KR100751847B1 - Synthesis and characterization of new antifolate having a furo[2,3-d]pyrimidine derivatives - Google Patents

Abstract

In order to synthesize a novel anticancer agent, the present invention introduces a Sandmeier reaction to replace a p-amino group of para aminobenzoic acid with a thio (S) group to obtain a compound (3), which is obtained in the presence of NaBH ₄ in ethanol. Diethyl L-glutamate using DCC or DEPC (diethylphosphoro-cyanidate) in the presence of a novel compound, a pteroic acid derivative (4) and DMF (dimethylformamide), by reacting with a midine derivative (2). To obtain an ester compound of formula (5), followed by = S ⁺ -CH ₃ reaction and hydrolysis if necessary to obtain a compound in free acid form of formula (I).

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Anticancer agent, MTX derivative synthesis

Description

Synthesis and characterization of new antifolate having a furo [2,3-d] pyrimidine derivatives}

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The present invention is a novel aminopteridine of the following structural formula [I] as a derivative of aminofoterin, methotrecetate (amthopterin: MTX) and homofolic acid, which acts as an antagonist of folic acid as a compound which is an anti-metabolant. A thio benzoic acid compound and its manufacturing method are related.

Where
R is hydrogen, methyl, -Et or -CH ₂ CH ₂ CH ₃ .
R ¹ and R ² are each hydrogen, Na, methyl, ethyl, propyl or isopropyl.
It functions as an anticancer agent, which is a malignant tumor therapeutic agent similar to the amino puteridine thiobenzophosphate compound of formula (I), and a similar known compound is methotrexate (MTX; The Merk Index 11, 5908, 1989). It has a structure as follows.

The mesotrexate compound has many side effects such as inhibition of bone marrow function, reduction of white blood cells, ulceration of digestive organs, liver and kidney function disorders, and toxicity (LD50 iv in rats: 14 mg / kg, scherf) is also serious. It is true that a lot of attention is required in the input.
Therefore, while the present inventors are studying an anticancer agent having a safer malignancy treatment, CH ₃ and H are introduced instead of the amino (NH ₂ ) group at the C ₂ position with a puteridine nucleus. Diethylglutamate was reacted with pteroic acid, an intermediate synthesized by substituting S (thio) group instead of N-CH _{3 at} position ₁₀ , to obtain a new stable anticancer agent of formula (I). It is filed.

Compounds with a pteridine nucleus have been reported for anticancer action and antimalarial action. Among them, aminopteridin and amethopteridin (MTX), whose chemical structures were identified in 1940 as an antagonist of folic acid, were clinically well known for about 40 years. It is the most representative anticancer agent. MTX, an anticancer agent, is a 7,8-dihydroxypol by dihydrofolate reductase (DHFR), which is a folic acid known as an antagonist of folic acid, known as a single carbon transfering agent. Rate, which in turn is reduced by DHFR to form 5,6,7,8-tetrahydrofolate. This compound has a 2,4-diaminopyrimidine structure similar in structure to the 2-amino-4-oxo (3H) pyrimidine known as the enzyme binidng site, and thus competitively binds to an enzyme-linking active site. It has been shown to exhibit anticancer activity by inhibiting the action of folic acid. As such, the mechanisms for cell division and growth of animals and plants obtained by the administration of folic acid have been revealed. When antagonists of folic acid are synthesized and administered, the function prevents the growth and division of normal cells and cancer cells. . The MTX has been widely used until 50 years since it was developed by Seeger and Montogomery and used as an anticancer drug.
Thus, while many researchers tried to synthesize new MTX, Tayler and Montogomery improved their synthesis using 6-formylpteridin, 6-hydroxymethylpteridine and pteridine 1-oxide.
However, MTX, which is known as an anticancer agent among anticancer drugs, has been required for the development of less toxic and powerful anticancer drugs since long-term administration of MTX-resistant cells results. Recently, the C-2 amino group of the pteridine parent nucleus was replaced with CH ₃ , H and other alkyl groups, and the pyrazine moiety and other N ¹⁰ − in C-5, C-6, C-7, and C-8. New derivatives are being developed by replacing positions. For example, N ¹⁰ -propargyl-5,8-deadeafolic acid, an antifolate based on quinazoline, is replaced with CH ₃ and H instead of C-2amino and propagyl is substituted for N ¹⁰ . It is a typical compound substituted with. Compared to conventional pteridine anticancer drugs, the drug has increased polar solubility and shows similar activity to MTX in suppressing thymidylate synthetase (TS) and suppressing growth of tumor cells culture.
Therefore, the present inventors have left the pyrimidine positional enzyme binding site, which is a part of the putridine nucleus as it is, and replaces the pyrazine moiety with a generally antibacterial and polar solubility furan form. Considering these factors, the anticancer effect is superior to methoxtrixate (methotrexate), but the toxic aminofuterine (aminpopterine) is substituted with X (thio) amino group of N ¹⁰ -CH ₃ , that is, S-methyl (= S ⁺ -CH ₃ ) to synthesize a new derivative having a molecular weight and a nearly similar form of molecular formula was proposed as a new material,

