Compounds useful as antiproliferative agents and garft inhibitors, the method of inhibition and proliferation and ether derivatives thiencarbazone acid

 

(57) Abstract:

Describes new compounds of General formula I, where a is CH2or S; Z IS C1-C3alkylene; X1-C6alkyl, and R1and R2- hydrogen. The compounds of formula I, in equilibrium with their 4-hydroxy-tautomers and are in the form diastereoisomeric mixtures and their pharmaceutically acceptable salts are strong GARFT inhibitors. These compounds and their salts are also useful antiproliferative agents. Describes how inhibition and proliferation and ether derivatives thiencarbazone acid. 3 S. and 11 C.p. f-crystals, 3 PL.

The invention relates to compounds of the following formula I which inhibit the enzyme glycinamide ribonucleotide the formyl transferase (GARFT). This invention also relates to pharmaceutical compositions containing the compounds of formula I, their use for the inhibition of GARFT and their use for inhibiting the growth and proliferation of cells of higher organisms or microorganisms, such as bacteria, yeast and fungi. This invention also relates to the preparation of such compounds and intermediate compounds used for their production.

GARF is m for cell division and proliferation. It is known that blocking this pathway has an antiproliferative effect, in particular anticancer. Thus, it was synthesized many analogs of folate and studied for their ability to inhibit GARFT. As a prototype known [1] specific forming a strong bond GARFT inhibitor, 5,10-daliasalabarria acid (DDATHF), exhibiting antitumor activity.

A large class of antiproliferative agents include compounds-antimetabolites. A concrete subclass of antimetabolites, known as antifolates or Antipolis, are antagonists of the vitamin folic acid. In a typical case, antifolates are strongly reminiscent of the structure of folic acid and include typical P-benzoyl glutamate part of folic acid. Glutamate part folic acid carries a double negative charge at physiological pH, and therefore, this compound and its analogs have an active energy-dependent transport system for passage through the cell membrane and have a metabolic effect. Many researchers have confirmed that folic acid in both its forms, reduced and oxidized, and its analogues actively transporterowych transport protein recovered folate, and he preferred restored folate, while also transports many derivatives of folic acid. Methotrexate (MTX) is transported through the transport system restored folate. Another transport protein folate called membrane folate-binding protein or mFBP, and it is preferred to folic acid [2].

Anticancer glutamate-containing antifolates, currently used in the clinic, including MTX, enter the cell via transport system restored folate with one significant exception, 5,10-daliasalabarria acid (DDATHF) is an antitumor GARFT inhibitor undergoing clinical trials. It was shown that DDATHF is transported into the cell through the transport system restored folate, and using mFBP [3].

It was hypothesized that unwanted toxicity, in particular for folate-depleted mammals, related to the fact that DDATHF, known GARFT inhibitor, has a high affinity for mFBP, uncontrolled during folate deficiency. It was further hypothesized that folic acid and other molecules that block mFBP, not allowing to transport other GARFT inhibitors can reduce taoudenni, strong GARFT inhibitors and having low toxicity. This task is solved in that the proposed antiproliferative agents given below formula 1, which are strong inhibitors GARFT, but do not form strong bonds with mFBP. Such compounds preferably have a constant link with mFBP at least 1000 times smaller than for DDATHF, while retaining the favorable properties of GARFT inhibition and transport of recovered folate for antitumor activity.

As indicated above, the compounds according to the invention possess anti-proliferative activity, that is, a property that may be in the form of antitumor activity. The connection according to the invention can be used by itself or act as a predecessor, in vivo turning into active connection. Preferred compounds according to the invention are particularly active in the inhibition of the enzyme GARFT. Particularly preferred compounds are active in the inhibition of growth of the cell line L1210, leukemia line of mouse cells that can be grown in tissue culture. Compounds according to the invention can also be active in inhibiting the growth of bacteria, calibrating, and their pharmaceutically acceptable salts, may be prepared in the traditional dose forms, such as capsules, tablets and injectables. Can also be used in solid or liquid pharmaceutically acceptable carriers, diluents or excipients.

Solid carriers include starch, lactose, dihydrate calcium sulphate, gypsum, sucrose, talc, gelatin, agar, pectin, gum Arabic, magnesium stearate and stearic acid. Liquid carriers include syrup, peanut oil, olive oil, saline solution and water.

The carrier or diluent may include any material for sustained release, such as glyceryl the monostearate or glyceryl distearate, single or with wax. When using a liquid medium, the drug can be in the form of a syrup, elixir, emulsion, soft gelatin capsule, sterile water for injection (e.g., solution or non-aqueous or aqueous liquid suspension.

The pharmaceutical preparations are prepared following the traditional technology of pharmaceutical chemistry, including such steps as mixing, granulating and kompresovana (if necessary) in tablet forms, or mixing, C is, parenteralnogo, local, intravaginal, intranasal, intrabronchial, intraocular, ear or rectal.

The composition of the invention can also include one or more pharmaceutically active compounds. For example, the composition can contain one of the following antineoplastic agents: mitotic inhibitors (e.g., vinblastine); alkylating agents; inhibitors of dihydrofolate reductase or TS inhibitors; antimetabolites (for example, 5-fluorouracil, citizenries); intercalating antibiotics, for example adriamycin, bleomycin), enzymes (for example asparaginase), topoisomerase inhibitors (e.g. etoposide; biological response modifiers (for example interferon). Compounds according to the invention can also be used in combination with one or more antiproliferative agents or GARFT inhibitors, in particular well-known [7-8] . The composition of the invention can also contain one or more antibacterial, antigenic, antiparasitic, antiviral, antipsoriatics or anticoccidial agents. Examples of antibacterial agents are sulfonamides such as sulfamethoxazole, sulfate and trimetrexate; penicillins; cephalosporins; and hinolan carboxylic acid and condensed with isothiazol counterparts.

Another aspect of the invention relates to therapeutic methods of inhibiting the growth or proliferation of cells of higher organisms or microorganisms, in which the host (recipient) influence the effective amount of the compounds according to the invention. Compounds according to the invention, particularly useful in the treatment of mammalian hosts (recipients), such as man, in the treatment of birds. Particularly preferred therapeutic method includes the impact on recipient effective for inhibition of GARFT number of compounds according to the invention.

Many described here antiproliferative compounds and their pharmaceutically acceptable salts can be used in therapeutic method according to the invention. These compounds can be applied in the form of a pharmaceutically acceptable composition containing a diluent or carrier, as described above.

The dose of the composition contains at least an effective amount of the active compound and preferably consists of one or more pharmaceutical dose units. "Effective amount" oboznachennomu effect in the application, for example, one or more pharmaceutical dose units.

