RU2289583C2 - Pyrimidotriazines, method for their preparing, pharmaceutical composition based on thereof and using - Google Patents

Abstract

FIELD: organic chemistry, medicine, biochemistry, pharmacy.

SUBSTANCE: invention describes novel pyrimidotriazines of the general formula (I):

wherein each R¹ and R² is chosen from the group comprising hydrogen atom, or R¹ and R² form in common chemical bond, -CH₂-Ar and Ar is chosen from the group comprising unsubstituted phenyl, unsubstituted naphthyl, phenyl, mono- or disubstituted with (lower)-alkoxy-group and naphthyl mono- or disubstituted with (lower)-alkyl, or their pharmaceutically acceptable salts. Also, invention relates to a method for synthesis of these compounds, pharmaceutical composition based on thereof and to using novel pyrimidotriazines for prophylaxis and/or treatment of diabetes mellitus as these compounds possess the strong expressed inhibitory effect on activity of protein tyrosine phosphatase PTP1B.

EFFECT: improved method of synthesis, valuable medicinal properties of compounds and pharmaceutical composition.

15 cl, 27 ex

Description

The invention relates to derivatives of pyrimido [5,4-e] [1,2,4] triazine-5,7-diamine, which are used to inhibit protein tyrosine phosphatases, especially PTP1B.

The invention relates primarily to compounds of the formula

where R ¹ and R ² each is selected from the group comprising hydrogen, or

R ¹ and R ² together mean a chemical bond, —CH ₂ -, —O—, —NH— or —NR ³ ,

R ³ means (ness.) Alkyl or-CH ₂ -Ar, and

Ar is selected from the group consisting of unsubstituted phenyl, unsubstituted naphthyl, phenyl, mono- or disubstituted by a group of lower, alkoxy, aryl, cycloalkyl, lower, alkylaryl, lower alkoxyaryl, lower. ) alkylcycloalkyl, (ness.) alkoxycycloalkyl, halogen, cyano or trifluoromethyl; and naphthyl, mono- or disubstituted by a group of lower alkyl, lower alkoxy, aryl, cycloalkyl, lower alkylaryl, lower alkoxyaryl lower alkyl cycloalkyl lower alkoxycycloalkyl or halogen, or their pharmaceutically acceptable salts.

Protein tyrosine phosphatases (PTP) are key enzymes in the processes of cell growth and differentiation. Inhibition of these enzymes can affect the modulation of multi-stage signaling pathways in which the tyrosine phosphorylation / dephosphorylation process plays a role. PTP1B is that specific protein tyrosine phosphatase that is often used as a prototype for this class of enzymes.

RTPase inhibitors are considered potential therapeutic agents for diabetes. See, e.g., Moeller et al., Current Opinion in Drug Discovery and Development, 3 (5), 527-540 (2000) or Zhang, Zhong-Yin, Current Opinion in Chemical Biology, 5, 416-423 (2001 )

It is established that the compounds of the formula

and their pharmaceutically acceptable salts, where R ¹ and R ² have the meanings indicated below, inhibit protein tyrosine phosphatases, especially PTP1B, and, therefore, can be used to reduce the concentration of glucose in plasma (blood) in mammals.

The term “lower alkyl”, used alone or in combination in the description of the application, means a linear or branched alkyl group containing a maximum of six carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec- butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl and the like. The (lower) alkyl groups are unsubstituted or substituted by one or more groups independently selected from the series cycloalkyl, nitro, aryloxy, aryl, hydroxy, halogen, cyano, (lower) alkoxy, (lower) alkanoyl, (lower) alkylthio, (ness.) alkylsulfinyl, (ness.) alkylsulfonyl and a substituted amino group. Examples of substituted lower alkyl groups include 2-hydroxyethyl, 3-oxobutyl, cyanomethyl and 2-nitropropyl.

The term “cycloalkyl” means an unsubstituted or substituted 3-7 membered cyclic hydrocarbon radical. Substituents used in the present invention include hydroxy, halogen, cyano, (ness.) Alkoxy, (ness.) Alkanoyl, (ness.) Alkyl, aroyl, (ness.) Alkylthio, (ness.) Alkylsulfinyl, (ness.) alkylsulfonyl, aryl, heteroaryl and a substituted amino group.

The term “lower alkoxy” means a straight or branched chain alkoxy group containing a maximum of 6 carbon atoms, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy and the like.

The term "(ness.) Alkylthio" means a (ness.) Alkyl group attached via a divalent sulfur atom, for example methyl mercapto or isopropyl mercapto group.

The term “aryl” means a mono- or bicyclic aromatic group, such as phenyl or naphthyl, unsubstituted or substituted with appropriate substituents. Preferred substituents include (ness.) Alkyl, (ness.) Alkoxy, hydroxy (ness.) Alkyl, hydroxy, hydroxyalkoxy, halogen, (ness.) Alkylthio, (ness.) Alkylsulfinyl, (ness.) Alkylsulfonyl, cyano, nitro, perfluoroalkyl, alkanoyl, aroyl, arylalkynyl, (ness.) alkynyl and (ness.) alkanoylamino. More preferred substituents are (ness.) Alkyl, (ness.) Alkoxy, hydroxy, halogen, cyano and perfluoro (ness.) Alkyl. Examples of the aryl groups used in the present invention are phenyl, para-tolyl, para-methoxyphenyl, para-chlorophenyl, meta-hydroxyphenyl, meta-methylthiophenyl, 2-methyl-5-nitrophenyl, 2,6-dichlorophenyl, 1-naphthyl etc.

The term “lower alkylaryl” means a lower alkyl group as defined above, in which one or more hydrogen atoms is replaced by an aryl group as defined above. Any lower alkylaryl, such as benzyl and the like, may be used in the present invention.

The term “lower alkoxyaryl” means a lower alkoxy group as defined above, in which one or more hydrogen atoms is replaced by an aryl group as defined above. Any lower alkoxyaryl, such as benzyloxy, may be used in the present invention.

The term “lower alkoxycarbonyl” means a lower alkoxy group attached through a carbonyl group. Examples of alkoxycarbonyl groups include ethoxycarbonyl and the like.

The term "pharmaceutically acceptable salts" means the corresponding acid addition salts or base addition salts which possess the biological effect and properties of the compounds of formula (I) and are formed using suitable non-toxic organic or inorganic acids or organic or inorganic bases. Examples of acid addition salts include salts of inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and salts of organic acids such as para-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, etc. Examples of base addition salts include ammonium, potassium, sodium, and quaternary ammonium salts, such as tetramethyl ammonium hydroxide. Chemical modification of a pharmaceutical compound (i.e., a drug) to form a salt is a well-known technique for one skilled in the art to obtain compounds with improved physical and chemical stability, hygroscopicity, fluidity and solubility. See, for example, H. Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 6 ^th Ed., 196 and 1456-1457 (1995).

The present invention relates to compounds of formula I

and their pharmaceutically acceptable salts, where

R ¹ and R ^{2 are} each selected from the group consisting of hydrogen, or

R ¹ and R ² together mean a chemical bond, —CH ₂ -, —O—, —NH— or —NR ³ ,

R ³ means (ness.) Alkyl or-CH ₂ -Ar, a

Ar is selected from the group consisting of unsubstituted phenyl, unsubstituted naphthyl, phenyl, mono- or disubstituted by a group of lower, alkoxy, aryl, cycloalkyl, lower, alkylaryl, lower alkoxyaryl, lower. ) alkylcycloalkyl, (ness.) alkoxycycloalkyl, halogen, cyano or trifluoromethyl; and naphthyl, mono- or disubstituted with a group of lower alkyl, lower alkoxy, aryl, cycloalkyl, lower alkaryl, lower alkoxy aryl lower alkyl cycloalkyl lower alkoxycycloalkyl or halogen.

