula (III) of the present invention is characterized in that

T is [3 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl] or [4 - ((4,6-dimethylpyrimidin-2yl) amino) phenyl]; L is a linear hydrocarbon chain of 2 to 20 carbon atoms; Z is selected from the group consisting of −CONH−, −NHCO−, −NH−, −S−,

Y

; Y

Q is selected from the group consisting of biotin, fluorescein or one

10 any of its derivatives, a cyanine fluorophore, a rhodamine fluorophore, a coumarin fluorophore, a ruthenium bipyryl, luciferin or any of its derivatives, an acridinium ester and micro-or colloidal gold microparticles or nanoparticles latex

According to another preferred embodiment, the compound of formula (III) of the present

The invention is characterized in that T is [6-methyl-2- (phenylamino) pyrimidin-4-yl]; L is a linear hydrocarbon chain of 2 to 20 carbon atoms; Z is selected from the group consisting of −CONH−, −NHCO−, −NH−, −S−,

Y

; Y

Q is selected from the group consisting of biotin, fluorescein or any one of its derivatives, a cyanine fluorophore, a rhodamine fluorophore, a coumarin fluorophore, a ruthenium bipyril, luciferin or one

Any of its derivatives, an acridinium ester and micro- or nanoparticles of colloidal gold, carbon or latex.

According to another preferred embodiment, the compound of formula (III) of the present invention is characterized in that T is [N- (4,6-dimethylpyrimidin-2-yl) -N- (phenyl) amino];

30 L is a linear hydrocarbon chain of 2 to 20 carbon atoms;

Z is selected from the group consisting of −CONH−, −NHCO−, −NH−, −S−, and

; Y

5 Q is selected from the group consisting of biotin, fluorescein or any one of its derivatives, a cyanine fluorophore, a rhodamine fluorophore, a coumarin fluorophore, a ruthenium bipyryl, luciferin or any one of its derivatives, an ester of acridinium and micro-or nanoparticles of colloidal gold, carbon or latex.

A fourth aspect of the present invention describes an antibody generated in response to a compound of formula (II) as described in this patent application.

According to a preferred embodiment of the invention, the compound of formula (II) that generates an antibody as described in this patent application is

15 characterizes because T is [3 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl] or [4 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl]; L is a linear hydrocarbon chain of 2 to 20 carbon atoms; Z is selected from the group consisting of −CONH−, −NHCO−, −NH−, −S−,

20 and; and

P is selected from the group consisting of albumin, thyroglobulin and hemocyanin.

Preferably, the compound of formula (II) that generates an antibody as described in this patent application, is characterized in that T is [3 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl] or [4 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl]; L is a linear hydrocarbon chain of 5 atoms of

carbon; Z is −CONH−; P is albumin; and n is a value selected from 1 to

fifty.

According to another preferred embodiment of the invention, the compound of formula (II)

which generates an antibody as described in this patent application is

5 characterizes why

T is [6-methyl-2- (phenylamino) pyrimidin-4-yl];

L is a linear hydrocarbon chain of 2 to 20 carbon atoms;

Z is selected from the group consisting of −CONH−, −NHCO−, −NH−, −S−,

 Y

;Y

P is selected from the group consisting of albumin, thyroglobulin and hemocyanin.

Preferably, the compound of formula (II) that generates such an antibody

As described in this patent application, it is characterized in that T is [6-methyl-2- (phenylamino) pyrimidin-4-yl]; L is a linear hydrocarbon chain of 4 carbon atoms; Z is −CONH−; P is albumin; and n is a value selected between 1 and 50.

According to another preferred embodiment of the invention, the compound of formula (II)

20 generating an antibody as described in this patent application is characterized in that T is [N- (4,6-dimethylpyrimidin-2-yl) -N- (phenyl) amino]; L is a linear hydrocarbon chain of 2 to 20 carbon atoms; Z is selected from the group consisting of −CONH−, −NHCO−, −NH−, −S−,

25 and; and

P is selected from the group consisting of albumin, thyroglobulin and hemocyanin.

Preferably, the compound of formula (II) that generates an antibody as described in this patent application is characterized in that T is [N (4,6-dimethylpyrimidin-2-yl) -N- (phenyl) amino]; L is a linear hydrocarbon chain of 4 carbon atoms; Z is −CONH−; P is albumin; and n is a value

5 selected between 1 and 50.

A fifth aspect of the present invention relates to a method of in vitro analysis of pyrimethanil in a sample comprising the following steps:

to.

contacting the sample with the antibody described in this patent application;

b. incubate the sample and the antibody of step (a) for a suitable period of time for an immunochemical reaction to take place; Y

C.

determine the existence of immunochemical reaction after incubation 15 of step (b).

The method of the present invention allows quantitative determination or qualitative analysis of the content of the fungicide pyrimethanil in a sample. Likewise, the method of the present invention allows to analyze the content of pyrimethanil in different types of samples, for example, samples of

20 foods, environmental samples such as water, soil or surface, and biological samples such as urine. Preferably, the present invention provides a method of in vitro analysis of pyrimethanil in a food.

According to a preferred embodiment, the determination of the immunochemical reaction in step (c) is performed by an immunoassay.

Competitive, using as a competitor a compound of formula (II) as described in this patent application. Preferably, the competitive immunoassay is of the ELISA type.

According to another preferred embodiment, the determination of the immunochemical reaction in step (c) is performed by a competitive immunoassay, using as a competitor a compound of formula (III) as described in this patent application. Preferably, the immunoassay

5 competitive is ELISA type.

The term "immunoassay" refers to an analytical assay in which an immunochemical reaction occurs for the detection or quantification of an analyte. Competitive immunoassays are those in which the analyte competes with another molecule for binding with the antibody.

The term "antigen" in this patent application refers to a molecule capable of interacting specifically with an antibody. The immunochemical interaction or reaction consists of the specific and non-covalent binding between an antibody and an antigen, which may be the analyte or a test antigen.

Here the term "test antigen, enzymatic antigen 15 or tracer" refers to a compound of formula (II) or of formula (III) that is used in the competitive assay.

A sixth aspect of the present invention also relates to a pyrimethanil detection kit that uses at least one antibody as described in this patent application. Additionally, the pyrimethanil detection kit

20 may comprise a compound selected from the group consisting of a compound of formula (II) and a compound of formula (III) as described in the present patent application.

The compound of formula (II) of the present invention can be obtained by a process comprising reacting a compound of formula (I) 25 with P, a natural or synthetic polypeptide of molecular weight greater than 2000

Daltons, by methods widely known in the art.

The process for obtaining the compound of formula (II) may also comprise, if necessary, an additional step of activating the functional group Y.

On the other hand, the compound of formula (III) of the present invention is

5 can be obtained by a method comprising reacting a compound of formula (I) with Q, a non-isotopic marker, by methods widely known in the art.

The process for obtaining the compound of formula (III) may also comprise, if necessary, an additional step of activating functional group 10 Y.

When the compound of formula (I) is characterized in that Y is a carboxylic group, the above-mentioned activation can take place by reacting the compound of formula (I) with N, N'disuccinimidylcarbonate. This activation procedure can take place in

An organic solvent such as acetonitrile, propanonitrile, dichloromethane, chloroform, tetrahydrofuran, benzene or toluene; in the presence of a base such as triethylamine, triisopropylamine, pyridine, 4-N, N-dimethylaminopyridine, picoline or diazo (1,3) bicyclo- [5.4.0] undecano; in a temperature range between 0 and 30 ° C.

The terms immunogenic and immunogenic as used in the present invention refer to a substance that is recognized as foreign to the living organism and therefore is capable of producing or generating an immune response in a host.

Likewise, the obtaining of antibodies from compounds of formula 25 (II) can also take place by immunization methods widely known in the art.

Antibodies generated from a compound of formula (II) as described in this patent application may be polyclonal antibodies, monoclonal antibodies, recombinant antibodies or antibody fragments.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives,

5 components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention.

