Title of Invention

METHOD FOR PREPARING UNSATURATED FATTY HYDROXY-ACIDS AND ESTERS THEREOF

Abstract Method for the preparation of unsaturated hydroxy fatty acids and their esters responding to the following general formula (Id): Formula (Id) with n = 1 to 4 m = 2 to 16 RI = OH, CI, Br, OR3 in which R3 represents a straight or branched alkyl, alkenyl or alkynyl radical of 1 to 16 carbons or glycerol esters, optionally substituted by one or more atoms of carbon, nitrogen, sulfur or halogens such as fluorine, chlorine, bromine and iodine; R2 = H, SiR'1R'2R'3 in which R'1 R'2 and R'3 can be identical or different from each other and represent a straight or branched alkyl, alkenyl or alkynyl radical of 1 to 16 carbons or glycerol esters, optionally substituted by one or more atoms of carbon, nitrogen, sulfur or halogens such as fluorine, chlorine, bromine and iodine; or R2 = C-Ar3 with Ar representing an aryl radical optionally substituted by one or more atoms of carbon, nitrogen, sulfur or halogens such as fluorine, chlorine, bromine and iodine; or R2 = the terrahydropyranyl of formula: 27 comprising the steps of 28 (1) Bromination of formula II to formula III (2) Oxidation in aldehyde of formula III (3) Witting-Horner reaction of formula la (4) Saponification of formula reacting la to lb (5) Protection of formula lb to lc (6) Esterification of formula lc to Id (7) deprotection of formula Id to le in which Ri, R2, m and n have the same meanings as in formula Id.
Full Text FORM 2
THE PATENTS ACT, 1970 (39 of 1970)
COMPLETE SPECIFICATION (See Section 10, rule 13)
METHOD FOR PREPARING UNSATURATED FATTY HYDROXY-ACIDS AND ESTERS THEREOF.
POTIER PIERRE of 14 AVENUE DE BRETEUIL, F-75007 PARIS, FRANCE, FRENCH National
The following specification particularly describes the nature of the invention and the manner in which it is to be performed : -

The present invention pertains to the field of chemical methods and to the use of the products obtained by these chemical methods.
The present invention pertains more particularly to a method for the preparation of unsaturated hydroxy, fatty acids and their esters responding to the following general formula (Id): Formula (Id)
Ferrule (Id)

avec n = 1 a 4
m = 2 a 16
with n = 1 to 4
m = 2 to 16
R1 == OH, CI, Br, OR3 in which R3 represents a straight or branched
alkyl, alkenyl or alkynyl radical of 1 to 16 carbons or glycerol esters,
optionally substituted by one or more atoms of carbon, nitrogen,
sulfur or halogens such as fluorine, chlorine, bromine and iodine;
R2 = H, SiR’1R’2R'3 in which R'1 R'2 and R'3 can be identical or different from each other and represent a straight or branched alkyl, alkenyl or alkynyl radical of 1 to 16 carbons or glycerol esters,
2

optionally substituted by one or more atoms of carbon, nitrogen, sulfur or halogens such as fluorine, chlorine, bromine and iodine; or R2 = C-Ar3 with Ar representing an aryl radical optionally substituted by one or more atoms of carbon, nitrogen, sulfur or halogens such as fluorine, chlorine, bromine and iodine; or R2 = the tetrahydropyranyl of formula:

ou R2- le tetrahydropyranyl de formule :

and the use of said products as anticollagenase agent, lipolytic agent or antiacne agent in a pharmaceutical and/or cosmetic preparation.
The products responding to the general formula (Id) are known and described in the literature for their biological properties and more particularly for their cosmetic and pharmacological properties. Moreover, the principal lipid constituent of honeybee royal jelly, which is trans-10-hydroxy-2-decenoic acid (or DHA) responds to the general formula (Id) in which R1 = OH, R2 = H, n = 1 and m = 3.
Various documents of the state of the art report methods for the preparation of unsaturated hydroxy fatty acids and their esters (Lee et al., 1993, J. Org. Chem., vol. 58, pages 2918-2919; Hurd and


3

Saunders, 1952, J. Am. Chem. Soc, vol. 74, pages 5324-5328; Krishnamurthy et al., 1989, Indian J. Chem. Sect. A, vol. 28, pages 288-291; Plettner et al., 1995, J. Chem. Ecol., vol. 21, pages 1017-1030). The methods that are already known in the state of the art present an oxidation step during which metallic salts such as chromium salts or manganese salts are employed. However, the use of metallic salts presents a certain number of disadvantages. First of all, at the level of the products obtained by these methods, these products can be contaminated by the metallic salts and thus their cosmetic and/or pharmacological application is limited due to this contamination. Second of all, the use of metallic salts leads to a contamination of the environment of the factories in which the synthesis is performed.
The preparation method therefore intends to resolve the problems cited above by proposing a novel and original route of synthesis capable of industrial transposition.
Moreover, the method of the invention is remarkable in that it enables a more rapid synthesis method with better yields that the methods known in the prior art. The method of the inventions makes it possible to eliminate from the first steps of said method the chromatographic procedures which are not industrial purification techniques.
The general synthesis diagram is the following:
4

