This invention relates to polyunsaturated fatty acid derivatives and their salts with tyrosine kinase inhibitor activity having the Formula (I) 0

11

R-. - C - X - - Y (I) ¹

wherein R-, is an alkyl chain consisting of 18 to 24 carbon atoms containing at least two unsaturated double bonds; is selected from oxygen, imiπo group, or nitrogen substituted with alkyl group consisting of 1 to 4 carbon atoms; is carboxyl, COOMe, or carboxa ide group, where

Me is metal; is either a side-group to the alpha carbon atom of any a ino acid found in living organisms or a group having the formula (II)

68689-5850-SI

where

A is OH and k = 0 or 1 or a moiety having the formula (III)

-CCH ₂ ) _n - X 2 m. - X - M (III)

where

X is as described above, n Is an integer from 0 to 3, inclusive, m is an integer from 0 to 4, inclusive,

M is either hydrogen or R-.-C0 group where R, is as described above, or a chain having the Formula (IV)

where n is as defined above; and to the method for preparation thereof.

Due to the immunostimulant and tyrosine kinase inhibitor activity, the compounds of the invention can be applied for stopping and suppressing the pathological cell proliferation, consequently, for treating the malignant neoplastic diseases.

According to recent investigations, the formation of malignant tumors is clearly the result of the abnormal activation of certain genes. The abnormal activation of these genes called proto-oncogenes and their transformation into oncogenes can be caused by several mechanisms independently of retroviruses. By definition, the term "oncogene" means that these genes permit the formation and survival of malignant neoplastic cells

/Bradshaw, T.K. : Mutagenesis 1_, 91 - 97 (1986:/.

In the present state of the art, the regulation of cell-division is carried out by a complex mechanism which consists of genomial information comprising proto-oncogenes and the finely adjusted interaction between different factors inducing growth and differentiation and endocrine and paracrine regulators. The close connection between oncogeπes and growth factors is also supported by the fact that the major part of oπcogenes encodes proteins which themselves are growth factors or growth factor receptors or which interact with the signal traπsduction mechanism induced by growth factors.

As each cell of an organism is part of a strictly regulated and systematic "cell society", it has long been presumed that, normally, cells only begin to devide as an effect of an extrinsic signal i.e. growth factor. Recent investigations have also proved that, in the permanently dividing neoplastic cells, a growth factor traπsductioπ pathway is always active but, in certain (pathological) cases, the exogene growth factor is replaced by an oπcogene product Winstein , B. ; J . Cell. Biochem. 33 , 213-224 (1987) and Paul, D.; Drug Res. 3∑~ 772-779 (1985)7.

Many of the consequent steps of the signal traπsduction mechanism are potential sites of oπcogene intervention.

Under the physiological conditions of regulated cell-division such as embriogenesis or regeneration of injured tissue, proto-oncogenes which take part in the regulation of cell-division are activated by growth factors. In transformed cells containing activated oπcogenes, the complex interaction signals and regulating mechanisms which normally appear in a tissue have stronger effects because the organism and the microenvironment tend to control the cells that "break loose".

In the present state of the art, all the tumors are monoclonal, that is, they originate from one single transformed cell. Tumor progression begins when these transformed cells become able to divide permanently in this special, "hostile" microenvironment, and the divisions result in viable variants. To be able to survive and divide in this competitive environment,neoplastic cells have to possess special division parameters and other favourable features such as resistance to immune effects. Thus, in the permanently dividing neoplastic cells, there is a signal transduction mechanism constantly "on" which induces cell division and with which inhibitory regulating signals of the environment are unable to interact /Nicolson, G.L.: Cancer Research £7, 1473 - 1487 (1987)7. In the present state of the art, the regulation of cell division is carried out by three main transduction mechanisms: the stimulation or inhibition of the tyrosine kinase pathway, the phospholipid metabolism protein kinase C pathway, and/or the CAMP protein kinase A pathway. The significance of the tyrosine kinase trans¬ duction pathway is demonstrated by the fact that a major part of the oncogenes encodes tyrosine kinases and that growth factor receptors and receptors of autocrine growth factors secreted by neoplastic cells are mainly tyrosine kinase as well Yarden et al.: Ann. Rev. Bioche . 57, 443 - 478 (1988)7.

