Patent Application
20140249105
The invention relates to novel ceramide analogues, processes for the preparation thereof and applications thereof in pharmaceutical and cosmetic compositions.
The ceramides represent a particular class of intraepidermal lipids naturally present in the skin and hair. They are formed from sphingosine or sphingosene, which combines with certain unsaturated fatty acids such as linoleic acid. They play a fundamental role in the structure of the epidermis and its functions, in particular in maintaining and controlling the hydration and cohesion of the stratum corneum. However, the content of ceramides in the skin varies under various conditions: it is higher when the epidermis is subjected to various aggressive factors such as injury, exposure to sunlight, and increased evaporation of water from the skin, and it decreases in the elderly and in subjects with atopic dermatitis.
Ageing of the subject therefore leads to a decrease thereof in the skin as well as the appearance of brown spots. The spots are treated with depigmenting products, in particular retinoic acid, azelaic acid, ascorbic acid and hydroquinone. All of these have side-effects. In particular, hydroquinone, one of the best-known depigmenting agents, leads to degeneration of collagen and elastin fibres, and has genotoxic and carcinogenic effects. It is now only used on medical prescription.
A subject of the present invention is novel cyclic diamides in which the two amide functions are carried by a ring comprising from 3 to 5 carbon atoms, based on the constituent elements of the lipid bilayers constituting the cell membranes, and processes for the preparation thereof.
Another purpose of the invention is the development of pharmaceutical and cosmetic preparations to take advantage of their biological effects, in particular as agents for combating ageing of the skin and as depigmenting agents.
A more particular subject of the invention is the compounds represented by general formula I shown below
in which
m=1, 2, 3 and n=0, 1
provided that m+n is different from 4,
X1 and X2 can be trans or cis relative to one another and represent, independently of one another, a group selected from
in which
in which δ varies from 1 to 12,
in which δ varies from 1 to 5,
in which
R4 represents a linear or branched alkyl group, comprising from 1 to 6 carbon atoms, in particular methyl, ethyl, isopropyl, tert-butyl, (OR4)2 optionally forming a ring between the two oxygen atoms, the (OR4)2 groups in particular originating from diols such as ethane-1,2-diol, propane-1,3-diol, 2,2-dimethylpropane-1,3-diol, 2,3-dimethylbutane-2,3-diol (pinacol), 2-methylbutane-2,3-diol, 1,2-diphenylethane-1,2-diol, 2-methylpentane-2,4-diol, 1,2-dihydroxybenzene (catechol), 2,2′-azanediyldiethanol, 2,2′-(butylazanediyl)diethanol, 2,3-dihydroxysuccinic acid (tartaric acid) and its esters, or (OR4)2 originates in particular from diacids such as 2,2′-(methylazanediyl)diacetic acid (mida),
A subject of the invention is the compounds in which X1 and X2 are identical or different and correspond to Formula IA or IB
in which
X1, X2, m and n have the meanings given above.
A subject of the invention is the compounds of Formula II
in which
R1, R2, Y1, Y2, m and n have the meanings given above,
R1, R2 are identical or different,
Y1 and Y2 are identical or different.
The compounds II have branchings on the ring at the nitrogen atom, for the 2 side chains. The symmetrical and asymmetrical compounds are included here.
For all the compounds II, it is possible to have the cis or trans configurations.
Advantageously, the compounds of the invention have Formula IIcis
in which
R1, R2, Y1, Y2, m and n have the meanings given above,
R1, R2 are identical or different,
Y1 and Y2 are identical or different,
m=1, 2, 3,
n=0, 1,
provided that m+n is different from 4.
The compounds IIcis are therefore exclusively of cis stereochemistry. They are constituted by a carbon ring with 3, 4 or 5 atoms, to which the 2 amide chains are attached.
Advantageously, the compounds of the invention have Formula IIA cis
in which
R1, Y1, m and n have the meanings given above,
m=1, 2, 3,
n=0, 1,
provided that m+n is different from 4.
The compounds IIA cis are therefore exclusively of cis stereochemistry. They are constituted by a carbon ring with 3, 4 or 5 atoms, to which the 2 amide chains are attached, these two chains being strictly identical; these compounds are symmetrical.
Advantageously, the compounds of the invention have Formula IIB cis
in which
R1, R2, Y1, Y2, m and n have the meanings given above,
provided that if R1 and R2 are identical, then Y1 and Y2 are different,
provided that if R1 and R2 are different, then Y1 and Y2 are identical or different,
m=1, 2, 3,
n=0, 1,
provided that m+n is different from 4.
The compounds IIB cis are therefore exclusively of cis stereochemistry. They are constituted by a carbon ring with 3, 4 or 5 atoms, to which the 2 amide chains are attached, these two chains being different; these compounds are asymmetrical.
Also advantageously, the compounds of the invention have Formula IItrans
in which
R1, R2, Y1, Y2, m and n have the meanings given above,
R1, R2 are identical or different,
Y1 and Y2 are identical or different,
m=1, 2, 3,
n=0, 1,
provided that m+n is different from 4.
The compounds IItrans are therefore exclusively of trans stereochemistry. They are constituted by a carbon ring with 3, 4 or 5 atoms, to which the 2 amide chains are attached.
Advantageously, the compounds of the invention have Formula IIA trans
in which
R1, Y1, m and n have the meanings given above,
m=1, 2, 3,
n=0, 1,
provided that m+n is different from 4.
The compounds IIA trans are therefore exclusively of trans stereochemistry. They are constituted by a carbon ring with 3, 4 or 5 atoms, to which the 2 amide chains are attached, these two chains being strictly identical; these compounds are symmetrical.
Advantageously, the compounds of the invention have Formula IIB trans
in which
R1, R2, Y1, Y2, m and n have the meanings given above,
provided that if R1 and R2 are identical, then Y1 and Y2 are different,
provided that if R1 and R2 are different, then Y1 and Y2 are identical or different,
m=1, 2, 3,
n=0, 1,
provided that m+n is different from 4.
The compounds IIB trans are therefore exclusively of trans stereochemistry. They are constituted by a carbon ring with 3, 4 or 5 atoms, to which the 2 amide chains are attached, these two chains being different; these compounds are asymmetrical.
A subject of the invention is the compounds of Formula I in which n is equal to 0 and m is equal to 1, and corresponding to Formulae VA or VB
in which
X1, X2, m and n have the meanings given above.
In this case, m=1 and n=0. The compounds VA and VB are therefore derivatives of a ring with three carbon atoms, rings optionally formed from an olefinic starting compound. They are therefore derivatives of cyclopropane. The two amide chains are each attached to a carbon atom of the ring.
The compounds VA are symmetrical; the compounds VB are asymmetrical.
For all the compounds VA and VB, it is possible to have the cis or trans configurations.
A subject of the invention is the compounds of Formula I in which n+m is equal to 2, and corresponding to general formula XXII
in which
X1, X2, n and m have the meanings given above.