4
Le schema general de synthase est le
suivant

HO

OH

HBr

HO' T-Sn "Br
m
m

o

H0^t4^ CHO (V



HO

jmL ^^n OH lb

R4

C»H»1

la


ou R1( R2, m et n ont la meme signification que dans la formule {Id) .
in which R1, R2, m and n have the same significance as in formula (Id).
The first step of this synthesis is a bromination; the initial compound of the reaction is a diol of formula (II). Numerous

5
techniques enabling bromination are known in the state of the art and can be used by the expert in the field in this step. This bromination requires the use of a solvent which can be, especially, toluene, benzene, dimethylformamide, tetrahydrofuran, cyclohexane, heptane, petroleum ether, etc. The reagent used in this bromination step can be aqueous or nonaqueous HBr, Ph3P, Br2, carbon triphenylphosphine tetrabromide or hydrobromic acid. As an example, we can cite the experimental bromination conditions using aqueous HBr described in Geresh et al., Tetrahedron Asymmetry, 1998, vol. 9, pages 89-96.
The second step is an oxidation of an aldehyde of formula (IV) in the presence of an optionally cyclic, optionally anhydrous tertiary amine N-oxide in the presence of DMSO. At the end of the reaction, the corresponding tertiary amine bromhydrate is eliminated by simple filtration. The optionally cyclic, optionally anhydrous tertiary amine N-oxides present in the second step are advantageously selected from among N-methyl morpholine oxide, trimethylamine oxide or triethylamine oxide or a mixture of these. Known in the prior art are other techniques enabling synthesis of aldehydes of general formula IV. However, step 2 of the method of the invention makes it possible to resolve the drawbacks of the techniques already known in the state of the art. As an example, we can cite the oxidation reaction in the presence of manganese salts of the corresponding cyclic alkenes (Lee et al., 1993, J. Org. Chem., vol. 10, pages 2918-2919). Step 2 of the method which is the object of the
6

invention thus makes it possible to avoid a step involving the presence of metallic salts. The article by Guindon et al. of 1984 (J. Org. Chem., vol. 49, pages 3912-3920) describes the synthesis of 8-hydroxy-octanal from 1,1- dimethoxy-8-methoxymethoxy-octane. However/ the synthesis yield of 8- hydroxy-octanal is relatively low (36%) whereas step 2 of the invention makes it possible to obtain higher yields. The other techniques known in the prior art enabling synthesis of aldehydes of general formula IV are lengthy synthesis methods involving more than 4 steps.
Step 3 of the method of the invention is a Witting-Homer reaction. This reaction is a reaction known by the expert in the field (Modern Synthetic Reaction. Second edition, Herbert 0. House, Witting Horner reaction, pages 682-703) and any experimental condition described in the state of the art can be used in the framework of the present invention. As an example, the Witting-Horner reaction can be performed in the presence of triethylphosphonoacetate and potassium carbonate.
Step 4 of the method of the invention is a saponification Step. No particular experimental condition is implemented in the method of the invention. The expert in the field on his own would be able to find suitable experimental conditions to use for this step.
Step 5 of the method is a step of specific protection of the alcohol functional group of the compound of general formula lb obtained in step 4. This reaction is performed in any enol ether in the presence of an acid catalyst. The reaction is advantageously
7

performed in dihydropyrane in the presence of PTSA (para toluene sulfonic acid). The product of general formula 1c obtained after step 5 is purified by simple aqueous washing and drying over sulfate. Steps 2 and 5 of the method of the invention are not described in the state of the art. They make it possible to resolve the technical problems described above while also increasing the yield and the rapidity of the method for the synthesis of the compounds of general formula I.
The product of formula (Id) obtained in step 5 of the method of the invention can be subjected to a final deprotection in order to obtain the compound of general formula (Ie). This deprotection is performed in a solution of methanol containing an acid catalyst. Any acid catalyst can be used in the invention. The acid catalyst employed is advantageously PTSA.
The product of formula (Id) obtained in step 5 of the method of the invention can be used in an esterificatioa reaction of the glycerol. Depending on the relative quantities of glycerol used, it is possible to obtain monoesters (2 possible isomers: in position 1 and 2), diesters (2 possible isomers: diesters 1.1 and 1.2) and triesters. After the step of esterification of the glycerol, the compound obtained can undergo a final deprotection under experimental conditions identical to the deprotection conditions of the product of formula (Id) cited above.
The products obtained by the method which is the object of the present invention are, as indicated above, used in the cosmetic and/or pharmaceutical field. However, the research of the applicant was able to demonstrate that the products obtained by the method of

