JPH0512347B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to quinazoline derivatives that are potent antitumor (anticancer) agents. Most of the drugs that have been used to treat tumors are based on the response of nucleic acids in rapidly dividing cells.
directly or indirectly prevented. Many drugs, called antimetabolites, are intended to kill tumors by depleting essential growth factors in competing with and displacing substrates for special enzymes involved in DNA synthesis. . Anti-metabolites can be classified according to their specific inhibitory action, and examples are analogues of purines and pyrimidines, and antifolates, such as aminopterin and amethopterin (methotrexate). Among the antimetabolites, which are antifolates, methotrexate is widely used in the treatment of human malignant tumor diseases, especially in the treatment of chorionic carcinoma and osteosarcoma and in the maintenance of acute lymphoblastic leukemia. The enzyme dihydrofluoride reductase (DHFR,
Suppression of EC1.5, 1.4) causes depletion of the pool of one carbon-supported tetrahydrofolate, resulting in
New synthesis of both thymidine and purine nucleotides required for DNA synthesis is blocked.
The "purine-poor" state induced by methotrexate has been shown to produce toxicity in the gastrointestinal tract of mice, so it does not contribute to the cytotoxic effects of methotrexate on one cultured tumor cell line and may be useful for other lines. may be neutralized. Methotrexate does not require metabolic activation and does not cause significant metabolic degeneration in most cell types. Known causes of methotrexate resistance include decreased membrane transport and increased cellular DHFR. In this latter case,
An effective target of methotrexate is thymidylate synthetase (TS, EC2, 1, 1, 4,
5), this enzyme catalyzes the final step in the de novo synthesis of cymidylate, which is also required for DNA synthesis. Direct inhibition of TS can be achieved with 5-fluorolaucyl, an active metabolite of 5-fluorodeoxyuridine monophosphate. However, resistance to 5-fluorouracil is often accompanied by a decrease in the appropriate activating enzyme, and the drug can also have toxic effects, which are due to its incorporation into RNA. Therefore, TS rather than pyrimidine substrates
An agent that competes with the tetrahydrofolate cosubstrate is needed. Because such compounds are effective against methotrexate-sensitive tumors as well as
They are also as active as or better than methotrexate in their resistance to increased cellular DHFR. Also, since the synthesis of purines is unaffected, it will be less toxic to the host. In addition, folate analogs
It is more effective than pyrimidines because it requires metabolic activation. Some typical quinazoline antifluorate agents are
have been shown to be inhibitors of TS and, for example, 2-amino-4-hydroxy and 2,4-
Although diamino derivatives have been shown to be quite effective inhibitors of TS, they also bind very strongly to DHFR. According to the present invention, it is now possible to obtain increased antitumor activity by substituting a certain group, especially an unsaturated hydrocarbon group, at the 10-position of a 2-amino-4-hydroxyquinazoline derivative. I found out. Unsaturated compounds, especially TS
, to a greater extent than their corresponding saturated analogs, and are therefore more effective. Therefore, according to the invention, the formula In the formula, R is 1 a linear or branched unsaturated hydrocarbon group containing 2 to 6 carbon atoms; or 2 CN, COOH or a salt or ester thereof, OH, =O, a halogen atom, NH ₂ ,
CONH ₂ , COC ₆ H ₅ , oxiranyl or 5-
A straight or branched chain containing up to 6 carbon atoms substituted with at least one substituent which is uracilyl (when said substituent is =O the hydrocarbon group R contains at least 2 carbon atoms) represents a branched saturated or unsaturated hydrocarbon group; n is 0 or an integer of 1 to 4; X is a halogen atom, _C1 to _C4 alkyl, aryl, aralkyl, _C1 to _C4 alkoxy or NO ₂
represents a group, provided that when n is 2 to 4,
n Xs can be the same or different; and Y is of the formula or Here, m represents an integer of 1 to 9, especially 1 (poly-L-glutamic acid residue),
or a pharmaceutically acceptable salt or ester thereof. The compounds of the invention are active as is;
Alternatively, it is useful as a suitable anticancer agent as a pro-drug that can be transformed in vivo to produce an active compound. Preferred substituents R defined in 1 are 2-
those containing 6 carbon atoms, such as 4, especially 3 carbon atoms, especially at least 2-
It has unsaturation in the or -position. Examples of such groups are alkenyl, alkadienyl, alkynyl and alkadiyninyl groups, and particular examples are allyl, propargyl, but-2-ynyl, but-3-ynyl and 1-methylprop-2-yl. The radical R defined under 2 preferably contains up to 6 carbon atoms in the carbon chain, in particular a total of up to 4 carbon atoms in the radical R as a whole. Saturated hydrocarbon chains preferably contain 1 to 3 carbon atoms and unsaturated hydrocarbon chains preferably contain 3 or 4 carbon atoms. Preferably one or two substituents are present on the chain. Preferred heteroatom substituents are halogeno, such as Cl, Br or F, and in the case of _C2 or higher hydrocarbon groups, the ═O is preferably bonded to a carbon atom other than the -carbon atom. Examples of such R groups are 2-oxopropyl, formylmethyl, 2,
2,2-trifluoroethyl, and 2-chloro-, 2-bromo- or 2-fluoroethyl. Preferred saturated carbocyclic substituents contain 3 to 6 ring carbon atoms, especially 3 to 5 ring carbon atoms. An example of such a radical R is cyclopropylmethyl. The saturated carbocyclic ring may itself have at least one _C1 - _C4 alkyl substituent, preferably methyl, preferably from 1 to 3, especially 1 such substituent. Substituents containing at least one heteroatom in definition 2) of R preferably have 1 to 6, in particular 1 to 4, heteroatoms, for example halogen atoms, O, N or S. Examples of such groups are OH, NH ₂ , CONH ₂ , COOH and their salts and esters, CN, NO ₂ , SH, methylsulfonyloxy, alkoxy and amino acid groups. Thus, the group R defined in 2)
is, for example, 2-hydroxyethyl, 3-hydroxypropyl, carbamoylmethyl, 2-aminoethyl, 2-mercaptoethyl, cyanomethyl, carboxymethyl, ethoxycarbonylmethyl, methylsulfonyloxyethyl, dimethylaminoethyl or methoxymethyl I can do it. Heteroatom-containing substituents can also contain rings that are carbocyclic or heterocyclic rings. A particular example of a group R containing a carbocyclic ring and a heteroatom is phenacyl. Alternatively, the carbocyclic ring can itself be substituted with heteroatom-containing groups, preferably 1 to 3 such groups, e.g.
Alternatively, it can be substituted with a group containing 1 to 3 heteroatoms such as S. Specific examples are _C1 - _C4 alkoxy (e.g. methoxy), OH, _NH2 ,
COOH, SH, keto, formyl and methylsulfonyloxy. Heterocyclic substituents preferably have 3 to 6 ring atoms, especially 3, 5 or 6 ring atoms.
and the ring heteroatoms are preferably 1 to 3 and can be N, S or O. An example of such an R group is oxiranylmethyl. These rings have at least one
It can be substituted with _C1 - _C4 alkyl groups, preferably methyl, or with heteroatom-containing groups, preferably with one or two of these groups. Substituents can be present on ring carbon atoms or heteroatoms. These heteroatom-containing groups preferably contain 1 to 3 O,
Contains heteroatoms that are N or S, and are, for example, _C1 - _C4 alkoxy (eg, methyl), =O, OH, _NH2 , COOH, SH, and formyl. A particular example of an R group in this category is 5-uracilmethyl, ie, the thyminyl group. The group X as a group containing at least one heteroatom preferably contains 1 to 3 heteroatoms, which can be, for example, N or O. Specific examples of such are _C1 - _C4 alkoxy and _NO2 . Other preferred groups X are halogen atoms, such as chlorine atoms, and methyl. The alkyl group in the aralkyl X substituent is
Usually 1 to 4 carbon atoms. Particularly effective anticancer agents according to the invention are those in which R represents propargyl, n=0, and Y represents a glutamic acid group or a salt or ester thereof. Furthermore, the corresponding compounds in which n=1 and X is chloro or methyl in the 2-position exhibit high activity. As indicated above, the present invention provides quinazoline derivatives in the free acid form and in the form of pharmaceutically acceptable salts or esters of those acids, which can be partially salts and esters. include. Thus, in the case of compounds containing glutamic acid and aspartic acid groups, monoester monoacid and diester derivatives and the corresponding salt forms are also included. Besides, Toba, COOH