In this case, the sum m+n=2. The compounds XXII are therefore derivatives of a ring with four carbon atoms. The compounds XXII are derivatives of cyclobutane. The two amide chains are each attached to a carbon atom of the ring. They can be carried by carbons that are adjacent or separated by another carbon atom of the ring.
The symmetrical and asymmetrical compounds are included.
For all the compounds XXII, it is possible to have the cis or trans configurations.
A subject of the invention is also the compounds of Formula I in which n is equal to 0 and m is equal to 2, and corresponding to Formulae XXIIA or XXIIB
in which
X1, X2, m and n have the meanings given above.
In this case, the sum m+n=2 with n=0 and m=2. The compounds XXIIA and XXIIB are therefore derivatives of a ring with four carbon atoms. The compounds XXII are derivatives of cyclobutane. The two amide chains are each attached to a carbon atom of the ring; they are carried by adjacent carbon atoms.
The symmetrical and asymmetrical compounds are included.
For all the compounds XXIIA, XXIIB, it is possible to have the cis or trans configurations.
A subject of the invention is also the compounds of Formula I in which n is equal to 1 and m is equal to 1 and corresponding to Formulae XXIIF or XXIIG
in which
X1, X2, m and n have the meanings designated above.
In this case, the sum m+n=2 with n=1 and m=1. The compounds XXIIF and XXIIG are therefore derivatives of a ring with four carbon atoms. The compounds XXII are derivatives of cyclobutane. The two amide chains are each attached to a carbon atom of the ring; they are carried by carbon atoms separated by another carbon atom, on the ring.
The symmetrical and asymmetrical compounds are included.
For all the compounds XXIIF and XXIIG, it is possible to have the cis or trans configurations.
A subject of the invention is also the compounds of Formula I in which n+m is equal to 3 and correspond to general formula VI
in which
X1, X2, n and m have the meanings given above.
In this case, the sum m+n=3. The compounds VI are therefore derivatives of a ring with five carbon atoms. The compounds VI are derivatives of cyclopentane. The two amide chains are each attached to a carbon atom of the ring. They can be carried by carbons that are adjacent or separated by another carbon atom of the ring.
The symmetrical and asymmetrical compounds are included.
For all the compounds VI, it is possible to have the cis or trans configurations.
The invention also relates to the compounds of Formula I in which n is equal to 0 and m is equal to 3 and corresponding to Formulae VIA and VIB shown below:
in which
X1, X2, m and n have the meanings given above.
In this case, the sum m+n=3 with n=0 and m=3. The compounds VIA and VIB are therefore derivatives of a ring with five carbon atoms. The compounds VI are derivatives of cyclopentane. The two amide chains are each attached to a carbon atom of the ring; they are carried by adjacent carbon atoms.
The symmetrical and asymmetrical compounds are included.
For all the compounds VIA and VIB, it is possible to have the cis or trans configurations.
A subject of the invention is also the compounds of Formula I in which n is equal to 1 and m is equal to 2 and corresponding to Formulae VIF and VIG shown below
in which
X1, X2, m and n have the meanings given above.
In this case, the sum m+n=3 with n=1 and m=2. The compounds VIF and VIG are therefore derivatives of a ring with five carbon atoms. The compounds VI are derivatives of cyclopentane. The two amide chains are each attached to a carbon atom of the ring; they are carried by carbon atoms separated by another carbon atom, on the ring.
The symmetrical and asymmetrical compounds are included.
For all the compounds VIF and VIG, it is possible to have the cis or trans configurations.
Advantageously, the compounds of the invention have Formula VIF cis
in which
R1 and Y1 have the meanings designated above.
The compounds VIF cis are therefore exclusively of cis stereochemistry. They are constituted by a carbon ring with 5 atoms, the 2 amide chains being attached to the ring by carbons that are not adjacent. These two chains are identical; these compounds are symmetrical.
Advantageously, the compounds of the invention have Formula VIG cis
in which
R1, R2, Y1, Y2 have the meanings given above,
provided that if R1 and R2 are identical, then Y1 and Y2 are different,
provided that if R1 and R2 are different, then Y1 and Y2 are identical or different,
m=1, 2, 3,
n=0, 1,
provided that m+n is different from 4.
The compounds VIG cis are therefore exclusively of cis stereochemistry. They are constituted by a carbon ring with 5 atoms, the 2 amide chains being attached to the ring by carbons that are not adjacent. These two chains are different; these compounds are asymmetrical.
Advantageously, the compounds of the invention have Formula VIF trans
in which
R1 and Y1 have the meanings designated above.
The compounds VIF trans are therefore exclusively of trans stereochemistry. They are constituted by a carbon ring with 5 atoms, the 2 amide chains being attached to the ring by carbons that are not adjacent. These two chains are identical; these compounds are symmetrical.
Advantageously, the compounds of the invention have Formula VIG trans
in which
R1, R2, Y1, Y2 have the meanings given above,
provided that if R1 and R2 are identical, then Y1 and Y2 are different,
provided that if R1 and R2 are different, then Y1 and Y2 are identical or different,
m=1, 2, 3,
n=0, 1,
provided that m+n is different from 4.
The compounds VIG trans are therefore exclusively of trans stereochemistry. They are constituted by a carbon ring with 5 atoms, the 2 amide chains being attached to the ring by carbons that are not adjacent. These two chains are different; these compounds are asymmetrical.
A subject of the invention is the compounds in which the X1 and X2 groups are cis to one another, X1 and X2 having the meanings given above.
These compounds are “cis” isomers as the two chains carried by the ring are located on the same side of the ring.
A subject of the invention is the compounds in which the X1 and X2 groups are trans to one another, X1 and X2 having the meanings given above.
These compounds are “trans” isomers as the two chains carried by the ring are located on the same side of the ring.
A subject of the invention is the compounds represented by general formula I in which X1 and X2 are represented as below:
R1 and R2 representing, independently of one another, linear or branched chains, having from 1 to 30 carbon atoms,
the R1—Y1 and R2—Y2 groups representing, independently of one another, one of the groups of the following formulae, the amine radical can optionally be substituted, the terminal hydroxyl radical can optionally be coupled to a glycoside residue selected from the α- or β-furanoses and the α- or β-pyranoses, or coupled to a linear aliphatic chain comprising one or more oxygen atoms, of formulae represented below,
in which
δ varies from 1 to 12, δ′ varies from 1 to 5,
or a radical that can optionally be protected,
Ra representing a linear or branched alkyl group comprising from 1 to 4 carbon atoms, optionally substituted by one or more halogen atoms,
in which
p varies from 1 to 28,
r varies from 1 to 29,
s+t varies from 2 to 27,
s+u varies from 2 to 24,
s+v varies from 2 to 21.
The different natures of X1 and X2 have been represented as above.
The two side chains each comprise an amide function. The length of the chains varies, these two chains being selected independently of one another.
The R1 and R2 portions are saturated or unsaturated, then containing from one to three carbon/carbon double bonds optionally bearing a halogen atom or a —CF3 group.
