PL151086B1 - Process for preparing condensed pyrimidine derivatives - Google Patents

Description

PATENT DESCRIPTION

Additional patent to patent no. - Pending: 87 12 30 / P. 280532 /

Priority: 86 12 30 Hungary

Application announced: ^{88 09 15}

Patent description published: 1990 12 31

Int.Cl. 'C07D 513/04

AND READING ROOM and PFN

And MsKir. Si- · -’d ei

Creators of the invention: Daniel Bózsing, GyOrgyi Kovanyi, Edit Berenyi, Kardy Magyar, Sandor Tuboly, Attila Mandi

The holder of the patent: Egis Gyógyszergyar, Budapest / Hungary /

METHOD OF MAKING NEW PYRIMIDINE DERIVATIVES

The subject of the invention is a process for the preparation of new pyrimidine derivatives of the general formula I, in which A is CH ₂ -CH ₂ or CH = CH, R 2 is hydrogen, C ₁ - C4 alkyl or optionally substituted phenyl, r is the number zero, 1 , 2, 3 or 4, optionally in the form of pharmacologically acceptable acid addition salts, or hydrates of compounds of general formula 1 or hydrates of acid addition salts of compounds of general formula 1.

British Patent No. 1,072,414 discloses thiazole (3,2-a) pyrimidine derivatives. These compounds have been reported to influence the circulation and have a sedative-calming, diuretic, cytostatic, bacteriostatic, anti-inflammatory and antipyretic effect.

It is known that one of the primary goals of infectious disease prevention is to increase the effectiveness of the induction of preventive immunity and to create uniform immunity conditions in large populations. Useful in this endeavor are compounds capable of stimulating the body's immune system.

The object of the invention was to provide a method for the preparation of new fused pyrimidine derivatives with enhanced immunostimulatory action.

This object was achieved by developing a process for the preparation of new compounds of general formula 1. Compounds of general formula 1 can exist in two tautomeric forms shown in scheme 1, in which A, R1 and r are as defined above.

It was found that the new compounds of the general formula I significantly increase the cellular resistance reaction, have a resistance stimulating effect and show no incompatibility with the vaccines used.

The term * C 1 -C ₄ -alkyl * denotes straight or branched, saturated aliphatic hydrocarbon groups / e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl and the like / R ^ as phenyl can

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151 086 optionally be substituted with one or more substituents (e.g., halogen, nitro, lower alkyl or lower alkoxy)

The compounds of the general formula I, in which A represents the group -CH ₂ -CH ₂ -, R 2 is hydrogen or methyl, and r is 1 and their pharmacologically acceptable salts are distinguished by particularly valuable pharmacological properties.

Particularly preferred compounds of general formula I include: 7-amino-5-imino-2,3-dihydro-5H-thiazolo (3,2-a J pyrimidine, 8-amino-6-imino-3-methyl-2H, 6H). -pyrimidine £ 2,1-b J / 1,3 / -thiazine and their pharmacologically acceptable acid addition salts

Pharmacologically acceptable acid addition salts of compounds of general formula I can be formed with inorganic and organic acids (e.g., they can be hydrochlorides such as hydrochlorides or hydrobromides, carbonates, bicarbonates, sulfates, acetates, fumarates, maleinates, citrates, ascorbates and the like / ·

The inventive process for the preparation of the compounds of general formula 1 and their pharmacologically acceptable salts and hydrates consists in that a compound of general formula 2 in which A is -CH ₂ -CH ₂ - or -CH = CH- and R ₄ and r are as defined above, reacting with the dinitrile of melonic acid of formula NC-CH ₂ -CN; and optionally converting the obtained compound of formula I into its pharmacologically acceptable acid addition salt, or releasing the free base compound of formula I from its acid addition salt or converting the compound of formula I or a pharmacologically acceptable acid addition salt thereof with with acid in hydrate □ as the reaction medium, solvents such as dialkylamide / e.g. dimethylformamide /, dialkylsulfoxide / preferably dimethylsulfoxide /, aliphatic alcohol / e.g. ethanol or isopropanol /, chlorinated aliphatic hydrocarbons / e.g. chloroform, carbon tetrachloride, methylene chloride are preferably used /, aromatic hydrocarbon / e.g. benzene, toluene, xylene /, aliphatic ether / e.g. diethyl ether, tetrahydrofuran, dioxane /, aliphatic hydrocarbon (e.g. hexane or gasoline) or a mixture of at least two of the above solvents. An aliphatic alcohol is particularly preferably used as the reaction medium. The reaction can preferably be carried out in the presence of a basic catalyst. For this purpose, alkali metal alkoxides (e.g. sodium methylate or potassium methylate), although other bases, e.g., alkali metal carbonate, may be used (e.g. sodium or potassium carbonate /, alkali metal bicarbonate / e.g. sodium or potassium bicarbonate /, alkali metal hydroxide / e.g. sodium hydroxide or potassium /, alkaline earth metal hydroxide / e.g. calcium hydroxide / or a tertiary amine / e.g. pyridine, triethylamine or other trialkylamine /.