Estimated daily dose for vertebrates contains up to 1 g of active ingredient per 1 kg of body weight of the recipient, preferably 0.5 g, more preferably 100 mg and most preferably about 50 mg and below. The selected dose can affect warm-blooded animal or a mammal, such as man, in need of treatment through inhibition of metabolic pathways of folate, using any suitable application method such doses, including local, for example in the form of ointment or cream; orally; rectally, for example in the form of suppositories; by parenteral injection; or continuous by intravaginal, intranasal, intrabronchial, custom or intraocular infusion.

Compounds according to the invention give any one or more effects selected from among the following: antiproliferative effect, antibacterial effect, antiparasitic effects, antiviral effects, antipsoriatic effect, Antiprotozoal effect, anticoccidial effect, anti-inflammatory, immunosuppressive effect and this makes it the effect. These compounds are particularly useful in obtaining of the compounds of the formula I

< / BR>
where A is CH2or S;

Z - C1-C3alkylen;

X - C1-C6alkyl; and

R1and R2- hydrogen.

This invention relates to pharmaceutically acceptable salts of compounds of formula I.

Although the compounds of formula I and shown in 4-oxforde and it's what is meant in this description, oxoprop still exists in tautomeric equilibrium with the corresponding 4 - hydroxy-group. Therefore, it should be clear that the compounds of formula I include as depicted in figures 4-oxo, and tautomeric 4-hydroxypoly. Thus, the invention also relates to pharmaceutically acceptable salts of 4-hydroxy of tautomers of the compounds represented by formula I.

The compounds of formula I are in the form of diastereoisomeric mixtures. It should be clear that the compounds having chiral centers are in the form of mixtures of diastereoisomers, unless otherwise indicated.

In the preferred case, A is sulfur or CH2. The most preferred Z is CH2. Preferred unsubstituted X. the Most preferred X, which stands or ethyl.

Preferably, when R1and R2each nesetsja CH2and X is the stands.

Preferred examples of compounds of formula 1 include:

N-(5-[2-(2-amino-4(3H)-oxo-5,6,7 8 tetrahydropyrido[2, 3-d] -pyrimidine-6-yl)ethyl] -4-methylthieno-2-yl)-L-glutamic acid; diethyl ether N-(5-[2-(2-amino-4-oxo-4,6,7 there, 8-tetrahydro-3H-pyrimido[5,4-6] [1,4] -thiazin-6-yl)ethyl] -4-methylthieno-2-yl)-L-glutamic acid; and

N-(5-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimido[5,4-6] [1,4]-thiazin-6-yl)ethyl]-4-methylthieno-2-yl)-L-glutamic acid.

The compounds of formula I are applicable as GARFT inhibitors. The compounds of formula I in which R1and R2each denotes hydrogen, are particularly active antitumor and antiproliferative agents.

Pharmaceutically acceptable salts according to the invention include, for example, salts of alkali metal, alkaline earth metal, other non-toxic metal, ammonium and substituted ammonium glutamic acid compounds according to the invention. Examples of salts are sodium, potassium, lithium, calcium, magnesium salt, a salt of pyridine and substituted pyridine compounds in the form of the free acid.

The compounds of formula I can be obtained as described below.

For R>
< / BR>
where R is halogen, preferably bromine;

X is defined above;

B - C1-C6alkoxy.

The compound of formula II is subjected to interaction with the compound of the formula III:

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where Y denotes CH2OH or a protected pyridopyrimidines formula IV

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Next, the synthesis can go one or two ways depending on whether Y is protected by pyridopyrimidines or CH2OH.

If Y is protected by pyridopyrimidines formula IV, a combination of compounds of formula II and III is preferably carried out in the presence of a catalyst of a transition metal, preferably palladium or Nickel, in the presence of a base, preferably dinucleophiles auxiliary base, in a solvent in which at least one of the reactants is at least partially soluble. Preferred solvents for the combination of the compounds of formulas II and III are diethylamin, acetonitrile, dimethylformamide, dimethylacetamide and triethylamine. Basic environment for combination preferably provide with dinucleophiles auxiliary base, which base can neutralize halogenation acid formed by the combination. It's about the ethylamine. If possible, instead of a separate solvent and base you can use the main solvent.

When Y is pyridopyrimidines, the combination of the compounds of formulas II and III gives compound of formula V:

< / BR>
where X, R1and R2such as defined above. The compound of formula V is subjected to interaction with gaseous hydrogen, preferably at 45-1000 psi, in the presence of a suitable catalyst of transition metal, preferably platinum, palladium or rhodium on coal or other suitable substrate, in a suitable solvent, preferably acetic acid or triperoxonane acid, to obtain the compounds of formula VI:

< / BR>
where X, R1and R2such as defined above.

And finally, the compound of formula VI hydrolyzing education free glutamic acid (R1and R2are both hydrogen) of formula I.

If Y is CH2OH, the interaction of the compounds of formulas II and III gives compound of formula VII:

< / BR>
where X are as defined above.

Compound of formula VII is subjected to interaction with gaseous hydrogen in the presence of a suitable metal catalyst, preferably palladium and the mules VIII is subjected to interaction with an oxidizing agent, preferably perruthenate of tetrapropylammonium, obtaining the compounds of formula IX:

< / BR>
where X are as defined above.

The compound of formula IX is subjected to interaction with the methylene donor, preferably methylene-triphenylphosphorane, in a suitable solvent, preferably tetrahydrofuran, to obtain the compounds of formula X

< / BR>
where X are as defined above.

The compound of formula X is subjected to interaction with dihydroxyvitamin agent, preferably by osmium tetroxide in the presence of a suitable oxidizing agent, preferably N-methylmorpholine-N-oxide, to obtain the compounds of formula XI:

< / BR>
where X are as defined above.

The compound of formula XI is converted into a compound of formula I, using one of the four following methods:

In the first method of making the compound of formula XI is subjected to interaction with sulfonylureas agent, preferably p-toluensulfonyl chloride or methanesulfonyl chloride, in the presence of dinucleophiles base, preferably triethylamine or diisopropylethylamine, to obtain the intermediate mono-sulfonylamino connection. It promezhutochnoye the compounds of formula XII:

< / BR>
where X are as defined above.

The epoxide of formula XII is subjected to interaction with a nitrogen-containing nucleophile, preferably sodium azide, in the presence of a mild catalyst of the Lewis acid, preferably lithium perchlorate or magnesium perchlorate, with the intermediate azide alcohol. Recovery azide alcohol, preferably with gaseous hydrogen in the presence of a metal catalyst, followed by appropriate protection attitudinal group, preferably tert-butoxycarbonyl, menthoxycarbonyl or benzyl, gives compound of formula XIII:

< / BR>
where X are as defined above, a R4and R5independently of one another are hydrogen or a suitable astonishing group. Preferred protective groups are tert-butoxycarbonyl, benzyl oxycarbonyl and benzyl.