The terminal portions Y1 and Y2 are hydrogens, protected or unprotected alcohol functions, protected or unprotected amines, in particular in the form —NHBoc and derivatives thereof, carboxylic acids or esters, as has been described above.
The invention relates to the compounds corresponding to one of the following formulae. Compounds according to claim 1, of general formula I, shown below:
The invention further relates to a process for the preparation of compounds of Formula I, cis and trans, represented by the following formula:
in which
m=1, 2, 3 and n=0, 1,
provided that m+n is different from 4,
X1 and X2 can be trans or cis relative to one another and represent, independently of one another, a group selected from
in which
in which δ varies from 1 to 12,
in which δ′ varies from 1 to 5,
in which
R4 represents a linear or branched alkyl group, comprising from 1 to 6 carbon atoms, in particular methyl, ethyl, isopropyl, tert-butyl, (OR4)2 optionally forming a ring between the two oxygen atoms, the (OR4)2 groups in particular originating from diols such as ethane-1,2-diol, propane-1,3-diol, 2,2-dimethylpropane-1,3-diol, 2,3-dimethylbutane-2,3-diol (pinacol), 2-methylbutane-2,3-diol, 1,2-diphenylethane-1,2-diol, 2-methylpentane-2,4-diol, 1,2-dihydroxybenzene (catechol), 2,2′-azanediyldiethanol, 2,2′-(butylazanediyl)diethanol, 2,3-dihydroxysuccinic acid (tartaric acid) and its esters, or (OR4)2 originates in particular from diacids such as 2,2′-(methylazanediyl)diacetic acid (mida),
in which
in which
Y2 has the same meaning as Y1,
R2 has the same meaning as R1,
Y1 and Y2 being able to be equal or different,
R1 and R2 being able to be equal or different,
D=—CO—R5
R5 representing
in particular derived from
in which
R9 and R10, different or equal, represent an alkyl group comprising from 1 to 10 carbon atoms, linear, branched or cyclic, optionally substituted by an amino group, in particular cyclohexyl, isopropyl, ethyl, dimethylpropylamino,
said carbodiimide in particular being selected from the following compounds
The invention relates to a process for the preparation of the compounds of Formula IA and IB, cis and trans, represented by the formulae shown below:
in which
X1 and X2 have the meanings given above,
which comprises an amide formation between a compound of Formula VII shown below:
in which
m=1, 2, 3 and n=0, 1, provided that m+n is different from 4,
A and B are such that:
in which
R2, R5 and Y2 have the meanings given above,
Y1 and Y2 being able to be equal or different,
R1 and R2 being able to be equal or different,
said process making it possible to obtain the compounds of Formula IA and IB represented above.
The symmetrical and asymmetrical compounds defined above are included. If the target product bears two X1 groups, it is symmetrical. If the target product bears an X1 group and an X2 group with X1 and X2 different, it is asymmetrical. The formulae of X1 and of X2 are shown below:
The invention relates in particular to a process for the preparation of the symmetrical compounds of Formula IIA, cis and trans, shown below:
in which
m=1, 2, 3 and n=0.1, provided that m+n is different from 4,
R1, Y1 have the meanings given above,
comprising coupling between a cis or trans diamine of Formula VIIA shown below:
in which
m and n have the meanings given above,
and a compound of Formula VIIIA
R1, R5 and Y1 having the meanings given above,
said process making it possible to obtain the compounds of Formula IIA represented above.
The amide formation is carried out in a standard manner, in particular
Therefore one stage is sufficient for preparing the compounds IIA.
Advantageously, the invention relates to a process for the preparation of the symmetrical compounds of Formula IIA cis shown below
in which
m=1, 2, 3 and n=0.1, provided that m+n is different from 4,
R1 and Y1 have the meanings given above,
said process comprising coupling between a diamine of Formula VIIA cis shown below:
in which
m and n have the meanings given above,
and a compound of Formula VIIIA
R5 having the meanings given above and in particular being equal to —OH,
R1 and Y1 having the meanings given above,
said process making it possible to obtain the compounds of Formula IIA cis represented above.
Particularly advantageously, the invention relates to a process for the preparation of the symmetrical compounds of Formula VIF cis represented below
in which
R1 and Y1 have the meanings given above,
said process comprising coupling between cis-1,3-diaminocyclopentane of the formula shown below
and a compound of Formula VIIIA
R5 having the meanings given above and in particular being equal to —OH,
R1 and Y1 having the meanings given above,
said process making it possible to obtain the compounds of Formula VIF cis represented above.
The invention relates in particular to a process for the preparation of compound 30 of the formula shown below
in which —OTHP is the group of formula,
said process comprising coupling between cis-1,3-diaminocyclopentane of the formula shown below
and the acid of the formula shown below
in which —OTHP has the meaning designated above,
said process making it possible to obtain compound 30 of the formula shown above.
The invention also relates in particular to a process for the preparation of compound 152 of the formula shown below
in which —OTHP has the meaning designated above,
said process comprising coupling between cis-1,3-diaminocyclopentane of the formula shown below:
and the acid of the formula shown below
in which —OTHP has the meaning designated above,
said process making it possible to obtain compound 152 of the formula shown above.
The invention also relates to a process for the preparation of the cis and trans asymmetrical compounds of Formula IIB shown below:
in which
provided that R1 and R2 are different from one another,
in which
R1, Y1, m and n have the meanings given above,
and a compound of Formula VIIIA
in which
R2, R5 and Y2 have the meanings given above,
said process making it possible to obtain the compounds of Formula IIB represented above.
Preparation of the asymmetrical molecules requires four reaction stages. The last stage is shown in the following diagram: it is the second amide formation, carried out in cis or trans series:
The compound obtained then has two amide chains attached to the ring by the nitrogen atom, but different in the respective natures of R1 and R2 and/or of Y1 and Y2. “R1—Y1” is supplied during the first amide formation whereas “R2—Y2” is supplied during the second amide formation. By proceeding in this way, it is possible to prepare asymmetrical compounds.
The invention relates in particular to a process for the preparation of compound VIID represented by the formula shown below:
said compound VIID being obtained by deprotection of the amine function of compound IX represented below
in which
The invention relates in particular to a process for the preparation of compound IX represented by the formula shown below:
said compound IX being obtained by monoacylation between the diamine X, one amine function of which is blocked by a protective group
in which
in which
R1, R5 and Y1 have the meanings given above,
said process making it possible to obtain the compounds of Formula IX represented above.
This reaction is the first one in the process for the preparation of the asymmetrical compounds. Since an amine function is blocked, the first coupling makes it possible to obtain compound IX. The chemical equation of this coupling is shown below:
The first side chain is thus attached to the ring.
The invention relates in particular to a process for the preparation of the compound X represented by the formula shown below:
said compound X being obtained by protection of the diamine of Formula VIIA shown below:
in which
m=1, 2, 3 and n=0, 1, provided that m+n is different from 4,
said process making it possible to obtain the compounds of Formula X represented above.
