FR2687664A1 - Autoxidisable compounds, their preparation, their application in asaying SOD activity and necessary for its implementation - Google Patents

Abstract

The subject of the invention is autoxidisable compounds of general formula I: in which: n = 1 or 2; R<1> = -OR<4> or -NR<5>R<6>; R<2> = H, -OR<4>, (1 to 6C)alkyl or else -CH2- or -CH2-CH2-, in order to form a ring by bonding with the phenyl substituent in the position meta with respect to R<1>; and R<3> = H, (1 to 6C)alkyl or -OR<4> (if R<2> is other than -OR<4>); with R<4> = H or (1 to 6C)alkyl, R<5> = H, (1 to 6C)alkyl, -CH2COOH, -C6H5COOH or -C6H5SO3H; and R<6> = H, (1 to 6C)alkyl or -CH2COOH. These compounds can be used for assaying SOD activity in a liquid medium by using the activation of their autoxidation by the SOD activity. Application to assaying SOD activity, especially in a biological medium.

Description

Autoxidizable compounds, their preparation, their application to the determination of the SOD activity and necessary for its implementation.

The present invention relates to novel autoxidizable chemical compounds and their application to the assay of superoxide dismutase (SOD) activity.

It relates more particularly to new autoxidizable chemical compounds, defined below, their preparation, a method for assaying the SOD activity, in particular in biological samples, using these compounds as reagents, as well as necessary or kits for the implementation of this assay method.

The tissues of aerobic organisms, and those of the human body in particular, are the site of a continuous production of superoxide 2 - ion, which results from the process of cellular respiration, as well as many essential metabolic pathways.

This superoxide ion production increases sharply when these organisms are subjected to "oxidative stress" of toxicological origin (hyperoxia, irradiation, intoxication by xenobiotics which generate free radicals, in particular) or physiopathological (inflammation, ischemia, post-ischemic reperfusion. , especially).

It is accepted by the entire scientific community that the superoxide ion is very toxic, although the mechanisms underlying this toxicity remain controversial (reference 1).

To protect against the deleterious effects of the superoxide ion, aerobic organisms have enzymatic systems, superoxide dismutases (SOD), which catalyze the degradation of the superoxide ion according to the following disproportionation reaction

<tb> 2 <SEP> 02 <SEP> e <SEP> - <SEP> + <SEP> 2 <SEP> H <SEP><<SEP><SEP> H202 <SEP> + <SEP> 2 <SEP>
<tb>
Given the major protective role of this disproportionation reaction with respect to the toxicity of the superoxide ion, the catalytic activity of intra- and extracellular SODs conditions the survival of the tissues of an aerobic organism (references 2 to 5 ).

The determination of SOD activity is therefore of major interest for biological researchers as well as for clinical chemistry laboratories, on condition that it can be carried out using a sensitive, specific, rapid and not very complex method.

Many methods of assaying SOD activity have been published and criticized (references 6 to 10), but it is generally accepted that there is still no satisfactory compromise between the reliability and the complexity of the existing methods, since the measurement of 'an SOD activity poses two major problems that none of the methods published to date have made it possible to solve in a simple way.

The first problem is that of the spontaneous decomposition of the sole substrate, the superoxide ion, which is very rapid at pH close to 7, and which although strongly slowed down, remains significant at pH 9, pH beyond which certain types natural SODs are quickly inactivated.

Obtaining a sufficiently stable stationary concentration therefore requires the presence of a dynamic source of superoxide ion in the assay solution.

Electrolytic sources require complex equipment and pre-extraction of the SOD catalyst, which is prohibitive in Clinical Chemistry.

Photochemical sources such as riboflavin lead to methods which cannot be standardized and which are complex to implement.

Enzyme sources such as xanthine oxidase require solutions to be prepared immediately.

They are expensive, a source of artifacts and difficult to automate.

The purely chemical sources are reduced to autoxidizable compounds such as 6-hydroxydopamine, pyrogallol, hydroxylamine or sulfite ion, the autoxidation of which is generally inhibited by SOD.

These chemical reagents rarely give precise and sensitive results on crude biological extracts, and they require the extemporaneous preparation of an anaerobic reagent solution; their use is therefore difficult to automate.

The second problem is due to the fact that it is difficult to directly measure ("direct" assay) the disappearance of the superoxide substrate 02 e - as a function of time, or the appearance of its disproportionation products, hydrogen peroxide H2 2 ( hydrogen peroxide) and oxygen, taking into account the concentrations and pH values available under the conditions of an assay for SOD activity.

The only "direct" spectrophotometric method is that of MARKLUND (references 11 and 12) which measures the speed of disappearance of the superoxide ion at pH 9.5, which does not allow the determination of all natural SODs.

This method requires a fast spectrophotometer. The high concentrations of superoxide ion and the wavelength (250 nm) used make the method imprecise, which forces the experimenter to take several measurements to obtain an average value. In addition, it is necessary to add catalase to avoid inactivation of SOD-Cu / Zn, and a new solution of potassium superoxide must be re-prepared immediately before each assay.

This direct spectrophotometric method is therefore difficult to implement, and it does not seem possible to improve it significantly.

The corollary is that the popular methods, that is to say those most often used, are generally indirect: they are based on the competition between the SOD activity that one seeks to measure, and the reaction of the superoxide ion with a chromogenic scavenger such as ferric cytochrome C or tetrazolium nitroblue (NBT), the reaction product of which can be measured by UV / visible spectrophotometry. A chemiluminescent trap, such as luminol or lucigenin, can also be used with greater measurement sensitivity, but problems of interference and instrument availability preclude the use of such methods by a layman laboratory.

In addition to the often insufficient specificity of chromogenic reagents with respect to superoxide, the main drawback of competitive assays is that they require linearization of the inhibition values (by
SOD), on 5 or 6 samples of increasing dilutions.

Finally, a certain number of immunoassays of SOD-Cu / Zn or of SOD-Mn are known.

Some are very sensitive, but they do not provide information on the enzymatic activity of the sample, which depends in particular on the degree of possible inactivation of the enzyme. They are also long to implement and relatively complex.

Measurement artefacts and assay multiplication are therefore intrinsically linked to all spectrophotometric methods accessible to most laboratories, which does not meet the needs of research laboratories and remains incompatible with the standardization requirements of Clinical Chemistry.

If UV / visible spectrophotometry is used, it seems difficult, if not impossible, to develop an assay method which is at the same time sensitive, more specific, simpler and more reliable than the existing methods, without having recourse to kinetic monitoring of a reaction activated by SOD activity rather than to that of a reaction inhibited by SOD activity or to a "direct" method.

In this regard, a recent method (references 13 and 14), using an autoxidizable dye, hematoxylin, differs from the others insofar as it exploits an activation of the autoxidation of the reagent by the SOD activity.

In fact, hematoxylin autoxidizes at pH> 6.5 (reference 13). The rate of autoxidation increases by a factor close to 100 between pH 7 and 9. SOD inhibits this autoxidation at pH <8.1, while it activates it at pH> 8.1.

This observation has led to the development of an assay, based on spectrophotometric monitoring of the formation of thematein at 560 nm, which is accelerated by SOD at alkaline pH. The implementation of this method is difficult due to the instability of the reagent and it remains dependent on a phase of preparation of the sample (dialysis) to eliminate the interference due to the presence of reducing compounds, such as for example glutathione.

In addition, the instability of the oxidation product measured, haematein, and the low sensitivity of the assay (at pH> 8.1), linked in part to the decomposition of thematein, but also to the presence of two sites of The potential oxidation (catechols) limit the interest and the use of this method.

In conclusion, the hematoxylin method, although it is of some interest, has drawbacks which do not allow its general use for the determination of SOD activity, in particular in biological samples.

The present invention aims in particular to - have a stable reagent outside the measurement conditions, and whose autoxidation product is stable during. the measurement - to increase the sensitivity of the assay compared to a method using hematoxylin (under conditions of activation of autoxidation) - to be able to eliminate the most probable possible interferences such as those linked to the presence of mercaptans such as glutathione , for example - to develop a process using a reaction medium with a pH less than or equal to 9, so as to allow a determination of the activity of known SODs, without risk of rapid inactivation.

These aims are achieved according to the invention which provides original chemical reagents, exhibiting the property of self-oxidising in an oxygenated solution, to lead to a chromophoric product whose appearance kinetics are increased in the presence of a catalyst of the SOD type. .

The structure of these reagents is characterized, in particular, by an aromatic ring with a catechol function, linked via a tertiary benzylic carbon to an aromatic ring, substituted by an electron-donor group.

The autoxidation product of these reagents exhibits an absorption wavelength very different from that characterizing the reagent itself and always greater than or equal to 500 nm.

The invention also provides a method using these reagents, which makes it possible to measure the SOD activity of any catalyst in an aqueous medium, using a single W / visible absorption measurement and whose simplicity of implementation. implementation is clearly superior to that of the other spectrophotometric methods previously described.

The invention further provides a method of assaying the SOD activity of the type described above, in the presence of mercaptans, by using a rapid reagent for thiol groups which makes it possible to eliminate the corresponding interferences, in particularly those which are due to the presence of glutathione or albumin in the sample.

dans laquelle n = 1 ou 2,
R1 = -OR4 ou -NR5R6,
R2 = hydrogène, -OR4, alkyle (avec 1 à 6 atomes de carbone),
ou bien -CH2- ou -CH2-CH2-, pour former un cycle par
liaison avec le substituant phényle, en position méta
par rapport à R1 et
R3 = hydrogène, alkyle (avec 1 à 6 atomes de carbone) ou
-OR4 (à condition que R2 soit différent de -OR4), avec
R4 = hydrogène ou alkyle (avec 1 à 6 atomes de carbone),
R5 = hydrogène, alkyle (avec 1 à 6 atomes de carbone),
-CH2COOH, -C6HsCOOH ou -C6HsSO3H et
R6 = hydrogène, alkyle (avec 1 à 6 atomes de carbone) ou
-CH2COOH.According to one of its aspects, the invention relates to the compounds corresponding to the general formula I

in which n = 1 or 2,
R1 = -OR4 or -NR5R6,
R2 = hydrogen, -OR4, alkyl (with 1 to 6 carbon atoms),
or -CH2- or -CH2-CH2-, to form a ring by
bond with the phenyl substituent, in the meta position
compared to R1 and
R3 = hydrogen, alkyl (with 1 to 6 carbon atoms) or
-OR4 (provided that R2 is different from -OR4), with
R4 = hydrogen or alkyl (with 1 to 6 carbon atoms),
R5 = hydrogen, alkyl (with 1 to 6 carbon atoms),
-CH2COOH, -C6HsCOOH or -C6HsSO3H and
R6 = hydrogen, alkyl (with 1 to 6 carbon atoms) or
-CH2COOH.

The compounds corresponding to the general formula I defined above can be prepared according to the general procedures described below, generally from commercial products.

However, the compounds in which R5 represents -C6HsCOOH or -C6HsSO3H are obtained from the appropriately substituted parabromodiphenylamine type building block. This building block is prepared according to the following general procedure.

Paranitrophenol is alkylated using an ester of chloroacetic acid. The derivative obtained is saponified, then amidated with parabromoaniline. The corresponding amide is subjected to the Smiles reaction (reference 15) to yield the corresponding diphenylamine derivative. The latter is reduced, diazotized and substituted in an appropriate manner to lead to the desired building block.

