“New derivatives of the déoxy-2'-uridine substituted in position 5,3 ' or 5 ' by acylés groupings x-amino, proceeded for their obtaining and drugs the container”.

Novel derivatives of the deoxy-2 '-uridine derivatives substituted in position 5, 5' or 5' Hydroquinone oramino acylated, process for their obtaining and medicaments containing them.

The present invention relates to novel derivatives of the deoxy-2 '-uridine derivatives substituted in position 5, 3' or 5" by groups with amino-acylated, a process for their preparation and pharmaceutical compositions containing them.

Already known some derivatives of the deoxy-2⁸ - uridine derivatives.

I. as well as have been described derivatives of the deoxy-2'-uridine derivatives substituted in position 5, and having the following general chemical formula:

R is an alkyl or alkenyl radical having 1 to 4 carbon atoms, an aryl radical or a halogen, said alkyl and aryl which may have at least one halogen substituent.

Also known, in particular by U.S. Patent 4,093 716, the amino-a 5'-dideoxy 2^{the L} , 5^Brméthyl-to-5-uridine derivatives of formula:

which is presented as a potential inhibitor of the herpes virus. The preparation of this compound is described in an article of Horwitz equation and Al, in the periodical " the j. The AM. Mal. Soc. " 27, 3045, (1962).

Also known, in particular by a publication flax and PRUSOFF (j pieces. The MED. Mal. 21 109 (1978) _), the amino-a 3 '" dideoxy-2 ', 3'-methyl 5-uridine derivatives of formula:

Also known, in particular by a publishing BELTZ and screwed (j pieces. Toxicol. Chem., 226, 1035, (1957)), the amino-a 5 a-deoxy-2 'uridine of formula:

The MED J the document. Mal. 1979, flight 22, no. 6, page 621 631 to generally takes a process for obtaining 5 thereof [[ür (Iodoacé " tamido) acyUamino] and 2' a-déoxyuridines, using, as synthesis intermediates to compounds of the formula:

in which R represents the residue of a Û) - amino acid.

These intermediate compounds are not presented as likely therapeutic application.

The present invention relates to a novel class of the deoxy-2'-uridine derivatives substituted at position 5, 3 'or 5' Hydroquinone oramino acylated, having the following general formula:

in which

. R is selected from an alkyl or alkenyl radical having 1 to 4 carbon atoms, an aryl radical or a halogen, said alkyl, alkenyl and aryl which may have at least one halogen substituent; and a radical of formula - NH-R-^, wherein rr.j represents an amino acid residue or a peptide comprising 2 to 6 amino acids;

. The R ' and R " is selected from a hydroxyl radical and a radical of formula - NH rr.j, wherein R ^ has the same meaning as above;

with the proviso that R * and R "are not simultaneously - NH-R-^ and that, when R is - NH-R-^, the R ' and R" are simultaneously a hydroxy group; and pharmaceutically-acceptable salt thereof.

The invention also includes within its scope all possible optical isomers of the compounds of formula (I in) as well as mixtures thereof.

In the general formula (I-), a halogen atom is preferably a chlorine or bromine atom;

The alkyl groups, alkenyl groups may be straight-chain or branched.

An alkyl group having 1 to 4 carbon atoms is for example methyl, ethyl, propyl, isopropyl, butyl, sec-butyl or tert.-butyl, preferably methyl or ethyl.

An alkenyl group having 1 to 4 carbon atoms is for example a vinyl group, propenyl or butenyl, preferably vinyl.

Pharmaceutically acceptable salts of the compounds of formula

(I-) include those formed with mineral acid, e.g. hydrochloric acid or sulfuric acid or with an organic acid, for example citric acid, tartaric, malic, maleic or fumaric acid.

It has been found, quite surprisingly of, that these new derivatives have valuable pharmacological property to be inhibitors of thymidine kinase present in the fetal human cancerous tissue and thus are inhibitors of DNA synthesis of proliferating cancer cells.

According to a particular characteristic, the invention encompasses the novel class of derivatives of the deoxy-2 '-uridine derivatives substituted at position 5', it is to say the compounds of general formula (I-), wherein R ' is hydroxy and R " is a group - NH-R-^.