The R group containing group can be in the form of a salt or an ester, for example an ethyl ester. The salts of the invention can be formed with physiologically acceptable inorganic or organic acids or bases and are preferably sodium salts, eg disodium salts. The ester is preferably a lipophilic ester, in which the compound is transported as a pro-drug to the enzyme site and then degraded to the corresponding acid form. Examples of preferred esters are esters with C ₁ -C ₂₀ aliphatic groups,
For example, an alkyl ester, preferably 1 to 4
Alkyl esters of carbon atoms, especially diethyl esters. Compounds of the invention have the formula where X and n are as defined above, R ₂ is a group R equal to R or in protected form, R ¹ is hydrogen or an amino protecting group, and Y' is as defined above Y represents a group of formula a), b) or c) as defined or a salt of said group or said group in partially or fully protected form, and the protecting group in Y′ is an ester group or other protected group, provided that the compound of formula a contains at least one protecting group and deprotection is carried out to remove more than one protecting group,
The deprotection can be carried out simultaneously or in any order; and can be prepared by completely or partially deprotecting the compound. Thus, the acids and salts of the invention can be prepared by complete deprotection of compounds of formula a, with partial esters containing the 2-amino group and
can be prepared by optional deprotection of the R ² group, where Y′ is either partially esterified or fully esterified, the latter form then partially and complete esters can be prepared by optionally deprotecting the 2-amino and ^R2 groups, where the Y' group is in the fully esterified form. It is. Deprotection of groups in the form of esters to generate acids can be carried out, for example, by gentle saponification using a base such as NaOH. The resulting acid can be subsequently reacted with an acid or base to form a pharmaceutically acceptable salt, if desired. Most preferably, the acid is reacted with NaOH to form the corresponding disodium salt. Alternatively, acid products can be esterified, salt products can be converted to acids or esters, or ester products can be transesterified. It may be desirable to generate the salt directly from the ester by deprotection methods;
wherein the compound of formula is in the form of a diester, and the disodium salt of the compound of formula is prepared by saponifying the diester with an aqueous solution of NaOH, 3 moles per mole of diester, and when the reaction is complete, 1 mole per mole of diester. HCl aqueous solution and preferably lyophilize the product salt. Salts can also be formed, for example at the 2-amino group, with acids such as HCl or HBr. The particular protecting group R ¹ is selected taking into account the R and Y' groups and the nature of the desired product, i.e. the conditions required to remove the R ¹ group do not affect the unprotected R group, and When a protecting ester group in Y' is required in the final product, it is usually chosen not to remove this ester group.
An example of an R ¹ group is a pivaloyl group, which can be removed in a saponification step to remove the ester group from Y′ using, for example, NaOH. R ² is equal to R or is the desired protected group R, as shown. Thus R ² in the latter case can contain, for example, a group COOH in the form of an ester or a group OH protected with an acid group. Examples of such groups ^R2 are ethoxycarbonylmethyl and 3-acetoxypropyl, which are deprotected to carboxymethyl or 3-hydroxymethyl, respectively. These R ² groups can, however, themselves be examples of R. Another protected example of R is
This is the case when the substituent simply does not contain an extra group, but is in a different form and the change is carried out as a deprotection step. This example will later be applied to the ^2-
The case is given in which hydroxy-3-bromopropyl undergoes a cyclization reaction under saponification conditions to remove the ester group from Y' and form the oxiranylmethyl group R. The protected compounds of formula a can themselves be prepared by the following coupling reaction: where R ¹ , R ² , X, n and Y′ are as defined for compounds of formula a, and
X'' is a leaving group, preferably halogeno, such as chloro or bromo, p-toluenesulfonate methylsulfonate or trifluoromethylsulfonate. This bond is generally an acid acceptor, such as calcium carbonate, potassium carbonate, in the presence of 2,6-dimethylpyridine or triethylamine and a solvent, e.g.
Performed in N,N-dimethylacetamide or cellosolve. The amine of the above formula is a compound of the formula R ² X′, where
R ² is as defined above, and It can be produced by direct reaction with (4-aminobenzoyl)-L-glutamate, -L-aspartate or -poly-L-glutamate. An alternative method of producing an amine of formula is to use the starting material 4-
(((4-methylphenyl)sulfonyl)amino)
^{Santi ( J} .Hetero-cyclic.
Chem. 4 475). The ester is then saponified to the free acid, converted to the acid chloride, and then combined with a protected Y' group, eg, in the form of a diester. The product is a protecting group
An amine of the formula CH ₃ -0/-SO2 on the amino group, which can then be removed. When it is desired to prepare a compound of the invention containing a substituent X, the corresponding N-(4-nitrobenzoyl)-L-glutamate, -L-aspar It is preferred to prepare the tate or poly-L-glutamate and convert it by hydrogenation or reduction to the corresponding 4-amino derivative, which is then reacted with ^R2X ' to produce the amine of formula. The reaction components of the above formula in which R ¹ =H and Hal are bromo or chloro are prepared from the corresponding 6-bromomethyl-2-trifluoromethylacetamide (pivaloyl) compounds in tetrahydrofuran, aqueous HBr and aqueous HBr, respectively. It can be produced by reacting with methanolic HCl. Bromination of 6-methyl-2-trimethylacetamide-quinazoline can be carried out using N-bromosuccinimide. An alternative method of producing protected compounds of formula a is
According to the following reaction formula, the 2-amino protected form and
It consists of reacting the N ¹⁰ -unsubstituted bond compound in the Y′ protected form with R ² X′, where R ² and X′ are of the form defined above: Compounds of formula b can themselves be prepared by the following reaction: Here, X″ is as defined above.
In this reaction, instead of the 6-CH ₂ -X'' compound of formula, the compound of formula a has the corresponding 6-CH 2 -
- formyl or 6-cyano compounds can be used. The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of an anti-cancer agent as defined for the invention and a pharmaceutically acceptable carrier or diluent. Experiments using mice artificially implanted with tumors have shown that intraperitoneal injection of the anticancer agents of the invention can increase the average survival time of mice related to administration. Furthermore, these animals do not lose significant amounts of weight or show other signs of toxicity during drug treatment. The compositions of the invention can be in a form suitable for, for example, parenteral (intravenous, intramuscular or intracavitary), oral, topical or rectal administration. The particular form of the composition may be, for example,
The composition is in the form of a solution, suspension, emulsion, cream, tablet, capsule, intracellular fat particle or microreservoir, especially in the form of a sterile injectable. Thus, another aspect of the present invention is to provide a method of treating cancer in the human or animal body by administering the anti-cancer agent of the present invention. A therapeutically effective dose in humans is believed to be in the range of 50-500 mg/Kg body weight. The compounds of the invention are expected to exhibit broad spectrum antitumor activity against a spectrum of leukemias, lymphoid malignancies and solid tumors, particularly cancers and sarcomas. Thus, in addition to use as a single agent, the compounds of the invention may be used, for example, as mitotic inhibitors (e.g., vincristine, vinblastine), alkylating agents (e.g., cyclophosphamide, melphalan, milleran, chlorambucil, mustin), anti-metabolites (e.g. 5-fluorouracil, 6-mercaptopurine, thioguanine, cytosine arabinoside, hydroxyurea), inter-culating antibiotics (e.g. adriamycin, bleomycin) or enzymes. (eg, asparaginase) or can be mixed with them. The compounds of the invention can also be used as a component of combined modality treatments, including, for example, radiation therapy and/or surgery. The following examples further illustrate the invention. Example 1 Diethyl N-(4-(prop-2-enylamino)benzoyl)-L-glutamate and diethyl N-(4-(prop-2-ynylamino)benzoyl)-L-glutamate were each converted to 2-amino-6 -Alkylated with chloromethyl-4-hydroxyquinazoline. 2-ethoxyethanol (2.5ml/mmol)