In order to prepare antibodies against pyrimethanil, the preparation of functionalized derivatives of said fungicide is required, that is, structural analogs of pyrimethanil incorporating a functional group capable of being used for conjugation to a P carrier or Q marker. This functional group is separated of the skeleton of the pyrimethanil molecule by a spacer L. The position of incorporation of the functional group into the structure of pyrimethanil for conjugation is not an obvious aspect and can be decisive for the

Viability of the compounds of formula (II) as inducers of the production of suitable affinity and selectivity antibodies against pyrimethanil and even of compounds of formula (II) or (III) that act as competing molecules and allow the development of a Sensitive and specific immunoassay for said fungicide, the ultimate object of the present invention.

The following illustrates with some examples and figures the manner in which the preparation of various pyrimethanil derivatives which are compounds of formula (I) and the corresponding compounds of formula (II), which are not intended to be limiting of the present can be carried out invention, and which serve to show not only the way in which the preparation of the

The same but also the importance that the structural nature of the compound of formula (II) may have for the production of antibodies of suitable affinity towards the analyte, suitable for the development of a good immunoassay.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Scheme of the synthesis of the compounds of formula (I) PMm6 and PMp6.

Figure 2. Scheme of the synthesis of the compound of formula (I) PMb5.

5 Figure 3. Scheme of the synthesis of the compound of formula (I) PMn5.

Figure 4. Scheme of the preparation of a compound of formula (II) from the corresponding compound of formula (I), where the term II-INM means compound of formula (II) that can be used to generate a pyrimethanil antibody, and the term II-ANT means compound of formula

10 (II) that can act as a competitor in a competitive immunological trial.

Figure 5. Title of the anti-pyrimethanil antibodies determined with 0.1 µg of compound of formula (II) immobilized homolog.

Figure 6. Standard curves for pyrimethanil in different formats of

15 competitive ELISA. The results obtained in the indirect test described in section 3.1 of the examples, and (▲) the results obtained in direct test described in section 3.2 of the examples are indicated by (●).

EXAMPLES

The invention will now be illustrated by tests carried out by the inventors, which shows the specificity and effectiveness of the compounds of formula (II) for obtaining anti-pyrimethanil antibodies. In the following examples the numbers in bold make reference to the corresponding structure shown in the diagrams of Figures 1 to

3. These examples are presented by way of demonstration but in no way can they constitute a limit to the invention.

1. Preparation of compounds of formula (II)

1.1. Synthesis of compounds of formula (I)

Example 1: Synthesis of PMm6 (2) and PMp6 (3) [radical of type T = R-I]

The synthesis of the compounds PMm6 (2) and PMp6 (3) is described in Figure

5 1 and involves the aromatic nucleophilic substitution reaction of the chlorine atom of 2-chloro-4,6-dimethyl-pyrimidine (1) by the amino group of the corresponding aminophenylalkanoic acid that incorporates the six-carbon hydrocarbon chain that constitutes the spacer arm (C. Suárez-Pantaleón et al., J. Agric. Food Chem. 2008, 56, 11122-11131). The reaction takes place without

The need for basic or acid catalysis, taking place effectively by prolonged heating of the two reactants in dioxane as a refluxing solvent. The details of the preparation and characterization of this compound of formula (I) are illustrated below.

Synthesis of 6- (3 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl) hexanoic acid

15 (2, compound PMm6)

A mixture of 2-chloro-4,6-dimethylpyrimidine (1, 71 mg, 0.5 mmol) and 6- (3-aminophenyl) hexanoic acid (103 mg, 0.5 mmol) in freshly distilled dry dioxane is heated to reflux with low stirring. nitrogen atmosphere for 24 hours. After this time, the reaction mixture is cooled to temperature

20 ambient and concentrated to dryness under reduced pressure on the rotary evaporator. The residue obtained is dissolved in the minimum amount of formic acid (approximately 0.2 mL) and the product is precipitated by slow addition of water (approximately 3 mL). The precipitated solid is collected by vacuum filtration and dried, obtaining the PMm6 hapten (2) as a white solid (109 mg,

25 70%). Mp: 199-202 ° C (crystallized from DMSO-H2O); 1H NMR (300 MHz, DMSO-d6) δ (ppm) 11.97 (1H, sa, COOH), 9.35 (1H, s, N-H), 7.64 (1H, da, J =

8.0 Hz, H-2 / H-6 Ph), 7.60 (2H, sa, H-2 Ph), 7.13 (1H, ta, J = 7.7 Hz, H-5 Ph),

6.72 (1H, da, J = 7.6 Hz, H-4 Ph), 6.60 (1H, s, H-5 Pirim), 2.30 (6H, s, Mex2), 2.50 (2H, t, J = 7.3 Hz, H -6), 2.20 (2H, t, J = 7.3 Hz, H-2), 1.54 (4H, m, H-3 and H5), 1.30 (2H, m, H-4); 13C NMR (75 MHz, DMSO-d6) δ (ppm) 174.4 (C-1), 166.7 (C-4 and C-2 Pirim), 159.7 (C-6 Pirim), 142.1 (C-3 Ph), 140.7 (C-1 Ph), 128.1 (C-5 Ph), 120.9 (C-6 Ph), 118.5 (C-4 Ph), 115.9 (C-2 Ph), 110.8 (C-5 Pirim),

5 35.2 (C-6), 33.5 (C-2), 30.5 (C-5), 28.1 (C-4), 24.3 (C-3), 23.4 (Mex2); IR (KBr) vmax / cm-1 3284, 3207, 3142, 3104, 2945, 2918, 2858, 2600-2400, 1689, 1618, 1596, 1558, 1492, 880, 776, 744, 552; MS (Electronic Impact, 70 Ev) m / z (%) 313 (M +, 77), 312 (20), 254 (12), 226 (81), 213 (80), 212 (20), 199 (12) , 198 (4), 73 (100); EMAR, m / z (M +) found for C18H23N3O2 313.17882, required

10 313.17903.

Synthesis of 6- (4 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl) hexanoic acid

(3, compound PMp6)

A mixture of 2-chloro-4,6-dimethylpyrimidine (1,142.8 mg, 1.00 mmol) and 6- (4-aminophenyl) hexanoic acid (207 mg, 1.00 mmol) in freshly distilled dry dioxane was heated under reflux with stirring. under nitrogen atmosphere for 30 h. After this time, the reaction mixture was cooled to room temperature and concentrated to dryness under reduced pressure on the rotary evaporator. The obtained residue was dissolved in approximately 0.5 mL of formic acid and the product was precipitated by slow addition of water (approximately 3 mL). The precipitated solid was collected by vacuum filtration and dried, obtaining the PMp6 compound (3) as a white powder (235 mg, 75%). Mp: 180-183 ° C (crystallized from DMSO-H2O); 1H NMR (300 MHz, DMSO-d6) δ (ppm): 11.98 (1H, sa, COOH), 9.33 (1H, s, NH), 7.67 (2H, d, J = 8.4 Hz, H-2 / H- 6 Ph), 7.06 (2H, d, J = 8.4 Hz, H-3 / H-5 Ph), 6.58 (1H, s, H-5 Pirim), 2.47 (2H, t, J = 7.5 Hz, 25 H -6), 2.29 (6H, s, Mex2), 2.19 (2H, t, J = 7.3 Hz, H-2), 1.52 (4H, m, H-3 and H-5),

1.28 (2H, m, H-4); 13C NMR (75 MHz, DMSO-d6) δ (ppm): 174.4 (C-1), 166.8 (C-4 / C-6 Pirim), 159.7 (C-2 Pirim), 138.5 (C-4 Ph), 134.5 (C-1 Ph), 128.1 (C3 / C-5 Ph), 118.5 (C-2 / C-6 Ph), 110.6 (C-5 Pirim), 34.3 (C-6), 33.6 (C-2 ), 30.8 (C-5), 28.1 (C-4), 24.3 (C-3), 23.4 (Mex2); IR (KBr) vmax / cm − 1: 3295, 3202, 3125,

30 3082, 2918, 2847, 2600-2400, 1698, 1618, 1585, 1547, 1410, 1377, 1274, 842, 733, 634, 547, 504; MS (Electronic Impact, 70 Ev) m / z (%): 313 (M +, 45), 312 (9), 238 (3), 226 (5), 213 (7), 212 (100), 211 (4 ); EMAR, m / z (M +): found for C18H23N3O2 313.17896, required 313.17903.