According to another particular characteristic, the invention encompasses the novel class of derivatives of the deoxy-2 '-uridine derivatives substituted in position 3', it is to say the compounds of general formula (I-), wherein R ' is a group - NH-R-^ and R " is a hydroxy group.

In yet another particular characteristic, the invention encompasses the novel class of derivatives of the deoxy-2 'uridine substituted in position 5, it is to say the compounds of general formula (I-), wherein R is a group - NH-R-^, the R' and R " being simultaneously hydroxy groups.

The novel compounds of formula (I-). according to the invention are, according to the structure of the aforementioned amino acid or peptide, as D or L, or LID.

The general method for the preparation of the compounds according to the invention of the general formula (I-) as defined above comprises reacting, enzymatically or chemically, of an amine derivative of general formula:

in which

R_grams is selected from an alkyl or alkenyl radical having 1 to 4 carbon atoms, an aryl radical or a halogen, said alkyl, alkenyl and aryl which may have at least one halogen substituent, and a radical - NH-₂ ;

The R * and R " is selected from a hydroxyl radical and a radical

the NH -₂ ,

with the proviso that R * and R "_has are not simultaneously - the n! - ^ and that, when R_grams ^ - is encompassed by the invention, the R '_grams and R "_has a hydroxy group are simultaneously;

with an amino acid or a peptide comprising 2 to 6 amino acids, and, if desired, the conversion^- d." a compound thus obtained into a pharmaceutically acceptable salt thereof.

The compounds of L-form are preferably prepared by enzymatic reaction of amine derivatives of formula (the) with an amino acid or a peptide of L or DL form, in the presence of an enzyme selected from papain and chymopapain, the reaction being carried out at a temperature of about 10 to about 70 °c in acid medium. Under these conditions, there is reaction between the amine function of the amino derivative in 5, in 3 'or 5' and function of the free acid of the amino acid or peptide, it being understood that the amine functions of said amino acid or peptide are previously blocked using known reagents such as benzyloxycarbonyl, the paraméthoxybenzyloxycarbonyle, t-butyloxycarbonyl or fluorénométhyloxycarbonyle.

The compounds of L-form, the d or dl can also be prepared using a method comprising chemical purely optional separation of a mixture of isomers of the compounds of formula (I in) in the isolated isomers.

Surprisingly, the compounds of formula (I-) according to the invention were found to be active in inhibiting DNA synthesis of cancer cells and certain viral particles.

These compounds are therefore particularly useful in the treatment of hormone dependent cancers and infections of viral origin.

It is often the oral route for all conditions requiring such compounds. To this end, the compounds of

the invention may be administered at doses by

2, 2

such as from about 30 mg/m to about 100 mg/m per day in the treatment of cancers, e.g. from about 50 mg and/kg to about 250 mg/kg per day, in the treatment of viral. Of course, these regimens may be adjusted to provide the optimal therapeutic response.

Thus, according to yet another aspect, the present invention relates to pharmaceutical compositions, in particular to inhibitor activity of the DNA synthesis of proliferating cancer cells, characterized in that they contain, as active ingredient, at least ' one of the compounds of general formula I defined above, in association with a pharmaceutically acceptable or non-toxic.

The pharmaceutical compositions of the invention are generally prepared according to conventional methods and are administered in a pharmaceutical form suitable, for example a solid oral dosage form may contain, with active diluents, e.g. lactose, cellulose; lubricants, such as stearic acid or polyethylene glycols; binding agents such as starches, methylcellulose; dyes; sweeteners; wetting agents such as polysorbates; and, in general, the pharmacologically inert and non-toxic materials used in the pharmaceutical compositions.

L¹ invention will be iltust ' to REE more in detail by the examples nbn-limiting following ii

Example 1: enzymatically

Synthesis of n - (L atanyl) - amino 5^, - dideoxy-2^,_/ 5^{, _}méthyi-a 5" one ' dihe

Transportable ii synthesis of n-th (n Bas ^ ytoxycarbonyl) - L-alanyl-L] a-alfiino and 5^, dideoxy ^ SSS-'-methyl-s uridinê

mainly in a 100 ml of tricol equipped with a magnetic stirrer, a thermometer and a refrigerant, is introduced successively ii.... -

16 ml of soda - normal

- 3.57 g of (n-benzyloxycarbonyl-) - alanine (0,016 moles)

- 3.86 grams of amino-a 5 '-dideoxy 2 ', 5'-methyl 5-uridine derivatives (0,016 moles)

- 188 mg of L-cysteine dissolved in 2.4 ml of distilled water

- 20 ml buffer solution at pH citric: 5

The reaction medium having an L ℮, ρΗ is heated to 50 °c is 8.98; from 30 °c, the mixture is clear.