Accordingly, it can be established that the key to the therapy of malignant tumors will be given by the knowledge and selective inhibition of the specific signal transduction mechanism used by the oncogenes and growth factors.

The aim of the invention is, therefore, to synthesize a novel tyrosine kinase inhibitor that stops and suppresses the pathological cell proliferation as well

as prevents the development of malignant tumors by the inhibition of the activity of tyrosine kinase enzyme.

The invention is based on the discovery that the said purpose can be realized completely by the application of polyunsaturated fatty acid derivatives of the invention, having the above Formula (I) wherein

R. is alkyl chain consisting of 18 to 24 carbon atoms containing at least two unsaturated double bonds;

X is selected from oxygen, i ino group, or nitrogen substituted with alkyl group consisting of 1 to 4 carbon atoms; is carboxyl, COOMe, or carboxamide group, where

Me is metal; is either a side-group to the alpha carbon atom of any amiπo acid found in living organisms or a group having the above Formula (II) where

A is hydroxyl and k = 0 or 1, or a moiety having the above Formula (III) where is as described above, is an integer from 0 to 3, inclusive, is an integer from 0 to 4, inclusive, is either hydrogen or R-.-C0 group where

R, is as described above, or a chain having the above Formula (IV) where π is as defined above.

Further, the invention is based on the discovery that compounds having the above Formula (I) when Incorporated-into the membranes of tumor cells are able to inhibit the signal transduction mechanisms activated by oncogenes and growth factors, consequently, the pathological cell proliferation is suppressed.

Compounds having the above Formula (I) have not been known in the literature up to now.

The known related compounds contain saturated fatty acid residues as R-, in the above Formula (I).

These compounds are excellent as detergents and skin food ingredients, mainly in cosmetics, due to their hydrating capability and skin softening activity. For example, such agents are described in the Japanese Patent No. 58.168,696 or in the Eur. Pat. No. 139,481. Hiroshi et al. Z~Chem. Pharm. Bull. Σ~ 2935 (1987)7 prepared palmitoyl serine applying to the preparation of liposo es. Several papers, such as Paquet et al. / ^" Can. 3. Biochem. 5J3, 573 (1980)7 or Berger et al. Tenside 2_3_, 156 (1986)7 covered the applications of palmitoyl threonine and palmitoyl methionine in the food industry.

It is worth to note that a fatty acylated derivative of diethylene triamine is described as one of the components of a chemically multicomponent system in the Hungarian Patent No. 4348/83, used for regeneration of neoplastic cells and tissues.

This invention, therefore, relates to the methods of preparing polyunsaturated fatty acid derivatives and their salts having the above Formula (I), wherein the substituents are as described above, comprising acylation of a compound having the Formula (VI)

where

Q is selected from hydroxyl, amiπo or NHR amino- alkyl groups with an alkyl group of 1 to 4 carbon atoms as R, Y and Ry are as described above with a polyunsaturated fatty acid derivative having the Formula (V) 0

(V)

where

Z is hydroxyl or R,-C0 group with R, being either as described above or a halogen atom, in the presence of an acid absorbent.

In accordance to this invention, the acylatiπg agents having the above Formula (V) can be prepared preferably from ^-linolenic acid (GLA) (18:3π-6(Z)-6,9,- 12-octadecatrienic acid) or from eicosapeπtaeπic acid (EPA) (20:5ou-3(Z)-5,8,11,14,17) or from docosahexaenic acid (DHA) (22:6α-3(Z)-4,7,10,13,16,19) as starting materials.

The main source of GLA may be the oil obtained from plant seeds, such as evening primrose (Oenothera biennis, Oenothera lamarkina) or Borago officiπalis e.g. by the method of Brit. Pat. No. 2,183,635 or from the Tetrahymeπa ciliates by the method of French Pat. No. 2,574,089. As feedstocks for DHA and EPA or other typically ^18-24°°"^ uπsaturated fatty acids, oils from various salt-water and fresh-water fishes, mainly mackerels, cods, herrings, sardines, calamaries, Hypophthalmyctis and from their livers, such as cod-liver oil or shark-liver oil may be used.