General procedure for the synthesis of compounds of series I (R2 = hydrogen or alkyl and RS = hydrogen or alkyl)
5,6-dihydroxindan-1-one or 6,7-dihydroxy-1tetralone (prepared from veratrole), suitably protected by an alkyl or aralkyl group such as for example the methyl or benzyl group, or by a group of the type silyl such as for example the terbutyldimethylsilyl group, is treated either directly or after prior mono- or bisalkylation (of the a-position of the ketone function) using an alkylating agent such as methyl iodide, for example, with the organomagnesium reagent corresponding to the phenyl bromide substituted in para by an oxygenated group, such as for example the methoxy group, or nitrogenous, such as for example the dimethylamino group.

The intermediates thus obtained are reduced and then deprotected to lead to the compounds of the I series.

The following reaction scheme illustrates this procedure.

i: R1PhMgBr
(R1 in para position) il: reduction then deprotection iii: base, R2X iiii: base, R3X
P: protective group
General procedure for the synthesis of compounds of series II (R2 or R3 = -OR4).

These compounds are obtained by hydration of alkenes, intermediates in the synthesis of compounds of the series
I, after hydroboration and oxidation; then the alcohols thus obtained are optionally alkylated with an alkyl halide such as, for example, methyl iodide, and finally deprotected.

The following reaction scheme illustrates this procedure.

i: 1) BH3: 2) H2O2, NaOH ii: deprotection iii: base, R4X
P: protective group
General procedure for the synthesis of compounds of series III (R2 = -CH2- or -CH2-CH2- forming a ring by bonding with the phenyl substituent in the meta position with respect to R1)
To synthesize these. compounds, the product of crotonization between the suitably protected 3,4-dihydroxybenzaldehyde and the suitably substituted 1-tetralone or indan-1-one is either directly or indirectly (after preparation of the higher homolog by the intermediate of a cyclopropane derivative) reduced by catalytic hydrogenation to the corresponding saturated derivative. The latter is cyclized in the presence of polyphosphoric acid and the alkene thus obtained is reduced and then deprotected to yield the desired compound of series III.

The following reaction scheme illustrates this procedure.

i: ii: reduction (or preparation of the higher homolog
then reduction) iii: polyphosphoric acid iiii: reduction iiiii: deprotection
P: protective group
According to another of its aspects, the invention relates to a method for assaying the superoxide dismutase (SOD) activity in a liquid medium, which method uses the activation of the autoxidation of a reagent by the activity.
SOD, characterized in that the said reagent is a compound corresponding to the general formula I defined above.

When the determination of the SOD activity must be carried out in a medium containing one or more compounds comprising one or more thiol groups, such as in particular a biological medium, the reducing power of the latter is likely to cause interference which should be determined. 'eliminate.

Such interferences can be easily avoided by adding to the medium in which the assay is carried out at least one reagent chosen from compounds of pyridinium and quinolinium types, the use of which as "mercaptan scavengers" is the subject of the application by French patent no. 91 14782 filed on November 29, 1991 in the name of BIOXYTECH and whose title is: “Mercaptan trapping reagents, their preparation and their applications”.

These. reagents meet the following criteria - specificity with respect to mercaptans under the operating conditions used, - rapidity of reaction, - non-interference with the other reagents used in the determination of the SOD activity.

dans laquelle
z1 = alkyle (avec 1 à 6 atomes de carbone), benzyle, p
nitrobenzyle, phényle, o,p-dinitrophényle,
-CH2-COOH ou -CH2-CH2-COOH;;
Z4 = hydrogène et
Z5 = hydrogène ou alkyle (avec 1 à 6 atomes de
carbone), ou
Z4 et Z5 forment ensemble, avec les deux atomes de
carbone intermédiaires, un cycle phényle;
X= halogénure, sulfonate, fluorosulfonate, phospho
nate, tétrafluoroborate ou tosylate; et soit z2 = vinyle, et
Z3 = hydrogène ou alkyle (avec 1 à 6 atomes de carbone)
(composés 2-vinyliques), soit Z3 : vinyle, et
z2 = hydrogène ou alkyle (avec 1 à 6 atomes de carbone)
(composés 4-vinyliques).More precisely, according to another of its aspects, the subject of the invention is a method for assaying superoxide dismutase (SOD) activity in a liquid medium containing one or more mercaptan (s), in particular a biological medium, which method uses the activation of the autoxidation of a reagent by the SOD activity, characterized in that the said reagent is a compound corresponding to the general formula I defined above and in that the medium also contains an amount capable of completely trapping said mercaptans by S-alkylation of at least one compound corresponding to the general formula II

in which
z1 = alkyl (with 1 to 6 carbon atoms), benzyl, p
nitrobenzyl, phenyl, o, p-dinitrophenyl,
-CH2-COOH or -CH2-CH2-COOH ;;
Z4 = hydrogen and
Z5 = hydrogen or alkyl (with 1 to 6 atoms of
carbon), or
Z4 and Z5 together form, with the two atoms of
intermediate carbon, a phenyl ring;
X = halide, sulfonate, fluorosulfonate, phospho
nate, tetrafluoroborate or tosylate; and let z2 = vinyl, and
Z3 = hydrogen or alkyl (with 1 to 6 carbon atoms)
(2-vinyl compounds), or Z3: vinyl, and
z2 = hydrogen or alkyl (with 1 to 6 carbon atoms)
(4-vinyl compounds).

The term “sulfonate” is understood to mean an anion of general formula R — SO3 in which R represents, for example, trifluoromethyl.

These compounds are in part known or can be synthesized by analogy to methods used for the synthesis of known compounds.

Thus, 2-vinyl compounds can be synthesized as follows.

The initial derivative (pyridine or substituted quinoline) is subjected to the Comins method (reference 16), using vinylmagnesium bromide. The compound thus obtained is oxidized with, for example, tetrachloro-1,4-benzoquinone (parachloranil) or sulfur Sg. The alkylation of heterocyclic nitrogen is carried out using an alkylating agent, for example trimethyloxonium tetrafluoroborate, methyl sulfate, benzyl bromide, fluorodinitrobenzene, sodium iodoacetate or 3-bromopyruvate sodium, chosen according to the meaning of Z1.

Example 9 of the experimental part which follows illustrates the preparation of a compound of this type.

On the other hand, 4-vinyl compounds can be synthesized as follows.

The corresponding heterocyclic aldehyde (aldehyde function, -CHO, in position 4) is subjected to the reaction of
Wittig, with, for example, triphenylmethylphosphonium bromide, or the Peterson reaction, with, for example, chloromethyltrimethylsilane. The alkylation of heterocyclic nitrogen is carried out using an alkylating agent, for example trimethyloxonium tetrafluoroborate, methyl sulfate, benzyl bromide, fluorodinitrobenzene, sodium iodoacetate or sodium 4bromopyruvate , chosen according to the meaning of Z1.

Example 10 of the experimental part which follows illustrates the preparation of a compound of this type.

The process according to the invention, in particular when it uses at least one “mercaptan scavenger” defined above, makes it possible to measure the SOD activity of any catalyst in an aqueous medium, using a single measurement of absorption
UV / visible, the ease of use of which is clearly superior to that of the spectrophotometric methods previously described.

This new assay process therefore constitutes a tool of choice for biological research in general as well as in clinical chemistry, in particular for the development of assay or diagnostic kits or kits.

In the latter case, this assay is likely to provide medically useful information in pathophysiological conditions such as diseases of inflammatory or ischemic origin, states of shock, infectious states, systemic diseases and autoimmune diseases, tumors. , states of malnutrition, diseases associated with aging, degenerative diseases, hemolytic anemias and syndromes of intoxication by environmental pollutants or by drugs.

Human samples on which this method can be used include erythrocytes, blood plasma, platelets, white blood cells, synovial fluid, cerebrospinal fluid, urine or any tissue extract.

In addition, this assay method can be used to carry out the pharmacokinetic studies necessary for the development and use of new drugs exhibiting SOD activity.

This assay method can also be used for the quality control of cosmetic or pharmaceutical preparations exhibiting SOD activity.

In general, this assay method can be used by researchers on any biological sample or any artificial solution likely to exhibit SOD activity.

According to a preferred embodiment, the method for assaying the superoxide dismutase activity of a sample, in particular biological, is based on a spectrophotometric measurement of the rate of autoxidation of a compound of general formula I above, in l 'absence and in the presence of said sample.

More precisely, according to a preferred embodiment, the subject of the invention is a method for assaying the superoxide dismutase (SOD) activity in a liquid sample, characterized in that it essentially comprises the steps consisting in 10) determining the maximum rate of autoxidation Vs of a compound of general formula I in the presence of the sample, by means of the change in absorbance as a function of time, at the wavelength characterizing the appearance of the product of autoxidation of the compound of general formula I used, in a reaction medium buffered to a pH value of 8.0 to 9.0, initiating the reaction by adding an aliquot of a stock solution of the compound of general formula I 20) determining, under the same conditions, the maximum rate of autoxidation Vc of the same compound in the absence of the sample; and 30) determining the SOD concentration of the sample by means of the difference between the measurements taken and a calibration curve established under the same operating conditions.

The reaction medium is advantageously constituted by an amino buffer, such as for example 2-amino-2methyl-1,3-propanediol (AMPD), containing 0.1 mM diethylenetriaminepenta-acetic acid (DTPA) and making it possible to bind the pH at a value in the range from 8.0 to 9.0, at the measurement temperature, for example by adding sodium hydroxide or hydrochloric acid, depending on the nature of the buffer considered.

This reaction medium must be equilibrated at the working temperature and saturated with air at this temperature.

The chosen working temperature, for example 370C, must be kept constant during the measurements.

The reaction is started by adding to the reaction medium (with or without SOD) an aliquot of a stock solution of one of the compounds of general formula I in an aqueous solution or in an organic solvent miscible with water. water, such as, for example, acetonitrile, dimethylformamide or dimethylsulfoxide, or alternatively in a mixture of such an organic solvent with distilled water.

To overcome an extemporaneous preparation of this stock solution or an anaerobic preparation thereof, the stock solution can be prepared in the presence of a boron derivative, such as boric acid for example, which makes it possible to obtain a reagent solution which is stable for a period of time which depends on the respective concentrations of the reagent and of the derivative of borde, on the respective nature of the reagent and of this derivative and on the storage conditions of the stock reagent solution. Thus a solution of the BXT 01050 reagent described below (example 7), prepared in a 50/50 (volume / volume) dimethylsulfoxide / distilled water mixture containing boric acid, such as the ratio of the boric acid concentrations and in reagent is equal to 150, is stable for at least 1 month if the solution is stored between 0 and 80C.

After homogenization of the solution, the change in absorbance is recorded as a function of time, at the wavelength characterizing the appearance of the autoxidation product of the reagent used (525 nm, for example, for the BXT 01050 reagent ), in order to determine the maximum rate of autoxidation.

The determination of the SOD activity in a biological sample is carried out by determining the difference in the rates measured in the presence (Vs) and in the absence of the sample (VC).

This difference can be reported for example on the ordinate of a calibration curve which was established under the same operating conditions from, for example, an SOD standard.

This calibration curve can be plotted by representing, as a function of the concentration of the standard used, the variation of the difference in the rate of autoxidation measured in the presence (VSOD) and in the absence of this standard (Vc).

However, according to a preferred embodiment, this calibration curve is plotted by representing the variation of the inverse of the difference in the speeds in question, as a function of the inverse of the activity of the standard.