Has 50 °c, is added a solution of 3.6 g of papain (2.9 with UL/mgs) in 8 ml of water. The pH evolves slowly toward 8.41.

By additon of citric acid powder (m=1.2 grams) the pH is adjusted to 4.6 and leaves under stirring for 24 hr bright.

Completion of the reaction, the reaction medium is cooled, filters and washes the precipitate with water.

After drying at 60 °c under vacuum, obtained 4.3 g of N - [(n-benzyloxycarbonyl-) - L aLanyU-amino 5^, - dideoxy-2^, , 5^, - methyl-to-5

- uridine derivatives.

The purification is carried out by crystallization in 50 ml of inéthanol.

m=3.9 grams BDR=55 % Purity: 95% Step b :

This compound is dissolved in methanol and hydrogenated in the presence of 400 mg of 10% palladium-on-carbon.

After filtration of the catalyst and concentration of solvent, the residue is taken up by ethyl ether and filter the precipitate obtained.

Obtained after drying 7.9 mmol of n - (L-alanyl-L) - amino 5^, - dideoxy-2^, , 5^, - methyl-5-uridine derivatives (TDR: 90%). In 20 ml of methanol the recrystallization of the previous product leads to 7.11 mmol of the desired optically pure compound. m=2.21 g of TDR overall ': 44%; F.=190 and 193 ° (Thermovar); [ODs ^²⁰ =(c=0.5 MeOH) + 28 °5; an IR (on KBr): 1670 cm "¹ (C=0); 1275 cm "¹ ,

1580 cm "¹ , 3250 cm '¹ . (N - h) NMR (200 MHz band, the CD₃ 0D): 1.30 (3:00, e. mth to Ala); 1.90 (3:00, e. Me); 2.22 to 2.28 (2:00, M., c 2 'H-); 3.47 to 3.51 (3:00, M., c 5' H-a, c-Ala-hr);

3.90 to 3.92 (1 hr, M., c 4 'H); 4.25 to 4.27 (1:00, M., c 3' H-); 6.18 (1 hr, T., c 1' H-a); 7.49 (1:00, e, c 6 H-a).

Example 2: chemically

Synthesis of n - (d-alanyl) - amino 5¹ - dideoxy-2 ', 5'-methyl 5-uridine derivatives

0

0

CH3

H₂ ONLY

CH-OC NH -

I

hM₃

Transportable : Synthesis of n a-c (n-benzyloxycarbonyl-) - d alanyU amino 5 'didéoxy-to-2 ', 5'-methyl 5-uridine derivatives.

In a knitted fabric of 100 ml equipped with a magnetic stirring, a thermometer and a hilt calcium chloride,

introduced:

the O

20 ml of dry dichloromethane - (4 sieve has)

- 893 mg of (n-benzyloxycarbonyl-) - d-alanine methyl (4 mmol)

- 0.6 ml of triethylamine (4.3 mmol)

The solution obtained is cooled -5 °c and adds 0.4 ml of ethyl chloroformate (4.2 mmol).

After 90 minutes of shaking, 964 mg d-added^, amino 5 '-dideoxy 2 ', 5'-methyl 5-uridine derivatives and leaves 60 minutes

-5 °c then overnight at room temperature.

The formed precipitate is filtered and washed with 15 ml of water permuted. After drying in a vacuum oven at 60 °c, obtained 1.33 g of n-a-c (n-benzyloxycarbonyl-) - d alanyl3-amino 5 '-dideoxy 2', 5'-methyl 5-uridine derivatives. 50 Ml of crystallization in methanol leads to 1.2 g of a compound of satisfactory purity (TDR: 67%).