The compound of formula XIII is subjected to interaction with allermuir or sulfonylureas agent, preferably methanesulfonyl chloride or p-toluensulfonyl chloride, in the presence of dinucleophiles base, preferably triethylamine or diisopropylethylamine, in a suitable solvent, in which at least one of the reagents, hydroxy group substituted with a suitable nucleophile, preferably ciocalteu salt, more preferably thioacetate potassium, obtaining the compounds of formula XIV:

< / BR>
where A, X, B, R4and R5such as defined above, and

Ac represents acetyl group.

Preferably, the AC is acetyl.

Alternatively the compound of formula XIII can be converted into a compound of formula XIV in one chemical reception using triphenylphosphine, diethyl or dimethyl of azodicarboxylate acid and a nucleophile, preferably teoksessa acid, in a suitable solvent.

The compound of formula XIV is treated with a nucleophilic base, preferably potassium carbonate, sodium carbonate, sodium hydroxide or potassium hydroxide, in an alcohol solvent, preferably methanol, ethanol or isopropanol, in the presence of an alkylating agent, preferably dimethyl or diethyl of chloromelanite, obtaining the compounds of formula XV:

< / BR>
where A, X, B, R4and R5such as defined above, and

R6each independently is hydrogen or a group which forms with attached group CO2easily hydrolyzable ester group. Preferably R6is C1-C2the alkyl.

The compound of formula XV is treated under conditions suitable to remove or R4or R5or both of the protective groups to obtain the compounds of formula XVI:

< / BR>
where A, X, and R6such as defined above.

If the protective groups used tert-butoxycarbonyl, suitable conditions is processing triperoxonane acid and subsequent neutralization.

The compound of formula XVI is subjected to interaction with an alkylating agent, preferably tetrafluoroborate trimethyl or triethyl hydronium, in a suitable solvent, preferably dichloromethane, with the formation of intermediate lachinova ether. Intermediate laktamny ether is subjected to interaction with guanidine in an alcoholic solvent, preferably methanol, ethanol or isopropanol, to form compounds of formula XVII:

< / BR>
where A, X are as defined above.

Alternatively the compound of formula XVI can be converted into a compound of formula XVII by reacting the compounds of formula XVI with Teollisuuden agent, preferably P2S5or 2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphate-2, 4-disulfide with obrazovaniye agent, preferably methyl iodide or tetrafluoroborate trimethyl or triethyl hydronium, and then guanidine in an alcoholic solvent, preferably methanol, ethanol or isopropanol, to obtain the compounds of formula XVII.

If is an alcohol function, i.e. if the group is attached to, forms an ester group, the compound of formula XVII hydrolyzing in basic conditions to obtain compounds of formula XVIII:

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where A and X are such as defined above.

The compound of formula XVIII connect [peptide bond] using known from the prior art funds hydrochloride diapir glutamic acid getting diapir formula XIX:

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where A, X, R1and R2such as defined above, except that neither R1or R2are not hydrogen.

And finally, if desired shape of the free glutamic acid, the compound of formula XIX hydrolyzing to form compounds of formula I.

The second method of making the compound of formula XIV receive as described above. This compound is treated with acid, preferably triperoxonane, hydrochloric or p-toluensulfonate, removal of all protective groups (R4, R5and Ac) is in a slightly alkaline buffer, preferably with pH 7 phosphate buffer, in a suitable solvent, preferably ethanol or methanol, is subjected to the interaction with the compound of the formula XXI:

< / BR>
obtaining the compounds of formula XVII. The end of the second method, the compounds of formula XVII to a compound of formula I, carried out similarly as described above.

In the third method of making the compound of formula XI is subjected to interaction with a suitable hydroxyl-protecting group, preferably trialkylsilyl group, more preferably tert-butyldimethylsilyl chloride, in the presence of soft dinucleophiles base, preferably triethylamine, to obtain the compounds of formula XXII:

< / BR>
where X are as defined above, a R7is a suitable hydroxyl-protecting group, preferably trialkylsilyl group.

The compound of formula XXII is then subjected to interaction with allermuir or sulfonylureas agent, preferably methanesulfonyl chloride or p-toluensulfonyl chloride, in the presence of dinucleophiles base, preferably triethylamine or diisopropylethylamine, in a suitable solvent, in which at least one of the reagents for me is xygraph replace with a suitable nucleophile, preferably ciocalteu salt, more preferably TIAA-cetecom potassium, obtaining the compounds of formula XXIII:

< / BR>
where A, X, B, R7and as such, as defined above. Alternatively the compound of formula XII can be converted into a compound of formula XIII in one chemical reception using triphenylphosphine, diethyl or dimethyl usedcar-barcelata acid and a nucleophile, preferably teoksessa acid, in a suitable solvent. The compound of formula XXIII is subjected to interaction with nucleophilic base or mild acid for the selective removal of acetyl group or groups A. the Obtained intermediate compound is subjected to interaction with the compound of the formula XXIV:

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in the presence of dinucleophiles base, preferably triethylamine, diisopropylethylamine or potassium carbonate, to obtain the compounds of formula XXV:

< / BR>
where A, X, and R7such as defined above.

The protective group R7the compound of formula XXV is removed by treatment with an appropriate reagent to obtain the compounds of formula XXVI:

< / BR>
where A, X are as defined above.

If R7is trialkylsilyl, data registered cesium.

The compound of formula XXVI cyclist with obtaining the compounds of formula XVII by activating the hydroxy-group activating agent, preferably methanesulfonyl chloride, followed by treatment with base. In the alternative case, the nitrogen of pyrimidinone first protects a suitable protecting group, preferably tert-butoxycarbonyl, followed by cyclization and removal of the protective group in acidic conditions. The ending of this method, the compounds of formula XVII to a compound of formula I, carried out similarly as described above.

In the fourth and preferred method of conversion of the alcohol compound of formula XXVI receive as described above. This alcohol is subjected to interaction with a suitable oxidizing agent to obtain aldehyde function, which cyclized to the compounds of formula XXVII

< / BR>
where A, X are as defined above.

The compound of formula XXVII is subjected to interaction with the regenerating agent, preferably lamborghini.com sodium, in the presence of a Lewis acid, preferably of etherate of boron TRIFLUORIDE, to obtain the compounds of formula XVII, as defined above. The ending of this method, the compounds of formula XVII to a compound of formula I, Khujand is to be obtained similarly to the case when Z is CH2. In particular, the compound of formula I, where Z is other than CH2can be obtained by using the olefin of formula XXXIV:

< / BR>
where X and R6such as defined above, and Z is as defined for formula I, but not CH2.