Polyunsaturated carboxylic acids are trans¬ formed into acyl halides having the above Formula (V) by reacting them with inorganic acyl halides, such as S0C1 ₉ ,

P0C1-,, PCI-,, PClc by any method well-known in the art of organic chemistry.

In the acylating reaction, polyunsaturated fatty acyl halides can be replaced by the corresponding anhydrides obtained by reacting the sodium salt of the corresponding fatty acid with said inorganic acyl halide.

The desired acylating agent may also be prepared directly from the carboxylic acid by any other methods known in the art of organic chemistry. In the method of this invention, the acylating agents having the above Formula (V), prepared as above, are reacted with compounds having the above Formula (VI) to obtain compounds having the above Formula (I) through N- or O-acylating. In the acylating reaction, organic bases such as pyridine or triethylamine are used as catalysts and acid absorbents. The reaction may be conducted at 10 to 120 °C with or without solvent. In some cases, the said organic bases may be used as solvents. Since polyunsaturated fatty acid derivatives are immiscible with water, the acylating reaction may be conducted in the aqueous alkalic medium by the Schotteπ- Baumann's method.

The biochemical and biological features of compounds having the above Formula (I) are as follows:

The effect of compounds having the above Formula (I) on the enzymatic activity of tyrosine kinase was measured by the method of Schwarup et al. /j. Biol. Che . 258, 10341 - 10347 (1984)7 using treated and untreated rat spleen homogeπisates.

For this test, 100 cm of stock solution containing 50 millimoles of TRIS-C1 (at pH 7.8), 50 millimoles of magnesium chloride, 10 micromoles of

sodium vanadate, 0.1 percent of Nonidet P-40 (from Fluka

A.G., Buchs, Switzerland), and 1 millimole of Angiotensin

II was mixed with 60 mm of rat spleen homogenisate.

The reaction was started by the addition of 0.5 πanomole of ( P)ATP . The reaction mixture as incubated at 30 °C for 10 min then the reaction was stopped by the addition of 150 mm of 1 percent solution of trichloroacetic acid.

3 10 mm of bovine serum albumiπe (at a concentration of

20 mg/cm ) was added to the mixture and the precipitated protein was removed by centrifuging (3200 g for 25 min). 3 50 mm aliquot of the supernatant liquid was dropped onto a Whatman P-81 phosphocellulose paper. The paper squares were washed six times with 0.5 percent phosphoric acid and once with acetone then dried. Their radioactivities were measured in 5 cm of scintillation liquid. The results are collected in the following Table:

Test substrate: rat spleen homogenisate

Activities of spleens: 0.7135 pmole 32P/mg proteiπ/miπ 1.0119 pmole 32P/mg protein/min 1.258 pmole 32P/mg protein/min

Materials tested Activity Tyrosine Matter

Seria l 32 number Number of pmole ?ι kinase content

Amount example /mg/min activity (mm ³ ) (mg) (%)

24 C 24 1 . 8

24 C 24 4 . 5

C = compound of Example 17 ¹⁶ 20 0.043 4.8 i

¹⁶ 50 o 0 _{2 5}

C = DMSO

^{18 20} 0.6917 62.18 1 ^{18 20} 0-3175 26.74 2.5

C = DMSO

^{20 20} 0.4088 36.98 1

20 20 2.5

14 14 14 14

15 15

ontrol

The immuπostimulating activity of compounds having the above Formula (I) was measured by the activation lymphocyte cells by virtue of polyclonal mytogenes as follows :

Two hundred thousend cells of a lymphocyte population botained on Ficoll Uromiro gradient A. Boyum: Scand. 3. Clin. Lab. Invest. 2__\_, 91 (1986)7 were pipetted into the reservoirs of flat-bottom icroplates to form 7 parallel cultures. 25 ,ug/cm of Concanavaliπ A (Con A, Pharmacia, Sweden) was added to each sample. The control solution was 25 /ug/cm of Con A ^' without any other ingredients. The plates were grown in an environment containing 5 percent of CO,- at 37 C for 72 hours. 8 hours before the end of growth, each sample was supplemented by 0.4 ,uCi of H-thymidiπe . Passing of 72 hours, the cultures were filtered on a glass- filter each. The filters were placed into scintillation cuvettes and 5 cm of Liquifluor-containing toluene solution was added. The samples were measured in a Nuclear Chicago Isocap 300 beta counter and activities were read in couπt/miπ (cpm).