2-amino-6-chloromethyl-4-hydroxyquinazoline hydrochloride and diethyl N-(4-prop-2-enylamino)benzoyl)-L-glutamate or diethyl N-(4-(prop-2-
The stirred mixture with inylamino)benzoyl)-L-glutamate (1 molar equivalent) was heated to 80<0>C. Triethylamine (2 molar equivalents) was then added to the clear solution, which was purified by TLC (10% EtOH
−CHCl ₃ /SiO ₂ ) is the best (but not perfect)
Heating was continued until the conversion was indicated. (Reaction times were 5 hours at 100° C. and 6 hours at 105° C., respectively.) The solvent was removed in vacuo, the residue was dissolved in CHCl ₃ and the solution was washed with water. The extract was dried (MgSO ₄ ) and concentrated to give the crude product. This was purified by HPLC using silica gel (180g), and 5%
Eluted with EtOH- _CHCl3 . The product is diethyl N-(4-(N-((2-amino-4-hydroxy-6-quinazolinyl)methyl)
Prop-2-enylamino)benzoyl)-L-
Glutamate (ester of CB3716) and diethyl N-(4-(N-((2-amino-4-hydroxy-6-quinazolinyl)methyl)prop-2-ynylamino)benzoyl)-L-glutamate (CB3721), amorphous It was a rubbery solid. Their structures were determined by elemental analysis and NMR (DMSO− _D6
The yield and melting point were as follows: Ester of CB3716: -21.5%, 107-112°C;
CB3721: −27.5%, 143−147℃. Example 2 Diethyl N-(4-(N((2-amino-4-hydroxy-6-quinazolinyl)methyl)prop-2
-ynylamino)benzoyl)-L-glutamate, the product of Example 1, was also prepared by the following method: 2 in N,N-dimethylacetamide (300 ml).
-amino-6-bromomethyl-4-hydroxy-
A solution of quinazoline hydrobromide (15.1 g) and diethyl N-(4-(prop-2-ynylamino)benzoyl)-L-glutamate (16.2 g) was prepared at 25°C in the presence of calcium carbonate (13.5 g). too
Stirred for 65 hours. Calcium carbonate for 15 minutes
Centrifuged at 2000 rpm (this can also be removed by passing through Celite). Then add the solvent to 40
It was removed at ℃/0.2mm. The crude product thus obtained was dissolved in 5% V/V ethanol in chloroform, except for a small amount of residue, and chromatographed on silica gel (Merck, Art 15111).
Eluted with EtOH- _CHCl3 . Product diethyl N
-(4-(N-((2-amino-4-hydroxy-6
-quinazolinyl)methyl)prop-2-ynylamino)benzoyl)-L-glutamate (CB3721) was a gray solid, mp 146-148°C (uncorrected), 69% yield. The structure was confirmed by elemental analysis. Actual value: C, 62.54; H,
5.65; N, 13.07. Calculated _{for C23H31N5O6} : C, 63.03: _H , 5.86; _N , 13.13%. The starting materials used in Examples 1 and 2 above were prepared as follows: Diethyl N-(4-(prop-2-enylamino)benzoyl)-L-glutamate was prepared from diethyl N-(4-aminobenzoyl). )-L-glutamate (5.00 g) in ethanol solution (100 ml) at 60°C.
by treatment with allyl bromide (5.4 ml) in the presence of potassium carbonate (2.14 g) for 8 hours.
After removing the solvent in vacuo, the residue was dissolved in chloroform and the solution was washed with water. The extract was dried (MgSO ₄ ) and concentrated to give the crude product. this
Purified by HPLC using 50:50 chloroform:petroleum ether as eluent. To produce diethyl N-(4-(prop-2-ynylamino)benzoyl)-L-glutamate, the reaction was run at 70 °C using 10 ml of an 80% solution of propargyl bromide in toluene instead of allyl bromide.
The above procedure was repeated, except for a 4 hour period. The structure was confirmed by elemental analysis and NMR spectroscopy, and the yield and melting point were: 76.5%, 60−61°C; and 55.5%, respectively.
%, 98−99℃. Example 3 N-(4-(N-((2-amino-4-hydroxy-6-quinazolinyl)methyl)prop-2-enylamino)benzoyl)-L-glutamic acid (CB3716) and N-(4-(N -((2-amino-
4-Hydroxy-6-quinazolinyl)methyl)prop-2-enylamino)benzoyl)-L-glutamic acid (CB3717) from the corresponding diethyl ester, which can be prepared as described in Example 1 or 2, in 25% aqueous EtOH (20ml/mmol)
and treatment with 1N aqueous NaOH (4 molar equivalents). After 1 hour, the solution was clarified by passing through "Hyflo" and the PH was brought to 4.7 with 1N aqueous HCl. A gel-like precipitate of product formed. The inorganic ions are then suspended for three cycles.
Removed by centrifugation-decantation. The amorphous gray hygroscopic solid was then vacuum dried at 100°C. It was divided into two systems: (i) n-butanol:acetic acid:water (5:2:3): (ii) chloroform:
Homogenized by TLC in methanol:acetic acid (75:20:5). The structure was confirmed by elemental analysis and UV spectroscopy (spectrum measured in 0.1N aqueous NaOH). Yield, melting point and UV data were as follows: CB3716:-46% yield mp207-9
℃, UV〓(nm)(〓) Max: 311.5 (29100), 276
(17600), 228.5 (49300), Minimum: 282 (16900), 252 (9060). CB3717: - Yield 81%, mp232 -5℃, UV〓(nm) (〓) Maximum: 301.5 (26600), 279 (23900), 229 (50700), Minimum: 284 (23700), 251.5 (9800). for use,
CB3717 was prepared using physiological saline (0.9% W/V
It can be prepared as the disodium salt at pH 8.5-9.0 in a solution of NaCl) at a concentration of 10 mg/ml. In Examples 1 and 3, melting points were measured and corrected on a Kofter block, and elemental analyzes were performed on the folate analogs using both a catalytic additive and an oxygen donor. Each compound should be satisfactory for C, H and N (±0.4%)
It had a trace analysis value. NMR spectra were obtained using a Perkin-Elmer R12B 60MHz spectrophotometer except that of the product of Example 1 on a Bruker at 50°C.
Obtained with HFX 90MHz Fourier conversion instrument. Preparative HPLC is a Jobin-Yvon chromatographic prep.
Chromatospac Prep10) with silica gel (Merck
The eluent was run through a Cecil 212A variable wavelength ultraviolet monitor. TLC is Polygram
PG23 pre-coated silica plate (Macherey−
Nagel & Co.). Centrifugation was performed at 20000g. Example 4 a Diethyl N-(4-(N-((2-amino-4-
Hydroxy-6-quinazolinyl)methyl)prop-2-ynylamino)benzoyl-L-glutamate (6.436 g) was suspended in water (130 ml) and 0.1N aqueous NaOH (36.19 ml), 3.00 molar equivalents).
Processed with. The mixture was shaken very vigorously for 45 minutes to give a solution. The slightly entrained material was processed by passing through grade 3 sinter. Six hours after the initial caustic addition, 0.1N aqueous HCl (13.00ml) was added to reach a pH of 8.45. This solution, containing a few precipitated particles, was lyophilized to give N-(4-(N
-((2-Amino-4-hydroxy-6-quinazolinyl)methyl)prop-2-ylamino)benzoyl-L-glutamic acid (CB3717) disodium salt and a mixture of sodium chloride,
Obtained as a cream-colored flake-like amorphous powder. HPLC analysis shows that N-(4-(N-((2-amino-4-hydroxy-6-quinazolinyl)methyl)prop-2-ynylamino)benzoyl-
L-glutamic acid (CB3717) is an organic substance 99
%. b In other preparations, diethyl N-(4-(N-
((2-Amino-4-hydroxy-6-quinazolinyl)methyl)prop-2-ynylamino)benzoyl)-L-glutamate (14.0 g) in water (400 ml), ethanol (68 ml) and 0.1N aqueous solution.
Stir with NaOH (78.7ml). This mixture was lyophilized to give a cream colored solid (15.5g). Since this mixture is highly hygroscopic, it was equilibrated with the atmosphere before microanalysis. Actual value: C, 43.41; H, 4.03; N,
10.91: Cl, 8.68; Na, 11.53; _H2O , 7.69;
C ₂₄ H ₂₁ N ₅ O ₆ Na ₂ +1.31 (NaCl) + 0.51 (HCl) +
_{2.5 ( H20 ) ( = C24H26.51N5O8.5Na3.31Cl1.82 ) _ _ _}
Calculated values for C, 43.56; H, 4.03; N,
10.58; Cl, 9.75; Na, 11.50; _H2O , 6.81%.
More accurate values for Cl and H ₂ O are:
Obtained from separately analyzed samples obtained by equilibrating dry material with air for 24 hours - actual values:
Cl, 9.48; _H2O , 7.12%. HPLC of this batch
Analysis showed that CB3717 constitutes 98% of the organic material. Example 5 2-Amino-6-bromomethyl-4-hydroxyquinazoline (1 molar equivalent), calcium carbonate (molar equivalent) and diethyl N-(4-( phenacylamino)benzoyl)-L-glutamate (1
molar equivalents) was stirred at room temperature for 60 hours. The mixture was filtered and the solvent removed in vacuo. The resulting oil was purified by chromatography on silica gel, eluting with 5% methanol-methylene chloride. The product diethyl N-(4-(N-((2
-amino-4-hydroxy-6-quinazolinyl)
methyl)phenacylamino)benzoyl)-L-
Glutamate (CB3744) has a melting point of 148-150°C, confirmed by elemental analysis and NMR. A second reaction using 2,6-dimethylpyridine instead of calcium carbonate gave the same product. In a similar manner, the following compounds were prepared by alkylation of the appropriate amine using calcium carbonate as the base:

TABLE The amine starting material used in Example 5 above can be prepared as follows. Diethyl N-(4-aminobenzoyl)- in N,N-dimethylacetamide (2.5 ml/mmol)
A mixture of L-glutamate (1 molar equivalent), 2,6-dimethylpyridine (1 molar equivalent) and ethyl bromoacetate (1 molar equivalent) was stirred at 90°C for 5 hours. The mixture was poured into water, extracted with ethyl acetate, and the organic phase was washed successively with water, 1N sulfuric acid, and brine. After drying over anhydrous sodium sulfate, the solvent was removed in vacuo and a colored oil was obtained which crystallized when triturated with cyclohexane. The product diethyl N-(4-(ethoxycarbonylmethylamino)benzoyl)-L-glutamate (
b: ^R2 = _-CH2COOC2H5 ) has a melting point of 84-85 _[ deg. _] C and was confirmed by elemental analysis. In a similar manner, the following compounds were prepared by treating the amine with the appropriate alkylating agent.

[Table] Note 1 In the reaction described in A) above, 2,6-
Anhydrous potassium carbonate (0.5 molar equivalent) can be used in place of dimethylpyridine (1 molar equivalent). 2 Stir at 110℃ for 4 hours. 3 Stir at room temperature for 16 hours. 4 Recrystallized from ethanol. Diethyl N-(4-amino-
2-methylbenzoyl)-L-glutamate (1
molar equivalent), 2,6-dimethylpyridine (1.1 molar equivalent) and propargyl bromide (1.1 molar equivalent)
The mixture was stirred at room temperature for 16 hours. The mixture was poured into water and extracted with ethyl acetate, the organic phase was washed with water, dried over anhydrous sodium sulfate and the solvent was removed in vacuo. The resulting oil was purified by chromatography on silica gel and elution with 3% ethyl acetate-methylene chloride. Product diethyl N-
(4-prop-2-ynylamino)-2-methylbenzoyl)-L-glutamate (c:R ² =-
_CH2C≡CH , X= _-CH3 , m=2) is 85-87℃
It has a melting point of , and was confirmed by elemental analysis and NMR. In a similar manner, the following compounds were prepared by acting on the relevant amines with appropriate alkylating agents.