If Z is C1-C3alkylene other than CH2the compound of formula XXXIV are rafinirovannom aldehyde of formula XXXVI:

< / BR>
where X and R6such as defined above, and Z is C1-C3alkylene other than CH2. The aldehyde of formula XXXVI can be obtained similarly to the method [9]. Rafinirovaniyu aldehyde can be carried out using methylene donor, preferably methylene-triphenylphosphorane.

The compound of formula XXXIV is subjected to interaction with dihydroxyvitamin agent, preferably by osmium tetroxide in the presence of a suitable oxidizing agent, preferably N-methylmorpholine-N-oxide, to obtain the compounds of formula XXXVII:

< / BR>
where X and R6such as defined above, and Z is as defined for formula I, but not CH2.

The compound of formula XXXVII is subjected to interaction with sulfonylureas agent, preferably p-Tolna of triethylamine or diisopropylethylamine, obtaining intermediate monosulfonamyl connection. This intermediate compound is subjected to interaction with a strong base, preferably sodium hydride, to obtain the compounds of formula XXXVIII:

< / BR>
where X and R6such as defined above, and Z is as defined for formula I, but not CH2.

The epoxide of formula XXXVIII is subjected to interaction with a nitrogen-containing nucleophile, preferably sodium azide, in the presence of a catalyst of a soft Lewis acid, preferably lithium perchlorate or magnesium, to obtain the intermediate azide alcohol. This intermediate connection restore, preferably with gaseous hydrogen in the presence of a metal catalyst, followed by appropriate protection attitudinal group, preferably tert-butoxycarbonyl, menthoxycarbonyl or benzyl, with obtaining the compounds of formula XVII':

< / BR>
where X, R6and R4and R5such as defined above, a Z is as defined for formula I, but not CH2.

The compound of formula XVII' then subjected to interaction with allermuir or sulfonylureas agent, preferably methanesulfonyl chloride or p-toluensulfonyl chlorine is chodashim solvent, in which at least one of the reactants is at least partially soluble, to obtain the activated hydroxy-group. The activated hydroxy-group is substituted with a suitable nucleophile, preferably ciocalteu salt, more preferably thioacetate potassium, obtaining the compounds of formula XVIII'

< / BR>
where A, X, R6, R4and R5and Ac such as defined above, a Z is as defined for formula I, but not CH2.

Alternatively the compound of formula XVII' is converted into a compound of formula XVIII' in one chemical reception using triphenylphosphine, diethyl or dimethyl of azodicarboxylate acid and a nucleophile, preferably teoksessa acid, in a suitable solvent.

The compound of formula XVIII' process nucleophilic base, preferably potassium carbonate, sodium carbonate, sodium hydroxide or potassium hydroxide, in an alcohol solvent, preferably methanol, ethanol or isopropanol, in the presence of an alkylating agent, preferably dimethyl or diethyl of chloromelanite, obtaining the compounds of formula XIX':,

< / BR>
where A, X, R6, R4and R5such as defined above, and Z is as Oprea removal or R4or R5or both of the protective groups to obtain the compounds of formula XX':

< / BR>
where A, X and R6such as defined above, a Z is as defined for formula I, but not CH2.

If the protective groups used tert-butoxycarbonyl, suitable conditions for the removal of this group is processing triperoxonane acid, followed by neutralization with obtaining the compounds of formula XX'.

The compound of formula XX' is subjected to interaction with an alkylating agent, preferably tetrafluoroborate trimethyl or triethyl hydronium, in a suitable solvent, preferably dichloromethane, with the formation of intermediate lachinova ether. This intermediate laktamny ether is subjected to interaction with guanidine in an alcoholic solvent, preferably methanol, ethanol or isopropanol, to form compounds of formula XXI':

< / BR>
where A, X and R6such as defined above, a Z is as defined for formula I, but not CH2.

Alternatively the compound of formula XX' is converted into a compound of formula XXI' by reacting the compounds of formula X' with Teollisuuden agent, preferably P2S5or 2,4-bis(4-methox the intermediate connection alkylate with an alkylating agent, preferably methyl iodide or tetrafluoroborate trimethyl or triethyl hydronium, and then guanidine in an alcoholic solvent, preferably methanol, ethanol or isopropanol, to obtain the compounds of formula XXI'.

The compound of formula XXI' hydrolyzing in basic conditions to obtain compounds of formula XXII':

< / BR>
where A and X are such as defined above, and Z is as defined for formula I, but not CH2. If R6is hydrogen in the compound of formula XXI', then the reaction of hydrolysis is not required, and the compound of formula XXI' combine peptide bond, as described below.

The compound of formula XXII' (or the compound of formula XXI', where R6is hydrogen) in the form of a free carboxylic acid, can be connected [peptide link] (with means known from the prior art) with the hydrochloride diapir glutamic acid with the formation of diapir formula XXIII':

< / BR>
where A, X are as defined above for formula XXII', a R1and R2each independently is a group which forms, together with the attached CO2easily hydrolyzable ester group, such as C1-C6alkyl, hydroxyalkyl, alkylaryl or arylalkyl.

1and R2each is hydrogen.

Detailed examples of the preparation of compounds of formula I are given below.

Example 1

N-(5- [2-(2-amino-4 (3H)-oxo-5, 6,7,8-tetrahydropyrido[2, 3-d]pyrimidine-6-yl)ethyl]-4-methylthieno-2-yl)-L-glutamic acid (compound 1)

< / BR>
Synthesis of

Compound 1 is synthesized as follows.

a) 5-bromo-4-methylthiophene-2-carboxylic acid:

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This connection get known [10] method.

b) 6-ethinyl-2-(paulolino)-4(3H)-occupied[2,3-d]pyrimidine

< / BR>
This connection get known [11] method.

C) diethyl N-(5-bromo-4-methylthieno-2-yl)-L-glutamate

< / BR>
To a stirred solution of 5-bromo-4-methyl-thiophene-2-carboxylic acid (3,32 g, 15 mmol), 1-hydroxybenzotriazole (2.24 g of 16.6 mmol), hydrochloride of diethyl ether L-glutamic acid (3.98 g, of 16.6 mmol) and diisopropylethylamine (2,9 ml, 2.15 g, of 16.6 mmol) in dimethylformamide (DMF) (40 ml) was added the hydrochloride of 1- (3-dimethylaminopropyl)-3-ethylcarbodiimide (3,18 g of 16.6 mmol). The resulting solution is stirred in an argon atmosphere at room temperature for 18 h, poured into brine (300 ml), diluted with water (100 ml) and extracted with ether (3 x 120 ml). United Ohm brown resin, you clean flash chromatography. Elution with hexane : EtOAc (2:1) gives the product in

an orange oil (of 5.05 g, 83% yield). Analyses show that this product is diethyl N-(5-bromo-4 - methylthieno-2-yl)-L-glutamate.