- i ? _

Spontaneous 5 _/ ug/ml Con A 25,ug/ml Con A

Basic level: 161 ⁺ -19 16 87 + -3017 28419 ■ _* ■ -3967 Product of Example 14 0.1 /ug/ml: 179 + -36 12287 + -2480 30749 + -4096 P: n.s . 0.01 n.s.

1.0 /ug/ml: 228 + -48 13513 + -2584 30728 + -3691 P: n.s. 0.01 n.s . 10 /Ug/ml: 183 + -25 10078 + -1951 25303 + -3500 p: n.s, 0.01 0.05

Product of Example 16 0.1 /ug/ml: 139 + -21 12932 + -2436 31470 + -3960 n.s. 0.001 0.05

1.0 /ug/ml: 219 + -38 133010 + -2560 32735 + -4293

P: n.s. 0.02 0.02

10 , ug/ml: 150 + -25 10608 + -2061 25551 + -3791

P: n.s. 0.01 n.s.

Product of Example 15 151 ⁺ -18 11759 + -1735 27780 ₊ -3245

P: n.s n.s n.s 1.0 /ug/ml ^: 172 + -26 12082 + -2065 27106 ₊ -3296 P ^: n.s. n.s. n.s

10 /ug/ml ^: 158 + -19 8336 + -1452 20316 ₊ -2827 ^{P: n} - ^s - 0.05 0.01

c.p.m. average + SEM n = 12 n.s. = not significant

The active ingredients having the above Formula (I) may be processed into capsules, tablets or other known pharmaceutical formulations along with pharmaceutically acceptable carriers and/or additives in the usual pharmaceutical ways.

The principal advantages of the compounds and pharmaceutical preparations of this invention are as follows

1. Through the inhibiton of the enzymatic activity of tyrosine kinase, a suppression of the signal transduction mechanisms activated by growth factors and/or oncogenes is provided leading to an inhibition of pathological cell proliferation processes.

The antioxidant and immunostimulating activity of the polyunsaturated fatty acid component gives a possibility to the complex tumor therapy.

Suppression of the pathological cell proliferation through the inhibition of tyrosine kinase is a much more gentle intervention than the conventional chemotherapy, such as a cytostatic treatment

The following examples are presented to illustrate the method of this invention but are not intended to limit its scope.

Example 1

Preparation of (Z)-5,8,11.14.17-eicosapenta- enoyl chloride (EPA-C1) 0,303 g (0.001 mole) of EPA was dissolved in 3 cm of cyclohexaπe. The solution was placed into a round-bottom flask equipped with a loading funnel and 0.092 g of PCI-,, dissolved in 1 cm of cyclohexane was introduced under a continuous N„ stream at 50 C. The mixture was stirred at room temperature for an hour. The reaction mixture was then clarified by activated charcoal and filtered. After evaporation, a yield of about 95 percent was obtained. Characteristic infrared absorption bands of the product: 1780 (CO), 1645 (C = C), 1460, 1440, 1370, 1340, 1290, 1240, 1170, 1095, 1015 cm ^-1 .

Example 2

Preparation of (Z)-4,7,10,13,16,19-docosahexa- enoyl chloride

(DHA Cl)

The same procedure as In Example 1 but EPA was replaced by 0,329 g (0.001 mole) of DHA. Yield was about 94 percent. Infrared bands: 1780 (CO) cm ^-1 .

Example 3

Preparation of (Z)-6,9,12-octadecatrienoyl chloride (GLA-C1)

The same procedure as in Example 1 but EPA was replaced by 0.278 g (0.001 mole) of GLA. Yield was about 88 percent.

Infrared bands 1780 (CO), 1650 (C = C) cm -1

Example 4

Preparation of a mixture of polyunsaturated fatty acyl chlorides containing 30.5 percent of EPA-C1 and 49.1 percent of DHA-CI 165 g (0.5 mole) of a mixture of polyunsaturate fatty acids containing 30.4 percent of EPA and 49.0 per¬ cent of DHA was dissolved in 500 cm of cyclohexane. The solution was placed into a three-necked round-bottom flask equipped with a reflux condenser and a loading funnel and was heated to 60 C with continuous stirring.

