FORM - 2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See Section 10 and Rule 13)


THfANINE DERIVATIVES, USES THEREOF AND PROCESSES FOR THE MANUFACTURE THEREOF
HINDUSTAN UNILEVER LIMITED, a company incorporated under
the Indian Companies Act, 1913 and having its registered office
a\ 1S5MS6. Backbay Reclamation, Mumto -400 020, MahaiashYra, totfta

The following specification particularly describes the invention and the manner in which it is to be performed


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THEANINE DERIVATIVES, USES THEREOF AND PROCESSES FOR THE
MANUFACTURE THEREOF
Technical Field of the Invention
5 The present invention relates to derivatives of the amino acid theanine. The present invention also relates to the use of such compounds in pharmaceutical, cosmetic and food compositions as well as to processes for their manufacture.
10 Background of the Invention
Food and beverage products which can enhance the mental and physical aspects of the human body are becoming increasingly popular. In particular, products which produce an enhanced state of mental acuity are especially commercially valuable.
15 Theanine is an amino acid which, within the plant kingdom, is uniquely found in tea (Camellia sinensis). Theanine has been found to have numerous beneficial effects on the human body and mind. For example, it is reported that theanine stimulates oc-waves in the mammalian brain and bestows a relaxed but alert
20 feeling to the individual. These physiological effects are
particularly apparent at specific dosage levels. For example,
International patent application published as WO 2006/061097
(Unilever PLC et al.) describes consumable compositions which
comprise specific levels of theanine and caffeine and which are
25 shown to provide noticeable improvements in concentration, mental focus and/or alertness of an individual consuming the compositions. European patent application published as EP 1 393 726 A (Taiyo Kagaku KK) also discloses compositions for improving mental concentration containing theanine.
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Although tea is relatively rich in theanine, in fact theanine only comprises about 1% by weight of the extractable tea solids in tea plant material. Thus, synthetic theanine (e.g. Suntheanine™ produced by Tayio Kagaku) has been developed to 5 meet the increasing demand for products with enhanced levels of theanine. Furthermore, theanine is unstable in aqueous solution and is easily hydrolysed, especially at low pH. Thus formulators are required to include very high levels of theanine in their products in order to ensure that they maintain efficacy over the 10 whole product shelf life.
Thus we have recognised that there is a need for new molecules that may deliver the physiological effects of theanine and/or offer enhanced storage stability. We have found that this object may be met by providing compounds that comprise theanine and/or 15 theanine-like moieties.
Definitions
Amino Acid Side Chain
Amino acids accord to the general formula (B):


20

As used herein, the term "sidechain of an amino acid" refers to that part of the amino acid represented by the radical R' in 25 formula (B).

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Theanine
Theanine is an amino acid having a glutamine side chain wherein the amide group on the side chain is ethyl-substituted. Thus theanine is also known as N-gamma-ethyl glutamine or 5-N-ethyl glutamine, and has the formula (A):

The naturally occurring form is L-theanine (N-gamma-ethyl-L-glutamine) wherein the chiral centre (i.e, the a-carbon denoted by * in fomula (A)) has (S)-stereochemistry. However, unless specified otherwise the term theanine as used herein encompasses L-theanine, D-theanine (i.e., wherein the chiral centre has (R)-stereochemistry), and mixtures thereof. L-theanine is preferred as this is believed to have the highest bio-activity.
By "theanine residue", is meant a moiety according to formula (C):


The group Xi is employed herein to represent a generic alkyl radical and is selected from linear, cyclic or branched alkyl and