Example 2: Synthesis of compound PMb5 (8) [radical of type T = R-IV]

The synthesis of the compound that incorporates the spacer arm by the pyrimidine ring is based on the previous preparation of a 1,3-dicarbonyl compound that incorporates in the proper position the carboxylated hydrocarbon chain that constitutes the spacer arm of the compound in question. Once this intermediate is prepared, the preparation of the pyrimidine ring is completed

10 through a condensation reaction of the carbonyl groups with the amino groups of the phenylguanidine. As illustrated in Figure 2, the preparation of 1,3-dicarbonyl compound (6) is carried out through the acylation reaction of allyl 3-oxobutanoate (4) with 6-chloro-6-oxohexanoate methyl, followed by catalyzed desalylation-decarboxylation reaction by

15 Pd (0). The condensation of 1,3-diketone (6) with phenylguanidinium nitrate in basic medium generates the complete skeleton of the compound of formula (I) whose synthesis ends with the hydrolysis of the methyl ester of (7) with base. The details of the preparation and characterization of this compound of formula (I) and all the intermediates of its synthesis are illustrated below.

20 i) Synthesis of 1-allyl 8-methyl 2-acetyl-3-oxooctanedioate (5)

Allyl 3-Oxobutanoate (4, 0.288 mL, 2.1 mmol) was added dropwise over a stirred suspension of anhydrous MgCl2 (200 mg, 2.1 mmol) in dry CH2Cl2 (2.5 mL) under a nitrogen atmosphere at room temperature. The mixture was cooled to 0 ° C in an ice bath and dried pyridine (0.339 was added

25 mL, 4.2 mmol). Stirring was continued at this temperature for 15 minutes and methyl 6-chloro-6-oxohexanoate (0.327 mL, 2.1 mmol) was added dropwise, after which stirring was maintained at 0 ° C for an additional 15 minutes. The ice bath was then removed and the mixture was allowed to stir at room temperature for 5 h. He then cooled off

Again the mixture in an ice bath and 6M HCl was added until acidic pH (approximately 1.5 mL), diluted with water and extracted with ethyl ether. The combined organic extracts were washed with saturated NaCl solution, dried over anhydrous MgSO4, filtered and concentrated under reduced pressure, obtaining compound 5 (580 mg, 97%) as a dense colorless oil that does not require purification and whose 1 H NMR spectrum shows that it is found exclusively in the keto-enol tautomeric form. 1H NMR (300 MHz, CDCl3) δ (ppm): 15.6 (1H, sa, OH), 5.98 (1H, ddt, J = 17.2, 10.3, 6.0 Hz, HC =), 5.37 (1H, dq, J = 17.2 , 1.4 Hz, = CH2), 5.29 (1H, dq, J = 10.3, 1.1 Hz, 10 = CH2), 4.70 (2H, dt, J = 6.0, 1.2 Hz, OCH2), 3.66 (3H, s, CO2CH3) , 2.66 (2H, t, J = 7.1 Hz, H-4), 2.36 (3H, s, CH3CO), 2.33 (2H, t, J = 7.1 Hz, H-7), 1.67 (4H, m, H- 5 and H-6); 13C NMR (75 MHz, CDCl3) δ (ppm): 198.8 (COCH3), 195.8 (C-3),

173.7 (CO2CH3), 166.8 (C-1), 131.8 (HC =), 119.3 (= CH2), 108.3 (C-2), 65.6 (OCH2), 51.5 (CO2CH3), 37.5 (C-4), 33.7 ( C-7), 25.7 (COCH3), 25.0 (C-6), 24.5

15 (C-5); IR (NaCl) vmax / cm − 1: 2951, 2869, 1732, 1711, 1563, 1437, 1219, 1077, 755; MS (Electronic Impact, 70 Ev) m / z (%): 284 (M +, 1.7), 226 (12), 211 (13), 194 (21), 177 (7), 169 (199), 153 (84 ), 143 (58); EMAR, m / z (M +): found for C14H20O6 284.12639, required 284.12599.

ii) Synthesis of methyl 6,8-dioxononanoate (6)

On a solution of Pd (OAc) 2 in dry THF (1.5 mL), formic acid (0.064 mL, 1.7 mL) and Et3N (0.113 mL, 0.81 mmol) were added consecutively under a nitrogen atmosphere at room temperature. After a few minutes of stirring, a solution of compound 5 (200 mg, 0.70 mmol) in dry THF (1.5 mL) was added and the resulting mixture was heated at reflux for 4 h. The

The reaction mixture was cooled to room temperature and filtered through a small silica column using ethyl ether for washing. The ethereal solution obtained was washed with water, saturated NaCl solution, dried, filtered and concentrated to dryness, obtaining compound 6 (127 mg, 90%) a viscous oil, whose 1 H NMR spectrum shows that it is a

30 equilibrium mixture of keto-enol tautomeric forms (72%) and

dicarbonyl (28%). 1 H NMR (300 MHz, CDCl 3) δ (ppm, only keto-enol tautomer signals): 15.45 (1H, sa, OH), 5.49 (1H, s, H-7), 3.67 (3H, s, CO2CH3), 2.29 (4H, m, H-2 and H-5), 2.05 (3H, s, CH3CO), 1.65 (4H, m, H-3 and H-4); 13C NMR (75 MHz, CDCl3) δ (ppm, only signals from the entoolic keto5 tautomer): 193.6 (C-8), 191.4 (C-6), 173.8 (C-1), 99.8 (C-7), 51.5 ( OCH3), 37.8 (C-5), 33.7 (C-2), 25.0 (C-3), 24.9 (C-9), 24.4 (C-4); IR (NaCl) vmax / cm − 1: 2945, 2869, 1738, 1618, 1432, 1203, 1170, 782; MS (Electronic Impact, 70 Ev) m / z (%): 200 (M +, 20), 182 (3), 169 (11), 168 (8), 143 (24), 127 (16), 126 (12 ), 115 (13), 114 (19), 113 (28), 111 (54), 100 (42), 85 (100); EMAR, m / z (M +):

10 found for C10H16O4 200.10514, required 200.10486.

iii) Synthesis of 5- (6-methyl-2- (phenylamino) pyrimidin-4-yl) pentanoate

methyl (7)

N-phenylguanidinium nitrate glass (175 mg, 0.88 mmol), Na2CO3 (47 mg, 0.44 mmol), diketone 6 (161 mg, 0.8 mmol) and a small PTFE coated magnetic rod were placed in a glass vial. The ampoule was purged with nitrogen, EtOH (3 mL) was added, closed in vacuo and heated in a bath at 80 ° C with good stirring for 18 h. After this time the vial was opened, the reaction mixture was transferred to a round bottom flask and the solvent was removed in the rotary evaporator. The obtained residue was chromatographed on silica gel, using hexane-EtOAc 9: 1 as eluent, to obtain compound 7 (169 mg, 70%) as an oil. 1 H NMR (300 MHz, CDCl 3) δ (ppm): 7.66 (2H, da, J = 8.2 Hz, H-2 / H-6 Ph), 7.31 (2H, ta, J = 7.5 Hz, H-3 / H -5 Ph), 7.04 (1H, sa, NH), 7.00 (1H, tt, J = 7.5, 1.2 Hz, H-4 Ph), 6.47 (1H, s, H-5 Pirim), 3.67 (3H, s , CO2CH3), 2.62 (2H, t, J = 7.2 Hz, H-5), 2.38 (3H, s, 25 CH3-Pirim), 2.37 (2H, t, J = 7.0 Hz, H-2), 1.74 ( 4H, m, H-3 and H-4); 13C NMR (75 MHz, CDCl3) δ (ppm): 173.9 (C-1), 170.7 (C-4 Pirim), 167.7 (C-6 Pirim), 159.7 (C-2 Pirim), 139.9 (C-1 Ph ), 128.8 (C-3 / C-5 Ph), 121.9 (C-4 Ph), 118.6 (C-2 / C-6 Ph), 111.1 (C-5 Pirim), 51.5 (OCH3), 37.2 (C -5), 33.9 (C-2), 27.9 (C-4), 24.6 (C3), 24.0 (CH3-Pirim); IR (NaCl) vmax / cm − 1: 3359, 2945, 2858, 1739, 1585, 1432,