A single amine function of compound VIIA is protected so as to be able to carry out the first amide formation. Protection is carried out by conversion to an amide or carbamate function. It is represented by the following equation:
The invention relates in particular to a process for the preparation of the cis and trans compounds of Formula IIB shown below:
in which
provided that R1 and R2 are different from one another,
m and n having the meanings given above,
to obtain compound X of the following formula:
in which
in which
R1, R5 and Y1 have the meanings given above,
to obtain the monomeric compound IX having the following formula:
in which
m, n, R1, Y1 and Rp′ have the meanings given above,
in which
m, n, R1, Y1 have the meanings given above,
provided that R1 and R2 are different from one another,
to obtain the target compound IIB:
m, n, R1, R2, Y1, Y2 having the meanings given above.
The process for the preparation of the family of asymmetrical compounds therefore involves four stages, carried out in cis or trans series.
The side chains are different as they originate from carboxylic acids or differently substituted derivatives, R5—CO—R1—Y1 and R5—CO—R2—Y2.
The invention also relates to a process for the specific preparation of the compounds of Formula IIC shown below:
in which
R3 having the meaning given above,
and a phosphonoacetamide of general formula XVIII
in which
The invention also relates to a process for the preparation of the phosphonoacetamide XVIII represented by the formula shown below
in which
m and n having the meanings given above,
and the phosphorylated carboxylic acid of Formula VIIIC
in which
V and R4 have the meanings given above,
said process making it possible to obtain the compounds of Formula XVIII represented above.
This reaction makes it possible to prepare the phosphonoacetamide represented by Formula XVIII by amide formation between the cyclic diamine of Formula VIIA and a phosphonoacetic acid. It is possible to work in halogenated series, V then representing fluorine, chlorine or bromine, carried by the carbon in the α position to the carboxyl group.
The reaction is carried out under standard amide formation conditions.
It is represented by the following equation:
The product obtained is thus a phosphonic amide that can be used in a reaction of the Wittig-Horner type, as described above. It is obtained with a yield of the order of 95%, after purification by silica chromatography.
The invention also relates to a process for the preparation of the compounds of Formula IIC shown below:
in which
m and n having the meanings given above,
and the phosphorylated carboxylic acid of Formula VIIIC
in which
in which
R3 has the meanings given above,
said process making it possible to obtain the compounds of Formula IIC represented above.
A second process is described for preparing the symmetrical compounds.
The compounds of Formula IIC are obtained in two stages with an excellent overall yield.
Another aspect of the invention consists of the pharmaceutical composition containing, as active ingredient, at least one of the compounds of Formula I, and in particular containing, as active ingredient, compound 30 of formula
and/or compound 152 of formula
together with a pharmaceutically acceptable vehicle.
Owing to their pharmacological properties, the compounds according to the invention are used in therapeutics as skin depigmenting agents, anti-ageing agents, tensing agents, anti-inflammatory agents.
For these purposes, they will be used in the form of pharmaceutical compositions containing, as active ingredient, at least one of the compounds of general formula I in combination with or mixed with an excipient or an inert, non-toxic, and pharmaceutically acceptable vehicle.
For therapeutic use, they will be presented in one of the pharmaceutical forms suitable administration by oral or topical route.
In this connection, we may mention plain or coated tablets, sugar-coated tablets, gelatin capsules, powders, as well as creams, ointments, lotions, emulsions, sprays, serums, milks.
Another aspect of the invention consists of the pharmaceutical composition containing, as active ingredient, several of the compounds of Formula I, and in particular containing, as active ingredient, several compounds including compound 30 and/or compound 152, in combination with a pharmaceutically acceptable vehicle.
It will be possible for the pharmaceutical compositions to contain mixtures of compounds of Formula I, in variable proportions.
According to a particular aspect of the invention, the pharmaceutical composition contains from 0.005% to 20 wt % of active ingredient per unit dose.
The dosage can vary depending on the pharmaceutical form and the subject's weight.
Another aspect of the invention consists of the cosmetic composition containing, as active ingredient, at least one of the compounds of Formula I, and in particular containing, as active ingredient, compound 30 of formula
and/or compound 152 of formula
in combination with a cosmetically acceptable vehicle.
Owing to their cosmetic properties, the compounds according to the invention are used in therapeutics as skin depigmenting agents, anti-ageing agents, tensing agents, healing agents.
For these purposes, they will be used in the form of cosmetic compositions containing, as active ingredient, at least one of the compounds of general formula I in combination with or mixed with an excipient or an inert, non-toxic, and cosmetically acceptable vehicle.
For therapeutic use, they will be presented in one of the cosmetic forms suitable for administration by cutaneous route.
In this connection we may mention creams, ointments, gels, oils, serums, milks, sprays, emulsions.
The excipients that are suitable for said administrations are oils, water and alcohol as well as surfactants, additives such as preservatives, antioxidants, colorants, perfumes.
Another aspect of the invention consists of the cosmetic composition containing, as active ingredient, several of the compounds of Formula I, and in particular containing, as active ingredient, several compounds including compound 30 and/or compound 152, in combination with a cosmetically acceptable vehicle.
The cosmetic compositions will be able to contain mixtures of compounds of Formula I, in variable proportions.
According to a particular aspect of the invention, the cosmetic composition contains from 0.005% to 20 wt % of active ingredient per unit dose.
The dosage can vary depending on the form.
The analytical techniques are as follows:
The NMR spectra were recorded at 300 MHz (Brücker spectrometer) for the proton. The chemical shifts are expressed in ppm, the residual chloroform being taken as internal reference (singlet at 7.28 ppm), or residual dimethylsulphoxide being taken as internal reference (multiplet at 2.50 ppm). The multiplicity of the signals is denoted by the following letters: s singlet, d doublet, dd doublet of doublets, t triplet, q quadruplet and m multiplet.
The melting points were measured by DSC (differential scanning calorimetry) on a Mettler Toledo instrument.
LC/MS analysis corresponds to coupling of HPLC analysis and analysis by mass spectrometry. It is carried out on an Alliance Waters 2695-ZQ2000 instrument.
Detector: DAD detector (Waters, ref.: 2996, λ=190 nm to 800 nm):
Mass detector (Waters, ref. ZQ2000): 100-1500 dalton; negative and positive ion
Temperature of HPLC oven: 40° C.
Flow: 1 mL/min
The methods used for HPLC are presented below. In the tables of analytical results, the gradient number is shown in the exponent, with the retention time.