Step b :

The hydrogenolysis and purification similar to those described in the example 1, lead to n - (d aianyl) - amino 5 'didéoxy-to-2 ', 5'-methyl 5-uridine derivatives optically pure cm=715 mg;

2.3 mmol; TDR 57 the X]=180 and 184 °F. (Thermovar) of O ^²⁰ =+ 22.6 degrees (c=1, MeOH) an IR (on KBr): 1670 cm "¹ (C=0); 1275 cm "¹ , 1580 cm "¹ , 3250 cm "¹ (N-H and); tic: (MeOH, hr₂ 0: 80/20) rf=0.21 silica^{60 F.} 254 *

NMR (200 MHz band, D.₂ 0): 1.23 - 1.30 (3:00, D., mth to Ala);

1.90 (3:00, E; mth); 2.35 to 2.41 (2:00, M., c 2 'H-); 3.52 to 3.57 (3:00, m-c - 5' hr, C. to Ala - hr); 4.04 to 4.02 (L-hr, M., c 4 'H); 4.37 to 4.40 (1:00, M., c 3 'H); 6.25 (1:00, T., c 1' H-a); 7.48 (11 hr, e, c 6 H-a) Example 3

Synthesis of n - (d-alanyl) amino 3 '-dideoxy 2 ', 3'-methyl 5-uridine derivatives

The operative procedure is identical to that described in the example 2.

Transportable : Synthesis of n - [(n-benzyloxycarbonyl-) - d alanyU-amino 3 'didéoxy and 2' and 3'-methyl 5-uridine derivatives (1)

The starting materials are as follows:

the O

20 ml of dry dichloromethane - (4 sieve has)

- 925 mg of (n-benzyloxycarbonyl-) - d-alanine methyl (4.15 mmol)

- 0.62 ml of triethylamine (4.4 mmol)

This gives 0.95 g of the compound (1) with a satisfactory purity (TDR: 64%).

F.=124 and 128 ° (thermovar): TDC=+ 25° (c=0, 1, MeOH)

NMR (200 MHz band, cd3 ODs): 1.2 to 1.3 (3:00, D., mth, to Ala); 1.7 (3:00, e.

Me); 2.29 to 2.32 (2:00, M., c 2 'H-);' 3.7 to 3.9 (3:00, M., c 5 'H, c4' H) 4 to 4.1 (1:00, D., c-Ala-hr); 6.2 (1:00, T., c 1' H-); 7.3 to 7.4 (5:00, the m); 7.9 (1:00, e, c 6 H-a)

Step b :

This compound (1) is placed in solution in methanol and hydrogenated in the presence of 200 mg of 10% palladium-on-carbon.

After filtration due catalyst and concentration of the solvent, the residue is taken up by ethyl ether and filter the precipitate obtained.

Obtained after drying 502 mg of n - (d-alanyl) - amino 3 'didéoxy and 2' and 3'-methyl 5-uridine derivatives (TDR 90%)=182 and 184 °F.

of OC²⁰₀ =+ 22.5 DEGREES (C=1, HR₂ 0)

tLC: (MeOH, hr₂ 0: 80/20) rf=0.26 silica 60 F.₂₅₄

NMR (200 MHz band, D.₂ 0): 1.2 to 1.3 (3:00, D., mth Ala); 1.9 (3:00, e, mth);

2.4 (2:00, M., c 2' H-); 3.7 (1 hr, M., C. Ala); 3.8 to 3.9 (2:00, M.,

5 - ℮ · - Η); 4 (1:00, M., c 4 '~H); 4.4 to 4.6 (1:00, M., c 3' H-a);

6.2 to 6.3 (1:00, T., C. T-hr); 7.7 (1:00, e, c 6 H-a)

Example 4 : Chemically

Synthesis of n - (L-alanyl-L) - amino 3 '-dideoxy 2 ', 3'-methyl 5-uridine derivatives

The operative procedure is identical to that described in the example 2.

Transportable : Synthesis of NC (n-benzyloxycarbonyl-) - L-alanyl-L]-amino 3 'dideoxy-2 ', 3'-methyl 5-uridine derivatives (2)

Used initially (20.7 mmol) 5 grams of amino-s-¹ - dideoxy-2 ', 3'-methyl 5-uridine derivatives.