Among the compounds of formula I, preferred are compounds of formula II

where Ar is selected from the group consisting of unsubstituted phenyl, unsubstituted naphthyl, phenyl mono- or disubstituted by a group of lower alkyl alkoxy aryl cycloalkyl lower alkylaryl lower alkoxyaryl lower .) alkylcycloalkyl, (ness.) alkoxycycloalkyl, halogen, cyano or trifluoromethyl; and naphthyl, mono- or disubstituted with a group of lower alkyl, lower alkoxy, aryl, cycloalkyl, lower alkaryl, lower alkoxy aryl lower alkyl cycloalkyl lower alkoxycycloalkyl or halogen.

In one preferred embodiment of the compounds of formula II, Ar is unsubstituted phenyl or unsubstituted naphthyl.

In another preferred embodiment of the compounds of formula II, Ar is phenyl monosubstituted with a group of lower alkyl, lower alkoxy, aryl, cycloalkyl, lower alkaryl, lower alkoxyaryl, halogen, cyano or trifluoromethyl.

In a further preferred embodiment of the compounds of formula II, Ar is phenyl monosubstituted with a group of lower alkyl, lower alkoxy, halogen, cyano or trifluoromethyl.

In a further preferred embodiment of the compounds of formula II, Ar is phenyl disubstituted by a group of lower alkyl, lower alkoxy, halogen or cyano.

In another preferred embodiment of the compounds of formula II, Ar is naphthyl, monosubstituted with a group of lower alkyl, lower alkoxy, lower alkylaryl, lower alkoxyaryl or halogen.

In yet another preferred embodiment of the compounds of formula II, Ar is naphthyl, monosubstituted by a group of lower alkyl, lower alkoxy or halogen.

In another preferred embodiment of the compounds of formula II, Ar is naphthyl disubstituted with a group of lower alkyl, lower alkoxy or halogen.

The compounds of the invention may exist as stereoisomers, especially enantiomers and diastereomers, each of which is included in the scope of the present invention.

Preferred compounds of the invention are selected from the range

1. 3-piperazin-1-ylmethylpyrimido [5,4-e] [1,2,4] triazin-5,7-diamine,

2. 3-diethylaminomethylpyrimido [5,4-e] [1,2,4] triazine-5,7-diamine,

3. 3-pyrrolidin-1-ylmethylpyrimido [5,4-e] [1,2,4] triazin-5,7-diamine,

4. 3-piperidin-1-ylmethylpyrimido [5,4-e] [1,2,4] triazin-5,7-diamine,

5. 3-morpholin-4-ylmethylpyrimido [5,4-e] [1,2,4] triazin-5,7-diamine,

6. 3- (4-methylpiperazin-1-ylmethyl) pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine,

7. 3- (4-benzylpiperazin-1-ylmethyl) pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine,

8. 3- (4-naphthalen-2-ylmethylpiperazin-1-ylmethyl) pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine,

9. 3- (4-naphthalen-1-ylmethylpiperazin-1-ylmethyl) pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine,

10. 3- (4-biphenyl-4-ylmethylpiperazin-1-ylmethyl) pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine,

11. 3- [4- (2-chlorobenzyl) piperazin-1-ylmethyl] pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine,

12. 3- [4- (3-chlorobenzyl) piperazin-1-ylmethyl] pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine,

13. 3- [4- (4-chlorobenzyl) piperazin-1-ylmethyl] pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine,

14. 3- [4- (3-methoxybenzyl) piperazin-1-ylmethyl] pyrimido [5,4th] [1,2,4] triazin-5,7-diamine,

15. 3- [4- (3-fluorobenzyl) piperazin-1-ylmethyl] pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine,

16. 3- [4- (3-trifluoromethylbenzyl) piperazin-1-ylmethyl] pyrimido [5.4-e] [1,2,4] triazine-5,7-diamine,

17. 3- [4- (4-trifluoromethylbenzyl) piperazin-1-ylmethyl] pyrimido [5.4-e] [1,2,4] triazin-5,7-diamine,

18. 3- [4- (3-bromobenzyl) piperazin-1-ylmethyl] pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine,

19. 3- [4- (3-cyanobenzyl) piperazin-1-ylmethyl] pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine,

20. 3- [4- (2,4-dimethylbenzyl) piperazin-1-ylmethyl] pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine,

21. 3- [4- (4-ethyl-2-methylnaphthalen-1-ylmethyl) piperazin-1-ylmethyl] pyrimido [5,4-e] [1,2,4] triazine-5,7-diamine and

22. 3- [4- (4-benzyloxybenzyl) piperazin-1-ylmethyl] pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine.

More preferred compounds of the invention are selected from the range

3-diethylaminomethylpyrimido [5,4-e] [1,2,4] triazine-5,7-diamine,

3- (4-benzylpiperazin-1-ylmethyl) pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine,

3- (4-naphthalen-2-ylmethylpiperazin-1-ylmethyl) pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine,

3- (4-naphthalen-1-ylmethylpiperazin-1-ylmethyl) pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine,

3- (4-biphenyl-4-ylmethylpiperazin-1-ylmethyl) pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine,

3- [4- (2,4-dimethylbenzyl) piperazin-1-ylmethyl] pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine,

3- [4- (4-ethyl-2-methylnaphthalen-1-ylmethyl) piperazin-1-ylmethyl] pyrimido [5,4-e] [1,2,4] triazine-5,7-diamine and

3- [4- (4-benzyloxybenzyl) piperazin-1-ylmethyl] pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine.

Further preferred is a process for preparing compounds of formula I, comprising reacting a compound of formula 3

with a compound of formula R ₄ NHR ₅ , where R ⁴ is —CH ₂ CH ₂ R ₁ , and R ⁵ is —CH ₂ CH ₂ R ₂ , where R ¹ and R ² are as defined above.

In addition, compounds of formula I are preferred for use as therapeutically active substances.

In addition, compounds of formula I are preferred for the manufacture of medicaments for the prophylaxis and / or treatment of diabetes.

Another preferred aspect of the invention is a pharmaceutical composition comprising a compound of formula I and a therapeutically inert carrier.

In addition, a preferred embodiment of the invention is a compound of formula I obtained by the method described above.

In addition, a method for the treatment and / or prevention of diseases associated with plasma glucose (blood) is preferred, the method comprising administering an effective amount of a compound of formula I. This method is presented as the use of a compound of formula I for the manufacture of a medicament for treatment diabetes prevention.

The compounds of the invention inhibit PTP1B in vitro and reduce plasma (blood) glucose in vivo. Therefore, the compounds of the invention can be used to treat diabetes.

The compounds of the invention can be administered by the oral, rectal, or parenteral route, for example, by the intravenous, intramuscular, subcutaneous, intrathecal or transdermal routes, or sublingually, or as ophthalmic preparations. Examples of dosage forms include capsules, tablets, suspensions or solutions for oral administration, suppositories, injection solutions, eye drops, ointments or spray solutions.