750, 689; EMAR, m / z (M +): found for C17H21N3O2 299.16321, required 299.16338.

iv) Synthesis of 5- (6-methyl-2- (phenylamino) pyrimidin-4-yl) pentanoic acid

(8, compound PMb5)

An aqueous solution of 2M NaOH (1 mL) was added over a solution of methyl ester 7 (167 mg, 0.56 mmol) in MeOH (3 mL) and the mixture was heated under reflux with stirring for 1.5 h. After this time the solvent in the rotary evaporator was removed and the dry residue obtained was dissolved in the minimum amount of HCO2H and subsequently water was added to the total

After precipitation of the product that was collected by vacuum filtration, the collected solid was washed with cold water and dried to obtain the PMb5 compound (143 mg, 90%) as a white solid. Mp: 201-204 ° C (crystallized from DMSO-H2O); 1H NMR (300 MHz, DMSO-d6) δ (ppm): 12.06 (1H, sa, COOH), 9.44 (1H, sa, NH), 7.80 (2H, da, J = 7.6 Hz, H-2 / H- 6 Ph), 7.24 (2H, ta, J = 7.9 Hz, H-3 / H-5

15 Ph), 6.89 (1H, tt, J = 7.3, 1.2 Hz, H-4 Ph), 6.62 (2H, t, J = 7.2 Hz, H-5 Pirim),

2.57 (2H, t, J = 7.4 Hz, H-5), 2.32 (3H, s, CH3-Pirim), 2.25 (2H, t, J = 7.2 Hz, H-2), 1.69 (2H, m. H -4), 1.54 (2H, m, H-3); 13C NMR (75 MHz, DMSO-d6) δ (ppm): 174.3 (C-1), 170.3 (C-4 Pirim), 167.0 (C-6 Pirim), 159.7 (C-2 Pirim),

140.9 (C-1 Ph), 128.3 (C-3 / C-5 Ph), 120.7 (C-4 Ph), 118.4 (C-2 / C-6 Ph), 110.4

20 (C-5 Pirim), 36.4 (C-5), 33.4 (C-2), 27.4 (C-4), 24.1 (C-3), 23.5 (CH3-Pirim); IR (KBr) vmax / cm − 1: 3295, 3202, 3082, 2918, 2852, 2503, 1694, 1618, 1585, 1552, 1508, 1372, 842, 727; MS (Electronic Impact, 70 Ev) m / z (%): 285 (M +, 10), 284 (2), 226 (7), 212 (5), 199 (40), 178 (7), 73 (100 ); EMAR, m / z (M +) found for C16H19N3O2 285.14819, required 285.14773.

Example 3: Synthesis of compound PMn5 (11) [radical of type T = R-VII]

The synthesis of the compound of formula (I) incorporating the spacer arm by the pyrimethanil amine nitrogen is carried out through an alkylation reaction of the amide anion derived therefrom with the ω-bromine ester of suitable chain length, followed of basic hydrolysis of the ester group to the corresponding carboxylic acid, as illustrated in Figure 3 and detailed below.

i) Synthesis of methyl 5 - ((4,6-dimethylpyrimidin-2-yl) (phenyl) amino) pentanoate (10)

In a round bottom flask 60% sodium hydride in oil was weighed

5 mineral (24.1 mg, 0.60 mmol) and then washed three times with dry pentane under a nitrogen atmosphere, then drying it with a gentle stream of the same gas. The resulting loose white powder was suspended in dry dimethylfomamide (1.5 mL) and a solution of pyrimethanil (9, 100 mg, 0.5 mmol) was added dropwise with stirring to the suspension obtained

10 dry dimethylformamide (1.5 mL) at room temperature. The mixture, which acquires a yellowish coloration, was stirred under nitrogen for 1 h and then 5-bromovalerate methyl syringe (0.148 mL, 1 mmol) was added via syringe. The resulting mixture was stirred at room temperature for approximately 72 h, poured into water and extracted with EtOAc. The

15 combined organic extracts were washed successively with water, 20% LiCl solution and saturated NaCl solution. They were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The obtained residue was purified by column chromatography on silica gel, using hexane-EtOAc 9: 1 as eluent, to obtain an approximately 1: 2 mixture of pyrimethanil.

Initial unreacted and 5 - ((4,6-dimethypyrimidin-2-yl) (phenyl) amino) methyl pentanoate (10) (145 mg).

ii) Synthesis of 5 - ((4,6-dimethylpyrimidin-2-yl) (phenyl) amino) pentanoic acid

(11, compound PMn5)

Water was added dropwise to a solution of the above mixture in THF

25 (0.85 mL) and subsequently solid LiOH · H2O (81.6 mg, 1.94 mmol). The resulting mixture was stirred at room temperature for 24 h and then the solvent was removed in the rotary evaporator. The obtained residue was diluted in H2O (3 mL) and carefully acidified with solid KHSO4 to pH 3-4. The resulting mixture was extracted with EtOAc, the combined organic phases were

dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The residue obtained was purified by chromatography on silica gel, using CHCl3-MeOH 9: 1 as eluent, to obtain unreacted pyrimethanil (36 mg) followed by compound PMn5 (11) as a dense oil 5 (89.8 mg, 60% from pyrimethanil). 1 H NMR (300 MHz, CDCl 3) δ (ppm): 7.36 (2H, m, H-3 / H-5 Ph), 7.27 (2H, m, H-2 / H-6 Ph), 7.18 (1H, tt , J = 7.2, 1.5 Hz, H-4 Ph), 6.32 (1H, s, H-5 Pirim), 4.04 (2H, t, J = 7.0 Hz, H-5), 2.37 (2H, t, J = 7.0 Hz, H-2), 2.24 (6H, s, Mex2), 1.66 (4H, m, H-3 and H-4); 13C NMR (75 MHz, CDCl3) δ (ppm): 179.0 (C-1), 166.9 (C-4 / C-6 Pirim), 161.7 (C-2 Pirim), 144.5 10 (C-1 Ph), 128.8 (C-3 / C-5 Ph), 127.5 (C-2 / C-6 Ph), 125.3 (C-4 Ph), 109.9 (C-5 Pirim), 49.5 (C-5), 33.7 (C- 2), 27.2 (C-4), 24.0 (Mex2), 21.8 (C-3); IR (NaCl) vmax / cm -1: 3044, 2928, 2864, 1708, 1578, 1563, 1498, 1475, 1376, 1339, 1219, 694; MS (Electronic Impact, 70 Ev) m / z (%): 299 (M +, 21), 298 (10), 226 (24), 224 (5), 213 (16), 212 (100), 209 (11 ), 208 (17), 207 (84), 200 (2), 199 (15), 198 15 (25); EMAR, m / z (M +): found for C17H21N3O2 299.16426, required

299.16338.

1.2. Activation of the compounds of formula (I)

Examples of compounds of formula (I) presented herein contain a carboxyl group as a functional chemical group for conjugation to carrier proteins, specifically by reaction with the free amino groups of the protein. The carboxyl group was activated using N, N’disuccinimidyl carbonate (DSC) following previously published protocols [F.A. Esteve-Turrillas et al., Anal. Chim. Minutes 2010, 682, 93-103]. In particular, 0.082 mmol of the corresponding compound of formula (I) of 25 pyrimethanil and 0.14 mmol of DSC was dissolved in 0.8 mL of dry acetonitrile under a nitrogen atmosphere. Then, 0.31 mmol of triethylamine was added and stirred at room temperature overnight. The solution was diluted in chloroform, washed with a saturated solution of NaHCO3 and brine and dried over anhydrous Na2SO4. After evaporating the solvent, the remaining residue was purified by column chromatography, using chloroform as

eluent, obtaining the N-hydroxysuccinimide ester of the compound of formula (I) of pyrimethanil in pure form.

1.3. Conjugation of the compounds of formula (I) to proteins to obtain compounds of formula (II)

5 Buffers and solutions: PB: 100 mM sodium phosphate buffer, pH 7.4; PBS: 10 mM sodium phosphate buffer, pH 7.4 with 140 mM NaCl; PBST: PBS buffer containing 0.05% (v / v) polyoxyethylene (20)

sorbitanmonolaurate (known as Tween 20); 10 CB: 50 mM sodium carbonate buffer, pH 9.6; Washing solution: 150 mM NaCl containing 0.05% (v / v) Tween 20.