상기 식에서,
R은 수소, 메틸, -Et 또는 -CH₂CH₂CH₃이고,
R¹ 및 R²는 각각 수소, Na, 메틸, 에틸, 프로필 또는 이소프로필이다.
본 발명에 따른 구조식(1)의 아미노 프테리딘 티오 벤조인산 화합물은 다음과 같은 경로에 의하여 제조한다.The present invention provides amino pteridine thio benzoic acid compounds of formula (I).

Where
R is hydrogen, methyl, -Et or -CH ₂ CH ₂ CH ₃ ,
R ¹ and R ² are each hydrogen, Na, methyl, ethyl, propyl or isopropyl.
The amino pteridine thiobenzoic acid compound of formula (1) according to the present invention is prepared by the following route.

1) Method 1

The Sandmayer reaction was introduced to replace the p-amino group of para aminobenzophosphoric acid with thio ((S) group to obtain dithiobisbenzophosphate compound (3), which was reacted with purpyrimidine compound (2) in the presence of NaBH ₄ in ethanol. By reacting to synthesize pteroic acid compound (4), which is a novel compound that is an intermediate, and using compound (4) in the presence of DMF (dimethylformamide) using DCC or DEPC (diethylphosphoro-cyanidate) Reaction with diethyl L-glutamate to obtain an ester compound of formula (5), followed by = S ⁺ -CH ₃ reaction and hydrolysis if necessary to obtain a compound in free acid form of formula (I). The schematic is as follows.

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Reaction Scheme

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In the above formula
R, R ¹ and R ² are as defined above.

The following compounds are mentioned as a preferable compound of the said Formula (1).

1) appliance

Infrared spectrometer used Perkin-Elmer 599B, and nuclear magnetic resonance spectrometer used Varian Model T60 and Bruker FT300 MHz. Mass spectra were measured by MS25 RTA GC-MS of Japan, and elemental analysis was carried out using the Italian Carlo Elba Elemental Analyzer CHNS-OEA 1108. Melting point was used Thomas Hoover model and temperature was not corrected. And T.L.C. Plates were used Merck DC-Fertig Plattenkeiselgel 60F 254.

2) reagents

p-aminobenzoic acid and diethylcyanophosphate used the product of Aldrich. Reagents such as acetamine hydrochloride, formamidine hydrochloride, sodium ethoxide and dimethyl framide were used by Tokyo Kasai Co., Ltd. and Aldrich Co., Ltd., and purified as necessary.

Hereinafter, an Example is given and described as follows.

Example 1

Preparation of 2,4-diamino-5-chloromethylpuro [2,3-d] pyrimidine (2)

20 ml of DMF was added to the flask, and 1.26 g (10 mmol) of 2,4-diamino-4-hydroxy pyrimidine compound was added to 20 ml of DMSO, followed by stirring at room temperature for 12 hours. The mixture was concentrated under reduced pressure, the crystals were collected, and then washed several times with ether and dried. The resulting compound was 1.36 g as a crystalline compound showing a purple color. Yield 68%.