Preferred routes of administration are intravenous, intramuscular, oral or inhalation methods.

Doses at which the compounds of the invention are administered in an effective amount depend on the nature of the particular active ingredient, the age and requirements of the patient, and the route of administration. Doses are determined by any conventional methods, for example, clinical trials to determine dose limits. In general, preferred are doses of from about 0.1 to 100 mg / kg body weight per day, more preferably 1-25 mg / kg body weight per day.

The invention also includes pharmaceutical compositions that comprise a pharmaceutically effective amount of a compound of the invention and a pharmaceutically acceptable carrier. Such compositions are prepared by conventional methods. Tablets or granules may contain binders, excipients, carriers or diluents. Liquid compositions can be prepared, for example, in the form of a sterile water-miscible solution. In addition to the active ingredients, the capsules may contain a filler. In addition, the compositions may include flavoring agents, as well as substances that are commonly used as preserving, stabilizing, water-retaining and emulsifying agents, as well as salts for regulating the osmotic pressure, buffering agents and other additives.

The above carriers and diluents may include appropriate pharmaceutically acceptable organic or inorganic substances, for example water, gelatin, lactose, starch, magnesium stearate, talc, gum arabic, polyalkylene glycols and the like.

Dosage units for oral administration, such as tablets and capsules, preferably contain from 25 mg to 1000 mg of the compound of the invention. Compounds of the invention are prepared by conventional methods. The specific method is shown in schemes 1-3.

Intermediate 3 containing a chloromethyl group is prepared, as shown in Scheme 1, from 2,4-diamino-2-mercaptopropyridine 1 hemisulphate, which is a commercial preparation. Compound 1 is S-methylated (e.g., with methyl iodide in the presence of sodium hydroxide) and then nitrosylated under standard conditions (e.g. sodium nitrate in acetic acid at approximately 50 ° C), whereby an intermediate aryl nitrosyl compound 2 is obtained. When replacing the thiomethyl group in the compound 2 hydrazine in a suitable solvent, such as dimethylformamide, at room temperature, followed by condensation with diethyl acetal of chloromethyl acetaldehyde, which is a commercial product, when heated (for example, at 85 ° C) in an acidic environment (for example, in the presence of HCl) receive compound 3 containing a chloromethyl group.

Then, the chloromethyl derivative 3 is reacted with various known amines in a suitable solvent, such as ethanol, by heating (for example, at about 80-100 ° C) to form the corresponding aminomethyl derivatives 4, as shown in Scheme 2. In amines of the formula R ⁴ NR ⁵ in Scheme 2, R ⁴ is —CH ₂ CH ₂ R ₁ , and R ⁵ is —CH ₂ CH ₂ R ₂ , where R ¹ and R ² are as defined above.

The piperazine derivative 5 (for example, compound 4, where R ⁴ and R ⁵ together form the —CH ₂ CH ₂ NHCH ₂ CH ₂ - fragment) is prepared from the chloromethyl derivative 3 and piperazine, as shown in Scheme 2. Alkylation of compound 5 with various known alkyl halides ( for example, R ³ Br or R ³ I, where R ³ is as defined above) is carried out in a suitable solvent, such as dimethylformamide, in the presence of a suitable base, such as potassium carbonate, at room temperature, whereby dialkylated piperazine derivatives are obtained 6 as shown in cx IU 3.

Examples

Example 1

6-methylthio-5-nitrosopyrimidine-2,4-diamine

Stage 1

To a solution of 105 g KOH in 1 l of water, 2,4-diamino-6-mercaptopopyrimidine 1 hemisulfate (70.0 g) was added with stirring, then methyl iodide (91 ml) and the resulting mixture was stirred vigorously for 4 hours. Solid separated by filtration, washed with water and dried in air overnight, to obtain 54.0 g of 6-methylthiopyrimidine-2,4-diamine as a tan solid.

¹ H NMR (DMSO-d ₆ ): 6.20 (s, 2H), 5.90 (s, 2H), 5.55 (s, 1H), 2.30 (s, 3H).

Stage 2

To a suspension of 6-methylthiopyrimidine-2,4-diamine (50.0 g, 321 mmol) in water (1000 ml) was added 500 ml of 2 N with stirring. acetic acid, the mixture was heated to 50 ° C, a solution of NaNO ₂ (24.0 g, 353 mmol in 200 ml of H ₂ O) was quickly added and the mixture was heated at 50 ° C for 1 h. Then the mixture was dark blue / purple cooled to room temperature and filtered. The blue / purple solid was washed several times with water and then ether, and dried in air to give 51.0 g of 6-methylthio-5-nitrosopyrimidin-2,4-diamine 2 as a blue / purple solid.

¹ H NMR (DMSO-d ₆ ): 9.70 (s, 1H), 8.10 (s, 1H), 7.95 (m, 2H), 2.43 (s, 3H).

Example 2

6-hydrazino-5-nitrosopyrimidine-2,4-diamine

Hydrazine hydrate (55% solution, 14.5 ml) was quickly added to a suspension (12.0 g, 64.9 mmol) of 6-methylthio-5-nitrosopyrimidin-2,4-diamine 2 in DMF at room temperature. The mixture was stirred overnight, then the bright pink mixture was filtered, the solid was washed several times with DMF, then with ether and dried in air, to give 9.53 g of 6-hydrazino-5-nitrosopyrimidine-2,4-diamine as solid pink.

¹ H NMR (DMSO-d ₆ ): 8.00 (s, 1H), 7.40 (s, 1H), 7.05 (m, 2H), 5.35 (m, 2H).

Example 3

3-Chloromethylpyrimido [5,4th] [1,2,4] triazine-5,7-diamine

To a DMF (350 ml) cooled in an ice bath (conc.) Was added with stirring. HCl (14 ml) and 7.14 g of 6-hydrazino-5-nitrosopyrimidine-2,4-diamine. After 5 minutes, chloroacetaldehyde diethyl acetal (15.4 ml) was added over approximately 2 minutes. The cooling bath was removed, the mixture was warmed to room temperature and held for 1 h, then the mixture was heated at 85 ° C for 1.5 h and cooled to room temperature for about 2.5 h. The mixture was filtered to remove a small amount of insoluble material brown, the filtrate was alkalinized conc. NH ₄ OH solution and diluted with an equal amount of water. The mixture was held for 1 h, the orange-brown solid was separated by filtration and dried in vacuo over P ₂ O ₅ to give 3.50 g of 3-chloromethylpyrimido [5.4-e] [1,2,4] triazine -5,7-diamine 3.

¹ H NMR (DMSO-d ₆ ): 8.25 (s, 2H), 7.95 (s, 1H), 7.30 (br s, 1H), 5.02 (s, 2H).

Example 4

3-piperazin-1-ylmethylpyrimido [5,4-e] [1,2,4] triazin-5,7-diamine

A mixture of 3-chloromethylpyrimido [5.4-e] [1,2,4] triazine-5,7-diamine 3 (2.00 g, 9.5 mmol) and piperazine (2.50 g, 29 mmol) in absolute ethanol was heated at 100 ° C in a closed tube for 4 hours. Then the mixture was cooled to room temperature and evaporated. The crude product was purified by reverse phase GHUR (Rainin C ₁₈ , eluent: gradient from 0% to 30% CH ₃ CH / H ₂ O / 0.1% TFA), bright yellow fractions containing the product were combined, CH ₃ CN removed in vacuo, the residue was dried lyophilically, to give 1.44 g of 3-piperazin-1-ylmethylpyrimido [5,4-e] [1,2,4] triazine-5,7-diamine 5 yellow trifluoroacetate.