8

the invention followed by a final deprotection step present an anticollagenase activity.
Collagen is the most abundant and important protein of the human body and the skin. This scleroprotein represents notably 75% of the proteins of the dermis to which it provides solidity. The fibroblasts create from the amino acids (hydroxyproline, lysine, proline) precollagen molecules which are transformed in the presence of vitamin C into collagen molecules. In order to form a network of fibrils, the collagen must create bonds among these different molecules.
Collagen renewal changes with age. The soluble collagen which bestows suppleness and resistance to the skin and the mucosa degrades increasingly rapidly under the influence of the proteolytic enzyme collagenase, which leads at the dermal level to an aging of the fibrous structure of the proteins. Moreover, the insoluble collagen which leads to a loss of elasticity becomes rigid as it polymerizes with the glucose molecules as a result of multiple bonds which are difficult to reverse (glycation phenomenon). These bonds make the collagen more resistant to attack by collagenases which leads to an increasing rigidity of the collagen fibers. This hardening phenomenon, characteristic of aged cutaneous tissues, must be combated as early as possible because it increases the destruction of fibroblasts by free radicals but also the denaturation of the dermal proteins.
9

The collagenases are enzymes that are weakly expressed under normal physiological conditions. Their over expression in aging and, in particular, during menopause in the female, lead to a greater denaturation of the dermal fibrous proteins.
However, the destruction of the collagen fibers can take place under circumstances other than aging. In fact, during a bacterial infection, the bacterial collagenases can destroy the collagen fibers of the infected host.
Moreover, tumoral invasion requires a degradation of the basal membrane and the extracellular matrix and of all of the structural proteins of these components which include collagen. There has therefore been demonstrated a very clear relationship between the invasive power of tumors and the presence of collagenase activity in human tumors. Collagenases are found at the level of tumor cells, but also in the fibroblasts surrounding the tumor. Normal epithelial cells secrete a very small amount of collagenases whereas these proteins are overexpressed by invasive or metastatic tumor cells.
Other degenerative diseases present a fibrinoid degeneration of collagen and are also referred to as "collagen diseases".
The invention therefore pertains to the use of products that can be obtained by the method of the invention as active anticollagenase agents. The research studies performed by the applicant particularly demonstrated the anticollagenase activity of trans-10-hydroxy-2-decenoic acid (DHA) and the glycerol ester of trans-10-hydroxy-2-decenoic acid (glycerol monoester in position 1). The invention also
10

pertains to the use of trans-10-hydroxy-2- decenoic acid (DHA) and the glycerol ester of trans-10-hydroxy-2-decenoic acid as an anticollagenase agent in a pharmaceutical and/or cosmetic preparation.
The invention pertains to the use of trans-10-hydroxy-2-decenoic acid (DHA) and the glycerol ester of trans-10-hydroxy-2-decenoic acid for the preparation of a drug intended for the prevention of or to cure the degradation of collagen. This drug is most particularly intended to prevent or cure the degradation of collagen by bacterial collagenases during a bacterial infection
The invention also pertains to the use of trans-10-hydroxy-2-decenoic acid (DHA) and/or the glycerol ester of trans-10-hydroxy-2-decenoic acid for the preparation of a drug intended for the regeneration of the skin and ligaments.
The invention also pertains to the use of trans-10-hydroxy-2-decenoic acid (DHA) and/or the glycerol ester of trans-10-hydroxy-2-decenoic acid for the preparation of a drug intended to prevent or cure tumoral invasion.
The invention also pertains to the use of trans-10-hydroxy-2-decenoic acid (DHA) and/or the glycerol ester of trans-10-hydroxy-2-decenoic acid for the preparation of a drug intended to prevent or cure degenerative diseases presenting a fibrinoid degeneration of the collagen and also referred to as "collagen diseases".
As stated above, the products obtained by the method which is the object of the present invention are used in the cosmetology
11

and/or pharmaceutical field. The applicant's research studies demonstrated that the products obtained by the method of the invention followed by a final deprotection step, and more particularly, trans-10-hydroxy-2-decenoic acid (DHA), present a lipolytic activity. Consequently, the products obtained by the method of the invention followed by a final deprotection step and, more particularly, DHA, can especially be employed in weight-reduction treatments and in any treatment known to require a lipolytic activity.
The products obtained by the method which is the object of the present invention are, as stated above, used in the cosmetology and/or pharmaceutical field. The applicant's research studies demonstrated that the products obtained by the method of the invention followed by a final deprotection step, more particularly, trans-10-hydroxy-2-decenoic acid (DHA) present an antiacne activity.
The treatment of acne requires the treatment of two major problems-which are, on the one hand, seborrhea, and, on the other hand, the bacterial proliferation responsible for the cutaneous inflammation. The applicant's research studies demonstrated that the method of the invention followed by a final deprotection step and, more particularly, DHA, present both a sebum- regulatory and an antibacterial activity.
DHA is capable of inhibiting cutaneous 5-alpha-reductase, the enzyme responsible for the production of di-hydro-testosterone. Treatment with 0.1% of DHA and treatment with 0.5% of DHA reduced by circa 70% and circa 90% respectively the 5-alpha-
12