Depending on the intensity of the SOD activity to be measured, the sensitivity of the assay in the pH range used (8.0 to 9.0) can be adapted by modifying, for example, one or more of the following parameters: concentration of compound of general formula I selected, buffer concentration and measurement temperature.

The rate of autoxidation of the reagent used can also be modulated by varying the concentration of a boron derivative in the reaction medium.

The existence, in a sample, in particular biological, of compounds interfering with the assay is most often manifested by a decrease in the speed measured in the presence of the sample, compared to that measured in its absence.

In the case of interference linked to the presence of mercaptans, such as for example glutathione, the introduction into the reaction medium of a “mercaptan trapping reagent” as defined above is then necessary.

Thus 1,4,6-trimethyl-2-vinylpyridinium tetrafluoroborate (compound of Example 9), for example, will be incorporated at a concentration fifty times greater than that of glutathione for the determination of the SOD activity in a lysate. erythrocyte.

If the existence, in a sample, in particular biological, of a compound capable of introducing an artefactual activation of the autoxidation of the compound of general formula I or chromogenic compound is suspected, a combination such as that described below, which involves a second measurement carried out under autoxidation inhibiting conditions, should be undertaken.

Likewise, in the case of the exploration of media, in particular biological, exhibiting low SOD activity, the sensitivity and precision of the assay can be increased by combining the measurement made under conditions of activation of autoxidation, a measurement carried out under conditions of inhibition of autoxidation.

A subject of the invention is therefore also a method of the type described above, characterized in that it also comprises a measurement carried out under conditions of inhibition of autoxidation.

The reaction medium used for this second measurement differs from the medium described above by the use of a buffer at a pH in the range of 7.2 to 7.8, in particular a phosphate buffer consisting, for example, of an adequate mixture of Potassium dihydrogenphosphate and dipotassium hydrogenphosphate hydrogenphosphate, all other things being equal.

The maximum rate of autoxidation of the compound of general formula is measured under these operating conditions.
I used in the absence (V'C) and in the presence of the sample (V's). The determination of the SOD activity in the sample is then obtained by means of (Vs - VC - V'S + V'C).

As previously, the value obtained can be plotted on the ordinate of a calibration curve, which was established under the same experimental conditions and which gives the variation of (VS-VC-V'S + V'C) as a function of the concentration of the standard used.

The new assay method according to the invention using as reagent a compound of general formula I is, by its principle, a specific method of measuring the SOD activity. It is sensitive, fast and easy to use, and is also fully automated, so it is suitable for processing large series of samples, as can be the case in chemistry.
Clinical.

In addition, the interferences linked to the presence of mercaptans, frequently encountered in biological samples, are easily eliminated by the use of at least one "mercaptan scavenging reagent" as defined above, without any additional phase of treatment.
I 'sample.

Finally, the very flexible operating conditions of this assay method give it flexibility of use allowing it to be adapted to very diverse samples, in particular biological samples, and therefore to take into account the possible particular needs of certain users, as is the case. the case in Research.

Examples of application of the new method for assaying the SOD activity using four compounds of general formula I, in the case of an erythrocyte lysate, are given, by way of illustration, in the experimental part which follows.

A subject of the invention is also a kit or kit for implementing the method for assaying SOD according to the invention.

This kit or kit essentially comprises, as reagent, a compound of general formula I, as defined above.

According to an advantageous embodiment, the kit or kit for carrying out the method for assaying the SOD activity according to the invention further comprises one or more compound (s) trapping mercaptans, of general formula II , as defined above.

According to a preferred embodiment, a subject of the invention is also a kit or kit for carrying out the method for assaying 'the SOD activity according to the invention, characterized in that it essentially comprises - a compound of general formula I in solution or in
powder, in the presence of boric acid or a derivative of this
last, - one or more compound (s) trapping mercaptans of
general formula II, in solution or powder, and - an AMPD-based buffer, buffering in the pH range
from 8.0 to 9.0.

EXPERIMENTAL PART
I Preparation of compound of general formula I
All reactions are carried out under an inert nitrogen atmosphere, unless otherwise indicated.

The mass spectra (MS) are recorded on an R10-10B apparatus from the company Nermag. The ionization mode used is either electronic impact (EI) at 70 electronvolts, or chemical ionization (IC).

The 1 H NMR spectra are recorded on a Gemini-200 type device from the company VARIAN. The chemical shifts are expressed in ppm relative to tetramethylsilane. The multiplicities are expressed as follows: "s" for singlet; "d" for doublet; "t" for triplet and "m" for byte.

The melting points (pF) are recorded with an apparatus from the Gallenkamp Company and are given uncorrected.

A. Compounds of the I series.

Example 1: Preparation of 5,6-dihydroxy-1- (4-hydroxyphenyl) indane (BXTOiO4l) 5, 6-Dimethoxy-3- (4-methoxyphenyl) indene
To a suspension of magnesium (0.24 g, 10 mmol) in 5 ml of anhydrous THF is added a small portion of 4bromoanisole. the mixture is heated slightly to initiate the reaction, then the remainder of brominated derivative (1.87 g, 10 mmol), dissolved in 5 ml of THF, is added dropwise over 15 min. After 1 hour of reflux, the magnesium has disappeared. The mixture thus obtained is cooled to room temperature and a solution of 5,6dimethoxyindan-1-one (1.92 g, 10 mmol) in 10 ml of anhydrous THF is added. The brown solution obtained is stirred for 20 h under these conditions and then hydrolyzed by adding a saturated solution of NH4Cl. The mixture is extracted with AcOEt. The combined organic phases are washed with a saturated aqueous solution of NaCl and dried over Na2SO4.

After evaporation of the solvent, a yellow oil (3.43 g) is obtained which is taken up in 5 ml of glacial acetic acid.

The solution is stirred for 15 min at room temperature then heated at 700C for 15 min. The solution cooled to OOC is poured slowly into 50 ml of a saturated solution of
NaHCO3. The mixture is extracted with AcOEt. The combined organic phases are washed with a saturated aqueous solution of NaHCO3, dried over MgSO4 and evaporated. The crude product (2.84 g) thus obtained is purified by chromatography, eluting with an AcOEt / hexane (1: 3) mixture, to provide a white solid (1.97 g, 70%).

Physical characteristics * pF: 111.0-112.00C (AcOEt / hexane = 1: 3).

* 1H NMR (CDCl3): 3.62 (d, J = 2.16Hz, 2H); 4.05 (s, 3H); 4.08 (s, 3H); 4.12 (s, 3H); 6.58 (t, J = 2.16Hz, 1H); 7.19 (d, J = 8.67Hz, 2H); 7.29 (s, 1H); 7.32 (s, 1H); 7.72 (d,
J = 8.67Hz, 2H).

* SM (IE): 282 (100), 267 (55), 251 (19), 181 (24), 165 (35), 152 (51), 84 (53).

5,6-Dimethoxy-1- (4-methoxyphenyl) indane
A solution of 5,6-dimethoxy-3- (4-methoxyphenyl) indene (1.5 g, 5.32 mmol) in 60 ml of ethanol is hydrogenated in an autoclave in the presence of palladium on carbon (10%, 25 mg) under 5 bars of hydrogen for 1.5 h. After filtration of the catalyst, evaporation of the solvent gives an oil which gradually solidifies (1.5 g, 100%).

Physical characteristics * 1H NMR (CDCl3): 1.99 (m, 1H); 2.45 (m, 1H); 2.90 (m, 2H); 3.72 (s, 3H); 3.79 (s, 3H); 3.87 (s, 3H); 4.24 (t, J = 7.97
Hz, 1H); 6.48 (s, 1H); 6.82 (s, 1H); 6.84 (d, J = 8.70Hz, 2H); 7.50 (d, J = 8.70Hz, 2H).

* SM: 284 (100), 269 (26), 253 (59), 241 (17), 177 (21), 165 (35), 152 (28), 115 (28), 84 (25), 77 ( 26) 5, 6-Dihydroxy-1- (4-hydroxyphenyl) indane
To a solution of the previously obtained phenylindane (50 mg, 0.86 mmol) in 2 ml of CH2Cl2, cooled to -700C, is added dropwise a solution of BBr3 in CH2Cl2 (1.0 M, 3.1 ml, 3 , 1 mmol). The red solution obtained is warmed to room temperature and stirred for 0.5 h at this temperature. The reaction mixture is poured into a mixture of ice water and AcOEt. The aqueous phase is saturated with NaCl then extracted twice with AcOEt. The combined organic phases are washed with a saturated aqueous solution of NaCl, dried over MgSO4 and evaporated to dryness to give a yellow oil (480 mg) which is chromatographed ( eluent: AcOEt / hexane = 1: 1), to yield the expected compound (white solid, 400 mg, 93%).

Physical characteristics * 1H NMR (acetone-d6): 1.90 (m, 1H); 2.41 (m, 1H); 2.79 (m, 2H); 4.08 (t, J = 7.97Hz, 1H); 6.39 (s, 1H); 6.75 (s, 1H); 6.77 (d, J = 8.70Hz, 2H); 6.98 (d, J = 8.70Hz, 2H); 7.77 (s, br, 3H).

* MS (IE): 242 (100), 225 (61).

Example 2: Preparation of 5,6-dihydroxy-1- (4 hydroxyphinyl) -2,2-dimAthylindane (BXT01045) 5, 6Dimethoxy-2-methylindan-1-one and 5, 6-dimethoxy-2 2-dimethylindan -1-one
A mixture of lithium diisopropylamide (LDA, 1.5 M in cyclohexane, 7.5 ml, 11.3 mmol) and 15 ml of anhydrous THE is cooled to -780C. A solution of 5,6-dimethoxyindan-1-one (1.92 g, 10 mmol) in 5 ml of THF is added dropwise thereto. The mixture is stirred for 30 min at -780C.

Methyl iodide (0.93 mL, 15 mmol) is added. After 30 min at -780C, the solution is warmed to room temperature and stirred at this temperature for 4 h. The solution is cooled again to -780C and the same amount of LDA is added dropwise as before. After 30 min, MeI (0.93 ml, 15 mmol) is added. Stirred 30 min at -780C then 4 h at room temperature. The reaction mixture is hydrolyzed by adding a saturated aqueous solution of NH4Cl. It is extracted with AcOEt. The organic phases are washed with a saturated aqueous solution of NaCl, dried over MgSO4 and evaporated to dryness to give a yellow solid (2.23 g). Chromatography, using an AcOEt / hexane (1: 9) mixture as eluent, gives 5,6-dimethoxy-2,2-dimethylindan-1-one (1.35 g, 61%) and 5,6-dimethoxy-2-methylindan-1-one (0.43 g, 21,% ).

Physical characteristics of 5,6-dimethoxy-2,2-dimethylindan-1-one * 1H NMR (CDCl3): 1.64 (s, 6H); 2.85 (s, 2H); 3.85 (s, 3H); 3.91 (s, 3H); 6.80 (s, 1H); 7.12 (s, 1H).

* pF: 134.0-134.50C (EtOH)
Physical characteristics of 5,6-dimethoxy-2 methylindan-1-one * 1H NMR (CDCl3): 1.22 (d, J = 7.41 Hz, 3H); 2.63 (m, 2H); 3.23 (dd, J = 9.24-16.85Hz, 1H); 3.84 (s, 3H); 3.90 (s, 3H); 6.81 (s, 1H); 7.11 (s, 1H).