Column: XTerra® MS C18: 4.6 mm×150 mm, 5 μm (Waters, ref. 186000490)
Eluent A: Water (HCOOH-0.02%); Eluent B=CH3CN) with elution gradient
Elution condition: gradient
Column: XTerra® MS C18: 4.6 mm×150 mm, 5 μm (Waters ref. 186000490)
Eluent A: Water (HCOOH-0.02%); Eluent B=CH3CN) with elution gradient
Elution condition: gradient
Column: XTerra® MS C18: 4.6 mm×150 mm, 5 μm (Waters, ref. 186000490)
Eluent A: Water (HCOOH-0.02%); Eluent B=CH3CN) with elution gradient
Elution condition: gradient
Column: XTerra® MS C18: 4.6 mm×150 mm, 5 μm (Waters, ref. 186000490)
Eluent A: Water (HCOOH-0.02%); Eluent B=CH3CN) with elution gradient
Elution condition: gradient
5. “SF—HCOOH ACN Grad7 30 mm” Method
Column: Sunfire™ C8: 4.6 mm×150 mm, 3.5 μm (Waters ref. 186002732)
Eluent A: Water (HCOOH-0.02%); Eluent B=CH3CN) with elution gradient
Elution condition: gradient
6. “SF—HCOOH ACN Grad12 45 mm” Method
Column: Sunfire™ C8: 4.6 mm×150 mm, 3.5 μm (Waters ref. 186002732)
Eluent A: Water (HCOOH-0.02%); Eluent B=CH3CN) with elution gradient
Elution condition: gradient
The cyclopropane rings, n=0 and m=1, of cis configuration are obtained from the anhydride described in the literature, commercial anhydride. The starting products are described in the various publications cited in the following list:
The trans diacid is commercially available from Aldrich. If they are prepared, the cyclopropanes of trans relative configuration can be obtained on the basis of condensation of the chloroacetate with an acrylic derivative.
The products used were synthesized on the basis of the procedures described in references (1) to (8) cited above, reference (8) relating in particular to the Curtius reaction for obtaining the trans diamine.
The various steps are shown in the following reaction diagram:
They are prepared by the same methods as those described above.
They are obtained in three steps starting from the corresponding cyclopentanediol. This sequence is used in the two series, cis and trans. The stereochemistry of the functional carbon is not altered by the later reactions providing the diamine.
The cis and trans sequential syntheses are described in the following references:
The 1,3-diaminocyclopentanes have been described since 1925, in particular by Diels and in Pfizer, AstraZeneca and Roche patents. The procedures used can be found in these patents and publications, the references of which are given below:
The fatty acids used, corresponding to the above formula with R2—Y2 an alkyl chain comprising from 7 to 29 carbon atoms, saturated or unsaturated having a variable number of double bonds, are commercially available: for example, oleic acid, myristic acid, and palmitic acid will be used.
These derivatives correspond to the above formula with:
30 g of lactone is dissolved in 10 volumes of toluene, under a nitrogen atmosphere. The mixture is cooled down to −78° C. and 1.01 equivalents of Dibal-H (ACROS) in solution at 20% in toluene are added dropwise, keeping the temperature at −78° C. The mixture is stirred for 2 hours at −78° C. Eight volumes of a saturated solution of Rozen salts (double tartrate salts; ACROS) are added at −78° C. After 18 hours of vigorous stirring at ambient temperature, the two-phase mixture is filtered on Celite, and then extracted with ethyl acetate. The organic phases are washed with a saturated NaCl solution, dried over MgSO4, filtered and concentrated under vacuum to give a crude product weighing 30 g (containing some traces of diol). The lactol, in open form-cyclic form equilibrium, is used as it is, without additional purification.
the following 8-hydroxyoctanal is prepared according to Example 1; its analytical characteristics are given below:
Characterization, step a, open form:
TLC: Rf=0.4 (heptane/ethyl acetate 6/4)
1H NMR (300 MHz, CDCl3): δ 1.34-1.68 (m, 10H); 2.45 (t, J=5.4 Hz, 2H); 3.66 (t, J=6.6 Hz, 2H); 9.78 (t, J=1.8 Hz, 1H).
19 g of lactol obtained in step a is diluted in 13 volumes of ethanol. 1.2 equivalents of triethylphosphonoacetate are added to the mixture in the presence of 1.5 equivalents of potassium carbonate. The reaction medium is heated at 40° C. for 18 hours. At ambient temperature, the mixture is hydrolysed with 10 volumes of distilled water and extracted with ethyl acetate. The organic phases are washed with a saturated NaCl solution, dried over MgSO4, filtered and concentrated under vacuum to give a crude product weighing 20 g.
The ester obtained is purified by chromatography with the eluent mixture heptane/ethyl acetate 7/3. 15 g of product is obtained (53% yield).
the following compound is prepared according to Example 3; its analytical characteristics are as follows:
Characterization, Step b, with V Equal to Hydrogen:
TLC: Rf=0.4 (heptane/ethyl acetate 7/3)
1H NMR (300 MHz, CDCl3): δ 1.24-1.38 (m, 9H); 1.43-1.50 (m, 2H); 1.51-1.57 (m, 2H); 2.15-2.21 (q, 2H); 3.60-3.64 (t, 2H); 4.14-4.20 (t, 2H); 5.77-5.82 (d, J=15.6 Hz, 1H); 6.91-6.98 (dt, J=15.6 Hz, 1H).
This step is also carried out in a fluorinated series starting from triethyl 2-fluoro-2-phosphonoacetate. Two isomers are then possible: E and/or Z. The equivalent of the above molecule in a fluorinated series is prepared according to Example 3 and is shown below:
Characterization, Step b, with V Equal to Fluorine:
TLC: Rf=0.43 (heptane/ethyl acetate 7/3)
1H NMR (300 MHz, CDCl3): δ 1.28 (t, 6H); 1.30-1.65 (m, 20H); 2.16 (q, 4H); 2.27 (m, 2H); 2.52 (m, 2H); 3.66-3.71 (t, 4H); 5.99-6.11 (dt, J=21.0 Hz configuration E, 1H); 6.18-6.34 (dt, J=33.0 Hz configuration Z, 1H).
0.60 g of hydroxy ester obtained in the preceding step b is dissolved in 10 volumes of tetrahydrofuran. 2.4 equivalents of a 2M soda solution are added slowly. The mixture is heated at 65° C. for 3 hours. Once the reaction has ended, the mixture is hydrolysed by adding a 3M hydrochloric acid solution, until pH=2 is obtained. The mixture is concentrated to dryness and the aqueous phase is then extracted with ethyl acetate. The organic phases are washed with a saturated NaCl solution, dried over MgSO4, filtered and concentrated under vacuum to give a crude product weighing 0.6 g.
The unsaturated hydroxy acid VIIIA is obtained, in the form of a white solid, by recrystallization from cold acetonitrile, m=0.37 g (yield equal to 71%).
it is prepared by the protocol described in Example 6. In the case of the following compound, the analytical characteristics are as follows:
Characterization, step c:
TLC: Rf=0.1 (heptane/ethyl acetate 6/4)
1H NMR (300 MHz, CDCl3): δ 1.33-1.37 (m, 6H); 1.45-1.49 (m, 2H); 1.55-1.58 (m, 2H); 2.20-2.25 (q, 2H); 3.62-3.66 (t, 2H); 5.79-5.84 (d, J=15.6 Hz, 1H); 7.03-7.10 (dt, J=15.6 Hz, 1H)
Mass spectroscopy: [M±Na]+ 209 (calculated 186)
Melting point: 62.5° C.±1° C.