¹ H NMR (DMSO-d ₆ ): 9.55 (s, 1H), 9.40 (s, 1H), 8.80 (s, 2H), 8.45 (s, 1H), 4.15 ( s, 2H), 3.10 (m, 4H), 2.80 (m, 4H).

Example 5

3-Diethylaminomethylpyrimido [5,4th] [1,2,4] triazine-5,7-diamine

A mixture of 3-chloromethylpyrimido [5.4-e] [1,2,4] triazine-5,7-diamine 3 (200 mg, 0.95 mmol) and diethylamine (2.00 ml) in absolute ethanol (2.0 ml) was heated at 100 ° C in a closed tube for 5 hours. Then the mixture was cooled to room temperature and concentrated in vacuo. The crude product was purified by reverse phase HPLC (Rainin C ₁₈ , eluent: gradient from 0% to 30% CH ₃ CN / H ₂ O / 0.1% TFA), bright yellow fractions containing the product were combined, CH ₃ CN removed in vacuo, the residue was dried lyophilically, to give 65 mg of yellow yellow 3-diethylaminomethylpyrimido [5,4-e] [1,2,4] triazine-5,7-diamine 4.

¹ H NMR (DMSO-d ₆ ): 9.10 (broad s, 1H), 8.90 (broad s, 1H), 8.15 (broad s, 2H), 4.80 (s, 2H ), 3.25 (q, 4H), 1.30 (t, 6H).

Example 6

3-Pyrrolidin-1-ylmethylpyrimido [5,4-e] [1,2,4] triazin-5,7-diamine

A mixture of 3-chloromethylpyrimido [5.4-e] [1,2,4] triazine-5,7-diamine 3 (70 mg, 0.33 mmol) and 1.0 ml of pyrrolidine was heated at 80 ° C in a closed tube in for 7 hours. Then the mixture was cooled to room temperature and concentrated in vacuo. The crude product was purified by reverse phase HPLC (Rainin C ₁₈ , eluent: gradient from 0% to 30% CH ₃ CN / H ₂ O / 0.1% TFA), bright yellow fractions containing the product were combined, CH ₃ CN evaporated in vacuo, the residue was lyophilized to give 50 mg of yellow 3-pyrrolidin-1-ylmethylpyrimido [5,4-e] [1,2,4] triazine-5,7-diamine trifluoroacetate.

¹ H NMR (DMSO-d ₆ ): 9.15 (broad s, 1H), 8.88 (broad s, 1H), 8.20 (broad s, 2H), 3.67 (s, 2H ), 3.20 (br.m, 2H), 1.85-2.10 (m, 4H).

Example 7

3-piperidin-1-ylmethylpyrimido [5,4th] [1,2,4] triazin-5.7-diamine

A mixture of 3-chloromethylpyrimido [5.4-e] [1,2,4] triazine-5,7-diamine 3 (70 mg, 0.33 mmol) and 1.0 ml of piperidine in a closed tube was heated at 80 ° C in for 7 hours. Then the mixture was cooled to room temperature and concentrated in vacuo. The crude product was purified by reverse phase GHUR (Rainin C ₁₈ , eluent: gradient from 0% to 30% CH ₃ CN / H ₂ O / 0.1% TFA), bright yellow fractions containing the product were combined, CH ₃ CN removed in vacuo, the residue was lyophilized to give 111 mg of yellow 3-piperidin-1-ylmethylpyrimido [5,4-e] [1,2,4] triazine-5,7-diamine trifluoroacetate.

¹ H NMR (DMSO-d ₆ ): 9.18 (broad s, 1H), 8.95 (broad s, 1H), 8.30 (broad s, 2H), 4.80 (s, 2H ), 3.50 (br.m, 2H), 3.10 (br.m, 2H), 1.35-1.90 (m, 6H).

Example 8

3-Morpholin-4-ylmethylpyrimido [5,4th] [1,2,4] triazin-5,7-diamine

A mixture of 3-chloromethylpyrimido [5.4-e] [1,2,4] triazine-5,7-diamine 3 (200 mg, 0.95 mmol) and 2.0 ml of morpholine was heated at 80 ° C in a closed tube in for 7 hours. Then the mixture was cooled to room temperature and concentrated in vacuo. The crude product was purified by reverse phase HPLC (Rainin C ₁₈ , eluent: gradient from 0% to 30% CH ₃ CN / H ₂ O / 0.1% TFA), bright yellow fractions containing the product were combined, CH ₃ CN removed in vacuo, the residue was lyophilized to give 261 mg of yellow 3-morpholin-4-ylmethylpyrimido [5,4-e] [1,2,4] triazine-5,7-diamine trifluoroacetate.

¹ H NMR (DMSO-d ₆ ): 9.25 (br s, 1H), 9.00 (br s, 1H), 8.30 (br s, 2H), 4.80 (s, 2H ), 3.85 (m, 4H), 3.30 (m, 4H).

Example 9

3- (4-Methylpiperazin-1-ylmethyl) pyrimido [5,4th] [1,2,4] triazin-5,7-diamine

A mixture of 3-chloromethylpyrimido [5.4-e] [1,2,4] triazine-5,7-diamine 3 (200 mg, 0.95 mmol) and N-methylpiperazine (2.00 ml) was heated at 80 ° C. in a closed tube for 7 hours. Then the mixture was cooled to room temperature and concentrated in vacuo. The crude product was purified by reverse phase HPLC (Rainin C ₁₈ , eluent: gradient from 0% to 30% CH ₃ CN / H ₂ O / 0.1% TFA) and bright yellow fractions containing the product after removal of CH ₃ CN under vacuum, lyophilized to give 178 mg of 3- (4-methylpiperazin-1-ylmethyl) pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine trifluoroacetate.

¹ H NMR (D ₂ O): 4.45 (s, 2H), 3.10-3.60 (m, 8H), 2.90 (s, 3H).

Example 10

3- (4-Benzylpiperazin-1-ylmethyl) pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine

A mixture of 3-chloromethylpyrimido [5.4-e] [1,2,4] triazine-5,7-diamine 3 (48 mg, 0.23 mmol) and N-benzylpiperazine (0.12 ml) in ethanol (0, 1 ml) was heated at 90 ° C in a closed tube for 2 hours. Then the mixture was cooled to room temperature and concentrated in vacuo. The crude product was purified by reverse phase GHUR (Rainin C ₁₈ , eluent: gradient from 0% to 50% CH ₃ CN / H ₂ O / 0.1% TFA), bright yellow fractions containing the product were combined, CH ₃ CN removed in vacuo, the residue was lyophilized to give 26 mg of 3- (4-benzylpiperazin-1-ylmethyl) pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine trifluoroacetate.

¹ H NMR (DMSO-d ₆ ): 7.50 (m, 5H), 4.34 (s, 2H), 4.25 (s, 2H), 2.90-3.40 (m, 8H).