* pF: 106.0-106.50C (EtOH) 1-Hydroxy-5 6-dimethoxy-1- (4-methoxyphenyl) -2, 2-dimethyl-indane
To a suspension of magnesium (72 mg, 3 mmol) in 6 ml of anhydrous THF is added a small portion of 4bromoanisole. The mixture is heated at slight reflux to initiate the reaction, then the remainder of brominated derivative (0.56 g, 3 mmol), dissolved in 5 ml of THF, is added dropwise over 15 min. After 1 hour of reflux, the magnesium is consumed. 5,6-Dimethoxy-2, 2-dimethylindan-1-one (0.66 g, 3 mmol) is added in small portions. The solution is stirred for 1 h at room temperature, then heated to reflux for 4 h. 10 ml of a saturated aqueous solution of NH4Cl are added to OOC. The mixture is extracted with AcOEt. the combined organic phases are washed with saturated aqueous NaCl solution, dried over MgSO4 and evaporated to give a yellow oil (1.20 g). Purification by chromatography (eluent: AcOEt / hexane = 1: 5) allows the expected product to be isolated (0.65 g, 66%).

Physical characteristics * 1H NMR (CDCl3): 0.62 (s, 3H); 1.13 (s, 3H); 2.60 (d
J = 15.10Hz, 1H); 2.86 (d, J = 15.10Hz, 1H); 3.77 (s, 3H); 3.79 (s, 3H); 3.89 (s, 3H); 3.91 (s, 1H); 6.71 (s, 1H); 6.79 (s, 1H); 6.83 (d, J = 9.00Hz, 2H); 7.19 (d, J = 9.00Hz, 2H).

5,6-Dimethoxy-1- (4-methoxyphenylY-2,2-dimethylindane
Sodium borohydride (1.40 g, 36.7 mmol) and aluminum trichloride (0.82 g, 6.1 mmol) are added to a solution of the previous product (0.39 g, 1.25 mmol). ) in 15 ml of anhydrous THF. The mixture is heated to reflux. It immediately turns pink, then red, and finally becomes colorless. After 20 h of reflux, the reaction mixture is poured into a mixture of 20 g of ice and 5 ml of HCl (1N). The aqueous phase is saturated with NaCl and extracted with AcOEt. The organic phases are washed with a saturated aqueous solution of NaCl, dried over MgSO4 and evaporated to dryness to give a colorless oil. Chromatography, eluting with an AcOEt-hexane mixture (1:10), gives the expected product (0.39 g, 66%).

Physical characteristics * 1H NMR (CDCl3): 0.63 (s, 3H); 1.19 (s, 3H); 2.72 (s, 2H); 3.74 (s, 3H); 3.79 (s, 3H); 3.87 (s, 3H); 3.91 (s, 1H); 6.56 (s, 1H); 6.78 (s, 1H); 6.83 (d, J = 8.86Hz, 2H); 6.98 (d,
J = 8.86Hz, 2H).

5,6-Dihydroxy-1- (4-hydroxyphenyl) -2,2-dimethylindane
To a solution of the derivative obtained previously (0.39 g, 1.25 mmol) in 2 ml of CH2Cl2, cooled to -700C, a solution of BBr3 in CH2Cl2 (1.0 M, 4.86 ml is added dropwise). , 4.86 mmol). The red solution obtained is warmed to room temperature and stirred for 1 h at this temperature. The reaction mixture is poured into a mixture of ice water and AcOEt. The aqueous phase is saturated with NaCl, then extracted twice with AcOEt. the combined organic phases are washed with a saturated aqueous Nazi solution, dried over MgSO4 and evaporated to dryness, to give a slightly red oil (410 mg) which is chromatographed (eluent: AcOEt / hexane = 1: 1), to lead to expected compound (0.29 mg, 86%).

Physical characteristics * 1H NMR (acetone-d6): 0.71 (s, 3H); 1.17 (s, 3H); 2.60 (s, 2H); 3.80 (s, 1H); 6.46 (s, 1H); 6.72 (s, 1H); 6.75 (d,
J = 8.74Hz, 2H); 6.89 (d, J = 8.74Hz, 2H).

* SM (IE): 270 (87), 227 (100), 107 (16).

Example 3: Preparation of 5,6-dihydroxy-1- (4-hydroxy phenyl) -2-methylindane (BXT01048) 5,6-Dimethoxy-3- (4-methoxyphenyl) -2-methylindene
To a suspension of magnesium (42 mg, 1.750 mmol) in 5 ml of anhydrous THF is added a small portion of 4-bromoanisole. The mixture is heated to slight reflux to initiate the reaction and then the remainder of brominated derivative (330 mg, 1.75 mmol), dissolved in 5 ml of THF, is added dropwise over 15 min. After 1 hour of reflux, the magnesium is consumed. The mixture thus obtained is cooled to room temperature and then a solution of 5,6dimethoxy-2-methylindan-1-one (300 mg, 1.46 mmol) in 5 ml of THF is added. The brown solution obtained is stirred for 18 h, then hydrolyzed by adding a saturated aqueous solution of NH4Cl. The mixture is extracted with AcOEt. The combined organic phases are washed with a saturated aqueous solution of NaCl, then dried over Na2SO4. After evaporation of the solvent, a yellow oil (0.54 g) is obtained which is taken up in 2 ml of glacial acetic acid. The solution obtained is stirred for 1 h at room temperature. The solution, cooled to OOC, is poured slowly into 50 ml of a saturated solution of NaHCO3. The mixture is extracted with AcOEt. The organic phase is washed with a saturated aqueous solution of NaHCO3, dried over MgSO4 and evaporated. The crude product (530 mg) thus obtained is purified by chromatography, eluting with an AcOEt / hexane (1: 9) mixture, to provide a colorless oil (0.25 g, 58%).

Physical characteristics * 1H NMR (CDCl3): 2.09 (s, 3H); 3.35 (s, 2H); 3.82 (s, 3H); 3.86 (s, 3H); 3.89 (s, 3H); 6.78 (s, 1H); 7.00 (d, J = 8.70
Hz, 2H); 7.04 (s, 1H); 7.33 (d, J = 8.70Hz, 2H).

5,6-Dimethoxy-1-t4-methoxyphenyl) -2-methylindane
A solution of the above phenylindene (240 mg, 0.81 mmol) in 5 ml of AcOEt is hydrogenated under approximately 106 Pa (10 atm) of hydrogen, in the presence of 20 mg of palladium on carbon (at 10%) for 2 h. The catalyst is removed by filtration, then the filtrate is evaporated to dryness, to give the expected product pure (0.24 g, 99%).

Physical characteristics * 1H NMR (CDCl3): 0.69 (d, J = 6.96Hz, 3H); 2.58 (dd, J = 6.9614.30Hz, 1H); 2.80 (m, 1H); 2.96 (dd, J = 7.04-14.30Hz, 1H); 3.75 (s, 3H); 3.77 (s, 3H); 3.88 (s, 3H); 4.25 (d, J = 7.68
Hz, 1H); 6.63 (s, 1H); 6.80 (d, J = 8.78Hz, 2H); 6.81 (s, 1H); 6.88 (d, J = 8.78Hz, 2H).

* MS (IE): 298 (100), 283 (25), 267 (44), 252 (7); 238 (12), 167 (42), 149 (42), 135 (33); 84 (62) 5,6-Dihydroxy-1- (4-hydroxyphenyl) -2-methylindane
To a solution of the previously obtained phenylindane (0.23 g, 0.77 mmol) in 2 ml of CH2C12, cooled to -700C, is added dropwise a solution of BBr3 in CH2C12 (1.0 m, 3.0 ml , 3.0 mmol). The red solution obtained is warmed to room temperature and stirred for 0.5 h at this temperature. The reaction mixture is poured into a mixture of ice water and AcOEt. The aqueous phase is saturated with NaCl, then extracted twice with AcOEt. The combined organic phases are washed with a saturated aqueous solution of Nazi, dried over MgSO4 and evaporated to dryness, to give a yellow oil (0.20 g) which is chromatographed (eluent: AcOEt / hexane = 1: 1), to yield the expected compound (0.18 mg, 91%).

Physical characteristics * 1H NMR (acetone-d6): 0.63 (d, J = 6.96Hz, 3H); 2.45 (dd,
J = 7.32-14.22Hz, 1H); 2.68 (m, 1H); 2.84 (dd, J = 7.04-14.22
Hz, 1H); 4.13 (d, J = 7.42Hz, 1H); 6.50 (s, 1H); 6.73 (m, 5H); 7.53 (s, 1H); 7.55 (s, 1H); 8.06 (s, 1H).

* SM (IE): 256 (100), 241 (23), 239 (46), 227 (27)
Example 4: Preparation of 1- (4-dimAthylaminophinyl) - 5,6-dihydroxyindane (BXT01049) 5, 6-Dihydroxyindan-1-one
5,6-Dimethylindan-1-one (3.13 g; 16.3 mmol) is dissolved in 10 ml of dichloromethane dried over a 3A sieve. To this light yellow solution is added, at -700C, a solution of boron tribromide in dichloromethane (1M; 28 ml, 28 mmol), dropwise. The reaction medium turns burgundy. The temperature of the medium is brought back to room temperature, then the solution is stirred for 2 h 30 min. The reaction medium is poured into 50 g of ice, then extracted with ethyl acetate. The organic phases are washed with a saturated aqueous solution of
NaCl, then dried over MgSO4. The solvent is evaporated off under reduced pressure.

The crude product (2.3 g; 87%) is used as it is for the next step.

5,6-Di (terbutyldimethylsilyloxy) indan-1-one
To a solution of 5,6-dihydroxyindan-1-one, previously described, (2 g; 12.2 mmol) in 40 ml of DMF dried on a 3A sieve, cooled to OOC, is added terbutyldimethylsilyl chloride (7, 35 g; 49.8 mmol) and imidazole (3.82 g; 56.1 mmol). The temperature is maintained for 10 min at OOC, then 16 h at room temperature. The product is extracted with ethyl acetate. The organic phase is washed with a saturated aqueous NaCl solution, then dried over MgSO4. The solvent is evaporated off under reduced pressure. The desired product is purified by chromatography with an eluent: hexane / ethyl acetate (8: 2) and obtained in the form of a beige solid (4.5 g; 94%).

Physical characteristics * 1H NMR (CDCl3): 0.195 (s, 6H); 0.205 (s, 6H); 0.92 (s, 18H); 2.60 (t, J = 7.63Hz, 2H); 2.95 (t, J = 7.63Hz, 2H); 6.85 (s, 1H); 7.15 (s, 1H).

5. 6-Di (terbutyldimethylsilyloxy) -3- (4-dimethylaminophenyl) indene
Magnesium (124 mg, 5.1 mmol) is covered with 1 ml of distilled THF and heated to 600 ° C. with stirring. A solution of 4-bromo-N, N-dimethylaniline (1.02 g, 5.1 mmol) in 5 ml of distilled THF is added dropwise over 20 min. The reaction mixture is heated under reflux of THF for 1 h. To this yellow-green solution, a solution of 5, 6-di (terbutyldimethylsilyloxy) indan-1-one (2 g, 5.1 mmol) in 10 ml of distilled THF is added, dropwise, over 5 min, at temperature. ambient. The medium is brought to reflux for 20 h. the reaction mixture, of violet color, is hydrolyzed with a saturated aqueous solution of ammonium chloride, then extracted with ethyl acetate. The organic phase is washed with a saturated aqueous NaCl solution, then dried over MgSO4. the solvent is evaporated off under reduced pressure. The desired product is purified by chromatography with an eluent: hexane / ethyl acetate (9/1) and obtained in the form of an oil (0.9 g; 36%).