Melting point: 179-183 ° C., TLCRf. 0.3 (chloroform: methanol = 90:10)

¹ H-NMR (DMSO-d6) δ: 4.9 (s, 2H, 8-CH2 ₂ ), 6.1 (s, 2H, 4-NH ₂ ), 6.57 (s, 2H, 2-NH ₂ ), 7.45 (s , 1H, 6-CH)

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Example 2

Synthesis of 4,4'-dithiobisbenzoic acid (3)

90 ml of distilled water was added to the flask, followed by dissolving 26 g (0.11 mol) of Na ₂ S 9H ₂ O. Then, 3.4 g (0.05 mol) of S ₂ was added thereto, stirred for 30 minutes, dissolved, and an ice bath was installed. Allow to cool before leaving. Separately, 13.7 g (0.1 mol) of p-aminobenzoic acid was added to 50 ml of distilled water, and then cooled to −10 ° C. using an ice bath. Then, 26 ml of C-HCl was added for 10 minutes to cool to 0 ° C., and 6.9 g of NaNO ₂ (0.1 mol) was dissolved in 30 ml of distilled water in small portions for 10 minutes and mixed with the solution left at the same temperature for 30 minutes while stirring, and stirred for 30 minutes, and then the mixture was filtered and the filtrate was adjusted to pH 3 to form crystals. The compound was filtered and dried to give 18.0 g of the title compound. Yield: 49.9%

Melting Point: 322 ～ 324 ℃

IR (KBr) cm ^-1 : 3500 (-OH), 1500 (C = O), 11.0 (S, 1H, CO, OH)

¹ H-NMR (DMSO-d ₆ ); 8.0 (q, 4H, -C ₆ H _5- ), 11.0 (s, 1H, CO OH)

Example 3

Synthesis of 2,4-diamino-5- [S- (phenyl-4-ylthio) methyl puro [2,3-d] pyrimidine (4)

0.147 g (0.74 mmol) of Compound (2) obtained in Example 1 was dissolved in anhydrous DMF, and 0.275 g (1.5 mmol) of 4-thiobenzoic acid compound (3) obtained in Example 2 was added, followed by anhydrous K ₂ CO ₃ . Put and reacted at 60 ℃ for 10 hours. 50 ml of acetonitrile solution was added to recover the crystal compound, which was then filtered and dried to obtain 0.09 g of the title compound. Yield: 79.6%

Melting Point: 179∼183 ° C, TLCRf 0.3 (Chloroform: Methanol = 90:10)

¹ H-NMR (DMSO-d ₆ ) δ; 4.18 (s, 2H, 8-CH2 _2- ), 5.89 (brs, 1H, 9-NH2), 6.21 (brs, 2H, 4-NH2), 6.78 (brs, 2H, 2-NH2), 7.44 (s, 1H, 6-CH)

Example 4

Synthesis of Tetraethyl 4,4'-dithiobis (N-benzoyl-L-glutamate) (38) Compound

To 200 ml of acetonitrile, 7.7 g (25 mmol) of 4,4'-dithiobis (benzoic acid) was added, followed by 10.3 g (50 mmol) of diethyl L-glutamate, which was stirred for 10 minutes, followed by 11.9 g (55 mmol) of DCC. The compound was stirred for 1 day at room temperature under dry drying. The resulting dicyclohexylurea was filtered off and the filtrate was concentrated under reduced pressure to remove the solvent and the resulting oil residue was dissolved in 300 ml of CHCl ₃ solution. Again the solution was washed with 10% NaHCO ₃ (2x50ml), 0.1N HCl (2x50ml) and distilled water, and then separated by a funnel. The separated organic layer was dehydrated by adding anhydrous MgSO ₄ , and then filtered and the filtrate was concentrated under reduced pressure. The resulting residue is dissolved in 300 ml of CH ₃ CN and insoluble compounds are filtered off. The filtrate was concentrated under reduced pressure again and cooled to give 7.9 g of the title compound. Yield 46%

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Melting point: 58 ～ 61 ℃,

IR (KBr) cm ^-1 : 3350 (NH), 1720 (C = O), 1620, 1510 (C = C)