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derivatives thereof. The derivatives atfe alkyl with heteroatom such as hydroxyalkyl, alkyl with amine functionality and/or alkyl with carboxyl functionality. Preferred alkyls are C1-C8 alkyIs. For example, the alkyl may be methyl, ethyl, propyl, isopropyl, 5 butyl, t-butyl, pentyl, cyclopentyl, hexylr cyclohexyl, heptyl or octyl.
The group X2 is employed herein to represent a generic alkenyl radical and is selected from linear, cyclic or branched alkenyl
10 and derivatives thereof. The derivatives are alkenyl with heteroatom such as hydroxyalkenyl, alkenyl with amine' functionality anH/or alkenyl with carboxyl functionality. Preferred alkenyls are C2-C8 alkenyls. For example, the alkenyl may be ethenyl, propenyl, isopropenyl, butenyl, t-butenyl,
15 pentenyl, cyclopentenyl, hexenyl, cyclohexenyl, heptenyl or octenyl. The term alkenyl also encompasses dienyl and higher levels of unsaturation.
The group - X3 is employed herein to represent a generic aryl 20 radical and is selected from aromatic radicals and derivatives thereof. The derivatives are aryl with heteroatom such as hydroxyaryl, aryl with amine functionality and/or aryl with carboxyl functionality. Preferred aryls are C6-C12 aryls. For example, the aryl may be phenyl, alkylphenyl, benzyl, 25 alkylbenzyl, or naphthyl.
Peptide
The term "peptide" as used herein means & compound comprising two 30 or more amino acid residues joined by at least one peptide bond.

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Summary of the Invention
In a first aspect, the present invention provides compounds of general formula (1):

10 wherein

15

Ri, R2, and R3 are each independently selected from the group consisting of H, G(Q).x snel G(Q)Q?

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In a further aspect, the present invention provides compositions comprising the compounds of the invention. These compositions are preferably suitable for administering the compound to an individual either as a pharmaceutical, cosmetic or foodstuff. 5
In a still further aspect, the invention provides use of the compounds or compositions of the invention as a medicament or in the preparation of a medicament. In particular, the compounds of the present invention may be administered to an individual to
10 provide a noticeable improvement in concentration, mental focus and/or alertness; and/or for giving a noticeable improvement in sleep quality; and/or for giving a noticeable improvement in creativity; and/or for maintaining or improving the performance of an immune system; and/or for improving digestion; and/or for
15 managing bodyweight; and/or for reducing mental stress; and/or for treating behavioural disorders; and/or for treating premenstrual syndrome; and/or for treating headache and/or migraine.
»
20 In a yet another aspect, the present invention provides a process for manufacturing the compounds of the invention. The process comprising the step of reacting theanine or a derivative thereof with at least one amino acid or a derivative thereof according to reaction scheme (I) herein below. This process allows for a
25 relatively simple route to the compounds of the invention and utilises readily available raw materials,- i.e. , amino acids and their derivatives.

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Detailed Description
Compounds
The present invention provides compounds of general formula (1)

1} -
10 These compounds contain a theanine residue, although the -residue TO&V be substituted, vjith labile groups that are easily converted in-vivo to yield the theanine residue structure. Thus Ri and R2 are each independently selected from the group consisting of H, C(0)X and C(0)OX, wherein for each of R\ and R2, X is
15 independently selected from the group consisting of H, Xif X2 and X3; and Yi and Y2 are each independently


selected from the group consisting of C=0 a
20
Q and Q' are each independently selected from the group
consisting of O, N, and S, and n is 1 or 2.
Formula (1) should also be understood to encompass salt forms of 25 any functional groups therein.

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In a preferred embodiment, the theanine residue is not substituted and the compound has formula (2):


10

15
20

The theanine residue of the compounds of formula (1) and/or (2) is coupled through a cyclic structure to a second amino acid residue such that R4 is the side chain of an amino acid or a specific derivative of theanine. Where R4 is the side chain of an amino acid, the amino acid is selected the group consisting of theanine, glycine, alanine, valine, leucine, isoleucine, glutamine, lysine, hydroxylysine, histidine, arginine, phenylalanine, asparagine, tyrosine, tryptophan, thyroxine, serine, threonine, cysteine, methionine, norvaline, ornithine, aspartic acid and glutamic acid.

5

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Where the second amino acid residue is a theanine residue or a derivative thereof, R4 is the side chain of theanine or a derivative thereof and has the formula (3):

(3) .

10

wherein R5 is selected from the group consisting of H, C{0)X and C{0)OX, wherein X is Xlf X2 or X3; and Y4 is selected
from the group consisting of C=0 and wherein Q and Q'
are each independently selected from the group consisting of 0, N, and S, and n is 1 or 2.