Physical characteristics * 1H NMR (CDCl3): 0.16 (s, 12H); 0.95 (s, 18H); 2.94 (s, 6H); 3.50 (d, J = 2.03Hz, 2H); 6.30 (t, J = 2.03Hz, 1H); 6.75 (m, 2H); 6.95 (s, 1H); 7.08 (s, 1H); 7.45 (d, J = 7.85Hz, 2H).

5,6-Di (terbutyldimethylsilyloxy) -1- (4-dimethylaminophenyl) - indane
A solution of the preceding compound (0.9 g; 1.82 mmol), in 10 ml of methanol and 2 ml of acetone, is stirred in the presence of palladium on charcoal (10%, 60 mg), at room temperature under hydrogen pressure (5 bars), for 3 h. The suspension is filtered, then the filtrate is evaporated to dryness under reduced pressure. The desired product is purified by chromatography with an eluent: hexane / ethyl acetate (9/1) and obtained in the form of a light beige solid (0.64 g; 71%).

Physical characteristics * 1H NMR (CDCl3): 0.08 (s, 6H); 0.18 (s, 6H); 0.90 (s, 9H); 0.95 (s, 9H); 1.90 (m, 2H); 2.45 (m, 2H); 2.90 (s, 6H); 4.17 (t, J = 8.07Hz, 1H); 6.4 (s, 1H); 6.7 (m, 2H); 7.05 (m, 2H); 7.20 (s, 1H).

5,6-Dihydroxy-1- (4-dimethylaminophenyl) indane
The product obtained previously (0.56 g; 1.12 mmol) is dissolved in 5 ml of distilled THE, then added with a molar solution of tetrabutylammonium fluoride (2.26 ml; 2.26 mmol) in THF, with stirring and at room temperature. Stirring is maintained for 1 h at this temperature. After addition of water, the product is extracted with ethyl acetate. The organic phase is washed with a saturated aqueous NaCl solution, then dried over
MgSO4. The solvent is evaporated off under reduced pressure. The desired product is purified by double chromatography 1st purification: eluent: hexane-ethyl acetate (6/4) 2nd purification: eluent: dichloromethane-methanol (95/5).

The desired product is obtained in the form of a purple oil (190 mg; 62%).

Physical characteristics * 1H NMR (CDCl3): 1.96 (m, 2H); 2.44 (m, 2H); 2.89 (s, 6H); 4.1 (t, J = 7.2Hz, 1H); 6.4 (s, 1H); 6.73 (d, J = 6.7Hz, 2H); 6.77 (s, 1H); 7.05 (d, J = 6.7Hz, 2H).

* SM (IE): 269 (100), 254 (10), 240 (9), 225 (28), 77 (10).

* HPLC (high performance liquid chromatography): (column: reverse phase RP18; length: 15 cm; eluent dichloromethane-methanol (94/6); flow rate: 1 ml / min, observation wavelength: 254 nm); retention time: 2.37 min; purity: 99.1 t.

B. Compounds of series II
Example 5: Preparation of 2,5,6-trihydroxy-1- (4 hydroxyphenyl) indane (BXT01035) 2-Hydroxy-5,6-dimethoxy-1- (4-methoxyphenyl) indane
To a solution of 5,6-dimethoxy-3- (4-methoxyphenyl) indene (1.0 g, 3.55 mmol) in 10 ml of anhydrous THF is added to OOC a solution of BH3.THF (1, 0 M, 4.26 mL, 4.26 mmol), in 15 min. The mixture obtained is stirred for 1 h at the same temperature then 4 h at room temperature.

The solution is cooled to OOC and 3.5 ml of water are added dropwise, then 7 ml of NaOH <10%). Then a saturated aqueous solution of H2O2 (30%, 7 ml) was added and the mixture was stirred for 2 h. The mixture is extracted with 50 ml of AcOEt. The organic phase is washed with 10 ml of water, then dried over MgSO4. Evaporation of the solvent gives a white solid (1.16 g) which is purified by chromatography, eluting with an AcOEt / hexane mixture (1: 3), to give the expected product (white solid, 0.97 g, 91 ).

Physical characteristics * pF: 128.0-129.00C (AcOEt / hexane = 1: 4).

* 1H NMR (CDCl3): 1.96 (d, J = 4.68 Hz, 1H, exchangeable with
D2O); 2.84 (dd, J = 6.79, 15.39Hz, 1H); 3.21 (dd, J = 6.9215.39Hz, 1H); 3.73 (s, 3H); 3.79 tus, 3H); 3.87 (s, 3H); 4.05 (d, J = 6.23Hz, 1H); 4.39 (m, 1H); 6.46 (s, 1H); 6.81 (s, 1H); 6.87 (d, J = 8.64Hz, 2H); 7.07 (d, J = 8.64Hz, 2H).

* SM (IE): 300 (100), 282 (18), 257 (27), 239 (5), 165 (7), 121 (15).

2 5,6-Trihydroxy-1- (4-hydroxyphenyl) indane
Aluminum tribromide (2.67 g, 10.0 mmol) is mixed with 24 ml of ethanethiol. The above phenylindane (300 mg, 1.0 mmol) is added in a single portion. After 62 h at room temperature, the ethanethiol is evaporated off, then the residue is treated with 15 ml of OOC water. The mixture is extracted with AcOEt. The combined organic phases are washed with saturated aqueous NaCl solution, dried over Na2SO4 and evaporated to dryness to yield a yellow oil (250 mg). Purification by chromatography, using an AcOEt / hexane mixture (1: 1) as eluent, gives the expected product (white solid, 140 mg, 54%).

Physical characteristics * 1H NMR (acetone-d6): 2.68 (dd, J = 6.31-15.02 Hz, 1H); 3.06 (dd, J = 6.59-15.30Hz, 1H); 3.90 (d, J = 6.53Hz, 1H); 4.20 (d,
J = 5.28Hz, 1H); 4.29 (m, 1H); 6.30 (s, 1H); 6.68 (s, 1H); 6.76 (d, J = 8.47Hz, 2H); 6.96 (d, J = 8.47Hz, 2H); 7.57 (s, 1H); 7.63 (s, 1H); 8.18 (s, 1H).

* SM (IE): 258 (100), 240 (40), 229 (13), 215 (39), 183 (27), 165 (32), 107 (46), 77 (28)
Example 6: Preparation of 5,6-dihydroxy-1- (4-hydroxyphenyl) - 2 -methoxyindane (BXT01040) 2,6-Dihydroxy-3- (4-hydroxyphenyl) indene
Has a solution of 5,6-dimethoxy-3- (4methoxyphenyl) indene (0.60 g, 2.23 mmol) in 2 ml of
CH2Cl2, cooled to -700C, a solution of BBr3 in CH2Cl2 (1.0 M, 9.60 ml, 9.60 mmol) is added dropwise. The red solution obtained is warmed to room temperature and stirred for 1 h at this temperature. The reaction mixture is poured into a mixture of ice water and AcOEt. The aqueous phase is saturated with NaCl, then extracted twice with AcOEt. The combined organic phases are washed with a saturated aqueous solution of NaCl, dried over MgSO4 and evaporated to dryness, to give a red solid (0.62 g) which is used directly in the next step.

Physical characteristics * 1H NMR (acetone-d6): 3.28 (d, J = 2.10Hz, 2H); 6.31 (t,
J = 2.10Hz, 1H); 6.88 (d, J = 8.45Hz, 2H); 7.03 (s, 1H); 7.07 (s, 1H); 7.44 (d, J = 8.45Hz, 2H); 7.72 (br s, 2H); 8.53 (br s, 1H).

* SM (IE): 240 (96), 222 (7), 194 (32), 181 (15), 165 (27), 152 (14), 43 (100).

5,6-Dibenzyloxy-1- (4-benzyloxyphenyl) indene
The crude product from the previous step (0.62 g) is dissolved in 8 ml of acetone. K2CO3 (1.85 g, 13.38 mmol) and benzyl bromide (1.73 g, 10.0 mmol) are added thereto. The mixture is heated at reflux for 18 h. The solid is filtered off, then the filtrate is evaporated to dryness. The residue is directly chromatographed, eluting with a mixture
AcOEt / hexane (1: 5), to give the expected product (0.82 g) which is used directly in the following step.

Physical characteristics * 1H NMR (CDCl3): 3.36 (d, J = 1.83Hz, 2H); 5.11 (s, 2H); 5.13 (s, 2H); 5.17 (s, 2H); 6.37 (t, J = 1.83Hz, 1H); 7.01 (d, J = 8.72Hz, 2H); 7.17 (s, 1H); 7.25-7.60 (m, 18H).

5,6-Dibenzyloxy-1- (4-benzyloxyphenyl) -2-hydroxyindane
To a solution of the above phenylindene (0.82 g, 1.61 mmol) in 8 ml of anhydrous THF is added, at room temperature, a solution of BH3.THF Hz THF (1.0 M, 3.20 ml, 3.20 mmol), over 15 min. The mixture obtained is stirred for 3.5 h at room temperature. The solution is cooled to 0 ° C. and 3.5 ml of water are added dropwise, then 7 ml of NaOH <10%). Then an aqueous solution of H2O2 <30%, 7 ml) was added and the mixture was stirred for 2 h. The mixture is extracted with AcOEt. The organic phase is washed with water, then dried over MgSO4. Evaporation of the solvent gives a colorless oil (0.95 g) which is purified by chromatography, eluting with an AcOEt / hexane mixture (1: 4), to give the expected product (white solid, 0.46 g, 54 ).

Physical characteristics * 1H NMR (CDCl3): 2.02 (broad s, exchangeable with D20, 1H); 2.81 (dd, J = 7.00-15.26Hz, 1H); 3.17 (dd, J = 6.64-15.26Hz, 1H); 4.03 (d, J = 6.28Hz, 1H); 4.34 (m, 1H); 5.01 (s, 2H); 5.06 (s, 2H); 5.15 (s, 2H): 6.57 (s, 1H); 6.86 (s, 1H); 6.92 (d, J = 8.72Hz, 2H) -; 7.40 (d, J = 8.72Hz, 2H); 7.24-7.50 (m, 15H) * MS (CI, NH3): 546 (MNH4 +).

5, 6-Dibenzyloxy-1- (4-benzyloxyphenyl) -2-methoxyindane
The above alcohol (0.26 g, 0.49 mmol) is mixed with powdered potassium hydroxide (0.11 g, 1.97 mmol). To this is added 3 ml of DMSO followed immediately by methyl iodide (0.14 g, 9.85 mmol). The mixture is stirred at room temperature for 3 h. 5 ml of water are added. The mixture is extracted with AcOEt. The combined organic phases are washed with a saturated aqueous solution of
NaCl, dried over MgSO4 and evaporated to give a yellow oil. Purification of this oil by chromatography (eluent: AcOEt / hexane = 1: 9) provides the expected product (0.26 g, 97%).

Physical characteristics * 1H NMR (CDCl3): 2.85 (dd, J = 7.01-15.35Hz, 1H); 3.23 (dd,
J = 6.78-15.35Hz, 1H); 3.38 (s, 3H); 4.04 (m, 1H); 4.12 (d,
J = 6.25Hz, 1H); 5.03 (s, 2H); 5.08 (s, 2H); 5.14 (s, 2H); 6.60 (s, 1H); 6.88 (s, 1H); 6.93 (d, J = 8.64Hz, 2H); 7.10 (d, J = 8.64Hz, 2H); 7.30-7.60 (m, 15H).