In this way the g (13.7 mmol) 6.14 of the compound (2), with a satisfactory purity (TDR: 66.5 %)

F. (thermovar): 145 and 148°

C.<x3²⁰_D. =+ 5 (c=1, MeOH) °9

NMR (200 MHz band, CDjOD): 1.30 to 1.34 (3:00, D., mth Ala)

1.68 (3:00, E, 05 - 4); 2.28 to 2.34 (2:00, T., c 2' H-a)

3.7 to 3.8 (3:00, M., c 4 'H, c 5 * - hr); 4.07 to 4.11 (1 Η, M., C. -) 4.47 to 4.51 (1:00, M., c 3' H-), 5.07 (2:00, S., HM₂ )

6.19 to 6.22 (1:00, T., c 1' H-); 7.33 to 7.27 (5:00, M., C. ^)

7.87 (1:00, E, c 6 H-a)

Th is obtained from the compound (2), (11.7 mmol) 3.66 g of n - (L-alanyl-L) - amino 3 '-dideoxy ', 2 3^, - methyl " 5-uridine derivatives

(TDR: 85.9%)

F. (thermovar): 180 degrees and 185 degrees Celsius

C." 3_D.²⁰ +=32 (c=1, MeOH) °4

NMR (200 megahertz, D.₂ 0): 1.28 to 1.31 (3:00, D., mth Ala)

1.90 (3:00, E, c 5 H); 2.41 to 2.50 (2:00, M., c 2' H-a);

3.54 to 3.58 (1:00, M., c-Ala-hr); 3.76 to 4.00 (3:00, M., C. "4 '" hr c 5' H); 4.49 to 4.53 (1:00, M., c 3 H-a);

6.22 to 6.26 (1:00, T., c 1' H-); 7.70 (1:00, e, c 6 H-a)

Example 5 : Chemically

Synthesis of n - (L-alanyl-L) - amino 5 a-deoxy-2^, human uridine -

Transportable : Synthesis of n "C. (the n~benzyloxycahbonyl)" L alanyüamino-a 5 a-deoxy-2'-uridine derivatives (3)

To a solution of 6 grams (21 mmol) of hydrochloride of amino-a 5 a-deoxy-2'-uridine derivatives in 220 ml of methanol and water (4:1) and 2.8 ml of triethylamine are added 10.38 grams (42 mmol) of n - (ethoxy-carbonyl) - 2 ethoxy 1.2 above dihydroquinoline derivative and 9.36 grams

(42 mmol) of (n-benzyloxycarbonyl-) - alanine.

The reaction medium is stored at room temperature 12 hours. After evaporation under vacuum at 20 °c solvents, the oil obtained is triturated thoroughly in the presence of petroleum ether to give a paste. The addition of chloroform gives rise to a precipitate which, after filtration, is carefully washed several times with chloroform and finally with ether.

After drying under vacuum at 20 °c obtained 8 grams (18 mmol) of n - [(n benzyloxycabonyl) - L alanyü amino 5 ~déoxy-a 2'-uridine derivatives. The recrystallization in a mixture of chloroform and inéthanol leads to 7.2 grams (16/5 mmol) of a compound of satisfactory purity (TDR: 77%)

Purity (by HPLC): the X 100

Ccdjj²⁰ =- 30° (C..=0.5, MeOH)

F. (thermovar)=104 and 107°

CCHs silica 60 F. ^ (CHClj, on EtOH: 90/10) rf=0,176 NMR (200 MHz band, CDjOO):

1.36 to 1.39 (3:00, D., HC₃ to Ala); 2.23 to 2.26 (2:00, M., c 2' H-a);

3.72 to 3.74 (2:00, D., c 5 'H); 3.91 to 3.93 (1:00, M., c 4' H);

4.31 to 4.35<2:00, M., c 'H 3, ch-Ala); 6.29 to 6.36 (1:00, T., c 'H 1);

8.61 (1:00, E, c 6h)

4 grams (9 mmol) of the compound (3) are dissolved in 300 ml of methanol and hydrogenated in the presence of 400 mg of palladium-on-carbon 10 7, during 3 hours. After filtration of the catalyst and concentration of solvent, the residue is taken up by ethyl ether and filter the precipitate obtained. Is obtained after drying the g (8.6 mmol) 2.7 n - (L-alanyl-L) - amino 5 a-deoxy-2'-uridine derivatives (TDR: 94%). The recrystallization in n-butanol results (6.7 mmol) 2.1 grams of the desired compound (BDR=73 7.)