The starting compounds of the general formulas 2 and NC-CH ₂ -CN can be used in equimolar amounts, and the malonic dinitrile of the formula NC-CH ₂ -CN can be used in an excess of 0.1-0.5 mol. The reaction is carried out at a temperature between room temperature and the boiling point of the reaction mixture depending on the starting compounds used. The reaction mixture can be worked up in a known manner.

The obtained compounds of formula I can be converted into acid addition salts by methods known per se. Thus, a compound of formula I can be reacted with a suitable organic or inorganic acid in an inert organic solvent.

Compounds of general formula I and their acid addition salts may form hydrates. These hydrates can be obtained either by adding water to the compound of general formula I or its acid addition salt, or they can be formed spontaneously due to the hygroscopic properties of the anhydrous compounds of general formula I.

The starting compounds of the formula 2 and the formula NC-CH ₂ -CN are known and readily available compounds, □ As mentioned previously, the compounds of the general formula 1 have valuable immunostimulatory properties.

The external regulation of the immune system and the correction of an overactive or underactive system has come to light in the last decade after a more thorough understanding of the immune processes.

In the microbiological sense, immunity means protecting the body against pathogens (such as bacteria, viruses, fungi or parasites) or against the toxic substances they release. Always to the current state of immunity and its development, in addition to the properties of

151 0-6 genetic, also influenced by biological environmental factors Cells of the immune system work according to a strictly defined division of labor, i.e. some of them have the ability to recognize foreign substances / antigens /, others produce antibodies or are differentiated into immunocytes of different reactivity, and some of them they can also coordinate and / or regulate the relationship between certain groups of cells. On this basis, immunity can also be characterized in such a way that the organism recognizes substances that are foreign to it and is able to produce an immune response against them through a strictly defined interplay of cells. .

From the point of view of prophylaxis against contagious diseases, the so-called active immunity induced artificially by inoculation, during which a weakened strain of pathogens, killed pathogens or their toxins are introduced into the body is very important

The effect of immunoprophylaxis in conferring immunity is very different. Some vaccines can achieve lifelong immunity, some offer protection for 1-2 years, and a very large proportion of them only develop a few months of immunity.

The knowledge and practical application in the last ten years of immunity modifying and stimulating materials has shown promising results and advances in correcting the lack of immunity and increasing the effectiveness of vaccines that induce only weak immunity.

The latter achievement is particularly important in the veterinary field, where the development of the immune identity of large animal populations is one of the basic requirements for achieving the goal that livestock should produce a level of production commensurate with its genetic capacity.

When investigating the immunostimulatory effect of certain compounds, it was found that the compounds of general formula I have a beneficial effect on the protective ability of the body's cells and, when used in practice, they may increase the immune effect induced by certain types of herpes vaccines having only a weak immune function / such as e.g. for the prevention of Aujeszky's disease /.

The results of the immunostimulatory activity of the compounds of general formula I obtained in the mice and pig model tests are shown in Tables 1 and 2.

Table 1

Model tests on mice

and Test compound at a dose of 0.1 mg / kg mouse body and the Vaccine 1 _l _ _ _ _ _ '"Ł 1 1 - J. 1 - L _ number animals 1 1 J - RCF CT Relationship with 1 | Ay weakened 1 AND 80 1 | 6-20 / 4-12 18-8 / 8-6 1 | example I. 1 -1. Ay closed - U - 80 1 4 - 3-17 / 3-9 7-16 / 6-8 - -1

Note: the numerator shows the values for the samples taken from the animals treated with the vaccine in combination with the test compound at 1st, 3rd and 4th weeks, and the denominator

gives the same values for animals treated alone vaccine; all values those expressed in percent Blackboard 2 Model tests on pigs Test compound - T. 1 ------- -I - Vaccine ₍ ------ - - r Number of animals _t RCF CT 1 Relationship by example 1 I, 1 weakened , 1 10. 14-24 / 6-14 18-13 / 5-11 1 5 mg / pig 1 | closed ¹ 10 ' 5-26 / 4-15 5-21 / 4-11 1 | β _ _ _ -------- w- . J.