5,6-Dihydroxy-1- (4-hydroxyphenyl) -2-methoxyindane
A solution of the preceding compound (0.26 g, 0.48 mmol) in 10 ml of AcOEt is hydrogenated under approximately 105 Pa (1 atm) of hydrogen in the presence of palladium on carbon (at 10%, 10 mg). The reaction is completed after 12 h. The catalyst is filtered off, then the solvent is evaporated off under reduced pressure to give a yellow oil. This crude product is purified by chromatography (eluent: AcOEt / hexane = 1: 4), to provide the expected product (white solid: 92 mg, 71%).

Physical characteristics * 1H NMR (acetone-d6): 2.68 (dd, J = 6.15-15.62 Hz, 1H); 3.15 (dd, J = 6.25-15.62Hz, 1H); 3.24 (s, 3H); 4.01 (m, 1H); 4.04 (d, J = 6.25Hz, 1H); 6.29 (s, 1H); 6.70 (s, 1H); 6.76 (d,
J = 8.24Hz, 2H); 6.98 (d, J = 8.24Hz, 2H), 7.62 (br s, 2H); 8.17 (br s, 1H).

* SM (IE): 272 (100), 240 (81), 227 (18).

C. Compounds of Series III
Example 7: Preparation of 5,6,6a, 11b-tetrahydro- 3,9,10-trihydfoxybenzolc] fluorene (BXT01050) 2- (3,4-Dimethoxybenzylidene) -6-methoxy-1-tetralone
6-methoxy-1-tetralone (7.04 g, 40 mmol) and 3,4-dimethoxybenzaldehyde (6.44 g, 40 mmol) are mixed with 100 ml of absolute ethanol. Stirred for 0.5 h to dissolve the maximum of reagents. We begin to introduce gaseous hydrogen chloride. An exothermic reaction is observed which makes it possible to completely dissolve all the reactants. The flask is cooled from time to time with an ice bath so that the temperature does not exceed 800C.

The mixture is thus saturated with hydrogen chloride after 1 h to give an intense red color. This mixture is left to stand at room temperature for 2 h. The reaction mixture is poured into a mixture of 400 ml of water and 40 ml of aqueous NaOH (3N). Vigorously stirred for 3 h at room temperature. The precipitated solid is filtered off on sintered glass, washed with water and finally dried under vacuum, over P2O5, to give a solid (12.35 g, 95%).

A small amount of product is recrystallized from AcOEt / hexane = 1: 2.

Physical characteristics * 1H NMR (CDCl3): 2.89 (t, J = 6.96Hz, 2H); 3.12 (dt, J = 1.706.96Hz, 2H), 3.85 (s, 3H); 3.89 (s, 3H); 3.90 (s, 3H); 6.68 (d, J = 2.48Hz, 1H); 6.85 (dd, J = 2.48-8.67Hz, 1H); 6.88 (d,
J = 8.14Hz, 1H); 6.95 (d, J = 1.64Hz, 1H); 7.04 (dd, J = 1,648.14Hz, 1H); 7.78 (s, 1H); 8.08 (d, J = 8.67Hz, 1H).

* SM (IE): 324 (87), 323 (100), 309 (38), 293 (39), 281 (11).

* mp: 109.0-110.00C (AcOEt / hexane = 1: 2).

2- (3,4-Dimethoxybenzyl) -6-methoxy-1-tetralone
2.0 g of palladium on carbon (10%) are added to a solution of the preceding compound (35.0 g, 0.107 mol) in 550 ml of AcOEt. The mixture is hydrogenated under approximately 2.105 Pa (2 atm) of hydrogen for 1 H. The catalyst is removed by filtration and the filtrate is evaporated to dryness, to give 38.4 g of a crude product which is dissolved hot in 100 ml of AcOEt. 130 ml of hexane are added. The crystals formed are filtered off (23.56 g). The filtrate is evaporated to dryness. The residue is chromatographed eluting with a mixture
AcOEt / hexane (1: 4), to give 6.03 g of the expected product, pure.

Yield: 29.59 g (84%).

Physical characteristics pF: 101.0-102.00C (AcOEt / hexane = 1: 2).

* 1H NMR (CDCl3): 1.67-1.83 (m, 1H); 2.00-2.14 (m, 1H); 2.51-2.72 (m, 2H); 2.84-2.96 (m, 2H); 3.34-3.48 (m, 1H); 3.82 (s, 3H); 3.84 (s, 3H); 3.85 (s, 3H); 6.64 (d, J = 2.36
Hz, 1H); 6.74-6.84 (m, 4H); 8.02 (d, J = 8.74Hz, 1H).

* SM (IE): 326 (35), 175 (9), 151 (100), 107 (13), 91 (17), 77 (11).

5,6-Dihydro-3,9,10-trimethoxybenzorclfluorene
15.0 g of polyphosphoric acid are mixed with the tetralone obtained previously (1.04 g, 3.2 mmol), then heated at 80-900C for 4 h. After cooling the mixture to room temperature, 10 g of ice-cold water and 20 ml of AcOEt are added. The organic phase is separated and the aqueous phase is extracted with AcOEt. The combined organic phases are washed with a saturated aqueous solution of NaHCO3 then with NaCl (sat.), Dried over MgSO4 and evaporated to dryness, to give a solid (0.91 g) which is purified by chromatography, eluting with a mixture.
AcOEt / hexane (1: 6). The expected product (0.61 g) and the starting product (0.24 g) are obtained. the yield of this reaction, relative to the product consumed, is 81%.

Physical characteristics * 1H NMR (CDCl3): 2.59 (t, J = 7.77 Hz, 2H); 2.89 (t, J = 7.77
Hz, 2H); 3.39 (s, 2H); 3.84 (s, 3H); 3.90 (s, 3H); 3.94 (s, 3H); 6.84 (m, 2H); 7.08 (s, 1H); 7.38 (s, 1H); 7.72 (d,
J = 9.24Hz, 1H).

* mp: 146.5-147.00C (AcOEt / hexane = 1: 4).

* MS (IE): 308 (100), 293 (14), 277 (38), 202 (24), 176 (33), 165 (17), 152 (21) 5,6, 6a1lb-Tetrahydro-3 9 , 10-trimethoxybenzoFcl fluorene
0.15 g of palladium on charcoal (10%) is added to a solution of the preceding product (0.90 g, 2.92 mmol) in 30 ml of AcOEt. The mixture is hydrogenated under a pressure of approximately 8.105 Pa (8 atm) of hydrogen for 5 h. The catalyst is removed by filtration and the filtrate is evaporated to dryness, to give the expected product, pure (0.90 g, 99%).

Physical characteristics * mp: 77.0-79.00C (AcOEt / hexane = 1: 4).

* 1H NMR (CDCl3): 1.45-1.65 (m, 1H); 1.66-1.80 (m, 1H); 2.75 (dd, J = 6.96-15.10Hz, 1H); 2.68-2.87 (m, 3H); 3.15 (dd,
J = 6.96-15.10Hz, 1H); 3.79 (s, 3H); 3.80 (s, 3H); 3.84 (s, 3H); 4.21 (d, J = 6.68Hz, 1H); 6.66 (d, J = 2.64Hz, 1H); 6.76 (s, 1H); 6.78 (s, 1H); 6.83 (dd, J = 2.64-8.38Hz, 1H); 7.33 (d, J = 8.38Hz, 1H).

* SM (IE): 310 (100), 295 (23), 279 (46), 267 (15), 189 (24), 121 (23).

5 6r6at11b-Tetrahydro-3,9,10-trihydroxybenzorclfluorene
To a solution of the compound obtained previously (0.57 g, 1.84 mmol) in 4 ml of CH2Cl2, cooled to -700C, a solution of BBr3 in CH2Cl2 (1.0 M, 6.5 ml is added dropwise). , 6.5 mmol). The red solution obtained is warmed to room temperature and stirred for 0.5 h at this temperature. The reaction mixture is poured into a mixture of ice water and AcOEt. The aqueous phase is saturated with NaCl, then extracted twice with AcOEt. The combined organic phases are washed with saturated aqueous NaCl solution, dried over MgSO4 and evaporated to dryness, to give a yellow oil (0.50 g). Chromatography of 0.28 g of this oil (eluent AcOEt / hexane = 1: 1) allows the expected compound to be isolated (0.24 g, 87%).

Physical characteristics * 1H NMR <acetone-d6): 1.46 (m, 1H); 1.61 (m, read); 2.46-2.81 (m, 4H); 3.01 (dd, J = 7.00, 14.98Hz, 1H); 4.05 (d, J = 6.68
Hz, 1H); 6.54 (d, J = 2.54Hz, 1H); 6.66 (s, 1H); 6.68 (s, 1H); 6.71 (dd, J = 2.54, 8.34Hz, 1H); 7.18 (d, J = 8.34Hz, 1H); 7.50 (br s, 2H); 7.83 (br s, 1H).

* SM (IE): 268 (82), 251 (31), 240 (100), 161 (21), 145 (26), 77 (30)
Example 8: Preparation of 5,6,6a, 7,8,12b-hexahydro 2,3,10-trihydroxy-benzo [c] phenanthrene (BXT010051) 2 '- (3,4-Dimethoxyphenyl) tetral-6-methoxy- 2-spiro-1'cyclopropan-1-one A a mixture of trimethylsulfoxonium iodide (4.40 g, 20 mmol) and sodium hydride (80% in mineral oil, 0.80 g, 20 mmol) are added 10 ml of
DMSO. The mixture is stirred. at room temperature for 1.5 h. 30 ml of DMSO are added, then the 2- (3,4-dimethoxybenzylidene) -6-methoxy-1-tetralone described above is added in small portions over 15 min. The mixture is stirred for 18 h at room temperature and then hydrolyzed by the addition of 50 ml of a saturated aqueous solution of NH4Cl.

The mixture is extracted with AcOEt. The combined organic phases are washed with water, dried over MgSO4 and evaporated under reduced pressure, to give a colorless oil (7.0 g). Purification of this oil by chromatography, eluting with an AcOEt / hexane mixture (1: 4), provides the desired product (5.41 g, 85%).

Physical characteristics * 1H NMR (CDCl3): 1.26 (dd, J = 4.24-7.08Hz, 1H); 1.68 (m, 1H); 1.85 (m, 2H); 2.70 (m, 2H); 2.94 (dd, J = 7.08-8.24Hz, 1H); 3.83 (s, 3H); 3.85 (s, 3H); 3.86 (s, 3H); 6.64 (d,
J = 2.52Hz, 1H); 6.77 (m, 3H); 6.82 (dd, J = 2.52-8.50Hz, 1H); 8.00 (d, J = 8.72Hz, 1H).

* MS (IE): 338 (100), 323 (12), 200 (20), 176 (19), 151 (73), 91 (29), 77 (51) 2- (3g4-Dimethoxyphenylethyll-6-methoxy -1-tetralone
A solution of the cyclopropanic derivative obtained previously (1.0 g, 2.96 mmol) and of paratoluenesulfonic acid (300 mg) in 20 ml of CH 2 Cl 2 is stirred at room temperature for 5 h. This solution is then washed with a saturated aqueous solution of
NaHCO3, dried over MgSO4 and evaporated under reduced pressure to give a yellow oil (1.05 g). Purification of this product by chromatography (eluent: AcOEt / hexane = 1: 4) gives a solid (0.57 g) which is a mixture of the two isomeric alkenes.