reductase activity compared to an untreated control. This inhibition results in a considerable reduction of the sebum level.
Furthermore, 0.5% of DHA presents after 14 or 28 days of treatment a bactericidal effect of circa 95 to 100% tested on Propionibacterium acnes, Staphylococcus aureus and Malassezia furfur.
Other advantages and characteristics of the invention will become apparent from the examples below pertaining, first of all, to the synthesis methods of the invention and then to the anticollagenase and lipolytic properties of the products that can be obtained by means of the synthesis methods of the invention.
Example 1. Operating mode for svnthesis of la (n = 6, m = 6, Rl = OEt) and Ie (glycerol triester) 1. Step 1: Bromination
Example 1 : Mode operatoire pour la
gynthese_ de la (n=1, m=6, Rl=OEt). et Ie (triester de glycerol)
V ■ auf/wiji&Be
HOv
-H20
M-14&23 M-209.I3
13

438 g (3 mol) of 1,8-octanediol was dissolved in 3 1 of toluene. 375 ml (3.3mol) of aqueous 48% HBr was then added. The medium was then heated to eliminate the water that was present and the water formed during the reaction by azeotropic distillation. After 13.5 h of contact, the medium was cooled and taken up with a saturated solution of NaHC03. The organic phase was separated and washed with a saturated solution of NaCI. After drying over MgSC4, the medium was concentrated to yield a crude product of 672 g.
The 8-bromooctanol was purified by distillation under reduced pressure, at 96°C under P - TLC: Rf = 0.8 (heptane/ether iso 8/2)
- 'H NMR (200 MHz, CDC13: 3.65 (t, 2H, J = 6.4 Hz); 3.43 (t, 2H, J = 6.8 Hz); 1.87 (m, 2H); 1.36-1.69 (m, 10H).
2. Step 2: Oxidation in aldehyde




708 g (5.87, 3 eq) of anhydrous N-methylmorpholine N-oxide was dissolved under N2 in 3 1 of DMSO. 410 g (1.96 mol) of 8-
14

bromooctanol dissolved in 11 of DMSO was then added over 30 minutes. The medium became clear. N-methylmorpholine ammonium bromide precipitated. After 65 h of agitation at ambient temperature, the salt was filtered and the medium was taken up with 4 1 of a saturated solution of NaCl. After extraction with 4 x 1 1 of ethyl acetate and drying, 320 g of crude product, constituted by 74% of aldehyde (yield = 83%) and 26% of 1,8-octanediol. Characterization
- TLC: Rf = 0.6 (heptane/ethyl acetate 8/2)
- 1H NMR (400 MHz, CDC13): 9.74 (t, 1H, J = 1.7 Hz); 3.61 (t, 2H, J = 6.6 Hz); 2.41 (dt, 2H, J = 1.7 and 7.3 Hz); 1.51-1.65 (m, 4H); 1.24-1.37 (m, 6H).
3. Step 3: Witting-Horner reaction

M*i*m
The preceding crude product (320 g) was dissolved in 3 1 of water. 800 ml (4.2 mol, 2.1 eq) of triethylphosphonoacetate was then added followed by 830 g (6 mol) of potassium carbonate. After 20 h of agitation, the reaction was A terminated. The medium was extracted by 4 x 1 1 of isopropyi ether. After drying over MgSO4 the organic phases were evaporated and yielded 650 g of crude product.
15

The product was purified either by distillation E = 120°C under P Or the product was purified by chromatography with a heptane/ethyl acetate 8/2 elution. In this case 119.6 g of products were obtained (28% from the brominated derivative). Characterization
- TLC: Rf = 0.8 (heptane/ethyl acetate 8/2)
- 1H NMR (400 MHz, CDC13): 6.91-6.99 (m, 1H); 5.78-5.82 (dt, 1H, J = 1.4 and 15.6 Hz); 4.17 (q, 2H, J = 7.1 Hz); 3.63 (t, 2H, J = 6.6 Hz); 2.18 (dq, 2H, J = 1.2 and 7.3 Hz); 1.22-1.65 (m, 11H).
4. Step 4. Saponification reaction

m

006

v ' ' mm
mm
119.6 g (0.56 mol) of hydroxyester was dissolved in 600 ml of ethanol and 400 ml of a 4.6 N solution of KOH was added. The medium was agitated for 8 h. The medium was extracted with isopropyi ether. The aqueous phase was acidified to pH = 1 and extracted with ethyl acetate. After drying and evaporation, 99.6 g of pink solids were obtained. Recrystallized in an isopropyi
16

ether/petroleum ether mixture, the product was obtained in the form
of a white solid (86 g, 83%).
Characterization
TLC: Rf = 0.2 (heptane/ethyl acetate 7/3)
Melting point: mp = 61.3°C
1H NMR (400 MHz, CDCI3): 7.06 (dt, 1H, J == 15,6 and 7 Hz); 5.81 (dt/ 1H, J = 1.5 and 15.6 Hz); 3.64 (t, 2H, J = 6.6 Hz); 2.22 (dq, 2H, J = 1.2 and 7.3 Hz); 1.52-1.58 (m, 2H); 1.45-1.48 (m, 2H); 1.33-1.3765 (m, 6H). 5. Step 5: Protection reaction