NMR (CDCl3) : 7.22 (1H, s), 6.86 (1H, d, J = 7.5 Hz), 4.69 (1H, ddd, J = 4.8, 7.5, 9.4 Hz), 4.23 (2H, q, J = 7.1 Hz), 4.12.(2H, q, J = 7.1 Hz), 2.55 - 2.39 (2H, m), 2.35 - 2.22 (1H, m), 2.19 (3H, s), 2.17 - 2.04 (1H, m), 1.29 (3H, t, J = 7.1 Hz), 1.23 (3H, t, J = 7.1 Hz).

Anal.: (C15H20NO5SBr) C, H, N, S, Br.

g), diethyl N-(5-[(2-[paulolino]-4(3H)-occupied[2,3-d] pyrimidine-6-yl)ethinyl]-4-methylthieno-2-yl) glutamate:

< / BR>
To a stirred solution of diethyl N-(5-bromo-4-methylthieno-2 - yl) glutamate (4,21 g, 10.4 mmol) in acetonitrile (55 ml) in an argon atmosphere add chloride bis (triphenylphosphine) palladium (702 mg, 1.0 mmol), copper iodide (200 mg, 1.1 mmol), triethylamine (1.5 ml, 1,09 g to 10.8 mmol) and 6-ethinyl-2-(paulolino)-4(3H)- occupied[2,3-d]pyrimidine (of 5.68 g, 21 mmol). The resulting suspension is heated under reflux for 6 hours After cooling to room temperature, the crude reaction mixture was filtered and the precipitate washed with acetonitrile (50 ml) and ethyl acetate (EtOAc) (2 x 50 ml). The combined filtrates concentrated in vacuo to obtain a brown resin, which cleaned the Analyses show, this product is diethyl N-(5-[(2-[paulolino]-4(3H)-occupied[2,3-d]pyrimidine-6-yl) ethinyl]-4-methylthieno- -2-yl) glutamate.

NMR (CDCl3) :

8.95 (1H, d, J = 2.2 Hz), 8.59 (1H, d, J = 2.2 Hz), 7.33 (1H, s), 7.03 (1H, d, J = 7.4 Hz), 4.73 (1H, ddd, J = 4.8, 7.4, 9.5 Hz), 4.24 (2H, q, J = 7.1 Hz), 4.13 (2H, q, J = 7.1 Hz), 2.55 - 2.41 (2H, m), 2.38 (3H, s), 2.35 - 2.24 (1H, m), 2.19 - 2.05 (1H, m), 1.34 (S, s), 1.30 (3H, t, J = 7.1 Hz), 1.24 (3H, t, J = 7.1 Hz).

Anal. (C29H33N5O7S. 0.75 H2O) C,H,N,S.

d) diethyl N-(5-[(2-[paulolino]-4(3H)-occupied[2,3-d] pyrimidine-6-yl)ethyl]-4-methylthieno-2-yl) glutamate:

< / BR>
A suspension of diethyl N-(5-[(2-[paulolino] -4(3H)-occupied [2,3-d]pyrimidine-6-yl)ethinyl] -4-methylthieno-2-yl) glutamate (959 mg, 1.6 mmol) and 10% palladium on coal (1.5 g, 150% of the mass equiv.) in triperoxonane acid (30 ml) is shaken under 50 psi of H2within 22 hours the Crude reaction mixture is diluted with CH2Cl2, filtered through a pad of Celite (diatomaceous earth) and concentrated in vacuo. The obtained residue was dissolved in CH2Cl2(120 ml), washed with saturated NaHCO3(2 x 100 ml), dried over Na2SO4and concentrated in vacuo to obtain a brown resin, which is purified flash chromatography. Elution with a mixture of CH2Cl2:CH3OH (49:1) to give a yellow solid about the 3-d]pyrimidine-6-yl)ethyl]-4-methylthieno-2-yl) glutamate.

(CDCl3) : 8.60 (1H, d, J > 2.2 Hz), 8.49 (1H, broad), 8.32 (1H, d, J = 2.2 Hz), 7.22 (1H, s), 6.78 (1H, d, J = 7.5 Hz), 4.72 (1H, ddd, J = 4.8, 7.5, 9.5 Hz), 4.23 (2H, q, J = 7.1 Hz), 4.11 (2H, q, J = 7.1 Hz), 3.12 - 3.00 (4H, m), 2.52 - 2.41 (2H, m), 2.37 - 2.22 (1H, m), 2.16 - 2.04 (1H, m), 2.02 (3H, s), 1.33 (S, s), 1.29 (3H, t, J = 7.1 Hz), 1.23 (3H, t, J = 7.1 Hz).

Anal. (C29H37N5O7S. 0.5 H2O) C,H,N,S.

e) diethyl N-(5-[(2-[paulolino]-4(3H)-oxo-5,6,7,8 - tetrahydropyrido[2,3-d]pyrimidine-6-yl)ethyl]-4-methylthieno-2-yl) glutamate:

< / BR>
A suspension of diethyl N-(5-[(2-[paulolino]-4(3H)-occupied [2,3-d] pyrimidine - 6-yl) ethyl]-4-methylthieno-2-yl) glutamate (2,98 g, 5 mmol), 10% Pt on coal (1.5 g, 50 wt% EQ.) in triperoxonane acid (170 ml) is shaken at 800 psi H2within 40 hours of the Crude reaction mixture is diluted with CH2Cl2, filtered through a pad of Celite and concentrated in vacuo. The obtained residue was dissolved in CH2Cl2(150 ml), washed with saturated of N (2 x 150 ml), dried over Na2SO4and concentrated in vacuo to obtain a brown resin, which is purified flash chromatography. Elution with a mixture of CH2Cl2: CH3OH (24: 1) first gives the unreacted substrate, and then the yellow solid product (293 mg, 10% yield). The analysis shows that this product is diethyl N-(5-[(2-[paulolino]-4( ) :

7.24 (1H, s), 6.75 (1H, d, J = 7.6 Hz), 5.57 (1H, broad), 4.72 (1H, ddd, J = 4.8. 7.6, 12.6 Hz), 4.22 (2H, q, J = 7.1 Hz), 4.11 (2H, q, J = 7.1 Hz), 3.43 - 3.36 (1H, m), 3.06 - 2.98 (1H, m), 2.89 - 2.68 (MN, m), 2.52 - 2.40 (3H, m),2.37 - 2.23 (1H, m), 2.15: (3H, s), 2.14 - 2.03 (1H, m), 1.94 - 1.83 (IH, m), 1.73 - 1.63 (2H, m), 1.32 (9H,s), 1.29 (3H, t, J= 7.1 Hz), 1.23 (3H, t, J = 7.1 Hz).