0.53 g of the latter mixture is taken up in 15 ml of AcOEt. The solution is hydrogenated, in the presence of palladium on carbon <10%, 50 mg), under a pressure of 2 bars of hydrogen for 1 h. The catalyst is filtered off and the filtrate is evaporated off under reduced pressure, to give the expected product (0.53 g, 57%).

Physical characteristics * 1H NMR (CDCl3): 1.80 (m, 2H); 2.24 (m, 2H); 2.42 (m, 1H), 2.66 (m, 2H); 2.92 (m, 2H); 3.81 (s, 3H); 3.82 (s, 3H); 3.84 (s, 3H); 6.64 (d, J = 2.36Hz, 1H); 6.77 (m, 3H); 6.79 (dd,
J = 2.36-8.80Hz, 1H); 7.97 (d, J = 8.72Hz, 1H).

* MS (IE): 340 (2.5), 176 (100), 164 (16), 151 (12).

5,6,7,8-Tetrahydro-2,3,10-trimethoxy-benzo rcl phenanthrene.

8 g of polyphosphoric acid are mixed with the tetralone obtained previously (0.41 g, 1.21 mmol), then heated at 80-900C for 2.5 h. After cooling the mixture to room temperature, 20 g of ice-cold water and 20 ml of AcOEt are added. The organic phase is decanted and the aqueous phase is extracted with AcOEt. The combined organic phases are washed with a saturated aqueous solution of NaHCO3 and then with a saturated aqueous solution of NaCl, dried over MgSO4 and evaporated to dryness to give a solid (0.38 g) which is purified by chromatography, eluting with an AcOEt / mixture. hexane (1: 9). The expected product is obtained (0.19 g; 49%).

Physical characteristics * 1H NMR (CDCl3): 2.31 (m, 4H); 2.66 (m, 4H); 3.80 (s, 3H); 3.82 (s, 3H); 3.89 (s, 3H); 6.77 (m, 3H); 7.08 (s, 1H); 7.44 (d, J = 8.42Hz, 1H).

* MS (IE): 322 (100), 307 (10), 291 (19), 189 (18), 161 (18) 5,6,6a, 7,8,12b-Hexahydro-2,3,10- trimethoxy-benzorcl phenanthrene
To a solution of the preceding product (170 mg, 0.50 mmol) in 10 ml of AcOEt, are added 20 mg of palladium on charcoal (at 10%). The mixture is hydrogenated under a pressure of 8.105 Pa, approximately (8 atm) for 2.5 h. The catalyst is removed by filtration and the filtrate is evaporated to dryness to give an oil. This oil is purified by chromatography, eluting with a mixture
AcOEt / hexane (1: 9), to give the desired product (150 mg, 88%).

Physical characteristics * 1H NMR (CDCl3): 1.53 (m, 2H); 1.81 (m, 1H); 2.00 (m, 1H); 2.32 (m, 1H); 2.75 (m, 4H); 3.77 (s, 3H); 3.78 (s, 3H); 3.81 (1H, hidden by OMe); 3.85 (s, 3H); 6.65 (m, 4H); 6.99 (d, J = 9.16Hz, 1H).

* SM (IE): 324 (100), 309 (5), 296 (33), 293 (37), 203 (36), 189 (86), 173 (39) 5,6,6a, 7,8, 12b-Hexahydro-2,3,10-trihydroxy-benzo rc1 phenanthrene
To a solution of the derivative obtained previously (150 mg, 0.37 mmol) in 1 ml of CH2C12, cooled to -700C, is added dropwise a solution of BBr3 in CH2C12 (1.0 M, 1.5 ml, 1 , 5 mmol). The red solution obtained is warmed to room temperature and stirred for 0.5 h at this temperature. The reaction mixture is poured into a mixture of ice water and AcOEt. The aqueous phase is saturated with NaCl, then extracted twice with AcOEt. The combined organic phases are washed with a saturated aqueous solution of NaCl, dried over MgSO4 and evaporated to dryness, to give a yellow oil (0.18 g). Chromatography of this oil (eluent: AcOEt / hexane = 1: 1 ) makes it possible to isolate the expected compound (91 mg, 87%).

Physical characteristics * 1H NMR (acetone -d6): 1.53 (m, 2H); 1.90 (m, 2H); 2.32 (m, 1H); 2.75 (m; 4H); 3.67 (d, J = 4.76Hz, 1H); 6.48 (s, 1H); 6.55 (s, 1H), 6.59 (m, 2H); 6.88 (d, J = 8.88Hz, 1H); 7.51 (br s, 2H); 7.95 (br s, 1H).

* MS (IE): 282 (100), 265 (16), 254 (53), 175 (26), 161 (36), 115 (31), 91 (22), 77 (24) II Preparation of compounds of general formula II ("mercaptan scavenging reagents11)
Example 9: Preparation of 1,4,6trimethyl-2-vinylpyridinium tetrafluoroborate
Synthesis of 4,6-dimethyl-2-vinylpyridine
Benzoyl chloride (29.12 g; 0.186 mol) is added to a solution of 2,4-dimethylpydirine (20 g; 0.186 mol; LANCASTER) in 200 ml of freshly distilled THF, cooled to -200 ° C. and under an inert atmosphere. ; JANSSEN) in 100 ml of THF. The reaction mixture is stirred at this temperature for 20 minutes. Then vinylmagnesium bromide (186 ml of a 1.0 M solution in THF) is added and the solution is stirred at room temperature for 0.5 h. The solvent is evaporated off under reduced pressure and the residue is taken up in 300 ml of terbutylmethyl ether. After washing the reaction medium with saturated NH4Cl solution, the organic phase is dried over MgSO4 and the desired product is purified by chromatography on a MERCK F254 silica column (eluent = hexane / AcOEt, 9: 1).

Yield = 39.5 g (83%), colorless oil.

The above product is taken up in 500 ml of toluene and added with p-chloranil (tetrachloro-1,4-benzoquinone) (41.7 g; 0.169 mol), then heated at reflux for 2 h. The reaction medium is concentrated under reduced pressure, then added with a sodium hydroxide solution (260 ml, 2N). The product is extracted with terbutylmethyl ether (300 ml) and purified by chromatography on a MERCK F254 silica column (eluent = hexane / AcOEt, 9: 1)
Yield = 11.1 g (54.2%), pale yellow oil.

Physical characteristics
* 1H NMR (200 MHz, CD3COCD3)
2.20 ppm (s, 3H); 2.50 ppm (s, 3H); 5.25-5.45 ppm (dd,
2H, J = 11.9-18.7Hz); 6.60-6.90 ppm (m, 3H).

* MS (DIC, NH3): MH + = 134 (100).

DIC = desorption by chemical ionization.

Synthesis of 1,4,6-trimethyl-2-vinylpyridinium tetrafluoroborate:
To a solution of 4,6-dimethyl-2-vinylpyridine (0.61 g; 4.13.10-3 mol) in 4.0 ml of methylene chloride under an inert atmosphere is added, in one portion, trimethyloxonium tetrafluoroborate ( 0.50 g; 3.76.10-3 mol). The suspension is left to react for 2 h at 250C.

The suspension is filtered and the product is dried under vacuum.

Yield = 0.162 g (18%), white crystals.

Physical characteristics
* 1H NMR (200 MHz, CD3COCD3)
2.60 ppm (s, 3H); 2.85 ppm (s, 3H); 4.20 ppm (s, 3H);
6.00 ppm (d, 1H, J = 12.8Hz); 6.25 ppm (d, 1H,
J = 19.2Hz); 7.27 ppm (dd, 1H, J = 12.8-19.2Hz); 7.82 ppm
(s, 1H); 7.95 ppm (s, 1H).

* MS (IE, 70eV): (M +) 148 (10); 147 (18); 133 (34);
121 (100); 106 (25); 91 (31); 77 (52); 49 (77).

* Microanalysis
Calculated (Jii): C 51.10; H 6.00; N 6.00
Found (%): C 49.80; H 6.08; N 5.96.

Example 10: Preparation of 1-methyl-4-vinylquinolinium tetrafluoroborate
Synthesis of 4-vinylquinoline
A solution of 4-quinolinecarboxaldehyde (5 g; 3,18.10-2 mol) in 32 ml of benzene, under an inert atmosphere, containing triphenylmethylphosphonium iodide (26 g; 6.36.10-2 mol) is added to a solution sodium hydroxide (96 ml; 5N). The two-phase system is stirred at 450C for 30 minutes. After decantation, the aqueous phase is extracted with ethyl acetate (3x100 ml). The organic phases are combined and washed with a 1N HCl solution (2x30 ml), then the aqueous phase is neutralized with NaHCO3 and extracted with methylene chloride (3x100 ml). The organic phase is dried over MgSO4. The desired product is purified by chromatography on a MERCK F254 silica column (eluent = cyclohexane / AcOEt, 8: 2).

Yield = 2.8 g (60%).

Physical characteristics
* 1H NMR (200 MHz, CDCl3)
5.60 ppm (d, 1H, J = 10Hz); 5.95 ppm (d, 1H,
J = 14Hz), 7.30-7.55 ppm (m, 4H); 7.60-7.75 ppm (t, 1H,
J = 4Hz), 8.07 ppm (t, 2H, J = 6Hz); 8.85 ppm (d, 1H,
J = 4 Hz).

Synthesis of 1-methyl-4-vinyl quinolinium tetrafluoroborate
To a solution of 4-vinylquinoline (0.72 g; 4.64.10-3 mol) in an acetonitrile / acetone (2.5 / 1) mixture is added under an inert atmosphere, in one portion, trimethyloxonium tetrafluoroborate (1, 58 g; 10.7.10-3 mol). The suspension is left to react for 1 h at 250C.

The solvent is evaporated off under reduced pressure, the residue is taken up in 2-propanol (25 ml) and then stirred for 30 minutes at room temperature. After addition of ethyl ether (25 ml), the suspension is filtered. The desired product is dried under vacuum.

Yield = 0.93 g (60.4%).

Physical characteristics
* 1H NMR (200 MHz, CD3COCD3)
4.30 ppm (s, 3H); 6.25 ppm (d, 1H, J = 12.5Hz); 6.65 ppm
(d, 1H, J = 17.5Hz); 7.85-8.00 ppm (dd, 1H, J = 12.5
17.5 Hz); 8.10 ppm (d, 1H, J = 7Hz); 8.35 ppm (dd, 2H,
J = 10-10 Hz); 8.57 ppm (d, 1H, J = 10Hz); 8.72 ppm (d,
1H, J = 10Hz); 9.35 ppm (d, 1H, J = 7Hz).

* MS (FAB) glycerol-NOBA: positive ion M + = 170 (100)
negative ion M- = 87 87 (100).

FAB = fast atom bombardment (Fast Atom
Bombardment).

NOBA = meta-nitrobenzyl alcohol.

* Microanalysis
Calculated (%): C 56.00; H 4.60; N 5.40.

Found (%): C 55.56; H 4.35; N 4.12.

III. Examples of applications
The operating protocols described below are examples of application of the assay method using respectively the BXT reagents 01041 (described in example 1), 01048 (described in example 3), 01049 (described in example 4 ) and 01050 (described in Example 7), to measure the activity
SOD in erythrocyte lysate.

Example 11: Assay procedure using the reagent
BXT 01041
A box containing the reagents described below is used. This reagent kit should be stored at a temperature between 0 and 40C.

Description of reagents R1: BXT 01041 2.10-3 M solution in the mixture
DMSO / H2O, 50/50 (v / v) containing boric acid
0.15 M.