BDR overall: 56%

Purity (by HPLC): 96 %

[x-<]_D²⁰ =(c=0.5 MeOH) + 15°

F. (thermovar): 166 and 168°

MSC 60f silica₂₅₄ (MeOH, hr₂ 0: 80/20) rf=0,303

An IR (or HBr): 1670 cm "¹ (C=0). ; 1275' citk¹ , 1580 cm "¹ , 3250 cm "¹ (N-H AND)

NMR (200 MHz band, the CD₃ 0D): 1.55 (3:00, D., CHjala), 2.20 to 2.31

(2:00, M., c 2 'H-); 3.72 to 3.74 (2:00, D., c 5' H->; 3.92 to 3.93 (1:00, M., c 4' H); 4.14 to 4.18 (1:00, M., c-Ala-hr); 4.35 to 4.38

(1:00, M., c 3 'H-); 6.28 to 6.34 (1:00, T., c 1' H-); 8.60

(1:00, E, c 6 H-a).

Examples 6 to 17

Using processes similar to those described in experimental examples 1 and 2 and that the skilled person can be easily found, we prepared the following compounds:

The n - (L-alanyl-L) - amine 5 '-dideoxy 2 ', 5 'ethyl 5-uridine derivatives of n - (L-alanyl-L) - amino 5'-dideoxy ', 5 2^, - butyl 5-uridine derivatives of n - (L-alanyl-L) - amino 5 '-dideoxy 2 ', 5 'propyl 5-uridine derivatives of n - (L-valyl) - amino 5'-dideoxy ', 5 2' a-méthy L 5-uridine derivatives of n - (L-leucyl) - amino 5 '-dideoxy 2 ', 5 '-methyl 5-uridine derivatives of n - (sarcosyl) - amino 5'-dideoxy 2 ', 5' a-méthy1-to-5-uridine derivatives of n - (P-alanyl) - amino 5 '-dideoxy 2 ', 5 '-methyl 5-uridine derivatives of n - (L-alanyl-L-alanyl-) - amino 5'-dideoxy 2 ', 5'-methyl-5~uridine ofN - (L-alanyl-L-alanyl-L-alanyl-L) - amino 5 '-dideoxy 2 ', 5' a-méthy1-to-5-uridine derivatives

The n - (L-alanyl-L) - amino 5 '-dideoxy 2 ', 5 '-vinyl 5-uridine derivatives of n - (L-alanyl-L) - amino 5'-dideoxy 2 ', 5' a-bromovinyl-to-5-uridine derivatives Examples 18 to 31

Using experimental processes similar to those described in the example 3 and that the skilled person can be easily found, we prepared the following compounds:

The n - (L-alanyl-L) - amino 3 '-dideoxy 2 ', 3 '-methyl 5-uridine derivatives of n - (L-alanyl-L) - amino 3'-dideoxy 2 ', 3' ethyl 5-uridine derivatives of n - (L-alanyl-L) - amino 3 '-dideoxy 2 ', 3'-butyl 5-uridine derivatives of n - (L-alanyl-L) - amino 3¹ - dideoxy-2^B ,3'-prôpyl-5-uri dine N-(L-valyl)-amino-3'-didéoxy-2',3'-méthyl-5-uridine N-(L-leucyl)-amino-3'-didéoxy-2',3'-méthyl-5-uridine N-(sarcosyL)-amino-3'-didéoxy-2',3'-méthyl-5-uridine N-(β-alanyl)-amino-3'-didéoxy-2',3'-méthyl-5-uridine N-(L-alanyl-L-alanyl)-amino-3'-didéoxy-2',3'-méthyl-5-uri dine N-(L-alanyl-L-alanyl-L-alanyl)-amino-3'-didéoxy-2',3'-méthy1-5-uridine