WV
,™
WVW

1/8)0


86 g (0.46 mol) of hydroxy acid was put in solution with 45 ml (0.48 mol) of 3,4-dihydro-2H-pyran in 500 ml of THF. 1 ml of concentrated HC1 was added and the medium was agitated for 24 h.
The THF was then concentrated, the crude product was taken up with ethyl acetate and washed with a saturated solution of NaCI until neutral pH. After drying over MgS04, 132 g of crude product was obtained (> 100%). Characterization
TLC: Rf = 0.4 (heptane/ethyl acetate 7/3)
17

1H NMR (400 MHz, CDC13: 7.06 (dt, 1H, J = 15.6 and 7 Hz); 5.81 (dd, 1H, J = 1.6 and 15.6 Hz); 4.58 (t, 1H, J = 2.8 Hz); 3.82-3.91 (m, 1H); .3.71-3.73 (m, 1H); 3.51-3.52 (m, 1H); 3.36-3.69 (m, 1H); 2.20 (m, 2H); 1.33-1.89 (m, 16H). 6.
Step 6: Esterification reaction of the glycerol

I.
:/Nj>'



I I
8.4 g (0.091 mol) of glycerol was put into solution in 500 ml of dichloromethane. The preceding crude product (0.46 mol), after elimination of traces of water by azeotropic distillation, was dissolved in 500 ml of dichloromethane and added to the medium. 56.8 g (0.46 mol) of dimethylamino pyridine was then added followed by 97 g (0.46 mol) of dicyclohexylcarbodiimide. The medium was agitated for 70 h. A precipitate appeared and was filtered.
The medium was concentrated and taken up in isopropyl ether. After filtration and concentration, 156 g of crude product was obtained and purified by chromatography and with a 7/3 heptane/ethyl acetate elution.
99 g of a fraction containing 2/3 product and 1/3 acyl urea was obtained.
18

Characterization
TLC: Rf = 0.7 (heptane/ethyl acetate 7/3)
1H NMR (400 MHz, CDC13: 6.96 (m, 3H); 5.80 (dd, 3H, J= 1.6 and 15.6 Hz); 5.29-5.31 (m, IH); 4.54-4.57 (m, 3H); 4.20-4.39 (m, 4H); 3.81-3.89 (m, 3H); 3.68-3.72 (m, 3H); 3.41-3.49 (m, 3H); 3.34-3.39 (m, 3H); 2.25-2.18 (m, 6H); 1.33-1.99 (m, 48H). 7. Step 7: Final deprotection



MS.

wmM

99 g (0.136 mol) of the preceding mixture was dissolved in 1 1 of methanol with 9.9 g of PTSA. The medium was agitated for 14 h. The reaction was terminated. The medium was then concentrated. The oil obtained was then taken up with H2O and brought to pH = 6 with a saturated solution of NaHCO3. The aqueous phase was extracted with dichloromethane. After drying of the organic phase and evaporation, 77 g of a yellow oil was obtained.
The product was purified by chromatography on silica CH2CI2/acetone 9/1 to 1/1 and CH2C12 / methanol 95/5.
19

27 g of product was obtained in the form of an oil which crystallized in the form of an amorphous yellowish white solid with a purity between 85 and 90%. Characterization
TLC: Rf = 0.2 (CH2C12/ acetone 9/1)
1H NMR (400 MHz, CDC13/): 6.94-7.01 (m, 3H); 5.78-5.84 (m, 3H); 5.29-5.31 (m, IH); 4.20-4.34 (m, 4H); 3.60-3.65 (m, 6H); 2.16-2.22 (m, 6H); 1.33-1.99 (m, 30H). Example 2. Operating mode for the synthesis of:




1. Step 1: Protection of 8-bromooctanol

ttWH^JK
M-92U
21 g (0.1 mol) of 8-bromooctanol was dissolved in 200 ml of dichloromethane. 16 g (0.104 mol) of terbutyldimethylsilyi chloride was then added at 0°C, followed by 7.5 g (0.11 mo) of imidazole. A precipitate was formed instantaneously. After 3 h of agitation, the
20

medium was filtered, concentrated and the crude product was distilled.
25.8 g of product was thus isolated at 99-104°C under P 1H NMR (400 MHz, CDC13): 3.59 (t, 2H, J = 6.6 Hz); 3.39 (t, 2H, J = 6.9 Hz); 1.82-1.89 (m, 2H); 1.30-1.50 (m, 10H); 0.88 (t, 9H, J = 2.7 Hz); 0.04 (s, 6H).
2. Step 2: Oxidation in aldehyde