A tube filled with CaC^ was connected to the top of the condenser and a slow N„ stream was applied. From the loading funnel, 45.8 g (0.33 mole) of PCI, was added dropwise to the solution with continuous stirring during 1 to 1.5 hour period. After the introduction ,the mixture was stirred for another 1 to 1.5 hours at 60 C. Deposited H,P0-, was removed from the cyclohexane solution by decaπtation. After clarification by activated charcoal, the solution was filtered then evaporated in vacuo. 166 g of acyl chloride was obtained at a yield of about 95 percent. Composition of the feedstock and the product was checked by HPLC.

Example 5

Preparation of N-eicosapentaenoyl (4-hydroxy- phenyD-glycine

1.67 g (0.01 mole) of D,L-4-hydroxypheπyl glycine then 15 cm of freshly distilled pyridine were placed into a round-bottom flask equipped with a reflux condenser and a loading funnel.

The mixture was heated to 60 to 65 C and at this temperature, 3.85 g (0.012 mole) of EPA-C1 was introduced dropwise with continuous stirring during an hour,

As the introduction had been finished, the reaction mixture was allowed to cool down and stirring was continued for 2 hours.

Pyridine was evaporated in vacuo, the residue was malaxated with water then extracted three times with ethyl acetate. The extract was washed twice with 0.5 per¬ cent hydrochloric acid then once with water, dried on dehydrated Na ₂ S0 ₄ and, finally, the solvent was evaporated. Yield was about 78 percent. Infrared bands: 1695 (C = 0), 1620 (C = 0 amide), 3200 (-NH, -OH) cm ^"1 .

Characteristic NMR signals in CDC1-, to the amino acid moiety: 8.8 (bs) 3H, 6.9 (d) and 6.7 (d) 4H; to the fatty acid moiety: 5.35 ( ), 2.85 (s), 2.50 - 1.40 (m), 0.98 (t) ppm.

Example 6

Preparation of N-eicosapentaenoyl L-tyrosine The same procedure as in Example 5 but D,L-4- hidroxyphenyl glycine was replaced by 1.81 g (0.01 mole) of L-tyrosiπe. Yield was about 62.5 percent.

Infrared bands: 1690 (CO), 1620 (CO amide), 3200

(-NH, -OH) cm ^-1 . NMR signals in CDC1 ₃ : 8.5 (bs) 3H, 6.7 (d) and

6.9 (d) 4H, 4.85 (q) 1H, 3.1 (d) 2H ppm while the signals to the fatty acid moiety were identical to those in Example 5.

- 17

Example 7

Preparation of N-eicosapentaenoyl L-(3,4-dihydroxyphenyl-c* -rnethyl)-alanine -.e same procedure as in Example 5 but

D,L- -r. droxypnenyl glycine was replaced by 2.11 g (0.01 mole) of 3,4-dihydroxyphenyl-o^-methylalanine.

Yield was about 63.5 percent.

Infrared bands: 1695 (CO), 1620 (CO amide),

3150 (-NH, -OH) cm ^"1 . NMR signals in CDC1 ₃ : 8.8 (bs) 4H 6.50 (d) and

6.62 (s) and 6.75 (d) 3H, 5.0 (q) 1H, 3.15 (dd 2H ppm.

The signals to the fatty acid moiety were identical to thos. in Example 5.

Example 8

Preparation of N-docosahexaenoyl (4-hydroxy- phenyD-glycine The same procedure as in Example 5 but EPA-C1 was replaced by 4.16 g j.012 mole) of DHA-C1 prepared as in Example 2. Yield was about 72.5 percent. IR and NMR spectra were identical to those in Example 5.

Exqmaple 9 * P oration of N-docosahexaenoyl L-tyrosine a e procedure as in Example 5 but 1.81 g . mole) -tyrosine -as u ^• '. ^* ! as the amino acid onent a* .PA-C1 was όpl? by 4.16 g (0.012 mole) DHA-C1. ield was about r / percent. IR and NMR spectra were identical to those in Example 6.