NR^- (L-glycyl) - amino~3 '-dideoxy 2 ', 3 '-methyl 5-uridine derivatives of n - (L-alanyl-L) ^ - amino 3'-dideoxy 2 ', 3'-methyl 5-uridine derivatives of n - (L-alanyl-L) - amino 3 '-dideoxy 2 ', 3 '-vinyl 5-uridine derivatives of n - (L-alanyl-L) - amino 3'-dideoxy 2.3 ' a-bromovinyl-to-5-uridine derivatives Examples 32 to 38

Using experimental process similar to that described in the example 5 and that the skilled person can be easily found, we prepared the following compounds:

The n - (L-valyl) amino 5 - deoxy-2'-uridine derivatives

The n - (L-leucyl) amino 5 - deoxy-2 '-uridine derivatives of n - (sarcosyl) - amino 5 a-deoxy-2' - human uridine Ν - (β θLanyU-amino 5 a-deoxy-2'-uridine derivatives

The n - (L-a-L-anyI-L-L-anyI) - amino 5 de-oxy 2 '-uridine derivatives Ν - (L alanyI) ^ - amino 5 a-deoxy-2'-uridine derivatives of n - (L-glycyl-L-alanyl-L) - amino 5 ~déoxy-a 2'-uridine derivatives

The activity of the compounds according to the invention, as inhibitors of DNA synthesis of cancer cells was evaluated:

"vitro" - Tf on the cytosolic (study 1)

- "in-vivo" on the MCF7 in culture (study 2)

- "in-vivo" immature females Wistar rats (study 3) Study 1

Of hormone-dependent breast cancer cells (MCF7 or) containing 95 - 96% fetal thymidine kinase, are treated by ultrasound and centrifuged at 105,000 grams.

The MCF7 originate from a pleural metastasis of a human breast cancer (PREV. H drunk, J VASQUEZ, along with A., sec. Rural Health BREUNAN m. J proc. Instit cancers ., _ 51, 1409 - 1416, 1973) and are for example available by A.T.' DC (American Type culture).

Concentration of the proteins from the cytosol obtained is measured and adjusted by dilution to 1.5 mg of protein per ml of cytosol.

It is incubated, to 37 °c during 10 min, in a

14 tris buffer (pH value: 7.6) in the presence of. a thymidine DC - 2d (40 pmolaire), ATP (8 mmolar) and magnesium chloride. The enzymatic reaction is stopped by the addition of perchloric acid.

The nucleotides formed are electrophoretically separated high voltage, stains are cut and counted.

Was given Table I the results of experiments carried out with the compounds of examples 1 and 2 as well as with controls without inhibitor, for comparison purposes.

The given values are expressed pi comoles per milliliter of cytosol per minute.

TABLE 1

This biochemical test is based on the insight that the thymidine kinase occupies a strategic position in the biochemical processes leading to the synthesis of DNA, which uses thymidine triphosphate of (TPP) which can be regarded as the end product of a complex response initiated by thymidine kinase.

As shown in the table 1, the compounds of the invention act as inhibitors of the synthesis TTP,, and thus presumably the DNA synthesis of proliferating cancer cells.

Study 2

Cells are cultured in MFC and 7 vials of

2, 6

75 cm. Each vial is seeded with cells suspended in 0, 6 x 10° in 10 ml of RPMI and 1640 supplemented with 2 mm L-glutamine, 2.5 pg/ml of Fungizone, 15 a U/ml penicillin,

50 pg/ml streptomycin and 5% fetal calf serum.

The cells are cultured for 72 hours at 37 °c in an incubator co ^ (110 to Eg, IR. Jouan case). The culture media is changed every 48 hours.

The harvesting of the cells is performed by peeling using a Genfoot-a beaker and the cell suspension is centrifuged at 200 g in a refrigerated centrifuge to obtain a cell pellet.

The various inhibitors to be tested are dissolved in RPMI and 1640 to the concentration of 1 mm. Their effects are tested during 48 hours and 72.

- Cell count : Cellular carpets are washed, 3 times, with 5 ml of phosphate buffered saline (PBS network). The contents of each flask culture is, then, suspended in 10 ml of PBS. The cells are counted using a d

H.C. Counter-Hycel counter 202.