20 g (61 mmol) of silyi derivative was put into solution in 200 ml of DMSO. 21.7 g (0.18 mol) of N-methylmorpholine N-oxide was then added. The medium was agitated for 72 h. A precipitate appeared. The medium was diluted with saturated NaCl then extracted with isopropyl ether. After drying and evaporation, 15.3 g of crude product was obtained.
The product was purified by distillation at 81 °C under P 21

1H NMR (400 MHz, CDC13: 9.76 (t, 1H, J = 1.9 Hz); 3.59 (t, 2H, J = 6.6 Hz); 2.42 (dt, 2H, J = 1.8 and 7.2 Hz); 1.49-1.68 (m, 4H); 1.30-1.32 (m, 6H); 0.88 (t, 9H, J = 2.7 Hz); 0.04 (t, 6H, J == 2.9 Hz). 3. Step 3: Witting reaction


wm&


1 vM^^^^wigsw^a^^

'S/

M"$M*
835 mg (21 mmol) of NaH was put into solution with 5 ml of THF and cooled to T 3.7 g of product was obtained by purification on silica gel (heptane/ethyl acetate 8/2 elution) (60%). Characterization
TLC: Rf = 0.6 (heptane/ethyl acetate 8/2)
'H NMR (400 MHz, CDC13: 6.95 (dt, 1H, J == 8.6 and 15.6 Hz); 5.79 (dt, 1H, J = 1.4 and 15.8 Hz); 4.17 (q, 2H, J == 7.1 Hz); 3.58 (dt, 2H, J = 6.6 and 9.8 Hz); 2.15-2.21 (m, 2H); 1.46-1.51 (m, 4H); 1.24-1.42 (m, 9H); 0.88 (t, 9H, J = 2.7 Hz); 0.04 (t, 6H, J ==2.9 Hz).
22

4. Step 4: Saponification
2 g (6 mmol) of ester was dissolved in 10 ml of ethanol and 5 ml of a 3.8 N solution of NaOH was added. The reaction was terminated in 4 h. The medium was acidified to pH = 1 and extracted with ethyl acetate. The product was thereby obtained without additional purification (1.5 g, 83%). Characterization
TLC: 0.2 (heptane/ethyl acetate 7.3)
'H NMR (400 MHz, CDC13: 7.07 (dt, 1H, J = 8.6 and 15.6 Hz); 5.81 (dt, 1H, J = 1.4 and 15.6 Hz); 3.59 (dt, 2H, J = 6.6 and 9.8 Hz); 2.21-2,27 (m, 2H); 1.46-1.51 (m, 4H); 1.24-1.42 (m, 6H); 0.89 (t, 9H, J = 2.7 Hz); 0.04 (t, 6H, J = 2.9 Hz).
Example 3. Evaluation of the anticollagenase activity of products
obtained by the method of the invention on frozen sections of human
skin
1. Operating mode
This study was performed with different solutions at the
concentration of 1 and 2% of active principles in comparison with the
23

excipient alone, buffer controls and collagenase. The active principles used were DHA, the 2-dimethylamino ethyl ester of trans-10-hydroxy-2-decenoic acid (ML40) and the glycerol ester of trans-10-hydroxy-2-decenoic acid (GM). Table 1 lists the different solutions tested.
Table 1
Tableau 1

Solution. 1 ; 2 3 4 5 S 7 9 ib in 12 13 I
Dim 21 j 1% AT! 1 1% |
ML40 |2% 2% 1% _ it J
j GM 2% j 11 j
! Collag&aase (U/ffllJ i 30 | 30 3C 30 30 30 30 [
key to column at left: Solution / DHA / ML40 /GM / Collagenase (U/ml)
Frozen 5-um-thick sections from a mammary plasty of a 54-year-old woman were placed on histological slides (4 sections per slide). Each solution was tested on one slide.
The sections were covered with the solutions to be tested then incubated for 2 hours at 37°C in a humid chamber. The solutions were eliminated by repeated rinsings and the sections were stained
24

with picrosirius. Microscopic examination was performed with the 2.5 objective and paper photographs were taken with Kodak Gold 100 ASA film. 2. Results
Table 2 summarizes the results of alteration of the collagen structure as a function of the tested solution. An absence of alteration of the collagen structure is indicated by 0 while a somewhat or markedly to very strongly altered collagen structure is indicated respectively by 1 or 2.
Table 2

Key to column at left: Solution / ML40 / GM / Collagenase U/ml / Alteration of the collagen structure
Moreover, application of the buffer control Tris or the excipient did not induce any alteration of the collagen structure.
25