15

6

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In a preferred embodiment, in order to maximise the theanine-like properties of the compound, it is a dimer of theanine and has the formula (4) :


10

4)

15

In the compounds of formulae -(1) _to (4), the chiral centres
(denoted * in formula (1) above) may be (S), (R) or a combination
thereof. Preferably, however, both chiral centres have (S)-
stereochemistry.

7

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Compositions
The compounds of the present invention may be employed in substantially pure form or may comprise part of a composition. These compositions are preferably suitable for administering the compound to an individual, e.g. in the form of a pharmaceutical or cosmetic composition. The preferred mode of administration is oral and so it is preferred that the composition is edible, most preferably as a foodstuff such as a beverage.
The effective amount of the compound of the invention which is included in a composition will depend on the exact nature of the composition and the intended benefit delivered by the composition. Typically, however, the composition comprises the compound in an amount from 0.000001 to 50% by- weight of the composition, preferably from 0.0001 to 20%, more preferably from 0.001 to 10% and most preferably from 0-01 to 5%.
The compositions preferably comprise other active components which can be derived from tea {although such active components need not actually be derived from tea) . Thus the compositions preferably comprise a component selected from the group consisting of caffeine, catechins, theaflavins, thearubigens, theanine, GABA (gamma-aminobutyric acid) and mixtures thereof.
Pharmaceutical and Cosmetic Compositions
The pharmaceutical and cosmetic compositions of the present invention may be suitable for any form of administration including oral, topical and/or intravenous administration. The

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form of the composition may, among others, be a tablet, pill, lozenge, paste, lotion, gel, cream, liquid (including emulsion), spray (including aerosol spray), foam or powder.
5 The pharmaceutical or cosmetic composition comprises a pharmaceutical^ acceptable vehicle which may act as a diluent, dispersant or carrier for the inventive compounds in the composition. The vehicle may be aqueous or anhydrous.
10 Water, when present, will be in amounts which may range from 5 to 99%, preferably from 20 to 70%, optimally between 40 and 70% by weight of the composition.
Besides water, relatively volatile solvents may also be included 15 within the vehicle. Most preferred are monohydric C1-C3 alkanols. These include ethyl alcohol, methyl alcohol and isopropyl alcohol.
Emollient materials may also be included in the vehicle. These 2 0 may be in the form of silicone oils and/or synthetic esters.
Humectants of the polyhydric alcohol type may also be employed in the vehicle. Typical polyhydric alcohols include glycerol, polyalkylene glycols and more preferably alkylene polyols and 25 their derivatives.
Thickeners may also be utilized as part of the vehicle, as may sugars, plasticizers, antioxidants, chelating agents, buffers, coloring agents, pigments, opacifiers, surfactants, propellants, 30 flavours and/or perfumes.
Preferred cosmetic compositions are those suitable for application to human skin and preferably include a skin benefit

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agent in addition to the inventive compound. Suitable skin benefit agents include anti-aging, wrinkle-reducing, skin whitening, anti-acne, sunscreen and sebum reduction agents. Examples of these include alpha-hydroxy acids, beta-hydroxy 5 acids, polyhydroxy acids, hydroquinone, t-butyl hydroquinone, vitamin B and C and their derivatives, micronised metal oxides, retinoids, betulinic acid, vanillic acid, allantoin, a placenta extract, hydrolactin, resorcinol derivatives, and mixtures thereof. 10
Food Compositions
A particularly preferred form of the composition is that of a foodstuff, as this allows for convenient and enjoyable
15 consumption of the compounds of the invention. The food composition may be, for example, a margarine, low fat spread, confectionery product (such as chocolate), ice cream, dressing, mayonnaise, sauce, bakery product, shortening or cheese. However, it is especially preferred that the food composition is a
20 beverage.
The food may be dried and contain less than 40% water by weight of the composition, preferably less than 25%, more preferably from 1 to 15%. Alternatively, the food may be substantially 25 aqueous and contain at least 40% water by weight of the composition, preferably at least 50%, more preferably from 65 to 99.9%.
The food preferably comprises nutrients including carbohydrate,
30 protein, fat, vitamins, minerals and mixtures thereof. The food
may be low calorie (e.g. have an energy content of less than 100
kCal per 100 g of the composition) or may have a high calorie