This step is also carried out in a fluorinated series. The molecule shown below is prepared according to Example 6, in a fluorinated series, and is characterized by:
TLC: Rf=0.12 (heptane/ethyl acetate 6/4)
1H NMR (300 MHz, CDCl3): δ 1.30-1.65 (m, 20H); 2.27 (m, 2H); 2.52 (m, 2H); 3.66-3.71 (t, 4H); 5.99-6.11 (dt, J=21.0 Hz configuration E, 1H); 6.18-6.34 (dt, J=33.0 Hz configuration Z, 1H).
The compounds of Formula IIA
are obtained by one of the two methods described below:
method A=amide formation;
method B=amide formation followed by a Wittig-Horner reaction.
This method comprises an amide formation reaction optionally followed by a step of deprotection of Y1. The acylation reaction is represented by the following equation:
1 equivalent of carboxylic acid VIIIA is dissolved in 10 volumes of tetrahydrofuran, under inert atmosphere. The diamine cyclic unit in trans series or cis series is added (0.5 equivalents), as well as 2.5 equivalents of 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride and 1.2 equivalents of 1-hydroxybenzotriazole. The suspension is cooled down to 0° C. and 3 equivalents of N,N-diisopropylethylamine are added slowly. The addition of a few drops of N,N′-dimethylformamide allows complete solubilization. The reaction medium is stirred for 16 h at ambient temperature. Analysis by thin-layer chromatography is used to check for the end of the reaction. The mixture is concentrated under vacuum. The residue is taken up in dichloromethane and distilled water. It is extracted with dichloromethane three times. The combined organic phases are washed with a 2M HCl solution and then with a saturated NaCl solution. They are dried over Na2SO4, filtered and concentrated, giving a brown oil, which is purified by silica gel chromatography (eluent dichloromethane/methanol 98/2). A pure product is obtained with a yield between 50% and 95%.
The compounds 1 to 161 are prepared according to the protocol of Example 9. The analytical characteristics of compound 27 are given below:
Characterization: compound 27
TLC: Rf=0.3 (dichloromethane/methanol 98/2)
1H NMR (300 MHz, CDCl3): δ 1.30-1.78 (m, 28H); 2.10 (m, 6H); 3.30 (m, 2H); 3.45 (m, 2H); 3.65 (m, 2H); 3.80 (m, 2H); 4.15 (m, 2H); 4.50 (m, 2H); 5.67-5.73 (d, J=15.3 Hz, 2H); 6.18 (d, 2H); 6.70-6.776 (dt, J=15.3 Hz, 2H)
Mass spectroscopy: [M+Na]+571.3 (calculated 548.77)
HPLC: method HCOOH_ACN_gradient 1, tR=7.2 min, 95% at 210 nm.
In certain cases, the acid derivative used bears a protective group in the form of —OTHP on the terminal alcohol function. The diprotected diamide compound is dissolved in 50 volumes of methanol. A catalytic quantity of p-toluenesulphonic acid is added and the mixture is stirred at 40° C. for 4 h. Monitoring by thin-layer chromatography provides a check for the end of the reaction. The mixture is then concentrated under vacuum; the residue is taken up in dichloromethane and distilled water. After several extractions with dichloromethane, the organic phases are washed with a saturated NaCl solution. They are dried over MgSO4, filtered and concentrated under vacuum. The residue is purified by trituration in a water/ethyl acetate mixture or on a silica column, giving a fraction of pure product with yields close to 70%.
In certain cases, the acid derivative used in the peptide coupling bears a protective group in the “tBdPhSiO—” form on the terminal alcohol function. The diprotected diamide compound is dissolved in 15 volumes of tetrahydrofuran. At 0° C., a solution of tetrabutylammonium fluoride (3 equivalents at 1M in THF) is added slowly. After stirring for 3 h at ambient temperature, monitoring by TLC provides a check for the end of the reaction. The reaction medium is then hydrolysed by adding a saturated NH4Cl solution. The mixture is extracted three times with ethyl acetate and the combined organic phases are washed with a saturated NaCl solution. After drying over MgSO4 and filtration, the organic solvent is removed under vacuum. The residue obtained is triturated in organic mixtures or is purified by silica gel chromatography.
In certain cases, the acid derivative used in the amide formation reaction bears a protective group in the —OAc form on the terminal alcohol function. The diprotected diamide compound is dissolved in 4 volumes of methanol. At ambient temperature, a freshly prepared aqueous solution of potassium carbonate (0.9 equivalent) and of potassium hydrogen carbonate (1.7 equivalents) is added. The reaction medium is stirred for 4 hours. Monitoring by TLC provides a check for complete deprotection. The mixture is then concentrated to dryness and then taken up in ethyl acetate and water. After trituration, the solid is filtered and dried under vacuum. The yield varies between 30% and 70%, depending on the length of the chain.
the deprotected product compound 36 is obtained by the protocol described in Example 13. Its analytical characteristics are as follows:
Characterization: compound 36
TLC: Rf=0.15 (dichloromethane/methanol 98/2)
1H NMR (300 MHz, CDCl3): δ 0.89 (m, 2H); 1.27-1.60 (m, 16H); 2.20 (m, 4H); 2.64 (m, 4H); 2.90 (m, 4H); 3.65 (t, 2H); 5.70-5.84 (dt, J=24.6 Hz, 2H); 6.21-6.04 (dt, J=37.8 Hz, 2H)
Mass spectroscopy: [M+Na]+ 467.2 (calculated 444.57)
HPLC: method HCOOH_ACN_gradient 1, tR=10.6 min, 97% at 240 nm
In certain cases, the acid derivative used in the amide formation reaction bears a protective group in the —NHBoc form on the terminal amine function. The diprotected diamide compound is dissolved in 2 volumes of diethyl ether. A solution of dry hydrochloric acid in diethyl ether (2M) is added and the reaction medium is stirred at ambient temperature for 2 h. Monitoring by TLC provides a check for the end of the reaction. After concentrating to dryness, the residue is triturated in dichloromethane, giving a dihydrochloride salt of the desired compound, with a yield between 70% and 95%.
the deprotected product, compound 101, is obtained by the protocol described in Example 15. Its analytical characteristics are as follows:
Characterization: compound 101
1H NMR (300 MHz, DMSO-d6): δ 0.89 (m, 2H); 1.30-1.57 (m, 12H); 2.12 (m, 4H); 2.51-2.76 (m, 8H); 5.62-5.82 (dt, J=15.0 Hz, 2H); 6.55-6.63 (dt, J=30.0 Hz, 2H); 7.93 (s, 4H); 8.15 (s, 2H).