The analytes were dissolved in anhydrous N, N-dimethylformamide (DMF) and stored at −20 ° C.

The compounds of formula (II) were prepared as outlined in Figure 4, according to the following procedures.

1.3.1. Compound of formula (II) where P is BSA

To 2.0 mL of a 15 mg / mL solution of bovine serum albumin protein (BSA) in CB buffer, 200 µL of a 50 mM solution of compound of formula (I) activated was obtained dropwise, obtained as indicated in he

20 section 1.2, in DMF. The reaction was carried out for 4 h at room temperature with gentle stirring and then, the compound of formula (II) was purified by molecular exclusion chromatography on Sephadex G-25 using PB buffer as eluent. The degree of conjugation, n as described in the compound of formula (II), measured spectrophotometrically was 16, 11,

25 14 and 14 for PMm6, PMp6, PMb5 and PMn5, respectively.

1.3.2. Compound of formula (II) where P is OVA

To 2.0 mL of a 15 mg / mL solution of egg albumin protein (OVA) in CB buffer, 100 µL of a 100 mM solution of the compound of formula (I) activated, obtained as indicated in the sample, was added dropwise Section 1.2, in DMF. The reaction was carried out for 2.5 h at temperature

Ambient with gentle agitation and then, the compound of formula (II) was purified by molecular exclusion chromatography on Sephadex G-25 using PB buffer as eluent. The degree of conjugation, n as described in the compound of formula (II), measured spectrophotometrically was 12, 8, 8 and 9 molecules for PMm6, PMp6, PMb5 and PMn5, respectively.

10 1.3.3. Compound of formula (II) where P is HRP

To 1.0 mL of a solution of 2.2 mg / mL of horseradish peroxidase protein (HRP) in CB buffer, 100 µL of a 10 mM (or 5 mM) solution of compound of formula (I) activated, obtained, was added dropwise as indicated in section 1.2, in DMF. The reaction was carried out for 4 h at

At room temperature with gentle stirring, then the compound of formula (II) was purified by molecular exclusion chromatography on Sephadex G-25 using PB buffer as eluent. The degree of conjugation, n as described in the compound of formula (II), measured spectrophotometrically was 7, 6, 5 and 3 for PMm6, PMp6, PMb5 and PMn5, respectively.

20 2. Immunization of rabbits

Two white rabbit females of the New Zealand breed were immunized following standardized protocols with each compound of formula (II) where P is BSA obtained as described in section 1.3.1. [C. Suárez-Pantaleón et al., J. Agric. Food Chem. 2010, 58, 8502-8511]. Each animal

25 received 0.3 mg of one of the compounds of formula (II) dissolved in 1 mL of a 1: 1 mixture of PB buffer and complete Freund's adjuvant. Immunization continued with the inoculation of a booster dose every 21 days with the same amount of conjugate but using incomplete Freund's adjuvant. Ten days after the fourth injection, the animals

they were bled and the blood obtained was allowed to clot at 4 ° C overnight. The next day, the sera were recovered by centrifugation, diluted to ½ with cold PBS and a volume of a saturated solution of ammonium sulfate was added. The resulting protein precipitate from each serum 5 was collected by centrifugation and redissolved in cold PBS buffer. Finally, the proteins were reprecipitated as before and stored in this state at 4 ° C. This precipitate contains an indeterminate mixture of proteins that we call antiserum, polyclonal antibody or simply antibody. Two antibodies were obtained from each compound of formula (II) where P is

10 BSA, identified as # 1 and # 2.

3. ELISA procedure

96-well polystyrene plates were used. Each antibody was evaluated in the two classic formats of competitive ELISA (that of immobilized antigen or conjugate with indirect detection and that of immobilized antibody

15 with direct detection) using both homologous test antigens, ie a test antigen from the same compound of formula (I) as used to obtain the immunogen; as heterologous test antigens, that is, obtained from a compound of formula (I) different from that used to obtain the immunogen. 8 electronic pipettes were used

20 channels for rapid and accurate dispensing of immunoreactive agents. After each incubation step, the plates were washed four times with a wash solution, using a 96 channel ELx405 washer (Biotek Instruments, Winooski, USA). The peroxidase activity used as a marker was revealed with 100 μL per well of a 2 mg / mL solution of o-phenylenediamine in

25 buffer 25 mM citrate, 62 mM phosphate, pH 5.4 containing 0.012% (v / v) H2O2. This development was developed for 10 min at room temperature and stopped using 100 μL per well of 2.5 M H2SO4. At the end of the tests, the absorbance of each well was read at 492 nm using a reference wavelength of 650 nm in a PowerWave HT microplate reader (Biotek

30 Instruments, Winooski, USA). The sigmoid standard curves obtained by representing absorbance versus analyte concentration were adjusted to a four-parameter logistic equation using the SPSS SigmaPlot software package (Chicago, USA). The antibody titer was defined as the reciprocal of the antibody dilution that provides a maximum signal.

5 (Amax) about 1.0 in the absence of free analyte in a competitive ELISA assay in the immobilized conjugate format homologous at 0.1 mg / mL with indirect detection. The affinity of the antibody (IC50) was estimated as the concentration of free analyte capable of halving the maximum signal.

3.1. Competitive ELISA assays in immobilized antigen or conjugate 10 format with indirect detection (indirect assay)

The plates were upholstered with 100 μL per well of a test antigen solution which is a compound of formula (II) where P is OVA at 0.01 or 0.1 μg / mL in CB buffer by overnight incubation at room temperature. After washing the plates, 50 μL was dispensed in each column

15 per well of a complete standard analyte curve in PBS followed by 50 μL per well of a specific dilution of a given antibody in PBST. The same reagent distribution was repeated for each plate with a different test antigen. The immunochemical reaction was carried out for 1 h at room temperature and then the plates were washed. Then each

20 well received 100 μL of a 1/104 dilution of GAR-HRP in PBST containing 10% fetal bovine serum. This reaction was left at room temperature for 1 h. After washing the plates, the retained peroxidase activity was revealed and the absorbance at 492 nm was read as described above.

25 3.2. Competitive ELISA assays in immobilized antibody format with direct detection (direct assay)

The plates were upholstered with 100 µL per well of a dilution of antibody in CB buffer by overnight incubation at room temperature. After washing the plates, 50 µL per well of a complete standard analyte curve in PBS was dispensed in each column followed by 50 µL per well of a specific dilution in PBST of a given enzyme tracer which is a compound of formula (II) where P is HRP. The same reagent distribution was repeated for each plate with a different antibody. The reaction

Immunochemistry was carried out for 1 h at room temperature and then the plates were washed. Finally, the retained peroxidase activity was revealed and the absorbance at 492 nm was read as described.

4. Results

4.1. Immune response

10 The four immunogens produced similar and equivalent immune responses, with titers around 105 (Figure 5).

4.1.1. Determination of antibody affinity

Each of the antibodies obtained was tested against its homologous test antigen using the competitive ELISA type assay, both in immobilized antigen assay format with indirect detection and in the immobilized antibody format with direct detection. Different concentrations of test antigen were tested against different concentrations of antibody in competitive assay using as competitor different concentrations of pyrimethanil prepared by serial dilution. Only from three of the compounds of formula (I) synthesized, PMm6, PMp6, and PMb5, was it possible to generate antibodies capable of recognizing pyrimethanil with high affinity. This result confirms that these structures are suitable for the objective pursued and demonstrates the difficulty in predicting the optimal functionalization position of the

25 compounds of formula (I). The values of the maximum signal, the IC50 and the slope of the resulting inhibition curve for each antibody with its homologous test antigen have been included in Tables 1 (a − d) and Tables 2 (a − d).