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The following test methods were used for the in vitro testing of peripheral lymphocytes:

Lymphocyte secretion

Leukocytes are secreted from blood samples treated with heparin as anticoagulant by the Ficall Paque method. The cell density in Hanks' solution containing 10% fetal serum is adjusted to 10 ⁶ / ml. 1.5 ml of each Leighton sample suspension is dispensed from the cell suspensions and then supplemented with antigens according to the tests.

Unrolling lmmunorozet / RCF / · After appropriate pretreatment, the 3% solution of sheep's red blood cells is adsorbed with Boiven antigens · 0.1 ml of the treated red blood cells are added to Leighton tubes 1 after 16 hours of incubation, they are determined under a microscope in the stained preparations ratio of rosette developing cells

Cytotoxic reactions / CT / · The test is carried out in an allogeneic system. · 0.1 ml of the suspension of lymphocytes with a cell density of 10 ⁶ / ml is added to the suspension of red sheep blood cells, sensitized with antigens. The values of the cytotoxic capacity are determined on the basis of haemolysis following incubation for 16 hours and measured on the Spekol device at 540 mm. The value of the reaction is expressed as a percentage of the control sample. lymphocyte blastogenesis, on the other hand, increases the effectiveness of cellular immune responses that can be demonstrated in the immuno-inducing phase, increasing the RCF value by 8-11% and the CT value by 10-12%.

Comparing the action of the compounds of general formula I with the action of the compounds designated TSl-3096, described in the journal Drugs of Futura / 1984, 9, 591 /, it can be seen that the difference between them is that while the latter only activate T-cells As suppressors, compounds of the general formula I increase the activity of T-killing lymphocytes, as well as other cells, so-called X lymphocytes, which manifests itself in increasing the cytotoxic capacity of the cellular immune system.

Trials of the stimulating effect of 7-amino-5-imino-2,3-dihydro-5H-thiazolo / 3,2-a J pyrimidine / compound from Example 2 / on the immunogenicity of IBR viral vaccines were also carried out, Marek, New Castle and also bovin adeno and toga / BVD /

The effect of different doses of 7-amino-5-imino-2,3-dihydro-5H-thiazole 3,2-a / pyrimidine hydrobromide mixed with the individual vaccines was investigated in model trials partly in mice and partly in chickens. Blood samples were taken from the test animals during the periods given in Tables 3 and 4 and the in vitro reactions (LST, RCF, CT, PHA /, of the secreted lymphocytes) as well as the amount of antibody detectable in the blood plasma / VN, HAG /

The LST, RCF and CT values obtained for the IBN / Boviphyl-IBR /, Bivalent Bovin Adenovirus and also Vedeac vaccines are given in Table 3.

The results for the Marek vaccine and the La Sota vaccine (against poultry) are given in Table 4.

Table 3

Results obtained with IBR, Adeno and Vedeac vaccines

_ _ _ Γ Vaccine and Groups and Numbers Dose * ₍ | _ST RCF u CT % 1 and mice 1 1 1 1 7 " 2 -Γ- 3 “Γ 4 ¹ 5th week i IBR ¹ a ¹ 50 ¹ 0.1 mg / mouse ¹ 7-20 5-17 ¹ 5 1 | 4 AND 4 1 ¹ 4 i inactive ¹ b ¹ 50 ¹ 0.5 mg / mouse 6-26 11-26 and 4 6 18 6 '2, / 0.2 ml / mouse / * c and 50 1 - 1 8-16 5 ~ 15 and 0 1 0 1 5 1 3 '1 i - | - - - “1“ ““ r - -— _n | - 1 - -1 -i - - - - - Adeno 1 ^a . ⁵⁰ J 0.1 mg / mouse ₍ 5-17 6-16 1 1 1 AND 3 1 14 and 0 ¹ inactive b . 50 0.5 mg / mouse 7-26 9-23 ¹ 2 1 6 1 8 1 3 and ^{0 1} / 0.2 ml / mouse / c 1 ¹ 50 1 and _ i 1 1 9-16 5-16 ¹ - _ ι 0 1 0 1 3 1 7 and ^{0 1} 1 1 I and 1 1 “L ~ 2 Ί “ 4 1 8 ¹ 12 weeks | Vede 1 1 0.5 mg / mouse ¹ 9-19 ' - r— Γ . and . 50 6-21 1 1 9 1 3 1 7 ι θ ¹ weakened b 50 ₍ 0.5 mg / mouse ' 9-27 11-26 ¹ 4 1 g 1 9 AND 11 and ^{2 1} / 0.2 ml / mouse / c _ 1 _ _ _ ¹ 50 J___ 1- ----- U 5.17 5-18 ' 1 1 3 3 1 1 0 and