Example 11

3- (4-Naphthalen-2-ylmethylpiperazin-1-ylmethyl) pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine

To a solution of trifluoroacetate 3-piperazin-1-ylmethylpyrimido [5,4-e] [1,2,4] triazin-5,7-diamine 5 (30 mg, 0.05 mmol) (obtained as described in example 4) in dry DMF (1.0 ml), 2-bromomethylnaphthalene (17 mg, 0.075 mmol) was added with stirring, followed by potassium carbonate (28 mg, 0.20 mmol). The mixture was stirred at room temperature for 24 hours, then transferred to CH ₃ CN / H ₂ O / 0.1% TFA and reverse phase HPLC (Rainin C ₁₈ , eluent: gradient from 0% to 30% CH ₃ CN / H ₂ O / 0.1% TFA), the bright yellow fractions containing the product were combined, CH ₃ CN was removed in vacuo, the residue was lyophilized to give 19 mg of 3- (4-naphthalen-2-ylmethylpiperazin-1- trifluoroacetate yellow methylmethyl) pyrimido [5,4th] [1,2,4] triazine-5,7-diamine.

¹ H NMR (DMSO-d ₆ ): 9.37 (br.s, 1H), 9.23 (br.s, 1H), 8.20 (br.s, 1H), 7.98 (m, 5H) ), 7.57 (m, 3H), 4.45 (broad s, 2H), 4.15 (broad s, 2H), 2.60-3.35 (m, 8H).

Example 12

3- (4-Naphthalen-1-ylmethylpiperazin-1-ylmethyl) pyrimido [5,4th] [1,2,4]] triazin-5,7-diamine

To a solution of trifluoroacetate 3-piperazin-1-ylmethylpyrimido [5,4-e] [1,2,4] triazin-5,7-diamine 5 (70 mg, 0.12 mmol) (obtained as described in example 4) in dry DMF (1.0 ml), 1-chloromethylnaphthalene (0.023 ml; 0.18 mmol) was added with stirring, followed by potassium carbonate (65 mg, 0.470 mmol). The mixture was allowed to stir at room temperature for 24 hours, then transferred to CH ₃ CN / H ₂ O / 0.1% TFA and purified by reverse phase HPLC (Rainin C ₁₈ , eluent: gradient from 0% to 30% CH ₃ CN (H ₂ O / 0.1% TFA), the bright yellow fractions containing the product were combined, CH ₃ CN was removed in vacuo, the residue was lyophilized, and 35 mg of 3- (4-naphthalen-1-ylmethylpiperazine- trifluoroacetate was obtained 1-ylmethyl) pyrimido [5,4-e] [1,2,4] yellow triazin-5,7-diamine.

¹ H NMR (DMSO-d ₆ ): 9.36 (br s, 1H), 9.23 (br.s, 1H), 8.53 (br.s, 1H), 8.32 (m, 1H) ), 8.23 (broad s, 1H), 8.00 (m, 2H), 7.60 (m, 4H), 4.68 (broad s, 2H), 4.23 (broad s, 2H), 2.80-3.40 (m, 8H).

Example 13

3- (4-Diphenyl-4-ylmethylpiperazin-1-ylmethyl) pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine

To a solution of trifluoroacetate 3-piperazin-1-ylmethylpyrimido [5,4-e] [1,2,4] triazin-5,7-diamine 5 (30 mg, 0.05 mmol) (obtained as described in example 4) in dry DMF (1.0 ml), 4-chloromethyldiphenyl (15 mg, 0.08 mmol) was added with stirring, followed by potassium carbonate (28 mg, 0.20 mmol). The mixture was stirred at room temperature for 24 hours, then transferred to CH ₃ CN / H ₂ O / 0.1% TFA and purified by reverse phase HPLC (Rainin C ₁₈ , eluent: gradient from 0% to 30% CH ₃ CN / H ₂ O / 0.1% TFA), the bright yellow fractions containing the product were combined, the CH ₃ CN was removed in vacuo, the residue was lyophilized to give 20 mg of 3- (4-diphenyl-4-ylmethylpiperazin-1 trifluoroacetate -ylmethyl) pyrimido [5,4-e] [1,2,4] yellow triazine-5,7-diamine.

¹ H NMR (DMSO-d ₆ ): 9.60 (s, 1H), 9.43 (s, 1H), 8.83 (br.s, 1H), 8.53 (br.s, 1H), 7.35-7.82 (m, 9H), 4.33 (s, 2H), 4.15 (s, 2H), 2.70-3.40 (m, 8H).

Example 14

3- [4- (2-Chlorobenzyl) piperazin-1-ylmethyl] pyrimido [5,4th] [1,2,4] triazin-5,7-diamine

To a solution of trifluoroacetate 3-piperazin-1-ylmethylpyrimido [5,4-e] [1,2,4] triazin-5,7-diamine 5 (50 mg, 0.08 mmol) (obtained as described in example 4) in dry DMF (1.0 ml), ortho-chlorobenzyl chloride (0.015 ml, 0.12 mmol) was added with stirring, followed by potassium carbonate (55 mg, 0.40 mmol). The mixture was stirred at room temperature for 24 hours, then transferred to CH ₃ CN / H ₂ O / 0.1% TFA and purified by reverse phase HPLC (Rainin C ₁₈ , eluent: gradient from 0% to 50% CH ₃ CN / H ₂ O / 0.1% TFA), the bright yellow fractions containing the product were combined, the CH ₃ CN was removed in vacuo, the residue was lyophilized to give 17 mg of 3- [4- (2-chlorobenzyl) piperazine- trifluoroacetate 1-ylmethyl] pyrimido [5.4-e] [1,2,4] yellow triazin-5,7-diamine.

¹ H NMR (MeOH-d ₄ ): 7.40-7.70 (m, 4H), 4.44 (s, 2H), 4.36 (s, 2H), 2.80-3.40 (m , 8H).

Example 15

3- [4- (3-Chlorobenzyl) piperazin-1-ylmethyl] pyrimido [5,4th] [1,2,4] triazin-5,7-diamine

To a solution of trifluoroacetate 3-piperazin-1-ylmethylpyrimido [5,4-e] [1,2,4] triazin-5,7-diamine 5 (50 mg, 0.08 mmol) (obtained as described in example 4) in dry DMF (1.0 ml), meta-chlorobenzyl chloride (0.015 ml, 0.12 mmol) was added with stirring, followed by potassium carbonate (55 mg, 0.40 mmol). The mixture was stirred at room temperature for 24 hours, then transferred to CH ₃ CN / H ₂ O / 0.1% TFA and purified by reverse phase HPLC (Rainin C ₁₈ , eluent: gradient from 0% to 50% CH ₃ CN / H ₂ O / 0.1% TFA), the bright yellow fractions containing the product were combined, CH ₃ CN was removed in vacuo, the residue was lyophilized to give 9 mg of 3- [4- (3-chlorobenzyl) piperazine- trifluoroacetate 1-ylmethyl] pyrimido [5.4-e] [1,2,4] yellow triazin-5,7-diamine.

¹ H NMR (MeOH-d ₄ ): 7.40-7.58 (m, 4H), 4.32 (s, 4H), 2.80-3.40 (m, 8H).