Result of tests in competitive ELISA format of immobilized antigen with indirect detection:

Table 1a

OVA − PMm6

Conjugate Title Ac. Antibody (μg / mL) (x103) Amax Pending

IC50 (nM) rPMm6 # 1 0.1 300 0.58 0.64 12.99

0.1 100 1.36 0.54 19.97 rPMm6 # 2 0.1 300 0.75 0.57 10.44

0.1 100 1.59 0.60 19.44

Table 1b

OVA − PMp6

Conjugate Title Ac. Antibody (μg / mL) (x103) Amax Pending

IC50 (nM) rPMp6 # 1 0.01 300 0.80 0.55 7.0

0.1 100 1.12 0.56 14.2 rPMp6 # 2 0.01 1000 0.74 0.44 5.6

0.1 300 1.09 0.57 22.6

Table 1c

OVA − PMb5

Conjugate Title Ac. Antibody (μg / ml) (x103) Amax Pending

IC50 (nM) rPMb5 # 1 0.01 100 1.13 0.64 2.2

0.1 300 1.30 0.57 5.3 rPMb5 # 2 0.01 100 0.92 0.57 3.0

0.1 300 0.95 0.50 9.0

1d table

OVA − PMn5

Conjugate Title Ac. Antibody (μg / mL) (x103) Amax Pending

IC50 (nM) rPMn5 # 1 0.01 300 1.13 0.64 57.7

0.1 1000 0.69 ------ rPMn5 # 2 0.01 1000 0.87 0.69 754.7

0.1 1000 0.82 -----

Result of tests in competitive ELISA format of immobilized antibody with direct detection:

Table 2a

HRP − PMm6

Tracer

Title Ac.

Antibody

(ng / mL) (x103) Amax Pending IC50 (nM)

rPMm6 # 1

10 30 0.81 0.67 5.75

10

10

1.30 1.00 7.32

rPMm6 # 2

30 3 0.59 0.43 194.65

100

3 1.07 0.43 592.47

Table 2b

HRP − PMp6

Tracer

Title Ac.

Antibody

(ng / mL) (x103) Amax Pending IC50 (nM)

rPMp6 # 1

3 30 1.11 0.61 6.9

10

30 1.50 0.72 14.8

rPMp6 # 2

one 30 1.98 0.60 58.6

3

100 1.14 0.50 12.8

5

Table 2c

HRP − PMb5

Tracer

Title Ac.

Antibody

(ng / mL) (x103) Amax Pending IC50 (nM)

rPMb5 # 1

3 10 0.86 0.75 0.9

10

30 0.65 0.84 1.9

rPMb5 # 2

3 3 0.62 0.80 1.1

10

3 1.45 1.02 0.6

2d table

HRP − PMn5

Tracer

Title Ac.

Antibody

(ng / mL) (x103) Amax Pending IC50 (nM)

rPMn5 # 1

3 30 0.76 0.74 46.2

10

30 1.97 0.79 63.1

rPMn5 # 2

3 30 0.78 0.63 144.0

10

30 2.18 0.72 205.4

Figure 6 shows the inhibition curves obtained with the rPMp6 # 1 antibody and the compound of formula (II) which is a homologous test antigen, both in test antigen format immobilized with

Indirect detection as with the immobilized antibody format with direct detection. For the indirect assay the wells were upholstered with 0.01 μg of compound of formula (II) which is a test antigen and the dilution of the antibody in the competition stage was 1/105. In the case of the direct format test, the well was upholstered with 100 µL of a dilution of the antibody to 1 / 2x104 and in the competitive stage 0.3 ng of homologous enzyme tracer was used.

Claims (36)

1. A compound of formula (I): T − L − Y 5 (I) characterized because T is selected from the group consisting of R-I, R-II, R-III, R-IV and R-V; HH N N NN N N R-I R-II H H NN NN N N R-III R-IV NN N R-V where RI is selected from the group consisting of [2 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl], [3 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl] and [4- ((4,6dimethylpyrimidin-2-yl) amino) phenyl]; R-II is [4,6-dimethyl-2- (phenylamino) pyrimidin-5-yl]; R-III is selected from the group consisting of [2 - ((4-methylpyrimidin-2yl) amino) phenyl], [3 - ((4-methylpyrimidin-2-yl) amino) phenyl] and [4 - ((4 -methylpyrimidin-2-yl) amino) phenyl]; R-IV is selected from the group consisting of [6-methyl-2- (phenylamino) pyrimidin-4 30 yl] and [6-methyl-2- (phenylamino) pyrimidin-5-yl]; and R-V is [N- (4,6-dimethylpyrimidin-2-yl) -N- (phenyl) amino]; L is a hydrocarbon chain of 2 to 40 carbon atoms, where the chain is linear or branched, saturated or unsaturated, and said hydrocarbon chain comprises between 0 and 10 heteroatoms that are selected from the 5 group consisting of S, O and N; Y is a functional group selected from the group consisting of −COOH, −NH2, −N3, −CH2Br, −CH2I, −CHO, −SH, −SO3H, −OSO2Ph, −NH − NH2 and −OSO2Ar; with the condition of: 10 a) when T is [4 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl], -L-Y is different from -CH2CH2-NH2; b) when T is [4 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl], -L-Y is different from −CONHCH2-CO2H; c) when T is [4 - ((4-methylpyrimidin-2-yl) amino) phenyl], -L-Y is different from 15 -CHCH3-CO2H; d) when T is [6-methyl-2- (phenylamino) pyrimidin-4-yl], -L-Y is different from −OCH2-CO2H; e) when T is [N- (4,6-dimethylpyrimidin-2-yl) -N- (phenyl) amino], L is a hydrocarbon chain of 2 to 40 carbon atoms, linear or branched, saturated or unsaturated, with the proviso that said chain hydrocarbon does not comprise any heteroatom; f) when T is [N- (4,6-dimethylpyrimidin-2-yl) -N- (phenyl) amino], -L-Y is different from - (CH2) 3-NH2.

2.

 A compound of formula (I) according to claim 1, characterized in that 25 L is a linear hydrocarbon chain of 2 to 20 carbon atoms.

3. A compound of formula (I) according to any one of the preceding claims, characterized in that Y is selected from the group consisting of −COOH, −CHO, −NH2 and −SH.

Four.

 A compound of formula (I) according to any one of the preceding claims, characterized in that T is [3 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl]

or [4 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl]; L is a linear hydrocarbon chain of 2 to 8 carbon atoms; and Y is selected within the group consisting of −COOH, −CHO, −NH2 and − SH. 5. A compound of formula (I) according to claim 4, characterized in that 5 is selected from the group consisting of H H NN (CH2) 5CO2H N N N N (CH2) 5CO2H Y 6. A compound of formula (I) according to any one of claims 1 10 to 3, characterized in that T is [6-methyl-2- (phenylamino) pyrimidin-4-yl]; L is a linear hydrocarbon chain of 2 to 8 carbon atoms; and Y is selected within the group consisting of −COOH, −CHO, −NH2 and −SH. 7. A compound of formula (I) according to claim 6, characterized in that is H HO2C (H2C) 4 N N N 8. A compound of formula (I) according to any one of claims 1 to 3, characterized in that T is [N- (4,6-dimethylpyrimidin-2-yl) -N- (phenyl) amino]; L is a linear hydrocarbon chain of 2 to 8 carbon atoms; and I know 20 select within the group consisting of −COOH, −CHO, −NH2 and −SH. 9. A compound of formula (I) according to claim 8, characterized in that it is (CH2) 4CO2H N N N 10. A compound of formula (II): [T − L − Z] n − P (II) characterized in that 5 T is selected from the group consisting of R-I, R-II, R-III, R-IV and R-V; H H NN NN N N R-I R-II H H NN NN N N R-III R-IV NN N R-V where RI is selected from the group consisting of [2 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl], [3 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl] and [4- ([(4.6 Dimethylpyrimidin-2-yl) amino) phenyl]; R-II is [4,6-dimethyl-2- (phenylamino) pyrimidin-5-yl]; R-III is selected from the group consisting of [2 - ((4-methylpyrimidin-2yl) amino) phenyl], [3 - ((4-methylpyrimidin-2-yl) amino) phenyl] and [4 - ((4 -methylpyrimidin-2-yl) amino) phenyl]; R-IV is selected from the group consisting of [6-methyl-2- (phenylamino) pyrimidin-4yl] and [6-methyl-2- (phenylamino) pyrimidin-5-yl]; and R-V is [N- (4,6-dimethylpyrimidin-2-yl) -N- (phenyl) amino]; L is a hydrocarbon chain of 2 to 40 carbon atoms, where the chain is linear or branched, saturated or unsaturated, and said chain hydrocarbon comprises between 0 and 10 heteroatoms that are selected from the group consisting of S, O and N; Z is a functional group selected from the group consisting of −CONH−, −NHCO−, −NHCONH−, −NHCSNH−, −OCONH−, −NHOCO−, −OCSNH−, −SCONH−, −S−, −S − S−, −NH (C = NH) -, −OCO−, −CO−, −CHOH−, −N = N−, −NH−, −NR−, Y  ; P is a natural or synthetic polypeptide of molecular weight greater than 2000 Daltons; Y n is a number with a value between 1 and 500.

eleven.