Diethylphosphonoacetic acid (4 equivalents) is diluted in dichloromethane (14 volumes) under inert atmosphere. The reagent O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (4.4 equivalents) as well as triethylamine (6.5 equivalents) are added. After stirring for five minutes at ambient temperature, the diamine cyclic unit (1 equivalent) is added and the reaction medium is heated at 50° C. for 30 min. Monitoring by thin-layer chromatography provides a check for the end of the reaction. The mixture is then hydrolysed by adding distilled water and then by adding a saturated NH4Cl solution. After two extractions with ethyl acetate, the organic phases are washed with a saturated NaCl solution, dried over MgSO4, filtered and concentrated under vacuum. The residue is purified on a silica column with a dichloromethane/methanol gradient. The pure product is obtained with a yield above 95%.
Compound 144 is obtained using the protocol of Example 17. The analytical characteristics of compound 144 are as follows:
Characterization: compound 144, diphosphonoacetamide intermediate:
TLC: Rf=0.2 (dichloromethane/methanol 95/5)
1H NMR (300 MHz, DMSO-d6): δ 1.12 (m, 2H); 1.22 (t, 12H); 1.42 (m, 2H); 1.83 (m, 2H); 2.72 (d, 4H); 3.42 (m, 2H); 4.0 (q, 8H); 8.04 (d, 2H)
Mass spectroscopy: [M+H]+=457.2; [M−H]−=455.2 (calculated 456.42)
HPLC: method HCOOH_ACN_gradient 1, tR=7.28 min, 94% at 210 nm.
The diphosphonoacetamide compound is used in a reaction of the Wittig-Horner type: under an inert atmosphere, 1 equivalent of diphosphonoacetamide product is dissolved in tetrahydrofuran (10 volumes). A base of the K2CO3 type (4 equivalents) and the aldehyde derivative (4 equivalents) are added to the reaction medium. The latter is heated at 50° C. overnight with stirring. Monitoring by TLC provides a check for the end of the reaction. At ambient temperature, the mixture is hydrolysed by adding distilled water. After three extractions with ethyl acetate, the organic phases are washed with a saturated NaCl solution, dried over MgSO4, filtered and concentrated under vacuum. The crude residue is purified by trituration in dichloromethane: the insoluble salts are removed by filtration whereas the filtrate is concentrated, giving the desired product.
Diethylphosphonofluoroacetic acid (3 equivalents) is diluted in dichloromethane (14 volumes) under inert atmosphere. The reagent O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (3 equivalents) as well as triethylamine (6.4 equivalents) are added. After stirring for five minutes at ambient temperature, the diamine cyclic unit (1 equivalent) is added and the reaction medium is heated at 50° C. for 30 min. Monitoring by thin-layer chromatography provides a check for the end of the reaction. The mixture is then hydrolysed by adding distilled water and then by adding a saturated NH4Cl solution. After two extractions with ethyl acetate, the organic phases are washed with a saturated NaCl solution, dried over MgSO4, filtered and concentrated under vacuum. The residue is purified on a silica column with a dichloromethane/methanol gradient. The pure product is obtained with a yield above 90%.
Compound 145 is obtained using the protocol of Example 20. The analytical characteristics of compound 145 are as follows:
Characterization of the fluorinated diphosphonoacetamide intermediate: compound 145
TLC: Rf=0.3 (dichloromethane/methanol 85/15)
1H NMR (300 MHz, DMSO-d6): δ 5.21-5.50 (m, 2H); 4.25 (m, 8H); 3.25 (m, 2H), 1.80-2.14 (m, 2H), 1.40 (t, 12H), 1.38 (m, 4H).
Mass spectroscopy: [M+H]+=493.1; [M−H]−=491.1 (calculated 492.4)
HPLC: method HCOOH_ACN_gradient 1, tR=8.18 min
The diphosphonoacetamide compound thus obtained is used in a reaction of the Wittig-Horner type: under an inert atmosphere, 1 equivalent of diphosphonoacetamide product is dissolved in tetrahydrofuran (10 volumes). A base of the K2CO3 type (4 equivalents) and the aldehyde derivative (4 equivalents) are added to the reaction medium. The latter is heated at 50° C. overnight with stirring. Monitoring by TLC provides a check for the end of the reaction. At ambient temperature, the mixture is hydrolysed by adding distilled water. After three extractions with ethyl acetate, the organic phases are washed with a saturated NaCl solution, dried over MgSO4, filtered and concentrated under vacuum. The crude residue is purified by trituration in dichloromethane: the insoluble salts are removed by filtration whereas the filtrate is concentrated, giving the desired product (description in Table 2).
The analytical description of the phosphonoacetamides XVIII obtained corresponding to the following diagram is given in Table 1 below. The reference of the compound is indicated by “c” followed by its number.
1H
a OK = coherent spectrum
1X-Terra column, gradient 1
The analytical characteristics of the symmetrical compounds of Formula IIA shown above, derived from α,β-unsaturated fatty acids described previously, are summarized in Table 2 below:
1H NMRa
aOK = coherent spectrum
bthe superscript after the retention time refers to the method used.
1Method HCOOH_ACN grad 1
7Method HCOOH_ACN grad 7
11Method HCOOH_ACN grad 11
The analytical characteristics of the symmetrical compounds of Formula IIA shown below in which —R1—Y1 is an alkyl group, derived from saturated fatty acids, are presented in Table 3 below:
r has from 6 to 29 carbon atoms.
a
1H NMRb
17.3 min12
19.5 min12
a nomenclature of the fatty acids (t:u(v)): t = number of carbons, u = number of double bonds, v = the carbon or carbons bearing the double bonds).
bOK = coherent spectrum; NA = not analysed
cthe superscript after the retention time refers to the method used.
12Method SF-HCOOH_ACN grad12
9Method HCOOH_ACN grad 9
7Method SF-HCOOH_ACN grad7
The synthesis comprises four steps: protection of one of the two amine functions, first amide formation carried out with the unprotected function by reaction with a fatty acid, deprotection of the blocked amine function and then second amide formation with a fatty acid different from that used in the first coupling.
The equations of the reaction diagram are shown below:
In this first step, the diamine compound VIIA is dissolved in tetrahydrofuran (10 volumes). Then at 0° C., 1.1 equivalent of Boc2O and 1 equivalent of triethylamine are added. The reaction medium is stirred overnight at ambient temperature. Monitoring by TLC provides a check for the end of the reaction. The mixture is then concentrated under vacuum and then purified on a silica gel column, giving the mono-protected compound.
The mono-protected diamine compound X obtained in the preceding step (Example 23) is used in a coupling reaction in the presence of N,N-diisopropylethylamine (DIEA), 1-hydroxybenzotriazole (HOBT), 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDCI), in order to obtain compound IX, according to the following protocol.