[Table] C Diethyl N-(4-aminobenzoyl)-L-glutamate (1 molar equivalent), calcium carbonate (2.5
molar equivalent), epibromohydrin (5 molar equivalent)
and N,N-dimethylacetamide (one of the amines)
2 ml/mmol) of the mixture was stirred at 90° C. for 40 hours. The mixture was filtered and the solvent removed in vacuo. The resulting oil was purified by chromatography on silica gel and fusion with 25% ethyl acetate-hexanes. Product diethyl 4-
(3-Hydroxy-1-azetidinyl)benzoyl-L-glutamate has a melting point of 87-90°C, confirmed by elemental analysis and NMR. D 55% ethanol-water (20ml/
diethyl N-(2-chloro-4
-Nitrobenzoyl)-L-glutamate was added sodium bicarbonate (6 molar equivalents), followed by sodium dithionate (3 molar equivalents) in portions over 20 minutes.
After stirring for an additional hour, the ethanol was removed in vacuo and the residue was diluted with brine. The compound was extracted with ethyl acetate, back-extracted into 2N hydrochloric acid, neutralized with sodium bicarbonate, and re-extracted into ether. The ether extract was dried over anhydrous sodium sulfate and the solvent was removed in vacuo to obtain an oil;
This was allowed to stand and crystallized. Product diethyl N-(2-chloro-4-aminobenzoyl)-L
- Glutamate has a melting point of 73-75°C, confirmed by elemental analysis and NMR. E Diethyl N-(2-methyl-4-nitrobenzoyl) in ethanol solution (8 ml/mmol)
-L-glutamate was hydrogenated using 10% palladium on charcoal until the theoretical amount of hydrogen was consumed. The reaction solution was filtered and the solvent removed in vacuo to give an oil which crystallized on standing. The product diethyl N-(2-methyl-4-aminobenzoyl)-L-glutamate has a melting point of 98-100°C and was analyzed by elemental analysis and
Confirmed by NMR. F Toluene (0.5 ml/mmol) cooled to 0°C
Pyridine (25 molar equivalents) was added to a stirred suspension of diethyl L-glutamate hydrochloride (1 molar equivalent) in 2-methyl-4-nitrobenzoyl chloride dissolved in toluene (0.7 ml/mmol). (1.5 molar equivalent) solution of
Added dropwise over 20 minutes. Stir for 30 more
Continued for 1 minute at 0°C and then 1 hour at room temperature.
After diluting with toluene, this mixture was diluted with water, 2N
Extracted with hydrochloric acid, 5% sodium bicarbonate solution and water. The organic phase was dried over anhydrous magnesium sulfate and the solvent was removed in vacuo. The resulting oil was chromatographed on silica gel and ethyl acetate.
Purified by elution with petroleum ether (1:1) at 60-80°C. Product diethyl N-
(4-Nitro-2-methylbenzoyl)-L-glutamate has a melting point of 67-68°C, confirmed by elemental analysis and NMR. Example 6 Diethyl N-(4-(N-((2-amino-4-hydroxy-6-quinazolinyl)methyl)phenacylamino)benzoyl)-L-glutamate (1
molar equivalents) were suspended in 33% ethanol-water (40 ml/mmol) and dissolved in 1N sodium hydroxide solution (3
millimolar equivalents) and this mixture was incubated at room temperature for 18
I stirred the time. 0.1N hydrochloric acid aqueous solution (3 molar equivalents)
A gel-like precipitate was obtained, which was purified by 6 cycles of centrifugation-decantation and resuspension in distilled water. The final aqueous suspension was lyophilized to yield an amorphous white solid. Product N
-(4-(N-((2-amino-4-hydroxy-6
-quinazolinyl)methyl)phenacylamino)benzoyl)-L-glutamic acid (d:

[Formula] No X group, m = 2. CB3733) is elemental analysis and proton NMR
Confirmed by (solution in DMSO-d ₆ ): 〓2.0
(2H, m), 2.3 (2H, m), 4.4 (1H, m), 4.7
(2H, s), 5.2 (2H, s), 6.6 (2H, d), 6.5−
7.0 (2H, b), 7.2 (1H, d, J7Hz), 7.4-7.8
(6H, m), 7.85 (1H, d, J3Hz), 8.05 (2H,
m), 8.15 (1H, d). The hydrolysis reaction in the above example proceeds from the diester to the monoacid to the monoester, and this intermediate is hydrolyzed in situ to the dibasic acid without isolation. In a similar manner, the following compounds were prepared by hydrolysis of the appropriate esters.

【table】

[Table] Example 7 6 in N,N-dimethylacetamide (25 ml) containing suspended sodium carbonate (15 mmol)
A solution of -bromomethyl-4-hydroxy-2-trimethylacetamide quinazoline (7.5 mmol) was prepared at 60<0>C. A solution of diethyl N-(4-(3-acetoxypropylamino)benzoyl)-L-glutamate (7.5 mmol) in N,N-dimethylacetamide (10 ml) was added over 90 minutes to the stirred reaction mixture. . Further at 60℃
After stirring for 12 hours, the reaction mixture was filtered and the liquid was extracted with ethyl acetate after addition of water (10 volumes). The organic phase was washed with water and the solvent was removed in vacuo to give an oil. The oil was chromatographed on silica gel (Merck 60) using a mixture of methylene chloride and ethyl acetate as eluent. The appropriate fractions were evaporated under reduced pressure to give an oil. This product is vacuum dried to form a foam-like solid diethyl N-(4-(N-
((4-hydroxy-2-trimethylacetamide-
6-quinazolinyl)-3-acetoxypropylamino)benzoyl)-L-glutamate, melting point 61
-64℃ was obtained. The amine starting material used in Example 7 above can be prepared as follows. Diethyl N-(4-aminobenzoyl)-L-glutamate (0.02M) and 3-acetoxypropyl bromide (0.02M) were dissolved in potassium carbonate ( 0.02M). The product was isolated as in Example 5B) and the resulting oil was chromatographed on silica gel (Merck 6) using a mixture of methylene chloride and ethyl acetate (2:1) as eluent to give an oil, which Crystallized on standing; mp 59-62°C. Example 8 Diethyl N-(4-(N-((4-hydroxy-2
-Trimethylacetamide-6-quinazolinyl)methyl)-3-acetoxypropylamino)benzoyl)-L-glutamate (0.74 mM) was dissolved in 5 equivalents of aqueous sodium hydroxide solution (0.2, 18.4 ml).
The mixture was hydrolyzed in ethanol (75 ml) at 50° C. overnight. PH the product with 0.2N aqueous HCl
4 and isolated as in Example 6 and then prepared as in Example 6 to prepare N-(4-(N-((2-amino-4-hydroxy-6-quinazolinyl)methyl)-3-hydroxypropylamino)benzoyl)-L - Glutamic acid (CB3729) was obtained; melting point 220°C (d). NMR
(90MHz, in DMSO-d ₆ ): 〓1.7-2.5, multiplet (6H); 〓3.6, multiplet (4H); 〓4.4, multiplet (1H); 〓4.7, singlet (2H); 〓6.5 ,, wide singlet (2H); 6.7, doublet J=8.5Hz (2H);
7.2, double line J = 8Hz (1H); 〓7.5, double line J =
1.5, 8Hz (1H) doublet; 7.7, singlet and doublet (3H); 8.1, doublet J = 7Hz (1H). Example 9 2- in N,N-dimethylacetamide (20ml)
Amino-6-bromomethyl-4-hydroxyquinazoline hydrobromide (1.00g) and diethyl N
A solution of -(4-(((5-uracilyl)methyl)amino)benzoyl)-L-glutamate (1.00 g) was stirred in the presence of calcium carbonate (0.67 g) at room temperature for 140 hours. Calcium carbonate was removed by filtration through celite, then the solvent was
It was removed at ℃/0.05mm. The crude product thus obtained was dissolved in 20% V/V ethanol in chloroform, except for a small amount of residue, and chromatographed on silica gel (Merck, Art 15111) and 20%
Elute with EtOH- _CHCl3 . The precipitated fractions were combined and concentrated to ethanol by repeated evaporation of the final solvent plus ethanol. volume
The resulting white crystals were made up to 50 ml and were obtained by centrifugation, washed by resuspension in ethanol, centrifugation and decantation. Drying (P ₂ O ₅ )
The impure product (32.7%) was obtained as a gray solid, melting point >350°C (with slight decomposition up to this temperature). NMR spectrophotometry reveals that the products are quinozoline, uracil and benzoyl-
The glutamate moiety is converted into diethyl N-(4
-(N-(2-amino-4-hydroxy-6-quinazolinyl)methyl-N-(5-uracilyl)methylamino)benzoyl)-L-glutamate in the correct 1:1:1 ratio. . The amine starting material used in this Example 9 can be prepared as follows. Diethyl N-(4-aminobenzoyl)-L-glutamate (3.22 g) was 5-diluted in N,N-dimethylacetamide solution (60 ml) for 70 hours at 23°C.
Treated with chloromethyluracil (1.93g) in the presence of calcium carbonate (3.00g). Calcium carbonate was removed by filtration through Celite, and the crude product was
Obtained by removal of solvent at °C/0.2mm. When an attempt was made to purify this oil by partitioning it between chloroform and water, an emulsion-like precipitate formed in the organic layer. The aqueous layer was decanted, the remaining suspension was filtered, and the white crystals retained were washed with hot ethanol. The combined liquid and washing liquid were evaporated to dryness. The resulting solid was extracted first with hot chloroform and then with dichloromethane to remove some starting amine. A final recrystallization of the remaining solid from ethanol yields fine needle-like crystals as the product,
A melting point of 183-200°C was obtained. The structure was confirmed by elemental analysis and NMR spectrophotometry. Example 10 Diethyl N-(4-(N-(2-amino-4-hydroxy-6-quinazolinyl)methyl-N-(5
-uracilyl)methyl-amino)benzoyl)-
L-glutamate (0.327g) was suspended in a mixture of water (7ml) and ethanol (2ml) and treated with 1N aqueous NaOH (6 molar equivalents). Upon vigorous shaking, a solution formed within 30 minutes. 1.5 more
After an hour, the solution was clarified by filtration and the PH
Bring to 4.0 with 1N aqueous HCl. A granular precipitate of product occurred. The inorganic ions were then separated by three cycles of suspension-centrifugation-decantation. Vacuum drying of the anhydrous white solid at 40°C yields the pure diacid N(4-(N-(2-amino-4-hydroxy-
6-quinazolinyl)methyl-N-(5-uracilyl)methyl-amino)benzoyl)-L-glutamic acid (CB3743) is dihydrate (68%), melting point 250
-255℃. The structure was determined by elemental analysis (C,
H,N) and UV spectrophotometry (0.1N aqueous NaOH
This was confirmed by the spectrum measured in 〓
(nm) (〓) Maximum: 310 (29200), 276.5 (22000),
230 (50700); Minimum: 282.5 (21500), 251.5
(11600). Example 11 2-Amino- in dimethylacetamide (8 ml)
6-bromomethyl-4-hydroxyquinazoline hydrobromide (0.420 g) and diethyl N-(4-
(2-fluoroethylamino)benzoyl)-L-
A solution of glutamate (0.370g) was stirred in the presence of calcium carbonate (0.417g) for 45 hours at room temperature (23°C). Calcium carbonate was removed by filtration through Celite and then the solvent was removed at 40-55°C/0.02mm. The crude product thus obtained was dissolved in 5% V/V ethanol in chloroform, except for a small amount of residue, and silica gel (Merch,
Chromatographed with Art15111, 250g), 5%
Eluted with EtOH- _CHCl3 . Product diethyl N
-(4-(N-((2-amino-4-hydroxy-6
-quinazolinyl)methyl)2-fluoroethylamino)benzoyl)-L-glutamate was a white solid, melting point 115-120°C (46.3%). Structure determined by elemental analysis (C, H, N and F) and NMR
Confirmed by spectrophotometry. The amine starting material used in this Example 11 was prepared as follows. Diethyl N-(4-aminobenzoyl)-L-glutamate (3.22 g) in a DMF solution (40 ml)
2-Fluoroethyl p-toluenesulfonate (4.36 g) (WFEdgell and
L.Parts, J.Am.Chem.Soc., 77 , 4899 (1955))
and treated in the presence of calcium carbonate (3.00 g). After 2 hours and 45 minutes it was necessary to add more reagent (4.36 g). Calcium carbonate was removed by filtration through Celite, and after removal of the solvent in vacuo, the residue was dissolved in chloroform and the solution was washed with water. The extract was dried (MgSO ₄ ) and concentrated to give the crude product. This is silica gel (Merck,
Purified by HPLC using 50:50 chloroform:petroleum ether as eluent. White crystals, (11.3%) melting point 105.5-106.5℃,
was gotten. The structure was confirmed by elemental analysis (C, H, N and F) and NMR spectrophotometry. Example 12 Diethyl N-(4-(N-((2-amino-4-hydroxy-6-quinazolinyl)methyl)-2-fluoroethylamino)benzoyl)-L-glutamate (0.206 g) was dissolved in water (4.5 ml) and ethanol (1 ml) and 1N aqueous NaOH.
(5 molar equivalents). If you shake it vigorously,
A solution formed within 5 minutes. After 2 hours, the solution was clarified by filtration and the pH was brought to 4.0 with 1N aqueous HCl.
A gelled precipitate of product occurred. Inorganic ions were then removed by three cycles of suspension-centrifugation-decantation. Vacuum drying of the amorphous white solid at 70°C (melting point >350°C, some darkening and slow decomposition from 245°C) yields N-(4-N-
((2-amino-4-hydroxy-6-xazolinyl)methyl)2-fluoroethylamino)benzoyl)-L-glutamic acid (CB3731), yield 60.1
%, obtained. The structure was determined by elemental analysis (C, H, N and F) and UV spectrophotometry (spectrum is 0.1N
(measured in aqueous NaOH). UV 〓 (nm) (〓) Max: 307 (29200), 275
(19500), 227.5 (49700); Minimum: 279 (19200), 250
(9100). Example 13 Diethyl N-(4-(N-) in glacial acetic acid (21 ml)
((2-amino-4-hydroxy-6-quinazolinyl)methyl)amino)benzoyl)-L-glutamate (SPAcharya and JB Hynes, J.
Heterocyclie Chem. 12 , 1283 (1975)) (2.111g)
To the solution was added paraformaldehyde (0.384 g, 3 molar equivalents), potassium cyanide (0.832 g, 3 molar equivalents) and anhydrous zinc chloride (2.119 g, 3.65 molar equivalents). The resulting slurry was stirred to remove moisture for 16.5 hours and partitioned between chloroform and water. The organic phase was removed, washed with water and dried (MgSO ₄ ). Evaporation of the solvent left the crude product as a yellow solid. This is silica gel (Merck, Art
11695), which was applied to the column each time in 6% ethanol in chloroform and eluted with this same solvent. This product is added to the third
The DMF solution was passed downward through a silica column (Merck, Art 15111), obtained by evaporation of the DMF solution at 70 °C (trace amounts of DMF were removed by vacuum drying (P ₂ O ₅ ) for 48 h).
Purification was achieved by coating on silica (Merck, Art 7734) (removed by method). Elution was with 6% ethanol in chloroform. The product diethyl N-(4-((2-amino-
4-Hydroxy-6-xazolinyl)methyl)cyanomethylamino)benzoyl)-L-glutamate was a gray solid, melting point 145-160°C.
Yield 44%. The structure was confirmed by elemental analysis and PMR spectrophotometry. Actual value: C, 60.53; H,
5.85; N, 15.86. Calculated value for C ₂₇ H ₃₀ N ₆ O ₆ C, 60.66; H,
5.66; N, 15.72%. −CH ₂ CN part (〓4.73, s,
2H) is bonded to N ¹⁰ , which means that the signals from the 3' and 5' protons (d, 2H) are from 〓6.62 to 〓
6.95, indicating the removal of electrons from the cross-linked region of the molecule. Example 14 Diethyl N-(4-(N-((2-amino-4-hydroxy-6-quinazolinyl)methyl)cyanomethylamino)benzoyl)-L-glutamate (0.108 g) was dissolved in water (2.5 ml). Ethanol (0.5ml)
and treated with 1N aqueous NaOH (3 molar equivalents). If you stir for a short time (10 minutes),
Dissolution was almost complete. After a further 1.25 hours, some entrained material was removed by filtration and the pH
4.1 with 1N aqueous HCl. A gel-like precipitate of product formed. The inorganic ions were then separated by two cycles of suspension-centrifugation-decantation. The clear solid product N-(4-(N-((2-amino-4-hydroxy-6-quinazolinyl)methyl(cyanomethylamino)benzoyl)-L-glutamic acid (CB3726) with P ₂ O ₅ Finally dried in vacuum at 80℃.No melting point.It is 210-250℃
It was made into a resin between. Yield 46%. Elemental analysis: Actual values: C, 57.89; H, 4.77; N, 17.59. _Calculated values _{for C23H22N6O6 C} , 57.74; H, 4.63; N, 17.56 _% . The following formulations are illustrative of the pharmaceutical compositions of the invention that can be prepared. W/W per tablet Active compound (microcrystalline) 83.3% “Avicel” – (microcrystalline cellulose)
12.7% Polyvinylpyrrolidone 1% Alginic Acid 2% Magnesium Stearate 1% The compound of the invention is mixed with "Avicel", polyvinylpyrrolidone is added and dissolved in a sufficient amount of industrial methylated spirit 74° OP. It can be melted into a mass suitable for granulation.
The agglomerate can be granulated by passing through a 20 mesh sieve (British sieve standard) and the resulting particles can be dried at temperatures below 50°C. The dried particles can be passed through a 20 mesh sieve and then the alginic acid and magnesium stearate can be added and mixed with the particles. The product can be compressed into tablets. The following formulations can be prepared by the same method. W/W per tablet Active compound 62.5% "Avicel" (microcrystalline cellulose) 33.5% Polyvinylpyrrolidone 1% Alginic acid 2% Magnesium stearate 1% Furthermore, tablets with the following composition can be produced. W/W per tablet Active compound (micronized) 83.3% Lactose (300 mesh) 6.3% Corn starch 5% Gelatin 3.3% Magnesium stearate 2% These tablets contain the compounds of this invention in combination with lactose and the required corn. Mix with half of the total amount of starch, and add gelatin to this mass.
It can be produced by adding a 50% W/V aqueous solution. The product can be granulated by passing through a 16 mesh sieve and the resulting particles dried to constant weight at temperatures below 60°C. The dried particles can be passed through a 20 mesh sieve, mixed with the magnesium stearate, and mixed with the remainder of the corn starch. This product can be compressed into tablets. Injectable solutions can be prepared as follows. When the compound of the invention is in the acid form, it (2 mmol) is treated with NaOH in an amount sufficient to neutralize it.
Alternatively, it can be dissolved in 100 ml of 0.9% NaOH aqueous solution containing a suitable base. The compound of Example 2 was added to TS in vitro.
and as an inhibitor of cell growth and as an inhibitor of L1210 tumors in vivo. Since methotrexate (MTX) resistance is an important issue in cancer treatment, the compounds of the present invention were also tested with this in mind. increased DHFR
The cross-resistance of the MTX-resistant cell line L1210/R71, which is known to have normal MTX transport, was measured in culture, and the cross-resistance of the MTX-resistant cell line L1210/R71, which is known to have normal MTX transport, was determined in culture and compared with labeled MTX or folic acid for transport into L1210 cells. The ability of compounds of the invention to compete was evaluated. Enzymology Compounds CB3716 and CB3717 were each dissolved in N/10NaOH and neutralized before use.
L1210 in log phase harvested from animals
Cell cytosol was partially purified for TS I ₅₀ and Ki measurements to remove phosphatases that degrade dUMP to deoxyuridine from the preparation. ( _I50 was defined as the concentration of inhibitor required to reduce the reaction rate of TS by 50%).
DHFR was purified by affinity chromatography and Ki measurements were performed. The results are shown in Table 1 below and comparisons are made for 10-propyl compounds corresponding to CB3716 and 3717.