- IB -

Example 10

Preparation of N-docosahexaenoyl L-(3,4- dihydroxyphenyl-pc-methyl)-alanine The same procedure as in Example 5 but 2.11 g (0.01 mole) of L-methyl dopa was used as amino acid and

EPA-Cl was replaced by 4.16 g (0.012 mole) of DHA-C1.

Yield was about 65.6 percent.

IR and NMR spectra were identical to those in Example 7.

Example 11

Preparation of N-octadecatrieπoyl (4-hydroxy- phenyl)-glycine

The same procedure as in Example 5 but EPA-Cl was replaced by 3.56 g (0.012 mole) of GLA-Cl prepared as in Example 3. Yield was about 73.8 percent. IR and NMR spectra were identical to those in Example 5.

Example 12

Preparation of N-octadecatrienoyl L-tyrosine The same procedure as in Example 5 but 1.81 g (0.01 mole) of L-tyrosine was used as amino acid and EPA-Cl was replaced by 3.56 g (0.012 mole) of GLA-Cl. Yield was about 64.7 percent.

IR and NMR spectra were Identical to those in Example 6.

Example 13 Preparation of N-octadecatrienoyl

L-(3,4-dihydroxy-phenyl-oC-methyl)-alanine The same procedure as in Example 5 but 2.11 g (0.01 mole) of σe-methyl dopa was used as amino acid and EPA-Cl was replaced by GLA-Cl. Yield was about 61-3 per- cent.

IR and NMR spectra were identical to those in Example 7

Example 14 Acylating of (4-hydroxyphenyl)-glycine

The same procedure as in Example 5 but EPA-Cl was replaced by 4.2 g (0.012 mole) of the mixture of polyunsaturated fatty acyl chlorides as prepared in Example

4. Yield of the mixed product was about 88 percent, containing N-eicosapentaeπoyl (4-hydroxypheπyl)-glycine and N-dicosahexaeπoyl (4-hydroxyphenyl)-glycine as the main components.

Infrared bands: 1695 (CO), 1620 (CO amide),

3200 (-NH, -OH) cm ^"1 . NMR signals in CDC1 ₃ : 8.5 (bs) 3H, 6. 9 (d) and

6.7 (d) 4H, 5.35 (m) 11H, 2.85 (s) 9H,

2.5 - 1.5 ( ) 8H, 0.98 (t) 3H characteristic to the ^' fatty acid.

Example 15

Acylating of L-tyrosine with the mixture of

Example 4

The same procedure as in Example 5 but 1.81 g (0.01 mole) of L-tyrosine was used as amino acid and EPA-Cl was replaced by 4.2 g of the mixture of poly¬ unsaturated fatty acyl chlorides as prepared in Example 4. Yield of the mixed product was about 66.2 percent, containing N-eicosapeπtaeπoyl tyrosine and N-docosa- hexaeπoyl tyrosine as the main components. Infrared bands: 1680, 1620, 3150 cm ^"1 . NMR signals in CDC1 ₃ : 8.50 (bs) 3H, 6.90 (d) 4H, 6.70 (d), 4.85 (q) 1H, 3.10 (d) 2H ppm. Signals characteristic to the fatty acid moiety were

identical to those In Example 14.

Example 16 Acylating of L-serine with the mixture of

Example 4

The same procedure as in Example 5 but 0.01 mole of L-serine was used as amino acid and 0.012 mole of the mixture of polyunsaturated fatty acyl chlorides prepared in Example 4 was applied as fatty acyl chloride.

Yield of the mixed product was about 56.7 percent, containing N-eicosapentaenoyl L-serine and N-docosa¬ hexaenoyl L-serine as the main components. Infrared bands: 1890, 1620, 3150 cm ^"1 . NMR signals in CDCl _j : 9.40, 8.40, 7.95 (s) 1H each, 4.50 ( ) 1H, 3.90 (dd) 2H.

Signals characteristic to the fatty acid moiety were identical to those in Example 5.

Example 17

Acylating of L-threonine

The same procedure as in Example 5 but 0.01 mole of L-threonine was used as amino acid and 0.012 mole of the mixture of polyunsaturated fatty acyl chlorides prepared in Example 4 was applied as fatty acyl chloride. Yield of the mixed product was about 62.5 percent, containing N-eicosapentaeπoyl L-threonine and N-docosa¬ hexaenoyl L-threonine as the main components. Infrared bands: 1690 (CO), 1620 (CO amide), 3150 (-NH, -OH) cm ^"1 .