Consequently, the product DHA at 1 and at 2% inhibited completely the activity of collagenase, whereas the products ML40 and GM at 2% only slightly inhibited the activity of collagenase.
Example 4: Evaluation of the anticollagenase activity of GM obtained by the method of the invention on human skin explants maintained in survival state 1. Operating mode
The study was performed on a 5% GM product in comparison with the excipient (hydrocerin), a positive control and a control in the presence of collagenase at 100 U/ml.
Hydrocerin was used as the excipient for the preparation of the product to be applied. This study was performed twice. In the first study, it was found that the action of collagenase on Day 2 remained very limited and insignificant. In the second study, the study time was extended and the collection of the explants was performed on Day 2 and on Day 4.
a. Preparation of the explants
Human skin explants prepared and distributed into 16 lots of three explants each were placed in survival state according to table 3.
26

Table 3

Tableau 3

J4

""""""" '"I



T^moin

3 ex.plaii.t& ;3 explants



Excipient

3 explants 3 explants



Produit a 5%
3 explants jj3 sxplants



Coatrdle p
3 explants; 3 explants



T^moiii + collaginase

3 explants 13 explants



| Excipient +• eollagenase

3 explants ]3 explants



Produit 1 5% (M + collageisase

3 explants 13 explants I



Oontrdle positif * collag&iase

3 explants 3 explants

Key: J = Day
Column at left: Control / Excipient / Product containing 5% GM / Positive control / Control + collagenase / Excipient + collagenase / Product containing 5% GM + collagenase / Positive control + collagenase
b. Application of the product containing 5% GM
The product was applied on Day 0 and on Day 2 at the rate of 20 mg per explant and collagenase was incorporated into the culture medium of the last 24 lots.
c. Histology
Three explants of each lot were collected on Day 2 and on Day 4, fixed in ordinary Bouin's fixative and subjected to histological processing.
27

The histological study comprised:
impregnation in paraffin,
sections,
staining with Sirius red F3B,
colorimetric measurements of the collagen by image analysis,
comparison with photographs.
2. Results
The samples collected on Day 2 did not reveal any significant collagenase activity in any of the lots examined. For this reason, the survival, the contact and the application were extended to Day 4. The collagenase action was monitored in two ways: intensity of the coloration of the collagen network and thickness of the dermal structure. With this study, the penetration of the active principle and its inhibitory activity were correlated in relation to collagenase. The results obtained were as follows:
- for the controls without collagenase, the dermis presented a
normal structure with regular bundles of collagen in all of the
compartments;
- for the controls with collagenase, the collagen bundles were
strongly degraded and the thickness of the dermis had diminished
by half;
- for the explants with excipient and collagenase, the collagen
bundles were strongly degraded but to a lesser degree than that
seen with the controls with collagenase, and the thickness of the
dermis had diminished by almost half;
28

- for the explants with the product containing 5% of GM and collagenase, the collagen bundles were very slightly degraded and the thickness of the dermis had diminished slightly;
- for the explants with positive control (phenanthroline) and collagenase, the dermal structure was identical to that of the controls without collagenase.
Under these experimental conditions, the GM product demonstrated a pronounced anticollagenase activity.
Example 5. Evaluation of the antilipolytic of DHA obtained by the method of the invention on adipose tissue explants ex vivo 1. Operating mode
The DHA was incorporated in the culture medium at the final concentration of 0.25 and of 0.5%. After a contact of 8 days, the activity was evaluated by quantitative determination of the lipids distributed in the culture medium.
a. Preparation of the explants
Twelve adipose tissue explants (plasty P202-AB31) were prepared and placed in survival state in BEM medium (BIG-EC'S Explants Medium). The explants were distributed in 4 lots of 3 explants: one control lot,
one positive control lot (caffeine at 0.1%), two product lots (DHA at 0.25 and 0.5%). b. Application of the products
29

On Day 0, the explants were placed in survival state in 2 ml of culture medium, in which the product to be tested was incorporated. This treatment was renewed on Day 2, Day 4 and Day 6.
c. Samples
On Day 2, Day 4, Day 6 and Day 8, the culture medium was collected. For each explant, the media collected on Day 2, Day 4, Day 6 and Day 8 were grouped together in the same tube and preserved at -20°C for quantitative determination of the lipids.
On Day 8, the adipose tissue explants were collected and fixed in ordinary Bouin's fixative for the histological study.
d. Histology
The fixed adipose tissue explants were dehydrated, impregnated with paraffin, but in block form, sectioned and stained with Masson's trichrome.
e. Quantitative determination of the lipids
After extraction of the culture medium/ the lipids were separated and quantitatively determined by TLC. 2. Results
The viability and morphology of the adipocytes was monitored by the histologic study.
The lipolytic activity was evaluated by analysis of the proportions of Monoglycerides, diglycerides, triglycerides and free fatty acids, a. Histology
30

After 8 days of maintenance in the survival state, the controls and treated explants presented no visible alterations nor cellular necroses.
b. Quantitative determination of the lipids
The results obtained are presented in table 4 below. The results are expressed in mass (mg) of each category of lipid released in the culture medium during the 8 days of treatment.