[Table] The presence of allyl and propargyl groups at the 10-position, compared to propyl groups, respectively
giving nearly 3-fold and 30-fold increased TS suppression values,
At the same time, DHFR inhibition remains at the same level. Suppression of TS by compound CB3717 was characterized and
It was found to be competitive with ^N5 , ^N10 -methylenetetrahydroforate at approximately 1.1 nM Ki. Cell culture L1210 and L1210:R71 cells were grown in RPMI1640 medium (Flow Flow) containing 10% horse serum.
Laboratories, Irvine, Scotland, UK) in continuous suspension medium. Cells were diluted to 10 ⁵ /ml and spiked (diluted in medium and sterilized by Millpore filtration) before the start of each culture.
Cell counts were performed after 24 and 48 hours using a modified Neu-bauer hemocytometer. _ID50 was defined as the drug concentration required to reduce the 48 hour counts to 50% of the control. The ID ₅₀ concentrations in cell culture for L1210 and L1210:R71 (MTX resistance) are shown in Table 2 below. The toxicity of the drug to both cell lines not only increased with increasing 10-substituted unsaturation;
The degree of cross-resistance of L1210:R71 is no longer noticeable. L1210:R71 is approximately 600 times more resistant to MTX.

[Table] *R = Compounds corresponding to propyl CB3716 and 3717. Reversal of the toxicity of compound CB3717 was induced by thymidine (10
〓M), but only partial reversal was achieved using folic acid concentrations up to 100〓M. These results are shown in FIG. 1 of the accompanying drawings. This figure shows 2〓M in culture.
Showing protection of L1210 cells from CB3717. ●CB3717 alone; OCB3717 + folic acid 5〓M; □
CB3717 + folic acid 50〓M; ■CB3717++ + folic acid 500〓
M；▲CB3717＋TdR1〓M；△CB3717＋TdR10
〓As these results show, CB3717 is TS
In particular, it acts as the main position. Because its affinity for DHFR is 10 times lower than that of TS, and adding thymidine to the culture medium can confer virtually complete protection of L1210 cells, whereas folic acid can be used at higher concentrations (100 μm). Even when they do, they do not reverse the effects of the drug. Compounds produced in other Examples were similarly evaluated in vitro as inhibitors of TS and cell growth, and the following results were obtained. (In these experiments, the TS used was >2000-fold from L1210 cells,
(purified by affinity chromatography). [Even if these compounds do not exhibit particularly good TS inhibition, they have significance as anticancer agents in that they exhibit toxicity against L1210 cells in tissue culture. ]