Compound X obtained previously (1 equivalent) is dissolved in 20 volumes of dichloromethane under an inert atmosphere. At ambient temperature, 1.1 equivalent of 1-hydroxybenzotriazole, 1.1 equivalent of 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride and 2 equivalents of N,N-diisopropylethylamine are added. After stirring for 5 minutes, compound VIIIA (1.2 equivalent) is added. The reaction medium is stirred for 18 h at ambient temperature. Monitoring by TLC provides a check for the end of the reaction. The mixture is then hydrolysed by adding water. After three extractions with ethyl acetate, the organic phases are washed with a saturated NaCl solution. They are then dried over MgSO4, filtered and concentrated under vacuum. The crude residue is purified on a silica gel column eluted with a heptane/ethyl acetate gradient, giving a white solid with a yield close to 30%.
The amine function protected by a Boc group is deprotected by the action of trifluoroacetic acid (2 equivalents) in solution in tetrahydrofuran (5 volumes), with stirring for 20 h. After concentrating to dryness, the compound VIID obtained is used directly in the next step without additional purification.
The amidoamine VIID obtained by Example 25 is used in peptide coupling with a carboxylic acid derivative different from that used for the first amide formation, in the presence of N,N-diisopropylethylamine (DIEA), 1-hydroxybenzotriazole (HOBT), 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDCI) in dichloromethane, following the conditions of the protocol described in Example 24.
The asymmetrical compounds IIB are obtained.
The NMR characteristics of the examples prepared are shown in the following tables.
The shifts of the protons in positions 1, 2, 3 referenced on the diagram are given as well as the vinylic proton shifts, if present.
It should be noted that R1a represents a linear or branched chain possessing from 1 to 30 carbon atoms, saturated or unsaturated, and in the case of an unsaturation, with the C═C double bond optionally substituted with a fluorine, chlorine, or bromine atom or with a —CF3 group.
1H NMR description (300 MHz, CDCl3, δ ppm)
1H NMR description (300 MHz, CDCl3, δ ppm)
1H NMR description (300 MHz, CDCl3, δ ppm)
Y1 and V have the meanings defined above.
1H NMR description (300 MHz, δ ppm)
1H NMR description (300 MHz, δ ppm)
1H NMR description (300 MHz, δ ppm)
The tests were carried out by reaction with DOPA oxidase on separated epidermis, compared with a DOPA control and with kojic acid at 0.06%. The products are tested in solution at 30 μg/mL in DMSO.
Procedure:
Epidermis samples originating from a frozen abdominoplasty (woman 33 years old) were separated by incubation in 2N NaBr for 1.0 h at 37° C.
They were then fixed in a buffered formolized fixing agent, rinsed and put in contact with the volume/volume mixture: solutions of L-DOPA/test formulation.
After incubation, they were rinsed and mounted between slide and cover slip with mounting liquid of the Aquatex type.
Observation was by optical microscopy with ×10 objective.
Images were obtained with a tri CCD Sony DXC 390P camera and stored using the Leica IM1000 data archiving software.
For each batch, several microscope fields were analysed using the LEICA QWin image analysis software.
For each field, the DOPA positive melanocytes were counted and the area of the zone was measured to determine the melanocyte count per mm2.
Results:
The observations of the epidermis samples tested with the different solutions of products dissolved in DMSO live the following results:
The variations are larger with compounds 40, 59 and 42 than with the compounds designated ref 1, ref 3, ref 4, ref 5 and ref 6, and indicated in the above table.
These compounds are therefore more effective than the reference compounds for limiting tyrosinase activity.
This was carried out for compounds in solution in DMSO. The synthesis of melanin was measured in a model of B16 melanocytes stimulated with a stable derivative of α-MSH (natural hormone that stimulates melanogenesis: Melanocyte Stimulating Hormone): NDP-MSH ([Nle4, DPhe7]-α-MSH).
Culture and Treatments:
Melanocytes were seeded on a 96-well plate and cultured for 24 h (37° C., 5% CO2, DMEM 1 g/L glucose without phenol red supplemented with glucose 3 g/L, L-glutamine 2 mM, penicillin 50 U/mL, streptomycin 50 μg/mL, fetal calf serum (FCS) 10%). After incubation, the culture medium was then replaced with supplemented or unsupplemented culture medium (non-stimulated control) with a stable derivative of α-MSH and with or without (controls) the test compounds or the reference (kojic acid at 25, 100, 400, 800 μg/mL). Each experimental condition was carried out with n=3, apart from the controls carried out with n=6. The cells were then incubated for 72 h. Wells without cells received in parallel the same quantities of medium, supplemented or not with NDP-MSH and with or without the test compounds or the reference in order to quantify the background noise associated with the presence of the compounds.
Melanin Assay
At the end of 72 hours of incubation, the total melanin (intra- and extracellular) was quantified by measuring the absorbance of each sample at 405 nm (direct reading of the culture plates) against a standard range of melanin (melanin concentrations tested from 0.78 to 100 μg/mL).
The background noise, measured in the wells without cells, was subtracted from the values measured so that only the effect connected with the production of melanin is taken into account, without including any interference connected with the presence of the compounds. The results were expressed in percentage of melanin relative to the control as well as in percentage inhibition.
Evaluation of the Viability of the Cells—Test of Reduction of MTT
At the end of the treatment, the cells were incubated in the presence of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), the conversion of which to blue crystals of formazan is proportional to the activity of succinate dehydrogenase (mitochondrial enzyme). After disruption of the cells, the formazan was dissolved in DMSO medium and the optical density (OD), representative of the number of live cells and of their metabolic reactivity, was measured with a microplate reader at 540 nm (VERSAmax, Molecular Devices).
The inhibition of melanogenesis by the claimed compounds is demonstrated at the cellular level. Their depigmenting properties are superior to those of the reference compounds, in particular those of the family of unsaturated α,β hydroxy acids (ref 1, ref 2, ref 3, ref 4).
The elastases are a sub-family of serine proteases responsible for the degradation of elastin. The numerous natural substrates of this enzyme include, in addition to elastin, the proteoglycans of cartilage, fibronectin and the type I, II, III and IV collagens. At the cutaneous level, inhibition of elastase can combat the effects of ageing, whether or not photo-induced, and limit the appearance of wrinkles and stretch marks.
In addition to a decrease in the production and an increase in the degradation of the extracellular matrix, skin ageing is accompanied by a decrease in proliferative capacity of the fibroblasts.
Thus, stimulation of the proliferation of the aged fibroblasts makes possible a partial reversal of the deleterious effects of ageing.
The phases of migration and proliferation of the cells are major phases in healing, which occur after the inflammation phase. They are necessary for recolonization of the wound.
An increase in migration and proliferation of the cells allows an improvement in healing.
The phases of migration and proliferation of cells are major phases of healing that occur after the inflammation phase and are necessary for recolonization of the wound.
An increase in the migration and proliferation of cells allows an improvement in healing.
| Number | Date | Country | Kind |
|---|---|---|---|
| 11/02317 | Jul 2011 | FR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/FR2012/051592 | 7/25/2011 | WO | 00 | 3/11/2014 |