Example 16

3- [4- (4-Chlorobenzyl) piperazin-1-ylmethyl] pyrimido [5,4th] [1,2,4] triazin-5,7-diamine

To a solution of trifluoroacetate 3-piperazin-1-ylmethylpyrimido [5,4-e] [1,2,4] triazin-5,7-diamine 5 (50 mg, 0.08 mmol) (obtained as described in example 4) in dry DMF (1.0 ml), para-chlorobenzyl chloride (29 mg, 0.18 mmol) was added with stirring, followed by potassium carbonate (55 mg, 0.40 mmol). The mixture was stirred at room temperature for 24 hours, then transferred to CH ₃ CN / H ₂ O / 0.1% TFA and purified by reverse phase HPLC (Rainin C ₁₈ , eluent: gradient from 0% to 50% CH ₃ CN / H ₂ O / 0.1% TFA), the bright yellow fractions containing the product were combined, CH ₂ CN was removed in vacuo, the residue was lyophilized to give 21 mg of 3- [4- (4-chlorobenzyl) piperazine- trifluoroacetate 1-ylmethyl] pyrimido [5.4-e] [1,2,4] yellow triazin-5,7-diamine.

¹ H NMR (MeOH-d ₄ ): 8.20 (m, 4H), 4.30 (s, 4H), 2.88-3.40 (m, 8H).

Example 17

3- [4- (3-Methoxybenzyl) piperazin-1-ylmethyl] pyrimido [5,4th] [1,2,4] triazin-5,7-diamine

To a solution of trifluoroacetate 3-piperazin-1-ylmethylpyrimido [5,4-e] [1,2,4] triazin-5,7-diamine 5 (50 mg, 0.08 mmol) (obtained as described in example 4) in dry DMF (1.0 ml) meta-methoxybenzyl chloride (19 mg, 0.12 mmol) was added with stirring, followed by potassium carbonate (55 mg, 0.40 mmol). The mixture was stirred at room temperature for 24 hours, then transferred to CH ₃ CN / H ₂ O / 0.1% TFA and purified by reverse phase HPLC (Rainin C ₁₈ , eluent: gradient from 0% to 50% CH ₃ CN / H ₂ O / 0.1% TFA), the bright yellow fractions containing the product were combined, CH ₃ CN was removed in vacuo, the residue was lyophilized to give 30 mg of 3- [4- (3-methoxybenzyl) piperazine- trifluoroacetate 1-ylmethyl] pyrimido [5.4-e] [1,2,4] yellow triazin-5,7-diamine.

¹ H NMR (MeOH-d ₄ ): 7.40 (m, 1H), 7.06 (m, 3H), 4.30 (s, 2H), 4.23 (s, 2H), 3.81 ( s, 3H), 2.80-3.40 (m, 8H).

Example 18

3- [4- (3-Fluorobenzyl) piperazin-1-ylmethyl] pyrimido [5,4th] [1,2,4] triazin-5,7-diamine

To a solution of trifluoroacetate 3-piperazin-1-ylmethylpyrimido [5,4-e] [1,2,4] triazin-5,7-diamine 5 (50 mg, 0.08 mmol) (obtained as described in example 4) in dry DMF (1.0 ml) meta-fluorobenzyl chloride (0.0143 ml, 0.12 mmol) was added with stirring, followed by potassium carbonate (55 mg, 0.40 mmol). The mixture was stirred at room temperature for 24 hours, then transferred to CH ₃ CN / H ₂ O / 0.1% TFA and purified by reverse phase HPLC (Rainin C ₁₈ , eluent: gradient from 0% to 50% CH ₃ CN / H ₂ O / 0.1% TFA), the bright yellow fractions containing the product were combined, CH ₃ CN was removed in vacuo, the residue was lyophilized to give 11 mg of 3- [4- (3-fluorobenzyl) piperazine- trifluoroacetate 1-ylmethyl] pyrimido [5.4-e] [1,2,4] yellow triazin-5,7-diamine.

¹ H NMR (MeOH-d ₄ ): 7.18-7.60 (m, 4H), 4.33 (s, 2H), 4.27 (s, 2H), 2.83-3.38 (m , 8H).

Example 19

3- [4- (3-Trifluoromethylbenzyl) piperazin-1-ylmethyl] pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine

To a solution of 3-piperazin-1-ylmethylpyrimido trifluoroacetate [5,4-e] [1,2,4] triazin-5,7-diamine 5 (50 mg, 0.08 mmol) (obtained as described in example 4) in dry DMF (1.0 ml), meta-trifluoromethylbenzyl bromide (0.021 ml, 0.14 mmol) was added with stirring, followed by potassium carbonate (55 mg, 0.40 mmol). The mixture was stirred at room temperature for 24 hours, then transferred to CH ₃ CN / H ₂ O / 0.1% TFA and purified by reverse phase HPLC (Rainin C ₁₈ , eluent: gradient from 0% to 50% CH ₃ CN / H ₂ O / 0.1% TFA), the bright yellow fractions containing the product were combined, CH ₃ CN was removed in vacuo, the residue was lyophilized to give 13 mg of 3- [4- (3-trifluoromethylbenzyl) piperazine- trifluoroacetate 1-ylmethyl] pyrimido [5.4-e] [1,2,4] yellow triazin-5,7-diamine.

¹ H NMR (MeOH-d ₄ ): 7.63-7.92 (m, 4H), 4.38 (s, 2H), 4.32 (s, 2H), 2.85-3.38 (m , 8H).

Example 20

3- [4- (4-Trifluoromethylbenzyl) piperazin-1-ylmethyl] pyrimido [5.4-e] [1,2,4] triazin-5,7-diamine

To a solution of trifluoroacetate 3-piperazin-1-ylmethylpyrimido [5,4-e] [1,2,4] triazin-5,7-diamine 5 (50 mg, 0.08 mmol) (obtained as described in example 4) in dry DMF (1.0 ml), para-trifluoromethylbenzyl bromide (0.021 ml, 0.14 mmol) was added with stirring, followed by potassium carbonate (55 mg, 0.40 mmol). The mixture was stirred at room temperature for 24 hours, then transferred to CH ₃ CN / H ₂ O / 0.1% TFA and purified by reverse phase HPLC (Rainin C ₁₈ , eluent: gradient from 0% to 50% CH ₃ CN / H ₂ O / 0.1% TFA), the bright yellow fractions containing the product were combined, CH ₃ CN was removed in vacuo, the residue was lyophilized to give 22 mg of 3- [4- (4-trifluoromethylbenzyl) piperazine- trifluoroacetate 1-ylmethyl] pyrimido [5.4-e] [1,2,4] yellow triazin-5,7-diamine.

¹ H NMR (MeOH-d ₄ ): 7.77 (m, 4H), 4.37 (s, 2H), 4.33 (s, 2H), 2.90-3.38 (m, 8H).

Example 21

3- [4- (3-Bromobenzyl) piperazin-1-ylmethyl] pyrimido [5,4th] [1,2,4] triazin-5-7-diamine

To a solution of trifluoroacetate 3-piperazin-1-ylmethylpyrimido [5,4-e] [1,2,4] triazin-5,7-diamine 5 (50 mg, 0.08 mmol) (obtained as described in example 4) in dry DMF (1.0 ml), meta-bromobenzyl bromide (34 mg, 0.14 mmol) was added with stirring, followed by potassium carbonate (55 mg, 0.40 mmol). The mixture was stirred at room temperature for 24 hours, then transferred to CH ₃ CN / H ₂ O / 0.1% TFA and purified by reverse phase HPLC (Rainin C _18, eluent: gradient from 0% to 50% CH ₃ CN / H ₂ O / 0.1% TFA), the bright yellow fractions containing the product were combined, CH ₃ CN was removed in vacuo, the residue was lyophilized to give 15 mg of 3- [4- (3-bromobenzyl) piperazine- trifluoroacetate 1-ylmethyl] pyrimido [5.4-e] [1,2,4] yellow triazin-5,7-diamine.