(Keep between 0 and 40C during measurements; stable
about 1 month after preparation, under the conditions
storage space mentioned above)
R2: 1,4,6-trimethyl tetrafluoroborate solution
2-vinylpyridinium 5.10-2 M in DMSO containing 25%
(w / v) ethylene glycol.

(Keep between 0 and 40C during measurements; stable
at least 1 month after preparation).

E: Absolute ethanol / chloroform, 62.5 / 37.5 (v / v).

(Store between 0 and 40C for the preparation of
sample).

T: 0.05 M AMPD / HCl buffer pH = 8.8, containing DTPA
0.1 mM.

(Let equilibrate in air and at 370C before measurements)
Preparation of the sample
At least 100 µl of whole blood is centrifuged at 40 ° C. and 3000 rpm for 10 minutes. The supernatant is removed.

The erythrocyte pellet is washed with 0.9% sodium chloride solution (cooled between 0 and 40C) and then centrifuged at 40C and 3000 rpm for 10 minutes. The supernatant is removed. The erythrocyte pellet is taken up with 4 times its volume of distilled water cooled between 0 and 40C and mixed.

To 125 μl of this erythrocyte lysate at 1 / 5th, on the one hand, and to 125 μl of distilled water, on the other hand, 200 μl of mixture E (cooled between 0 and 40C) are added. Each of the solutions is mixed well and centrifuged at 40 ° C. and 3000 rpm for 10 minutes. The supernatant thus obtained will be used for the assay, respectively for the sample (free of hemoglobin) and for the control.

Supernatants should be stored between 0 and 40C for measurements.

Dosing procedure
The measurements are carried out at 370C in a spectrophotometer, the measuring cells of which are thermostatically controlled.

The reaction mixture corresponding to each assay is prepared extemporaneously according to the proportions given in the table below for 1 ml tanks.

Each reaction is triggered by the addition of 10 µl of reagent R1. After homogenization of the medium, the change in absorbance is recorded immediately and followed for 1 min 30 s.

The absorbance measurement is carried out at 501 nm in cuvettes with an optical path of 1 cm, against air.

For each measurement, the reaction medium defined in the table below must be incubated for 1 minute at 370C before adding the reagent R1.

<tb>

<SEP> WITNESS <SEP> SAMPLE <SEP>
<tb><SEP> R2 <SEP> 20 <SEP> Rl <SEP> 20 <SEP> pil <SEP>
<tb> White
<tb> sample
<tb><SEP> * <SEP> 40 <SEP> 1 <SEP>
<tb> SAMPLES <SEP> 40 <SEP> pil <SEP>
<tb><SEP> T <SEP> 930 <SEP> pLl <SEP> 930 <SEP> Rl <SEP>
<tb> (*): Distilled water having undergone the same procedure
extraction than the erythrocyte lysate.

For each measurement, the maximum autoxidation rate, expressed in absorbance units per minute, is determined by subtracting the absorbance value recorded after 20 s from that recorded after 80 s, which corresponds to a linear oxidation phase, as a function of time, of the reagent BXT 01041.

Expression of results
Let VC and Vs be the speeds corresponding respectively to the control and to the sample, the SOD activity of the latter is calculated using the equation of the calibration line presented in FIG. 1 which represents the variations of the inverse of the difference (Vs-Vc) expressed in min / variation in absorbance (min / A abs.), as a function of the inverse of the concentration of SOD activity, expressed in ml / U. This straight line makes it possible to determine the value of the abscissa corresponding to an experimentally measured ordinate value.

The inverse of this abscissa is then multiplied by the dilution factor D.

The results are then expressed in units of SOD activity per ml of sample, according to
SOD activity units = 1 / {0.026 x [1 / (VS-Vc)] - 0.075} x D
The unit U of SOD activity defined in this method has the unique advantage of providing an invariable reference, given the absence of unstable protein or enzymatic reagents, such as xanthine oxidase.

The use of this unit is strongly recommended, but the user can translate it into a weight value of a purified SOD with which he will have previously constructed a calibration curve. Such a weight estimate will be strongly affected by the percentage of inactive enzyme present in the preparation.

The results can also be expressed, for example, in U / ml of whole blood or in U / mg of erythrocyte proteins.

Remarks
The control of the assay procedure can be carried out by assaying the SOD activity of an erythrocyte lysate prepared as described previously and stored at -700 ° C. as aliquots to serve as a control. The SOD activity of such aliquots is stable for at least 6 months at -700C.

The assay should be performed on a thawed aliquot extemporaneously.

Example 12: Assay procedure using the reagent
BXT 01048
Same as Example 11, except that
- In R1, reagent BXT 01041 is replaced by reagent BXT 01048.

- The change in absorbance is followed at 505 nm.

The attached FIG. 2 represents the variations of the inverse of the difference (Vs-Vc), expressed in min / variation of the absorbance (min / Nabs.) As a function of the inverse of the concentration of SOD activity, expressed in ml / U, in this example.

SOD activity units = 1 / {0.028 x [1 / (VS-Vc)] - 0.077} x D
Example 13: Assay procedure using the reagent
BXT 01049
Same as Example 11, except that
- R1 is replaced by R3 which is defined as follows
BXT 01049 2.10-3M solution in the mixture
50/50 (v / v) DMSO / H2O, containing 0.1M boric acid.

- The change in absorbance is followed at 522 nm for 1 min.

- The maximum autoxidation rate is obtained by subtracting the absorbance value recorded after 20 s from that recorded after 50 s.

The attached FIG. 3 represents the variations of the inverse of the difference (Vs-Vc), expressed in min / variation of the absorbance (min / A abs.) As a function of the inverse of the concentration of SOD activity, expressed in ml / U, in this example.

SOD activity units = 1 / (0.023 x [l / (VS-VC)] - 0.063} x D.

Example 14: Assay procedure using the reagent
BXT 01050
Same as Example 11, except that
- R1 is replaced by R4 which is defined as follows
BXT 01050 2.10-3 M solution in the mixture
50/50 (v / v) DMSO / H2O, containing 0.3M boric acid.

- The change in absorbance is followed at 525 nm for 1 min.

- The maximum autoxidation rate is obtained by subtracting the absorbance value recorded after 20 s from that recorded after 50 s.

The attached FIG. 4 represents the variations of the inverse of the difference (Vs-Vc), expressed in min / variation of the absorbance (min / A abs.) As a function of the inverse of the concentration of SOD activity, expressed in ml / U, in this example.

SOD activity units = 1 / {0.070 x [1 / (VS-VC)] - 0.102} x D.

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3. FRIDOVICH I. (1989), Superoxide dismutases; year
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4. KARLSSON K. and MARKLUND SL (1988), Extracellular
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5. CZAPSKI G. and GOLDSTEIN S. (1988), The ubiquiness both
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6. FRIDOVICH I. (1982b), Measuring the activity of
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7. FLOHE L. and OTTING F. (1984), Superoxide dismutase
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8. BANNISTER JV and CALABRESE (1987), Assays for
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9. FLOHE L. BECKER R., BRIGELIUS R., LENGFELDER E. and
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Claims (13)

1.- Compounds of general formula I

in which n = 1 or 2, R1 = -OR4 or -NR5R6, R2 = hydrogen, -OR4, alkyl (with 1 to 6 carbon atoms), or -CH2- or -CH2-CH2-, to form a ring by bond with the phenyl substituent, in the meta position compared to R1 and R3 = hydrogen, alkyl (with 1 to 6 carbon atoms) or -OR4 (provided that R2 is different from -OR4), with R4 = hydrogen or alkyl (with 1 to 6 carbon atoms), R5 = hydrogen, alkyl (with 1 to 6 carbon atoms), -CH2COOH, -C6HsCOOH or -C6HsSO3H and R6 = hydrogen, alkyl (with 1 to 6 carbon atoms) or -CH2COOH. 2.- Method for assaying superoxide dismutase (SOD) activity in a liquid medium, a method which uses the activation of the autoxidation of a reagent by the activity SOD, characterized in that the said reagent is a compound corresponding to the general formula I, defined in claim 1. 3.- Method for assaying the superoxide dismutase (SOD) activity in a liquid medium containing one or more mercaptan (s), in particular a biological medium, a method which uses the activation of the autoxidation of a reagent by the SOD activity , characterized in that said reagent is a compound corresponding to the general formula I defined in claim 1 and in that the medium also contains an amount, capable of completely trapping said mercaptans by S-alkylation, of at least one compound responding to the general formula He

in which Z1 = alkyl (with 1 to 6 carbon atoms), benzyl, p nitrobenzyl, phenyl, o, p-dinitrophenyl, -CH2-COOH or -CH2-CH2-COOH ;; Z4 = hydrogen and Z5 = hydrogen or alkyl (with 1 to 6 atoms of carbon), or Z4 and Z5 together form, with the two atoms of intermediate carbon, a phenyl ring; X = halide, sulfonate, fluorosulfonate, phospho nate, tetrafluoroborate or tosylate; and let z2 = vinyl and Z3 = hydrogen or alkyl (with 1 to 6 atoms of carbon); or Z3: vinyl, and z2 = hydrogen or alkyl (with 1 to 6 carbon atoms) 4. A method according to claim 3, characterized in that, in general formula II, X = trifluoromethylsulfonate. 5. A method according to any one of claims 2 to 4, characterized in that it is based on a spectrophotometric measurement of the autoxidation rate of a compound of general formula I, in the absence and in the presence of l sample to be assayed. 6.- A method of assaying superoxide dismutase (SOD) activity in a liquid sample, characterized in that it essentially comprises the steps of 10) determining the maximum rate of autoxidation Vs of a compound of general formula I in the presence of the sample, by means of the change in absorbance as a function of time, at the wavelength characterizing the appearance of the autoxidation product of the compound of general formula I used, in a buffered reaction medium at a pH value of 8.0 to 9.0, initiating the reaction by adding an aliquot of a stock solution of the compound of general formula I; 20) determining, under the same conditions, the maximum rate of autoxidation Vc of the same compound in the absence of the sample; and 30) determining the SOD concentration of the sample by means of the difference between the measurements taken and a calibration curve established under the same operating conditions. 7. A method according to any one of claims 2 to 6, characterized in that it comprises the addition of a boron derivative, in particular boric acid, to modulate the rate of autoxidation of the compound of general formula I. 8. A method according to any one of claims 2 to 7, characterized in that an aqueous solution of a compound of general formula I in the presence of a boron derivative, in particular boric acid, is used to stabilize the compound. compound of general formula I outside the measurement conditions. 9. A method according to any one of claims 6 to 8, characterized in that the measurement made under conditions of activation of the autoxidation of the compound of general formula I is combined with a measurement carried out under conditions of inhibition of this autoxidation, in a pH range of 7.2 to 7.8. 10. A method according to any one of claims 2 to 9, characterized in that the compound of general formula I is S, 6,6a, lîb-tetrahydro-3,9,10-trihydroxybenzo [c] fluorene. 11.- Kit or kit for carrying out the method according to any one of claims 2 to 9, characterized in that it essentially comprises, as reagent, a compound of general formula I. 12. A kit or kit according to claim 11, characterized in that it further comprises one or more compound (s) scavenger (s) of mercaptans of general formula II. 13.- Kit or kit according to claim 12, characterized in that it essentially comprises: - a compound of general formula I in solution or in powder, in the presence of boric acid or a derivative of this last, - one or more compound (s) trapping mercaptans of general formula II, in solution or powder, and - an AMPD-based buffer, buffering in the pH range from 8.0 to 9.0.