Anal. (C29H41N5O7S. 0.5 H2O C,H,N,S.

W) N-(5-[2-(2-amino-4(3H)-oxo-5,6,7,8-tetrahydropyrido [2,3-d]pyrimidine-6-yl)ethyl]-4-methylthieno-2-yl) glutamic acid (compound 1):

A solution of diethyl N-(5-[(2-[paulolino] -4(3H)-oxo-5,6,7,8-tetrahydropyrido[2,3-d] pyrimidine-6-yl)ethyl] -4-methylthieno-2-yl) glutamate (293 mg, 0.5 mmol) in 1 N NaOH (25 ml) was stirred at room temperature for 90 h, and then neutralized 6 N Hcl. The resulting precipitate is collected by filtration and washed with water (4 x 10 ml) to give a yellow solid product (63 mg, 28% yield). The analysis shows that this product is diethyl N-(5-[2- (2-amino-4(3H)-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-6-yl)ethyl]-4-methylthieno-2-yl) glutamic acid.

NMR (DMSO-d6) : 12.44 (2H, broad), 9.89 (1H, broad), 8.42 (1H, d, J= 7.8 Hz), 7.57 (1H, s), 6.39 (1H, br s), 6.12 (2H, br s), 4.30 (1H, ddd, J = 4.8, 7.8, 9.6 Hz), 3.26 - 3.18 (2H, m), 2.83 - 2.74 (3H, m), 2.31 (2H, t, J = 7.4 Hz), 2.12 (3H, s), 2.09 - 2.01 (1H, m), 1.94 - 1.80 (2H, m), 1.68 - 1.47 (3H,m).

Anal. (C20H25N5O6S. 1.1 H2
Known analysis method GAR-transformylase (GARFT) [12] was modified and used as described below. The reaction mixture contained the catalytic domain of human GARFT, 0-250 nm test compound, 20 μm glycinamide of ribonucleotide (GAR), 10 or 20 μm of N10-formyl-5,8-ideasabout (FDDF), 50 mm HEPES-KOH (pH 7.5) and 50 mm KC1. The reaction was initiated by addition of enzyme to a final concentration of 11 nm, followed by monitoring the absorption increases at 294 nm at 20oC (e294=18.9 mm-1cm-1).

The GARFT inhibition constant (K1determined by the dependence of the steady-state rate of the catalytic reaction on the concentration of inhibitor and substrate. Based on the apparent Ki(Ki,app) concentration FDDF it was determined that the observed type of inhibition is competitive with respect to FDDF and is described by the equality Ki,app=Ki+(Ki/Km) [FDDF]. The Michaelis constant for FDDF, Kmwas determined independently by the dependence of the catalytic rate on the concentration of FDDF. These two definitions, Kmand Kiwere fitted nonlinear methods to the equation of Michaelis or the equation of Michaelis for competitive inhibition, respectively. Would the fitting of these data nonlinear methods to the equation a strong connection Morrison [13].

Determination of dissociation constants for folate-binding protein human

The dissociation constant (Kd) for folate binding protein (FBP) of the person identified in the analysis of competitive binding using United with membrane FBP obtained from cultured KB cells.

Preparation of the membrane fraction of KB cells:

Stuck KB cells were scraped from the flasks, washed in ice-cold PBS and centrifuged at 5000 x g for 5 min at 4oC. the Precipitated cells (2108cells) resuspendable in 10 ml of suspension buffer (KH2PO4-KOH pH 7,4: 10 μm EDTA: 10 μm 2-mercaptoethanol) was rapidly destroyed by ultrasound for complete cell lysis and centrifuged at 12000 x g for 10 min at 4oC. the Precipitate was released from endogenous linked folate by resuspendable in 20 ml of acidic buffer (50 mm KH2PO4-KOH pH 3.5: 10 μm EDTA: 10 μm 2-mercaptoethanol) and centrifuged as before. Then the precipitate resuspendable in 5 ml of suspension buffer at pH 7,4 without EDTA. The protein content was quantified using the method of Bradford, using BSA as a standard. Typical output in this way was 4-5 mg total membrane protein 2108cells. This is the end is but a competitive binding of FBP:

Inhibitor competed with3H-folic acid for binding to FBP. The reaction mixture contained (in 1 ml of 50 mm KH2PO4-KOH pH 7,4: 10 mm 2-mercaptoethanol) 50-100 mg of membrane protein cells containing 3-6 picomol (3-6 nm) FBP, 17,25 pmol3H-folic acid (17,25 nm of 0.5 µci), and various concentrations of competitors. The reaction was carried out at 25oC. because Of the very slow release of the associated3H-folic acid competitor was subjected to preliminary binding for 30 min in the absence of 3H-folic acid. Then add3H-folic acid and the mixture was left to come to equilibrium for 2.5 hours the Reaction mixture, the whole was passed through nitrocellulose filters under vacuum to separate the cell membranes with an associated3H-folic acid. Isolated membranes were then washed 4 times in 1 ml of reaction buffer. The amount of bound3H-folic acid was measured by counting the scintillations nitrocellulose membrane. The data obtained nonlinear customized, as described above. FBPd3H-folic acid used to calculate Kdcompetitor was obtained by direct titration FBP3H-folate with subsequent nonlinear fit what their origins are listed in table 1. Growing conditions and environmental requirements for each cell line are shown in table 2. All cultures were maintained at 37oC , 5% CO2in the air, moisturizing incubator.

Inhibition of growth in vitro:

Stock solutions of inhibitors were prepared in 10 mm sodium bicarbonate in water and stored in aliquot 1 ml at -20oC for experiments with cell cultures. Inhibition of cell growth was measured using a modification of the well known [14] method.