¹ H NMR (MeOH-d ₄ ): 7.38-7.80 (m, 4H), 4.30 (s, 4H), 2.85-3.38 (m, 8H).

Example 22

3- [4- (3-Cyanobenzyl) piperazin-1-ylmethyl] pyrimido [5,4th] [1,2,4] triazin-5,7-diamine

To a solution of 3-piperazin-1-ylmethylpyrimido trifluoroacetate [5,4-e] [1,2,4] triazin-5,7-diamine 5 (50 mg, 0.08 mmol) (obtained as described in example 4) in dry DMF (1.0 ml), meta-cyanobenzyl bromide (23 mg, 0.18 mmol) was added with stirring, followed by potassium carbonate (55 mg, 0.40 mmol). The mixture was stirred at room temperature for 24 hours, then transferred to CH ₃ CN / H ₂ O / 0.1% TFA and purified by reverse phase HPLC (Rainin C ₁₈ , eluent: gradient from 0% to 50% CH ₃ CN / H ₂ O / 0.1% TFA), the bright yellow fractions containing the product were combined, the CH ₃ CN was removed in vacuo, the residue was lyophilized, and 12 mg of 3- [4- (3-cyanobenzyl) piperazine- trifluoroacetate was obtained 1-ylmethyl] pyrimido [5.4-e] [1,2,4] yellow triazin-5,7-diamine.

¹ H NMR (MeOH-d ₄ ): 7.60-7.92 (m, 4H), 4.37 (s, 2H), 4.27 (s, 2H), 2.95-3.35 (m , 8H).

Example 23

3- [4- (2,4-Dimethylbenzyl) piperazin-1-ylmethyl] pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine

To a solution of trifluoroacetate 3-piperazin-1-ylmethylpyrimido [5,4-e] [1,2,4] triazin-5,7-diamine 5 (50 mg, 0.08 mmol) (obtained as described in example 4) in dry DMF (1.0 ml), 2,4-dimethylbenzyl chloride (0.020 ml, 0.13 mmol) was added with stirring, followed by potassium carbonate (55 mg, 0.40 mmol). The mixture was stirred at room temperature for 24 hours, then transferred to CH ₃ CN / H ₂ O / 0.1% TFA and purified by reverse phase HPLC (Rainin C ₁₈ , eluent: gradient from 0% to 50% CH ₃ CN / H ₂ O / 0.1% TFA), the bright yellow fractions containing the product were combined, the CH ₃ CN was removed in vacuo, the residue was lyophilized to give 10 mg of 3- [4- (2,4-dimethylbenzyl) trifluoroacetate) piperazin-1-ylmethyl] pyrimido [5,4-e] [1,2,4] yellow triazin-5,7-diamine.

¹ H NMR (DMSO-d ₆ ): 9.40 (br s, 1H), 8.17 (br s, 2H), 7.00-7.40 (m, 3H), 4.28 (br .s, 2H), 4.10 (broad s, 2H), 2.95-3.35 (m, 8H), 2.34 (s, 3H), 2.28 (s, 3H).

Example 24

3- [4- (4-Ethyl-2-methylnaphthalen-1-ylmethyl) piperazin-1-ylmethyl] pyrimido [5,4-e] [1,2,4] triazine-5,7-diamine

To a solution of 3-piperazin-1-ylmethylpyrimido trifluoroacetate [5,4-e] [1,2,4] triazin-5,7-diamine 5 (50 mg, 0.08 mmol) (obtained as described in example 4) in dry DMF (1.0 ml), 1-chloromethyl-2-methylnaphthalene (34 mg, 0.18 mmol) was added with stirring, followed by potassium carbonate (55 mg, 0.40 mmol). The mixture was stirred at room temperature for 24 hours, then transferred to CH ₃ CN / H ₂ O / 0.1% TFA and purified by reverse phase HPLC (Rainin C ₁₈ , eluent: gradient from 0% to 50% CH ₃ CN / H ₂ O / 0.1% TFA) and bright yellow fractions containing the product, after removal of CH ₃ CN in vacuo, were lyophilized to give 23 mg of 3- [4- (4-ethyl-2-methylnaphthalene- 1-ylmethyl) piperazin-1-ylmethyl] pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine yellow.

¹ H NMR (MeOH-d ₄ ): 8.07 (m, 1H), 7.92 (m, 2H), 7.40-7.50 (m, 3H), 4.90 (s, 2H), 4.37 (s, 2H), 2.70 (s, 3H), 2.80-3.45 (m, 8H).

Example 25

3- [4- (4-Benzyloxybenzyl) piperazin-1-ylmethyl] pyrimido [5.4-e] [1,2,4] triazin-5,7-diamine

To a solution of 3-piperazin-1-ylmethylpyrimido trifluoroacetate [5,4-e] [1,2,4] triazin-5,7-diamine 5 (50 mg, 0.08 mmol) (obtained as described in example 4) in dry DMF (1.0 ml), 1-chloromethyl-2-methylnaphthalene (27 ml, 0.12 mmol) was added with stirring, followed by potassium carbonate (58 mg, 0.42 mmol). The mixture was stirred at room temperature for 24 hours, then transferred to CH ₃ CN / H ₂ O / 0.1% TFA and purified by reverse phase HPLC (Rainin C ₁₈ , eluent: gradient from 0% to 50% CH ₃ CN / H ₂ O / 0.1% TFA), the bright yellow fractions containing the product were combined, CH ₃ CN was removed in vacuo, the residue was lyophilized to give 23 mg of 3- [4- (4-benzyloxybenzyl) piperazine- trifluoroacetate 1-ylmethyl] pyrimido [5.4-e] [1,2,4] yellow triazin-5,7-diamine.

¹ H NMR (DMSO-d ₆ ): 9.63 (s, 1H), 9.50 (s, 1H), 8.91 (br.s, 1H), 8.77 (br.s, 1H), 7.40 (m, 7H), 7.05 (m, 2H), 5.12 (s, 2H), 4.25 (s, 2H), 4.17 (s, 2H), 2.60-3 40 (m, 8H).

Example 26

Inhibition of PTP1B in vitro

Human PTP1B (1-321) was cloned from a human cDNA library using conventional molecular biology techniques. The cDNA sequence was identical to the published human PTP1B sequence (registration number M33689). The protein was expressed in E. coli and purified as described by Barford D. et al., J. Mol. Biol., 239, 726-730 (1994).

Determination of inhibition of PTPase activity

The measurement of RTPase activity was carried out according to one of two methods.

In the first method for determining the inhibitory activity against PTP1B, a peptide containing phosphotyrosine, the amino acid sequence of which corresponds to section 1146 (TRDI (pY) E) of the insulin receptor containing autophosphorylated tyrosine, was used as a substrate. The reaction was carried out under the following conditions:

PTP1B (0.5-2 nM) was incubated with the compound for 15 min in a buffer solution containing 37.5 mM MES, pH 6.2, 140 mM NaCl, 0.05% BSA and 300 nM DTT. The reaction was initiated by the addition of 50 μM substrate. After incubation at room temperature (22-25 ° C) for 20 min, the reaction was stopped by the addition of KOH and the amount of free phosphate was measured using malachite green, as described in the literature (see Harder et al., Biochem. J., 298, 395 (1994).