 A compound of formula (II) according to claim 10, characterized in that L is a linear hydrocarbon chain of 2 to 20 carbon atoms.

12.

 A compound of formula (II) according to any one of the claims

15 10 or 11, characterized in that Z is selected from the group consisting of −CONH−, −NHCO−, −NH−, −S−, Y . 13. A compound of formula (II) according to any one of the claims 10 to 12, characterized in that P is selected from the group consisting of albumin, thyroglobulin, hemocyanin, β-galactosidase, peroxidase, phosphatase and oxidase. 14. A compound of formula (II) according to any one of claims 10 to 13, characterized in that T is [3 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl] or [4 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl]; L is a linear hydrocarbon chain of 2 to 20 carbon atoms; Z is selected from the group consisting of −CONH−, −NHCO−, −NH−, −S−, and ; Y 5 P is selected from the group consisting of albumin, thyroglobulin, hemocyanin, β-galactosidase, peroxidase, phosphatase and oxidase. 15. A compound of formula (II) according to claim 14, characterized in that T is [3 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl] or [4 - ((4,6-dimethylpyrimidin-2-yl ) amino) phenyl]; L is a linear hydrocarbon chain of 5 10 carbon atoms; Z is −CONH−; P is selected from the group consisting of albumin and peroxidase; and n is a value selected between 1 and 50. 16. A compound of formula (II) according to any one of claims 10 to 13, characterized in that T is [6-methyl-2- (phenylamino) pyrimidin-4-yl]; 15 L is a linear hydrocarbon chain of 2 to 20 carbon atoms; Z is selected from the group consisting of −CONH−, −NHCO−, −NH−, −S−, and ; Y 20 P is selected from the group consisting of albumin, thyroglobulin, hemocyanin, β-galactosidase, peroxidase, phosphatase and oxidase. 17. A compound of formula (II) according to claim 16, characterized in that T is [6-methyl-2- (phenylamino) pyrimidin-4-yl]; L is a linear hydrocarbon chain of 4 carbon atoms; Z is −CONH−; P is selected 25 of the group consisting of albumin and peroxidase; and n is a value selected between 1 and 50. 18. A compound of formula (II) according to any one of claims 10 to 13, characterized in that T is [N- (4,6-dimethylpyrimidin-2-yl) -N- (phenyl) amino]; L is a linear hydrocarbon chain of 2 to 20 carbon atoms; Z is selected from the group consisting of −CONH−, −NHCO−, −NH−, −S−, and ; Y P is selected from the group consisting of albumin, thyroglobulin, hemocyanin, β-galactosidase, peroxidase, phosphatase and oxidase. 19. A compound of formula (II) according to claim 18, characterized in that T is [N- (4,6-dimethylpyrimidin-2-yl) -N- (phenyl) amino]; L is a chain 10 linear hydrocarbon of 4 carbon atoms; Z is −CONH−; P is selected from the group consisting of albumin and peroxidase; and n is a value selected between 1 and 50. 20. A compound of formula (III): [T − L − Z] m − Q 15 (III) characterized in that T is selected from the group consisting of R-I, R-II, R-III, R-IV and R-V; H H NN NN N N R-I R-II H H NN NN N N R-III R-IV NN N R-V where RI is selected from the group consisting of 2 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl], [3 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl] and [4- ( (4,6-dimethylpyrimidin-2-yl) amino) phenyl]; R-II is [4,6-dimethyl-2- (phenylamino) pyrimidin-5-yl]; R-III is selected from the group consisting of [2 - ((4-methylpyrimidin-2yl) amino) phenyl], [3 - ((4-methylpyrimidin-2-yl) amino) phenyl] and [4 - ((4 -methylpyrimidin-2-yl) amino) phenyl]; R-IV is selected from the group consisting of [6-methyl-2- (phenylamino) pyrimidin-4 10 yl] and [6-methyl-2- (phenylamino) pyrimidin-5-yl], and R-V is [N- (4,6-dimethylpyrimidin-2-yl) -N- (phenyl) amino]; L is a hydrocarbon chain of 2 to 40 carbon atoms, where the chain is linear or branched, saturated or unsaturated, and said hydrocarbon chain comprises between 0 and 10 heteroatoms that are selected from the 15 group consisting of S, O and N; Z is a functional group selected from the group consisting of −CONH−, −NHCO−, −NHCONH−, −NHCSNH−, −OCONH−, −NHOCO−, −OCSNH−, −SCONH−, −S−, −S− S−, −NH (C = NH) -, −OCO−, −CO−, −CHOH−, −N = N−, −NH−, −NR−, Q is a non-isotopic marker; Y m is an integer with an integer value between 1 and 1000. 21. A compound of formula (III) according to claim 20, characterized in that L is a linear hydrocarbon chain of 2 to 20 carbon atoms. 22. A compound of formula (III) according to any one of claims 20 or 21, characterized in that Z is selected from the group consisting of −CONH−, −NHCO−, −NH−, −S−, Y . 23. A compound of formula (III) according to any one of claims 20 to 22, characterized in that Q is selected from the group consisting of biotin, fluorescein or any of its derivatives, a cyanine fluorophore, a rhodamine fluorophore, a Coumarin fluorophore, a bipyril of Ruthenium, luciferin or any of its derivatives, an acridinium ester and micro- or nanoparticles of colloidal gold, carbon or latex. 24. A compound of formula (III) according to any one of claims 20 to 23, characterized in that T is [3 - ((4,6-dimethylpyrimidin-2-yl) amino) phenyl] or [4 - ((4, 6-dimethylpyrimidin-2 10 il) amino) phenyl]; L is a linear hydrocarbon chain of 2 to 20 carbon atoms; Z is selected from the group consisting of −CONH−, −NHCO−, −NH−, −S−, Y ; Y Q is selected from the group consisting of biotin, fluorescein or any one of its derivatives, a cyanine fluorophore, a rhodamine fluorophore, a coumarin fluorophore, a ruthenium bipyryl, luciferin or any one of its derivatives, an acridinium ester and micro-or nanoparticles of 20 colloidal gold, carbon or latex. 25. A compound of formula (III) according to any one of the claims 20 to 23, characterized in that T is [6-methyl-2- (phenylamino) pyrimidin-4-yl]; L is a linear hydrocarbon chain of 2 to 20 carbon atoms; 25 Z is selected from the group consisting of −CONH−, −NHCO−, −NH−, −S−, and ; Y Q is selected from the group consisting of biotin, fluorescein or any one of its derivatives, a cyanine fluorophore, a rhodamine fluorophore, a coumarin fluorophore, a ruthenium bipyryl, luciferin or any one of its derivatives, an acridinium ester and micro-or nanoparticles of 5 colloidal gold, carbon or latex. 26. A compound of formula (III) according to any one of the claims 20 to 23, characterized in that T is [N- (4,6-dimethylpyrimidin-2-yl) -N- (phenyl) amino]; L is a linear hydrocarbon chain of 2 to 20 carbon atoms; 10 Z is selected from the group consisting of −CONH−, −NHCO−, −NH−, −S−, and; Y Q is selected from the group consisting of biotin, fluorescein or one 15 any of its derivatives, a cyanine fluorophore, a rhodamine fluorophore, a coumarin fluorophore, a ruthenium bipyryl, luciferin or any of its derivatives, an acridinium ester and micro or nanoparticles of colloidal gold, carbon or latex. 27. An antibody generated in response to a compound of formula (II) as described in any one of claims 10 to 19. 28. Method of in vitro analysis of pyrimethanil in a sample comprising the following steps: to. contacting the sample with the antibody described in claim 27; 25 b. incubate the sample and the antibody of step (a) for a suitable period of time for an immunochemical reaction to take place; Y C. determine the existence of immunochemical reaction after the incubation of step (b). 29. Pyrimethanil analysis method according to claim 28, characterized in that the determination of the immunochemical reaction in step (c) is performed 5 by a competitive immunoassay, using as a competitor a compound of formula (II) as described in any one of claims 10 to 19. 30. Pyrimethanil analysis method according to claim 28, characterized in that the determination of the immunochemical reaction in step (c) is performed 10 by a competitive immunoassay, using as a competitor a compound of formula (III) as described in any one of claims 20 to 26. 31. Pyrimethanil detection kit, characterized in that it comprises at least one antibody as defined in claim 27 and a compound 15 selected from the group consisting of a compound of formula (II) as defined in any of claims 10 to 19 and a compound of formula (III) as defined in any of claims 20 to 26. H (CH2) 5CO2H NN m-NH2-Ph- (CH2) 5CO2H N 8h 70% 2 PMm6 N N Cl H NN p-NH2-Ph- (CH2) 5CO2H N (CH2) 5CO2H 30h 75% 3 PMp6 Fig. 1 O (CH2) 4CO2CH3 (CH2) 4CO2CH3 OR ClCO (CH2) 4CO2CH3 Pd (AcO) 2O OR OR Or MgCl2, Py-Cl2CH2 Et3N, THF OR OO 4 90% 97% 56 H HN N H H .HNO3 N N i) NaOH, MeOH N N NH2 60 ° C, 1h N N Na2CO3, CH3OH ii) HCOOH 75 ° C, 24h (CH2) 4CO2CH3 (CH2) 4CO2H 90% 70% 78 PMb5 Fig 2 i) LiOH, THF-H2O H Br (CH2) 4CO2CH3 (CH2) 4CO2CH3 (CH2) 4CO2H 25 ° C, 24h NN ii) KHSO4-H2O NN NaH NN DMF, 25 ° C, 72h N N N 60% 9 10 11 PMn5 Fig. 3 Fig. 4 PMm6 # 1PMm6 # 2PMp6 # 1 PMp6 # 2 PMb5 # 1 PMb5 # 2 PMn5 # 1 PMn5 # 2 Antibody Fig. 5 Absorbance 492 nm 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 [Pyrimethanil] (nM) Fig. 6 SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 201230098 SPAIN Date of submission of the application: 25.01.2012 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: See Additional Sheet RELEVANT DOCUMENTS