Cells of each cell line in the middle of the logarithmic phase of growth were diluted to a concentration of 18500 cells/ml in fresh RPMI growth medium (Mediatech, Washington, DC) with the addition of detalizirovannoi fetal calf serum (Hyclone Laboratories Inc., Logan, UT), and then aliquots were placed in a column from the 2nd to the 12th 96-well plates for micrometrology. Column 1 filled with the same volume, 135 ml of fresh medium without cells, for use as a blank. The tablets were placed in the incubator at 37oC with 5% CO2in the air. After 1-4 h tablets were removed from the incubator, added test compound in 10 x final concentration of 15 ml/well in a binary dilutions in columns from 12th to 4th. For the reverse experiment gipoksantin (1,75 mm) or AICA (1,75 mm) wklug compounds were prepared on each tablet in four copies. Fifteen milliliters of the medium without the test compounds were added to the wells of column 1 tablets. Then the cells were returned to the incubator and left there alone for the full incubation period. On the 3rd day for L1210 and L1210/C1920 cells or 5th day for CCRF-CEM cells to all wells of all tablets was added 50 ml of 0.8 ml/mg MTT bromide (4,5 - dimethylthiazol-2-yl)-2,5-diphenyl of tetrazole (Sigma catalog NoM) dissolved in the medium for culturing the tissue, after which the cells were returned to the incubator. After 4 h, all the tablets were removed from the incubator and centrifuged at 1200 rpm for 7 minutes Medium was pumped out and all wells of all tablets was added 150 ml of DMSO. Then the tablets were mixed at low speed on a vortex mixer for 1 h in the dark at room temperature. Share metabolized MTT was measured spectrophotometrically at 540 nm on a Molecular Devices Vmax kinetic microplate reader for. The concentration of drug required to reduce cell growth by 50%, which is measured by the MTT metabolism was determined by interpolating between OD (minus blank) directly above and below 50% of the control OD (minus blank).

As shown by the above comparative data, compound 1 has Kdin respect to the co-4,6,7,8-tetrahydro-3H-pyrimido[5,4-6] [1,4]thiazin-6-yl)ethyl]-4-methylthieno-2-yl)-L-glutamic acid (compound 2):

< / BR>
Compound 2 was prepared as follows.

a) methyl ester of 5-bromo-4-methylthiophene-2-carboxylic acid

< / BR>
To a solution of 5-bromo-4-methylthiophene-2-carboxylic acid (20,32 g, 92 mmol) in CH3OH (450 ml) is added concentrated H2SO4(4 ml). The resulting solution was heated under reflux for 18 hours the Solvent is removed by concentration in vacuo and the resulting residue is separated in a mixture of saturated NaHCO3(350 ml) and ether (350 ml). The layers are separated and the aqueous phase extracted with ether (3 x 150 ml). The combined organic extracts are dried over MgSO4and concentrated in vacuo to obtain a red oil, which was purified flash chromatography. Elution with a mixture of hexane:ethyl acetate (9:1) gives the product as a yellow oil hardened with standing (MT 18: 34 g, 85% yield). The analysis shows that this product is the methyl ester of 5-bromo-4 - methylthiophene-2-carboxylic acid.

NMR (CDCl3) : 7,47 (1H, s), 3,86 (3H, s), measuring 2.20 (3H, s).

Anal. (C7H7O2SBr) C,H,S,Br.

b) methyl ester of 5-(3-hydroxypropyl)-4-methylthiophene-2- -carboxylic acid

< / BR>
To a stirred solution of methyl ester 5-bromo-4-methylthiophene-2-carboxylic acid (5,18 g, 22 Malmedy (42 mg, 0.22 mmol) and propargilovyh alcohol (1.5 ml, 1.44 g, 26 mmol). The resulting mixture was stirred at room temperature for 18 hours the Solvent is removed by concentration in vacuo and the resulting residue diluted with water (200 ml) and then extracted with ethyl acetate (3 x 100 ml). The combined organic extracts washed with 0.5 N HCl (100 ml), dried over MgSO4and concentrated in vacuo to obtain a brown oil, which was purified flash chromatography. Elution with hexane:EtOAc (2:1) gives the product as an orange oil hardened with standing (4,07 g, 88% yield). The analysis showed that this product is the methyl ester of 5-(3-hydroxypropyl)-4-methylthiophene-2-carboxylic acid.

NMR (CDCl3) : 7.52 (1H, s), 4,55 (2H, s), a 3.87 (3H, s) to 2.29 (3H, s)

Anal. (C10H10O3S) C, N, s

C) methyl ester of 5-(3-hydroxypropyl)-4-methylthiophene-2 - carboxylic acid

< / BR>
A suspension of methyl ester 5-(3-hydroxypropyl)-4 - methylthiophene-2-carboxylic acid (3,86 g, 18 mmol) and 5% palladium on coal (0,72 g, 19% of the mass equiv.) in EtOAc (110 ml) is shaken under 50 psi of H2within 20 hours the Crude reaction mixture was filtered through a pad of Celite and the filtrate was concentrated in vacuo to obtain the product as a yellow oil (3,8 carboxylic acid.

NMR (CDCl5) : 7,51 (1H, s), of 3.84 (3H, s), 3,71 (2H, t, J=6.2 Hz), of 2.86 (2H, t, J=7,6 Hz) of 2.16 (3H, s), with 1.92 (2H, tt, J=6,2 and 7.6 Hz)

Anal. (C10H14O3S) C, H, s

d) methyl ester of 4-methyl-5-(3-oksipropil)-thiophene-2 - carboxylic acid

< / BR>
To a stirred suspension of methyl ester 5-(3 - hydroxypropyl)-4-methylthiophene-2-carboxylic acid (3,74 g, 17 mmol), N-methylmorpholin-N-oxide (3.00 g, 26 mmol) and powdered 4 a molecular sieves (4.5 g) in CH2Cl2(50 ml) add perruthenate tetrapropyl ammonium (300 mg, 0.85 mmol). The resulting suspension is stirred at room temperature for 40 minutes, the Solvent is removed by concentration in vacuo and the resulting residue purified flash chromatography. Elution with a mixture of hexane: tOAc (4:1) gives the product as a yellow oil (1,82 g, 49% yield). The analysis shows that this product is the methyl ether of 4-methyl-5-(3-oksipropil)-thiophene-2-carboxylic acid.

NMR (CDCl3) : 9,83 (1H, t, J=0.8 Hz), to 7.50 (1H, s), of 3.84 (3H, s), of 3.07 (2H, t, J=7.4 Hz), and 2.83 (2H, dt, J=0.8 and 7.4 Hz), 2,17 (3H, s).

Anal. (C10H12O3S) C, H, s

d) methyl ester of 5-(3-butenyl)-4 - methylthiophene-2-carboxylic acid

< / BR>
To a stirred suspension of bromide methyltriphenylphosphonium (3,14 g, 8,8 mmol) in THF (30 ml) in an argon atmosphere, pri 0oC for 75 min at room temperature, and then cooled to -65oC before adding dropwise a solution of methyl ester 4-methyl-5- (3-oksipropil)-thiophene-2-carboxylic acid (1,71 g, 8.1 mmol) in THF (30 ml). The cooling bath removed and the reaction mixture stirred for 90 min, until she gradually warm to room temperature. The crude reaction mixture was concentrated in vacuo to a volume of 20 ml, diluted with ether (200 ml), filtered through a pad of celite [brownmillerite]. The filtrate was concentrated in vacuo to obtain an orange oil, which was purified flash chromatography. Elution with hexane:EtOAc (95:5) gives the product as a yellow oil (772 mg, 46%). The analysis shows that this product is the methyl ester of 5-(3-butenyl)-4-methylthiophene - 2-carboxylic acid.