In the second method, the total inhibitory activity against PTPases was determined using a PTPase kit. The substrate (6.8-difluoro-4-methylumbelliferyl phosphate, DiFMUP, Molecular Probes) was used at the concentration used to measure K _m for each enzyme. The composition of the buffer solution was identical to that indicated above in the analysis using malachite green, except that instead of MES, 37.5 mM diethylglutarate, pH 6.2, was used. The reaction was stopped by the addition of KOH. In this case, the fluorescence of the dephosphorylated product was measured (λ _exc 360 nm, λ _isp 460 nm).

When determining the kinetic parameters of the reaction, the indicated buffer solutions were used, except that the reaction was initiated by the addition of the enzyme and stopped after 10 minutes.

According to the above in vitro analysis, the IC ₅₀ value for PTP1B of the compounds of the present invention is less than 500 μM, preferably less than 100 μM.

According to the results of the in vitro analysis, all the compounds described in examples 4-25 with inhibition of PTP1B have an IC ₅₀ value of less than 30 μM.

Example 27

The effect of compounds on the level of glucose in plasma (blood) when tested in a mouse model

To measure the antidiabetic effect, the compounds were tested in vivo using a known model of type 2 diabetes and rodent obesity.

Obesity in ob / ob mice

To evaluate the effect of the compounds on lowering glucose levels simultaneously with lowering triglycerides, male / female ob / ob mice (line C57BL6 / J) weighing 40-50 g were used (see Diabetologia, 14, 141-148 (1978)) (Jackson Labs) . Animals were preliminarily divided into groups of 10-12 individuals, depending on glucose level and body weight. Animals received normal food and had free access to water. The test compound (suspended in 1% Na-CMC) was administered daily through a gastric tube for five days. Immediately prior to administration, part of the tail was incised in animals, blood was taken from the tail vein, and the plasma (blood) glucose concentration was determined on the first day of the experiment. On the fifth day, 2 hours after administration, blood was collected in a similar manner and the glucose concentration was determined. Then the animals were anesthetized and killed by bleeding. Blood and tissues were collected for analysis. Compounds were considered active if they caused a statistically significant (p≤0.05) decrease in blood glucose compared to glucose in mice treated with vehicle.

Diet-induced obesity in C57BL6 / J mice (DIO mice)

Type 2 diabetes was induced in mice when kept on a high fat diet for 4-6 months (Diabetes, 37, 1163-1167 (Sept., 1988)). The C57BL6 / J males (3–4 weeks old) were kept on a high fat diet for 4–6 weeks. During this time, animals developed hyperglycemia and hyperinsulinemia, and body weight was 40-50 g. DIO mice (n = 6) were weighed and stripped of food 2 hours before oral administration of the analyzed compounds. Immediately prior to the administration of substances through a gastric tube, blood was taken from the tail vein and the glucose concentration (time 0) was determined as described above. The test compound was administered to the animals once a day for five days; the control animals (receiving the vehicle) were not tested. On the fifth day, the glucose level was measured before administration (time 0) and 2 and 4 hours after administration. 4 hours after administration, insulin and triglycerides were also measured. Compounds were considered active if in experimental animals they caused a statistically significant (p≤0.05) decrease in blood glucose, insulin and triglycerides compared to animals treated with a vehicle.

Testing of the compounds described in examples 5, 10 and 13 in mice in vivo was carried out according to the method described in example 27. These compounds reduced the level of glucose in the blood by at least 15%.

Claims (15)

1. Pyrimidotriazines of the General formula

where R ¹ and R ² each means hydrogen or R ¹ and R ² together form a chemical bond, —CH ₂ -, —O—, —NH— or —NR ³ ; R ³ means ness. Alkyl or a group-CH ₂ -Ar, where Ar is selected from the group consisting of unsubstituted phenyl, unsubstituted naphthyl; phenyl mono- or disubstituted with lower alkyl, halogen, cyano, trifluoromethyl, phenyl, benzyloxy or lower alkoxy; naphthyl mono- or disubstituted with lower alkyl; or their pharmaceutically acceptable salts. 2. Compounds according to claim 1 of the formula

where Ar is selected from the group consisting of unsubstituted phenyl, unsubstituted naphthyl; phenyl mono- or disubstituted with lower alkyl, halogen, cyano, trifluoromethyl, phenyl, benzyloxy or lower alkoxy; naphthyl, mono- or disubstituted with lower alkyl, or pharmaceutically acceptable salts of the compounds of formula II. 3. The compounds according to claim 1, where Ar means unsubstituted phenyl or unsubstituted naphthyl. 4. The compounds according to claim 1 or 2, where Ar is phenyl monosubstituted with lower alkyl, halogen, cyano, trifluoromethyl, phenyl, benzyloxy or lower alkoxy. 5. The compounds of claim 1, wherein Ar is phenyl monosubstituted with lower alkyl, lower alkoxy, halogen, cyano or trifluoromethyl. 6. The compounds according to claim 1, where Ar means phenyl disubstituted by lower alkyl, lower alkoxy, halogen or cyano. 7. The compounds of claim 1, wherein Ar is naphthyl monosubstituted with lower alkyl. 8. The compounds of claim 1, wherein Ar is naphthyl disubstituted with lower alkyl. 9. The compounds according to claim 1, selected from the series 3-diethylaminomethylpyrimido [5,4-e] [1,2,4] triazine-5,7-diamine, 3- (4-benzylpiperazin-1-ylmethyl) pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine, 3- (4-naphthalen-2-ylmethylpiperazin-1-ylmethyl) pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine, 3- (4-naphthalen-1-ylmethylpiperazin-1-ylmethyl) pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine, 3- (4-biphenyl-4-ylmethylpiperazin-1-ylmethyl) pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine, 3- [4- (2,4-dimethylbenzyl) piperazin-1-ylmethyl] pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine. 3- [4- (4-ethyl-2-methylnaphthalen-1-ylmethyl) piperazin-1-ylmethyl] pyrimido [5,4-e] [1,2,4] triazine-5,7-diamine and 3- [4- (4-benzyloxybenzyl) piperazin-1-ylmethyl] pyrimido [5,4-e] [1,2,4] triazin-5,7-diamine. 10. A method of obtaining a compound according to any one of claims 1 to 10, comprising reacting a compound of formula 3

with a compound of the formula R ⁴ NHR ⁵ , where R ⁴ is —CH ₂ CH ₂ R ₁ , and R ⁵ is —CH ₂ CH ₂ R ₂ , where R ¹ and R ² are as defined in claim 1. 11. Compounds according to any one of claims 1 to 9 for use as a therapeutically active substance. 12. Compounds according to any one of claims 1 to 9 for the manufacture of medications intended for the prevention and / or treatment of diabetes. 13. A pharmaceutical composition having inhibitory activity against protein tyrosine phosphatase PTP1B and containing a compound according to any one of claims 1 to 9 and a therapeutically inert carrier. 14. Compounds according to any one of claims 1 to 9, obtained by the method according to claim 10. 15. The use of a compound according to any one of claims 1 to 9 for the manufacture of a medicament for the prophylaxis and / or treatment of diabetes.