Acides teas
KoftQ-'Slyciridas ;Di"8Iydeiride6
0,00
1,06
rm,/ms
a O.Str

tableau 4
.,,,,,,,,,„™™i™„ i,SS j 0,00

11,53

.-.v.-.iv.v.v.v.w.-.v»-j-*J

Key: Top column headings: Monoglycerides / Diglycerides / Fatty
acids / Triglycerides
Second level of column headings (repeated 4 times): Mean /
Standard deviation
Column at left: Control / Caffeine 0.1% / DHA/JLB at 0.25% /
DHA/JLB at O.5%
31

Table 5 below represents the statistical analysis using Students test of the results of the quantitative determination of the fatty acids released in the culture medium during the 8 days of treatment
Table 5

Tableau 5
T&aoin daf&ins DHA a o,as% om £ 0,5%
1,10 33, 80 9,9S 12,61
0,94 .^"i j *i»-v: 11,67 11, €4
0, 87 25,9721,99 12, €6 10,14
tieymim 0,$7 U/43 J* JL g "J J^J
Ecart-type 0,1 4,2 1,1 1,1
% 2780,6 1075,6 lose,8
&> atigmeatation/Timoin
Probabi' „lt& « |> » 0,012 0, 005 0,005
Key: Column headings: Control / Caffeine / DHA at 0.25% / DHA at
0.5% At middle left: Mean / Standard deviation
At bottom left: % augmentation/Control / Probability "p"
The essential parameter in this study was the variation of the quantity and the percentage of the fatty acids released in the culture medium after the treatment period.
Compared to the untreated control, an augmentation by a factor of circa 29 (2780%) for the positive control (caffeine at 0.1%) was observed.
32

DHA at 0.25 and 0.5% led to a significant augmentation respectively of 1075 and 1087%. The augmentation of the concentration used did not have an effect on the efficacy. It would appear that the maximum effective dose is on the order of 0.25%.
Under the operating conditions described above and according to Student's test, DHA applied at 0.25 and 0.5% presents a significant lipolytic activity to that of caffenine.
33

Documents:

147-mumnp-2004-cancelled page(27-2-2004).pdf

147-mumnp-2004-claim(granted)-(27-2-2004).pdf

147-mumnp-2004-claims(granted)-(27-2-2004).doc

147-mumnp-2004-correspondence(30-3-2005).pdf

147-mumnp-2004-correspondence(ipo)-(1-2-2005).pdf

147-mumnp-2004-correspondence-others.pdf

147-mumnp-2004-correspondence-received-010704.pdf

147-mumnp-2004-correspondence-received-260204.pdf

147-mumnp-2004-correspondence-received-300305.pdf

147-mumnp-2004-correspondence-received.pdf

147-mumnp-2004-descripiton (complete).pdf

147-mumnp-2004-diagram.doc

147-mumnp-2004-form 19(27-2-2004).pdf

147-mumnp-2004-form 1a(1-7-2004).pdf

147-mumnp-2004-form 1a(27-2-2004).pdf

147-mumnp-2004-form 2(granted)-(27-2-2004).doc

147-mumnp-2004-form 2(granted)-(27-2-2004).pdf

147-mumnp-2004-form 3(26-2-2004).pdf

147-mumnp-2004-form 5(26-2-2004).pdf

147-mumnp-2004-form-19-270204.pdf

147-mumnp-2004-form-19.pdf

147-mumnp-2004-form-1a.pdf

147-mumnp-2004-form-2.doc

147-mumnp-2004-form-2.pdf

147-mumnp-2004-form-26.pdf

147-mumnp-2004-form-3.pdf

147-mumnp-2004-form-5.pdf

147-mumnp-2004-pct-search report.pdf

147-mumnp-2004-power of attorney(2-6-2004).pdf


Patent Number 210959
Indian Patent Application Number 147/MUMNP/2004
PG Journal Number 41/2008
Publication Date 10-Oct-2008
Grant Date 16-Oct-2007
Date of Filing 27-Feb-2004
Name of Patentee POTIER PIERRE
Applicant Address 14, AVEUE DE BRETEUIL, F-75007 PARIS
Inventors:
# Inventor's Name Inventor's Address
1 POTIER PIERRE 14, AVEUE DE BRETEUIL, F-75007 PARIS
2 PICOT Françoise 50, rue de Dampierre, F-78460 Chevreuse
3 BRAYER JEAN-LOUIS 42, rue Jules Dubrulle, F-60440 Nanteuil Le Haudouin
PCT International Classification Number C07C45/28
PCT International Application Number PCT/FR02/03094
PCT International Filing date 2002-09-11
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 01/11815 2001-09-12 France