NMR (CDCl3) : 7.50 (1H, s), 5.84 (1H, ddt, J=10.2, 17.0, 6.6 Hz), 5.07 (1H, dd, J=1.6, 17.0 Hz), 5.02 (1H, dd, J=1.6, 10.2 Hz), 3.84 (3H, s).

Anal. (C11H14O2S) C, H, s

e) methyl ester 5-(3,4-digitaluhr)-4-methylthiophene-2-carboxylic acid

< / BR>
To a stirred solution of N-methylmorpholin-N-oxide (735 mg, 6.3 mmol) and osmium tetroxide (5 mg, 0.02 mmol) in acetone (30 ml) was added a solution of methyl ester 5-(3-butenyl)-4-methylthiophene-2 - to atoi temperature for 48 h, then filtered through a pad of Celite. The filtrate was acidified by the addition of 0.5 M H2SO4(10 ml), and acetone was removed by concentration in vacuo. The aqueous residue was diluted with water (20 ml) and was extracted with EtOAc (3 x 25 ml). The combined organic extracts were washed with water (3 x 25 ml), dried over Na2SO4and concentrated in vacuum to obtain a brown resin, which was purified flash chromatography. Elution with a mixture of CH2Cl2: EtOAc (2:3) gave the product in the form of a whitish solid (577 mg, 71% yield). The analysis showed that this product is a methyl ester of 5-(3,4-digitaluhr)-4-methylthiophene-2-carboxylic acid.

NMR (CDCl3) : 7,50 (1H, s), of 3.84 (3H, s), 3,79-and 3.72 (1H, m), 3,86 (1H, dd, J=3.2, and the 10.9 Hz), 3,48 (1H, dd, J=7,4, up 10.9 Hz), 3.00 and is 2.80 (2H, m).

Anal. (C11H16O4S) C, N, s

The above examples illustrate various aspects of the invention. It is clear that the possible appropriate modifications available to the specialist in this field.

Listed here chemical groups can be substituted, as is customary in chemistry. In some cases, this capability is specified by reference, for example, substituted or unsubstituted C1-C3alkyl group.

If UCO series.

Literature

1. F. M. Muggia, "Folate antimetabolites inhibitor to de novo purine synthesis," New Drugs, concepts and Results in Cancer Chemotherapy, Kluwer Academic Publishers, Boston (1992), 65-87.

2. A. C. Antony, "The Biological Chemistry of Folate Receptors," Blood, The Journal of The American Society of Hematology, vol. 79 (1992), 2807-2820.

3. G. Pizzorno et al. , "5,10-Dideazatetrahydrofolic Acid (DDATHF) Transport in CCRF-CEM and MA104 Cell Lines," The Journal of Biological Chemistry, vol. 268 (1993), 1017-1023.

4. T. Alati et al., "Evaluation of the Mechanism(s) of Inhibition of the Toxicity, But Not the Antitumor Activity of Lometrexol (DDATHF) by Folic Acid," Proceedings of the American Association for Cancer Research, vol. 33 (1992), Abstract 2432, 407.

5. L. L. Habeck et al. "A Novel Class of Monoglutamated Antifolates Exibits Tight-binding Inhibition of Human Glycinamide Ribonucleotide Formyltransferase and Potent Activity against Solid Tumors," Cancer Research, vol. 54 (1994), 1021-1026.

6. U.S. patent 5217974 (Grindey).

7. WO 94/13295 published 23.06.94.

8. WO 92/05153 published 02.04.92.

9. Chuan Shih et al. Journal of Medicinal Chemistry, vol. 35 (1992), 1109-1116.

10. N. Nemec, Collection Czechoslov. Chem. Commit., vol. 39 (1974), 3527.

11. E. C. Taylor & G. S. K. Wong, J. Org.Chem., vol.54 (1989), 3618.

12. Young et al. Biochemistry 23 (1984), 3979-3986.

13. Morrison, Biochem Biophys Acta 185 (1969), 269-286.

14. Mosmann, J. Immunol. Methods 65 (1983), 55 to 63.

1. The compound of the formula I

< / BR>
where A is CH2or S;

Z - C1- C3alkylen;

X - C1- C6alkyl;

R1and R2is hydrogen,

or SUB>2
CH2.

3. The compound or salt according to p. 1, wherein X is methyl or ethyl.

4. The compound or salt according to p. 1, characterized in that A - CH2or S, Z is CH2and X is methyl.

5. The compound or salt according to p. 1, characterized in that it is chosen from N-(5-[2-(2-amino-4(3H)-oxo-5,6,7,8-tetrahydropyrido[2,3-d] pyrimidine-6-yl)ethyl] -4-methylthieno-2-yl)-L-glutamic acid and its pharmaceutically acceptable salts; diethyl ether N-(5-[2-(2-amino-4-oxo-5,6,7,8-tetrahydro-3H-pyrimido[5,4-6] [1,4] -thiazin-6-yl)ethyl] -4-methylthieno-2-yl)-L-glutamic acid and its pharmaceutically acceptable salts and N-(5-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimido[5,4-6] [1,4] -thiazin-6-yl)ethyl]-4-methylthieno-2-yl)-L-glutamic acid and its pharmaceutically acceptable salts.

6. Connection on p. 1, characterized in that it is a N-(5-[2-(2-amino-4(3H)-oxo-5,6,7,8-tetrahydropyrido[2,3-d] pyrimidine-6-yl)ethyl]-4-methylthieno-2-yl)-L-glutamic acid.

7. Method of inhibiting the growth or proliferation of cells of microorganisms or higher organisms impact on the recipient mammal or bird active compound, characterized in that active compound using an effective amount of compounds f the data connection of the formula I Z - CH2or CH2CH2.

9. The method according to p. 7, characterized in that the above compound of formula I X is methyl or ethyl.

10. The method according to p. 7, characterized in that the above compound of the formula I A - CH2or S and X is methyl.

11. The method according to p. 7, characterized in that the compound of formula I is a N-(5-[2-(2-amino-4(3H)-oxo-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-6-yl)ethyl]-4-methylthieno-2-yl)-L-glutamic acid.

12. Compound of formula VII

< / BR>
X - C1- C6alkyl;

B - C1- C6alkoxy.

13. Connection on p. 12, wherein X is methyl or ethyl.

14. Connection on p. 12, characterized in that B is methoxy or ethoxy.

 

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