- DNA assay : It is performed on an aliquot of the sonicate cells and spectrofluorimetrically according to the technique of C. Brunk and Al, Analyst Senate. Biochem., 92, 497 - 500 1979.

TABLE 2

Study 3

PROTOCOL

Animals females Wistar rats (IFFA creed) at age 22 days of average weight of between 30 and 50 grams.

They are separated from the mother 2 days before the start of the test.

They have free access to food and drinking water for the duration of the test.

Products. 0.9% NaCl

. Absolute alcohol

. 17 Β estradiol dipropionate (sigma-reference e. 9125)

A solution to 2 mg ml in 0.9% NaCl. is performed

. 6 - C ^ deoxythymidine-H3(Amersham's reference TRK61 to 24 IC/mmol

The working solution is prepared by dilution of the commercial solution at 1/11ème " its activity concentration is 10 of the PCI/0, F. ml.

. Example 1

. Example 2

The products are placed in solution in the NaCl 0.9 T- to deliver a volume of 0.1 ml/animal in

. Diphenylamine (sigma-reference d. 2285) in solution in glacial acetic acid (1%)

. H .S0, concentrate

H

" Calf thymus DNA (Borhinger 104,175 reference) in the NaOH solution 5 mm

. Instagel®

Procedure

1. Treatments and sacrifice

They are performed according to the following scheme:

to verbs

t23h

t24h

Estradiol

naCI or

+ C.³ Hr] DTH-

Sacrificial inhibitor

similar to the batch "absolute control" 0.1 ml 0.9% NaCl receives intraperitoneally. The other batches receive

800 nanograms/animal of estradiol in a volume of 0.1 ml.

all animals receive t23h 10 PCI C.³ Hr] in a volume of 0.1 ml DTH-intraperitoneally.

The batch "cookie estradiol" and the batch "absolute control" receive 0.1 ml 0.9% NaCl.

The batches "inhibitors" receive the products to be tested in a volume of 0.1 ml intraperitoneally.

t24h all animals are sacrificed by cervical dislocation.

The uterus are taken ' ^ wiped on filter paper, weighed and frozen.

2. Treatment of the uterus

The uterus is ground in 0.5 ml of perchloric acid 0.6 n to using the putter. The piston of the mill is rinsed with 3 times with 0.3 ml of perchloric acid 0.6 n homogenate and the flushing liquids are organized into glass conical tubes, and centrifuged at 800 g for 10 mins. The pellet is washed 2 times with 1 ml of perchloric acid 0.3 n-.

The socket is resumed by KOH 0.3 0.6 ml of n; the tubes are placed in a water bath at 37 °c during 2 hours 30.

After alkaline hydrolysis (hydrolysis of RNA) 30 μ L of perchloric acid 11 n are added in order to precipitate the DNA.

The tubes are cooled in ice and centrifuged to 4200 g for 10 mins.

The pellet is washed 2 times with 1 ml of perchloric acid 0.3 n-.

The last residue is placed again by 0.6 ml of perchloric acid 0.6 n the tubes are placed 10 minutes to 80 °c. After cooling in ice for several minutes they are centrifuged for 10 minutes at 4200 grams.

3. Determining the radioactivity incorporated into the DNA

50 μ L of supernatant are placed in vials of counting. After addition of 5 ml of Instagel, the counting is performed on 10 minute.

The cc.p.m. (counts per minute) are converted into d.p.m.:

cc.p.m.

d.p.m.=(d.p.m. disintegrations per minute=)

E

I=counting efficiency=0.37

dd.p.m.

The d.p.m. in picomoles of DTH-according to the formula

2220x25

3

specific tee generally H] DTH-

25 picomole/IAS

1nci=2220 dd.p.m.

The results are expressed in pmol of deoxythymidine incorporated by uterus.

4. Dosage of the d.n.a..

100 Μ L of supernatant has, diluted at 1/6 in perchloric acid 0.6 n are added 1.2 ml of a solution of 05 diphenylamine in glacial acetic acid (1 g/100 ml).

The tubes are placed 10 minutes boiling water bath. After cooling, the optical density is read at 595 nm. The calibration is performed using a DNA solution of 10 calf thymus.

The results are expressed in pg/uterus.

TABLE 3