【table】

[Table] Transfer studies Transfer studies were carried out for compound CB3717 as a representative compound of the present invention by culturing cells in the presence of a labeled substance, removing a portion, separating and washing the cells by centrifugation, and pelleting. It was carried out by counting. The effect of varying concentrations of compound CB3717 in the medium on the transport of tritiated methotrexate and tritiated folic acid is shown in Figure 2 of the accompanying drawings. ●4〓M MTX; △4〓M folic acid Transport of both of these compounds
Suppressed by CB3717. Thus, the compound clearly eliminates the problem of using methotrexate, where resistance to methotrexate can arise due to increased cellular DHFR content and decreased cell membrane transport machinery that transports both methotrexate and tetrahydrofolate derivatives. The site of cytotoxicity of CB3717 is TS and not DHFR, and transport studies show that this compound can clearly be transported for methotrexate as well as for folic acid depending on the route used. CB3717 produced a dose-dependent decrease in the transport of both of these substances. As is clear from the results of the above in vitro and in vivo tests, the compound of the present invention has a large TS suppressing effect and tumor cell growth suppressing effect, and has an excellent antitumor effect. Treatment of L1210 Tumor-Containing Animals L1210 tumors were routinely contained in DBA2 mice. For the experiment, take 5 x ¹⁰⁴ female C57B1 cells.
×DBA2F1 hybrid mice were implanted intraperitoneally 3 days before treatment, and at the time of treatment, approximately 2×
gave an established tumor of 10 ⁶ cells. Compound
CB3717 was dissolved in N/10NaOH, diluted to the appropriate volume, and the PH was adjusted to 8.5 before intraperitoneal injection. The results are summarized in Table 3 below.

[Table] *See Figure 3 Figure 3 shows the survival rate of L1210-bearing mice injected with CB3717. ■ No treatment (10 animals) - MTX control □ No treatment (20 animals) - CB3717 control (animals
0.4 ml of 20 mM NaHCO ₃ buffer was injected daily x day. ) ●MTX 3mg/Kg daily x 5 (10 animals) ▲CB3717 125mg/Kg daily x 5 (10 animals) △CB3717 200mg/Kg daily x 5 (10 animals) Differences between CB3717 and MTX treated animals teeth,
Using the logarithmic ranking test, highly significant P<
It is 0.001. A single dose of compound CB3717 up to 160mg/Kg
It was not toxic and had very little therapeutic effect. The 5 daily dosing schedule increased mean survival times, and these increases were scaled by dose (see Table 3). 9 out of 10 long-term (>120 days) survivors at doses of 125 and 200 mg/Kg.
There was one. The effectiveness and solubility of the drug precluded the use of higher doses. Appropriate controls were injected with bicarbonate at PH 8.5, since the injection solution can be neutralized to PH 8.5 while preserving solubility. None of the control groups showed an increase in lifespan. For comparison purposes, other groups of animals were treated with various doses of methotrexate based on the same schedule. The optimal dose of methotrexate, 3 mg/Kg, reduces the average lifespan to 71
% increase, but gave only 2/10 long-term survivors. These animals did not exhibit significant amounts of weight loss or other signs of toxicity during drug treatment. This is illustrated in Figure 4 of the accompanying drawings, which shows 10 animals treated with 5 daily doses of CB3717, 125 mg/Kg/day (■) and 200 mg/Kg/day (▲). The body weight changes of mice; untreated tumor-containing controls (●); and untreated tumor-free controls (○) are shown. Results of studies involving treatment of L1210 tumor-containing animals show a significant increase in long-term survival compared to animals optimally treated with methotrexate. Toxicity Compounds produced in Examples 3 and 4 above
When CB3717 was intraperitoneally injected into mice for 5 consecutive days, the LD ₅₀ value was 330 mg/Kg/day (male) and
It was 380 mg/Kg/day (female).

[Brief explanation of the drawing]

Figure 1 shows the protection of L1210 cells from 2〓M CB3717 in culture. Figure 2 shows the effect of varying concentrations of compound CB3717 in the medium on the transport of tritiated methotrexate and tritiated folic acid. Figure 3 shows the survival rate of L1210-bearing mice injected with CB3717. FIG. 4 shows changes in body weight of mice.

Claims (1)

[Claims] 1 formula In the formula, R is 1) a linear or branched unsaturated hydrocarbon group containing 2 to 6 carbon atoms; or 2) CN, COOH or a salt or ester thereof, OH, =O, a halogen atom , _NH2 ,
CONH ₂ , COC ₆ H ₅ , oxiranyl or 5-
A straight or branched chain containing up to 6 carbon atoms substituted with at least one substituent which is uracilyl (when the substituent is =O the hydrocarbon group R contains at least 2 carbon atoms) represents a branched saturated or unsaturated hydrocarbon group; n is 0 or an integer of 1 to 4; X is a halogen atom, _C1 to _C4 alkyl, aryl, aralkyl, _C1 to _C4 alkoxy or represents a NO ₂ group, provided that when n is 2 to 4, the n Xs can be the same or different;
And Y is the formula or Here, m is an integer from 1 to 9 (poly-L
A 2-amino-4-hydroxyquinazoline derivative or a pharmaceutically acceptable salt or ester thereof, which represents a group of -glutamic acid residue). 2. The quinazoline derivative according to claim 1, wherein Y is a glutamic acid residue (b). 3. The quinazoline derivative according to claim 1 or 2, wherein n is 0. 4. The quinazoline derivative according to any one of claims 1 to 3, wherein R is a _C3 linear unsaturated hydrocarbon group. 5 N-(4-(N-((2-amino-4-hydroxy-6-quinazolinyl)-methyl)prop-2-
The quinazoline derivative according to claim 4, which is inylamino)benzoyl)-L-glutamic acid. 6 formula In the formula, R is 1) a linear or branched unsaturated hydrocarbon group containing 2 to 6 carbon atoms; or 2) CN, COOH or a salt or ester thereof, OH, =O, a halogen atom , _NH2 ,
CONH ₂ , COC ₆ H ₅ , oxiranyl or 5-
A straight or branched chain containing up to 6 carbon atoms substituted with at least one substituent which is uracilyl (when the substituent is =O the hydrocarbon group R contains at least 2 carbon atoms) represents a branched saturated or unsaturated hydrocarbon group; n is 0 or an integer of 1 to 4; X is a halogen atom, _C1 to _C4 alkyl, aryl, aralkyl, _C1 to _C4 alkoxy or represents a NO ₂ group, provided that when n is 2 to 4, the n Xs can be the same or different;
And Y is the formula or Here, m is an integer from 1 to 9 (poly-L-
An anticancer agent comprising, as an active ingredient, a 2-amino-4-hydroxyquinazoline derivative or a pharmaceutically acceptable salt or ester thereof, which represents a group of glutamic acid residues. 7. The anticancer agent according to claim 6, wherein Y is a glutamic acid residue (b). 8. The anticancer agent according to claim 6 or 7, wherein n is 0. 9. The anticancer agent according to any one of claims 6 to 8, wherein R is a _C3 linear unsaturated hydrocarbon group. 10 The active ingredient is N-(4-(N-((2-amino-
4-hydroxy-6-quinazolinyl)-methyl)
Prop-2-ynylamino)benzoyl)-L-
The anticancer agent according to claim 9, which is glutamic acid.