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content (e.g. have an energy content of more than 100 kCal per 100 g of the composition, preferably between 150 and 1000 kCal).
The food may also contain salt, flavours, colours, preservatives, 5 antioxidants and mixtures thereof.
When the food composition is a beverage, it will typically comprise at least 85% water, more preferably at least 90%, optimally between 95 and 99.9% by weight of the beverage. 10 Preferably the beverage is a coffee-based beverage, a tea-based beverage and/or a cocoa-based beverage.
The pH of the beverage may, for example, be from 2,5 to 8, preferably 3 to 6, more preferably from 3.5 to 5.
15
Most preferably the beverage is tea and comprises tea-derived solids such as catechins, theaflavins, thearubigens and mixtures thereof. The tea solids may, for example, comprise from 0.005 to 3% by weight of the beverage, more preferably from 0.01 to 2%,
20 most preferably from 0.05 to 1% by weight of the beverage.
Use of the Compounds or Compositions
The compounds and compositions of the present invention may be used to deliver any of the benefits associated with theanine. In 25 particular, the compounds and/or compositions may be used as a medicament or in the manufacture of a medicament for providing at least one of the following benefits:
-improving concentration, mental focus and/or alertness;
-improving sleep quality; 30 -improving creativity;
-improving the immune system;
-improving digestion;

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-managing bodyweight; -reducing mental stress; -treating behavioural disorders; -treating premenstrual syndrome; 5 -treating headache and/or migraine.
For example, the compound or composition may be used in a method of providing at least one of these benefits to an individual, the method comprising administering to the individual the compound or 10 composition. Preferably the compound and/or composition is administered orally.
Manufacture of the Compounds
The compounds of the present invention may be manufactured by any 15 suitable process. However, a preferred process for manufacturing the compound comprises the step of reacting theanine or a derivative thereof with at least one amino acid or a derivative thereof.
20 Suitably, the reaction involves coupling of the amino-acid backbones of the theanine and the at least one amino acid. Thus it is preferred that the reaction comprises forming at least one peptide bond between the theanine or derivative thereof and the at least one amino acid or derivative thereof. More preferably,
25 the reaction comprises forming two peptide bonds between the theanine or derivative thereof and the at least one amino acid or derivative thereof. The peptide bonds may, for example, be formed as a result of nucleophilic attack at the terminal carbonyl group of theanine by the amino group of the amino acid and nucleophilic
3 0 attack at the carbonyl of the amino acid by the amino group of theanine.

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The formation of the peptide bonds may, for example, be achieved by heating a mixture of theanine and amino acid to high temperature (170 to 200CC) in an organic solvent with a silica catalyst, as described in V.A. Basiuk et al., Synthesis-
5 Stuttgart, 1992, 5, pp. 449-451. However, in order to avoid such harsh reaction conditions it is preferred that the terminal carbonyl of the theanine and/or the amino acid is activated. Suitable activation means are well-known to those in the art and usually involve replacing the OH group of the carboxylic acid
0 functionality with a group such as acyl chloride, anhydride, ester and the like. Thus the theanine or derivative thereof for use in the process is preferably according to formula (6):
5
wherein R6 is selected from the group consisting of CI, OC(0)X, 0 OX, OC (NX) NHX' , OC (CH2> OX, OC (0) OX, OP (0) Ph2, 0P+ (X) (X') X", OP (0) (OX) OX', N(X)N(X')X", and N3 and wherein each of X, X' and X" is independently selected from the group consisting of H, Xi, X2 or X3.
5 For preparing compounds of formula (1), the process preferably comprises the reaction shown in scheme (I):


0

wherein R7 is selected from the group consisting of CI, OC(0)X, OX, OC (NX) NHX' , OC (CH2) OX, OC (0) OX, OP (0) Ph2, 0P+ (X) (X' ) X", OP (0) (OX) OX' , N(X)N(X')X", and N3 and wherein each of X, X' and X" is independently selected from the group consisting of H, Xi, X2 or X3.
Nucleophilic substitution to form peptide bonds is promoted at high pH, therefore it is preferred that the reaction of the theanine or derivative thereof with the at least one amino acid or derivative thereof occurs at a pH of from 8 to 14, more preferably 10 to 12.