Category

56 Documents cited Claims Affected

TO

KIM, H.-J. et al. “Automated flow fluorescent immunoassay for part per trillion detection of the neonicotinoid insecticide thiamethoxam.” Analtytica Chimica Acta 2006, Volume 571, pages 66-73. [Available online 05.05.2006]. See page 66, summary; page 69, figure 2; page 67, experimental part. 1-31

TO

ARGARATE, N. et al. “Evaluation of Immunoassays as an Alternative for the Rapid Determination of Pesticides in Wine and Grape Samples.” Journal of AOAC International 2010, Volume 93, Number 1, pages 2-11. 1-31

TO

BAGGIANI, C. et al. “Molecularly imprinted solid-phase extraction method for the high performance liquid chromatographic analysis or fungicide pyrimethanil in wine”. Journal of Chromatography A 2007, Volume 1141, pages 158-164. [Available online on 18.12.2006]. See page 158, summary. 1-31

TO

EP 0270111 A1 (KUMIAI CHEMICAL INDUSTRY, CO., LTD. & IHARA CHEMICAL INDUSTRY, CO., LTD.) 03.12.1987, page 3, lines 4-6; page 4, example (I); pages 6-9, example 1. 1-31

TO

US 6376548 A1 (MULVIHILL, M.J. & SHABER, S.H.) 04/23/2002, column 1, lines 3-10; column 3, line 27; column 6, example (I). 1-31

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 29.05.2013

Examiner G. Esteban García Page 1/4

REPORT OF THE STATE OF THE TECHNIQUE Application number: 201230098 CLASSIFICATION OBJECT OF THE APPLICATION C07K14 / 00 (2006.01) C07D239 / 22 (2006.01) G01N33 / 531 (2006.01) G01N33 / 532 (2006.01) C12N9 / 02 (2006.01) C12N9 / 08 (2006.01) C12N9 / 14 (2006.01) C12N9 / 38 (2006.01) Minimum documentation sought (classification system followed by classification symbols) C07K, C07D, G01N, C12N Electronic databases consulted during the search (name of the database and, if possible, search terms used) EPODOC, WPI, REGISTRY, CAPLUS, MEDLINE, BIOSIS, XPESP, EMBASE, NPL, PUBMED, CHEMSPIDER State of the Art Report Page 2/4  WRITTEN OPINION Application number: 201230098 Date of Completion of Written Opinion: 05.29.2013 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims 1-31 Claims IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims 1-31 Claims IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/4  WRITTEN OPINION Application number: 201230098 1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

KIM, H.-J. et al. Analtytica Chimica Acta 2006, Vol. 571, pp. 66-73. 05.05.2006

D02

ARGARATE, N. et al. Journal of AOAC International 2010, Vol. 93, No. 1, pp. 2-11. 2010

D03

BAGGIANI, C. et al. Journal of Chromatography A 2007, Vol. 1141, pp. 158-164. 18.12.2006

D04

EP 0270111 A1 03.12.1987

2. Statement motivated according to articles 29.6 and 29.7 of the Regulation of execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The object of the invention is a compound of formula (I), which comprises a phenylaminopyrimidine moiety; a compound of formula (II), derived from (I) that includes a polypeptide; a compound of formula (III), derived from (I) having a non-isotopic marker; an antibody generated in response to a compound of formula (II), a method of in vitro analysis of pyrimethanil using said antibody; and a pyrimethanil detection kit comprising the antibody and a compound of formula (II) or (III). Document D01 discloses an ultrasensitive fluorescent immunoassay that allows quantitative analysis of the thiamethoxane neonicotinoid insecticide (see page 66, summary). To carry out the test, the corresponding hapten was prepared, which has a carboxylic acid functional group (see page 69, figure 2), the hapten-BSA conjugates, which were immobilized on PMMA, and the corresponding monoclonal antibodies specific for thiamethoxane (see page 67, experimental part). Document D02 discloses a series of immunochemical tests for the detection of three pesticides (atrazine, bromopropylate and 2,4,6-trichlorophenol) in wine, grapes and must samples (see page 2, summary). Documents D01 and D02 use a strategy similar to that of the application for the detection of an insecticide (thiamethoxane), but it differs in the nature of the compound on which said strategy is applied, which in the case of the application is the fungicide pyrimethanil Document D03 discloses a method for solid phase extraction of pyrimethanil from wine samples using a molecular printing polymer (see page 158, summary). This document discloses a chromatographic procedure for the analysis of pyrimethanil which, unlike the application procedure does not include immunoassays. The pyrimethanil derivatives obtained by printing the polymer are also different from the conjugates collected in the patent application. Document D04 discloses pyrimidine derivatives of formula (I) that have activity as fungicides (see page 3, lines 4-6; page 4, formula (I)). In particular, numerous compounds that are substituted in the pyrimidine ring, in the benzene ring and / or in the nitrogen that binds them are disclosed (see pages 6-9, table 1). Although these compounds contain the same fundamental skeleton of the compound (I) of the invention, none of them have the substituents that appear therein and which are set out in claim 1. The cited documents show only the state of the art of the field to which the invention belongs. None of them, taken alone or in combination with the others, disclose or contain any suggestion that could direct the person skilled in the art to a compound of formula (I) (independent claim 1); and, therefore, also towards a compound of formula (II), derived from (I) that includes a polypeptide (independent claim 10); a compound of formula (III), derived from (I) having a non-isotopic marker (independent claim 20); an antibody generated in response to a compound of formula (II) (independent claim 27); a method of in vitro analysis of pyrimethanil using said antibody (independent claim 28); and a pyrimethanil detection kit comprising the antibody and a compound of formula (II) or (III) (independent claim 31). Therefore, it is considered that the object of claims 1-31 meets the requirements of novelty and inventive activity set forth in Articles 6.1 and 8.1 of the Patent Law. State of the Art Report Page 4/4