^x Dose of 7-Amino-5-imino-2,3-dihydro-5H-thiazole / 3,2-a / pyrimidine hydrobromide LST and RCF values represent results obtained in weeks 1 and 5

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Table 4

The results obtained for the Marek and Phylavac vaccines

1 "1 ----- _q and --- Ί r and - · , Vaccine 1 Group number Dose and LST i RCF 1 1 and 2 and 3 and 4 5th week. 1. 1 chickens 1 1 1 1 1 1 and a ¹ 50 1 mg ¹ 3-16 ¹ 1 | 4-17 1 and 0 ¹ 0 ¹ 6 I ¹ 0 I 1 ^{2 1} Marek 1 b J ⁵⁰ 3 mg 1 ^5-22 .1 5-26 0 and 5 , 27 | 10 Ί 1 ’ 1 c 1 50 And 2-14 l 4-15 1 0 0 • 9 ¹ 1 ^{1 4} 1 and-------- - “1 - - - - | - - - - " * 1 - - 1 '1 * 1 a i 50 1 9 and 5-19 i 3-18 1 0 1 0 and 9 and 9 and 1 ¹ and Phylavac 1 b i 50 3 mg and 5-23 i 5-25 1 0 1 > 11 • 11 ^{1 3} i 1 c ¹ 50 - ¹ 4-15 ¹ 2-15 1 0 ¹ 3 ¹ 3 ¹ 2 ¹ 1 1

and ^x 7-Amino-5-imino-2,3-dihydro-5H-thiazolo / '3,2-a) pyrimidine hydrobromide L5T and RCF values represent the results obtained in weeks 1 and 5

On the basis of the presented results, the following conclusions can be drawn:

A dose of 7-amino-5-imino-2,3-dihydro-5H-thiazole / 3,2-a / pyrimidine hydrobromide of 0.5 mg / mouse given with IBR, Adeno and Vedavac vaccines increases LST values by 10, 10 and 12%, and the cytotoxic capacity of lymphoid cells by 12.91.9% compared with control animals / treated with venison /

The amount of virus-neutralizing antibodies in the blood plasma quadruples with IBR 1 and twice with adeno

A 3 mg / chicken dose of 7-amino-5-imino-2,3-dihydro-5H-thiazole / '3,2-a / pyrimidine hydrobromide in a mixture with Marek vaccine and Phylavac vaccine increases the LST by 8 and 8% and RCF values by 11 and 10%, respectively, and cytotoxic capacity by 8 and 18% respectively

The compounds of general formula I are used in the form of pharmacological agents containing as active ingredient at least one compound of general formula I or a pharmacologically acceptable salt thereof, or a hydrate of the compound of general formula I or a salt thereof, in admixture with suitable solid or liquid carriers. Pharmacological preparations. These compositions can be prepared in a manner known per se for the preparation of agents acting on the immune system. Thus, the vaccine and the compound of general formula I can be introduced into the body at different sites. It is also possible to use the compound of general formula I in combination with the vaccine.

The daily dose of the compounds of formula I varies widely. The daily parenteral dose is usually from about 0.1 mg / kg to about 30 mg / kg, preferably about 0.3-15 mg / kg, especially about 1-2 mg / kg body of a pig The active substance can be advantageously put into a vaccine and injected intramuscularly

The invention is further illustrated by the following examples:

Example I. Preparation of 7-amino-5-imino-2,3-dihydro-5H-thiazole-3,2-a) pyrimidine hydrobromide. A mixture of 10.21 g (0.1 mol) of 2-aminothiazoline, 6.61 g (0.1 mol) of malonic acid dinitrile, 10.12 g (0.1 mol) of trimethylamine is reacted at room temperature for 2 hours. and 125 ml of ethanol, then the reaction mixture is clarified and its pH is adjusted to 6 by the addition of 48% hydrogen brine solution while cooling it in ice water. 18 g (72% yield) of the desired compound with a melting point above 300 ° C are obtained.