NMR signals in DC1 ₃ : 7.35 (bs) 3H, 4.50 (d) 1H, 4.40 ( ) 1H, 1.20 (d) 3H ppm, characteristic to the amino acid residue. Signals to the fatty acid moiety were

identical to those in Example 5

Example IB Acylating of L-ornithine.HC1

The same procedure as in Example 5 but 1.63 g (0.01 mole) of L-ornithine hydrochloride was used as amino acid and 0.012 mole of the mixture of polyunsaturated fatty acyl chlorides prepared in Example 4 was applied as fatty acyl chloride. Yield of the mixed product was about

2 45.5 percent, containing N -eicosapeπtaeπoyl L-ornithine.

2 HC1 and N -docosahexaenoyl L-orπithise•HC1 as the main components.

Infrared bands: 1690 (CO), 3100 (-NH), 1630 (C = 0 amide) cm ^" .

NMR signals characteristic to the amino acid moiety in

CDC1 ₃ : 8.30 - 7.30 (bs) 5H, 4.50 (m) 1H, 3.25 (m

2H, 2.35 (m) 2H, 1.60 ( ) 2H ppm.

Example 19

Acylating of L-lysine.HCl

The same procedure as in Example 5 but 1.83 g

(0.01 mole) of L-lysine hydrochloride was used as amino acid and 0.012 mole of the mixture of polyunsaturated fatty acyl chlorides prepared in Example 4 was applied as fatty acyl chloride. Yield of the mixed product was about

2 43.2 percent, containing N -eicosapentaenoyl L-lysine.HCl

2 and N -docosahexaenoyl L-lysine.HCl as the main components. Infrared bands: 1690 (CO), 1630 (CO amide), 3150

NMR signals in CDCl 8.30 - 7.30 (bs) 5H, 4.50 (m)

1H, 3.25 (m) 2H, 2.30 (m) 2H, 1.50 (m) 4H ppm.

-> ->

Example 20

Acylating of L-arginiπe.HCl

The same procedure as in Example 5 but 2.11 g

(0.01 mole) of L-arginine hydrochloride was used as amino acid and the mixture of polyunsaturated fatty acyl chlorides prepared in Example 4 was applied as fatty acyl chloride.

In the extraction step, ethyl acetate was replaced by n-butanol. Yield of the mixed product was about 57.1 per-

2 cent, containing N -eicosapentaenoyl L-argiπine.HC1 and

2 N -docosahexaenoyl L-arginine.HC1 as the main components..

Infrared bands: 1695 (CO), 1625 (CO amide), 3200

(-NH, -OH) cm ^"1 .

NMR signals in CDC1 ₃ : 8.50 (bs) and 7.60 (bs) and

6.BO (bs) 7H, 4.40 (bs) 1H, 3.30 (bs) 2H, 1.50 (m) 4H ppm.

Signals characteristic to the fatty acid moiety were identical to those in Example 5.

Example 21

Acylating of p -methyl dopa

The same procedure as in Example 5 but 2.11 g

(0.01 mole) of (3,4-dihydroxyphenyl-©C-methyl)-alanine was used as amino acid and the mixture of polyunsaturated fatty acyl chlorides prepared in Example 4 was applied as fatty acyl chloride. Yield of the mixed product was about 67.8 percent, containing N-eicosapentaenoyl < .-methyl-dopa and N-docosahexaenoyl ^•» ' ^■ •■■■—methyl-dopa as the main components. NMR signals characteristic to the amino acid moiety in CDC1, ^: 8.80 (bs) 4H, 6.75 (d) and 6.62 (s) and 6.50 (d) 3H, 5.00 (q) 1H, 3.15 (dd) 2H. Signals characteristic to the fatty acid moiety were identical to those in Example 5.

Example 22

Acylating of diethylene tria ine

2.12 g (0.04 mole) of diethylene triamine, 8.88 g (0.088 mole) of triethylamine and 150 cm ³ of anhydrous toluene were placed into a four-necked round- bottom flask. The mixture was heated to 85 to 90 °C then 29.08 g (0.08 mole) of the mixture of fatty acyl chlorides prepared as in Example 4 was added dropwise with continuous stirring. The mixture was continued to stir at this temperature for 4 hours then 2.9 g (0.008 mole) of the said fatty acyl chloride mixture was additionally dropped in and stirring was continued for 2 hours more.