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Examples
EXAMPLE 1
This example demonstrates the manufacture of a compound of the 5 invention (N-ethyl-3-[5-(2-ethylcarbamoyl-ethyl)-3, 6-dioxo-piperazin-2-yl]-propionamide; see formula (4)).
L-Theanine (5 g) (Suntheanine™) was slurried in dry MeOH (50 ml). Then 5.145 g of CI2SO were added drop-wise over 5 minutes 10 (part way through the addition all of the solids dissolve to give a clear colourless solution) and the resulting solution was held for 12 hours at 20°C to allow the reaction shown in scheme (II) to occur:

25

MeOH, SO2 and HC1 were then removed under vacuum to leave a thick clear oil to which dry MeOH (50 ml) was added. The pH was then raised by adding MeONa while testing pH by intermittently spotting onto wet indicator paper. After adding 3.17 g of MeONa, the pH had increased to >11, which resulted in the reaction shown in scheme (III):

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The resulting solution was held for 12 hours at 20°C to allow the reaction shown in scheme (IV) to occur:

The resulting suspension was then filtered to remove NaCl and the 2 0 filtrate subjected to vacuum to remove MeOH. The product was a white solid.
The product was analysed with LOMS (Liquid-Chromatography coupled Mass Spectroscopy) under the following conditions:
25

Column:

Phenomonex Gemini 5|i. C18 110A 150mm x 2.00mm

30
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Solvents: A: 0.1% Formic Acid / Water
B: 0.1% Formic Acid / Acetonitrile
Flow rate: 0.2 ml / minute
Column Temperature: 30°C
Stop Time: 25 minutes

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Solvent Gradient:

Time (minutes) %A %B
0 80 20
10 99 1
11 0 100
14 0 100
15 99 1

Mass Spectrometer Detection:
Selected Ion Recording at m/z 175.6 for Theanine.
10 Selected Ion Recording at m/z 313.5 for Theanine
dimer.
The results showed that the product was free from theanine (retention time of theanine = 3.7 min) and had a characteristic 15 new peak with a retention time of around 7.4 min.
The high resolution proton NMR spectrum of the product in MeOD-oU is shown in Figure 1.
20 EXAMPLE 2
This example demonstrates the superior stability of the theanine dimer (N-ethyl-3-[5-(2-ethylcarbamoyl-ethyl)-3,6-dioxo-piperazin-2-yl]-propionamide) prepared in Example 1 compared with that of theanine.
25
0.05 M citrate buffer with a pH of 4.0 at 20.3°C was prepared. To this buffer were added 5.35 mM theanine and 2.675 mM of the theanine dimer. The resulting solution was held at 92°C for two weeks with small samples being taken regularly for analysis with
30 HPLC. The results are shown in Table 1:

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TABLE 1

Storage Time (hours) Theanine Remaining (mM) Theanine Dimer Remaining (mM)
0.0 5.35 2.68
2.5 4.83 2.75
17.5 2.54 2.80
26.3 1.83 2.78
42.0 0.81 2.99
50.0 0.05 3.31
121.3 0.04 3.36
137.3 0.03 3.38
145.8 0.02 3.51
161.6 0.01 3.54
170.2 0.01 3.59
185.8 0.01 3.63
193.3 0.01 3.80
The slight increase in the concentration of theanine dimer with time was due to gradual evaporation of water from the container 5 which could not be completely sealed during the experiment. However, it is clear from the data in [Table 1 that the theanine dimer is much more stable than theanine.

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Claims
1. A compound of formula (1

wherein
10

15
20
25

Ri/ R2/ &n