For CgKgBrN ^ S with a molecular weight of 249.136, the following results of elemental analysis are obtained:

Calculated: C 28.93% H 3.64% N 22.49% S 12.87% Br 32.07%

Found: C 29.23% H 3.70% N 22.46% S 12.85% Br, 32.30%.

Example II. Preparation of 7-amino-5-imino-2,3-dihydro-5H-thiazole? 3,2-a) pyrimidine. A mixture of 10.21 g / 0.1 mol / 2-aminothiazoline, 6.61 g / 0.1 mol / malonic acid dinitrile and 200 ml of dimethylformamide is reacted at a temperature of 20 minutes.

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155 ° C. The reaction mixture is cooled to room temperature, allowed to stand and evaporated. The oily residue is crystallized from acetonitrile and filtered to obtain 5.1 g of the desired compound (yield 30%) with a melting point above 300 ° C.

Ola C _g H _g N ₄ S with a molecular weight of 168.218, the following results of elemental analysis are obtained:

Calculated: C 42.84% H 4.79% N 33.31% S 19.06%

Found: C 42.81% H 4.69% N 33.29% S 19.03%

Example 3: Preparation of 7-amino-5-imino-2,3-dihydro-5H-thiazole (3,2-a) pyrimidine bicarbonate dihydrate. About a solution of 224 g of sodium bicarbonate in 2500 ml of water, 24.9 g (0.1 mol) of 7-amino-5-imino-2,3-dihydro-5H-thiazole hydrobromide (3,2-a J pyrimidine) are added. The reaction mixture is stirred for 8 hours at room temperature, filtered, washed with water and dried to give 17.2 g (75% yield) of the desired compound, mp 146-148 ° C.

For ^C 7 ^H x3 ^N 4 ° 5 ^S ° ^m w 65.266 the following elemental analysis results are obtained:

Calculated: C 31.70% Found: C 32.68%

Example

H 4.94% N 21.12% S 12.09%

H 4.70% N 20.69% S 12.18%.

IV. Preparation of 8-amino-6-imino-3,4-dihydro-2H-6H-pyrimidine (2,1-b) thiazine bicarbonate dihydrate. 25.3 g (0.1 mole) of 8-amino-6-imino-3,4-dihydro-2H-6H-pyrimidino hydrobromide (2H-6H-pyrimidino) (2,1-b) thiazines are treated as described in Example 3 to give 17 g of the desired compound / 70% yield, m.p. 196-1S8 ° C.

Ola CgH ^ gN ^ OgS with a molecular weight of 279.293, the following results of elemental analysis are obtained:

Calculated: C 34.40% H 5.41% N 20.06% S 11.48%

Found: C 33.65% H 5.52% N 19.57% S 11.32%

Example 5 Preparation of 7-amino-5-imino-2,3-dihydro-5H-thiazole (3,2-a) pyrimidine diacetate. In 30 ml of acetic acid is stirred for? hours 5.3 g (0.02 mol) of 7-amino-5-imino-2,3-dihydro-5H-thiazole / 3,2-a) pyrimidine bicarbonate dihydrate. The solution is evaporated, the remaining crystals are suspended in ethanol, then the suspension was filtered and dried to give 3.6 g of the desired compound (yield 59.5%), mp 158-160 ° C.

For a molecular weight of 320.351, the following results of elemental analysis are obtained:

Calculated: C 43.70% H 6.00% N 18.53% S 10.60%

Found: C 43.68% H 5.97% N 18.17% S 10.56%

Claims (1)

Patent claim A process for the preparation of new pyrimidine derivatives of general formula I, in which A is -C1 -CB2-, or -CH-CH-, R2 is hydrogen, C1 -C4 alkyl or optionally substituted phenyl, and r is the number zero, 1, 2, 3 or 4, optionally in the form of pharmacologically acceptable acid addition salts or hydrates of compounds of general formula 1 or hydrates of acid addition salts of compounds of general formula 1, characterized in that the compound of general formula 2, where A is a -CB1 -CB2- group or a -C1CH- group and R6 and r are as defined above, reacted with malonic acid dinitrile of formula NC-CF4 -CN; and optionally converting the obtained compound of formula I into its pharmacologically acceptable acid addition salt, or releasing the free base compound of formula I from its acid addition salt, or converting the compound of formula I or its pharmacologically acceptable acid addition salt with acid to hydrate. 151 0Θ6 NH ΑΝ Η Α, Λν ^Λ ΝΗ ₂ From ^s (R ₄ ) r Formula 2 NH% Nh r Scheme