Precipitated triethylamine hydrochloride was filtered at room temperature and filtrate was evaporated

2 under N ₂ atmosphere at 4000 N/m . The residue was washed with petroleum ether. Yield of the mixed product was about

62.4 per cent, containing N, ' - bis(aminoethylene)-imino7- ^' bis-docosahexaeπoate and N,N '-/bis(aminoethylene)-irniπo7- bis-eicosapeπtaenoate as the main components. Infrared bands: 1645 (CO amide), 3250 (-NH) cm ^' -1

NMR signals in CDC1 ₃ : 3.55 (m) 8H (N-CH ₂ ~) ppm and twice of the signals characteristic to the fatty acid moiety as in Example 5.

Example 23

Acylation of diethanolamine

2.83 g (0.02 mole) of diethanolamine hydro- 3 chloride then 15 cm of dioxaπe were placed into a round- bottom flask equipped with a reflux condenser and a loading funnel. The mixture was heated to 55 to 60 °C and 13.6 g (0.04 mole) of the mixture of fatty acyl chlorides prepared as in Example 4 was added at that temperature during 1 hour period.

The reaction mixture was continued to stir for

2 2 hours followed by evaporation at a pressure of 5400 N/m .

The residue was washed with petroleum ether. Yield of the mixed product was about 92.6 percent, containing 0,0'-(diethanolamine)-bis-docosahexaenoate.HCl and , 0- ( diethanolamine) -bis-docosahexaeπoate.HCl as the main components,

Infrared bands : 1720 (CO ester ) , 3100 (-NH ) cm ^"1 .

NMR signals in CDC1 ₃ : 10 . 0 ( s ) 1H , 4 . 0 - 4 . 4 (m)

4H ₅ 3 .3 - 3 . 7 (m) 4H ppm . Signals characteristic to the fatty acid moiety were identical to those in Example 5.

Example 24 Acylation of L-lysine

5.61 g (0.034 mole) of L-lysine monohydrate was dissolved in 150 cm of distilled water in a round- bottom flask equipped with magnetic stirrer. The solution was heated to 80 C and, with continuous stirring, Cu(0H) ₂ .CuC0,. H ₂ 0 was added in small portions until it appeared to be dissolved. The possible excess of Cu(0H) ₂ .CuC0-,.xH ₂ 0, remained after stirring for 15 minutes, was filtered out. The solution was cooled to room temperature with continued stirring and 1.8 g (0.017 mole) of Na ₂ C0 ₃ then 12.2 g (0.034 mole) of the mixture of fatty acyl chlorides prepared as in Example 4 were added dropwise. After 1-hour stirring, the acylated copper complex precipitate was filtered then washed once with methanol and several times with water until a colourless wash liquor had been obtained. In a round-bottom flask, 200 cm of methanol was added to the copper complex and the complex was decomposed by bubbling gaseous H _? S through the mixture.

The reaction mixture was filtered and the solvent was evaporated under N _? atmosphere at a pressure of 5400 N/m not above 50 C. The residue was washed with petroleum ether, malaxated with acetone, filtered and dried under N„ atmosphere in both cases. Yield of the mixed product was 17.24 percent. M.p. : 116 C (with decomposition).

N -eicosapentaenoyl L-lysine and N -docaso- hexaenoyl L-lysine were the main components of the product.

Calculated Found

C 73.12 72.98 H 9.71 9.92 N 6.33 6.18

Example 25 Preparation of N -docosahexaenoyl L-ornithine

The same procedure as in Example 24 but L-lysine was replaced by 0.034 mole of L-ornithine and the mixture of acyl chlorides was replaced by DHA-C1 prepared as in Example 2. Yield was about 15.6 percent.

Calculated Found

72.84

9.61 6.51

Example 26

Preparation of N -docosahexaenoyl L-lysine The same procedure as in Example 24 but the mixture of acyl chlorides was replaced by DHA-Cl prepared as in Example 2. Yield was 20.42 percent.

Found

74.10 9.72 6.21