¹ H-NMR (CDCl ₃ ); 1.23 (m, 6H, 2-0CH ₂ CH3 _3- ) 4.08 (m, 4H, 2-0CH2 ₂ CH ₃ ) 7.44 and 7.7 (aromatic quartet 4H, J = 8.3 Hz) 8.9 (d, 1H, -CoNH = CH )

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Example 5

Diethyl N- [4- [N-[-[(2,4-diaminopuro [2,3-d] pyrimidin-5-yl) methyl] thio]
Synthesis of Benzoyl] -L-Glutamate (5)

[A] method

Puteric Acid Derivative Compound (4) 0.252 g (0.75 mmol) of diethyl [p- (thio) benzoyl] -L-glutamate, a compound obtained by adding 0.148 g (0.79 mmol) to 10 ml of DMF and then reducing with NaBH 4 in an alcohol solution ) And then add 0.207 g (1.5 mmol) of K2CO3. After reacting at about 50 ° C. for 6 hours, the mixture is concentrated under reduced pressure and cooled. The resulting crystals are collected, washed with alcohol and washed again with ether. And dried to obtain 0.225 g of the resulting compound. Yield 52.3%

[B] method

To 15 ml of re-distilled DMF, 0.148 g (0.75 mmol) of Compound (2) was added in small portions for 5 minutes, followed by 98 ml (0.6 mol) of diethylphosphorocyanate and 202 mg (2 mol) of Et ₃ N, followed by 30 minutes at room temperature. Stir while. 117 mg (0.6 mol) × diethyl L-glutamate × 2 times was added and stirred at once, checked by TLC (91: 1 CHCl ₃ -MeOH), and the insoluble compound was filtered off. The filtrate was concentrated under reduced pressure, distilled water was added, crystallized, and then stirred for 30 minutes. The filtrate was filtered, and the crystal compound was washed with distilled water and IPE to obtain 0.13 g of the compound.

IR (KBr) cm ^-1 : 3300,3200 (NH ₂ ), 1750, 1645 (C = O), 1630, 1550 (C = C)

Yield 68%, Rf. 0.67

Example 6

Diethyl N- [4- [N-[-[(2,4-diaminopuro [2,3-d] pyrimidin-5-yl) methyl] thio-
Methylinium] benzoyl] -L-glutamate synthesis [I-ester compound]

0.174 g (0.39 mmol) of the compound (5) was added to 2 ml of DMF, followed by 3 ml of acetic acid, and slowly added with excess of 0.5 g of CH ₃ I (methyladioxide). 0.108 g (0.00052 mole) of AgClO4 was reacted at about 80 ° C. for 6 hours, and then concentrated under reduced pressure and cooled. The resulting crystals are collected, washed with alcohol and washed again with ether. And dried to obtain 0.15 g of the title compound.

Yield: 75%, Rf. 0.46

Example 7

Diethyl N- [4- [N-[-[(2,4-diaminopuro [2,3-d] pyrimidin-5-yl) methyl] thio
-Methylinium] benzoyl] -L-glutamic acid synthesis [I]

0.174 g (0.336 mmol) of the [I] ester compound was added to 2 ml of DMF, followed by 3 ml of acetic acid, and slowly added 0.5 g of CH ₃ I (methylidoid). 0.108 g (0.00052 mole) of AgClO4 was reacted at about 80 ° C. for 6 hours, and then concentrated under reduced pressure and cooled. The resulting crystals are collected, washed with alcohol and washed again with ether. And dried to obtain 0.09 g of the title compound.

Yield: 58.1%, Rf. 0.46

1) Antitumor Activity Test

Antitumor activity against the compounds of the formulas (1-1), (1-2), (1-3) and (1-4) according to the present invention was evaluated as an increase in survival. 1 * 10 leukemia p388 cells are injected intraperitoneally into BDF1, mice. Each group consists of six mice. The next day, the four compounds were each suspended in 0.2% Tween 80-solution in saline and administered intraperitoneally to determine the survival in days. The results are shown in Table 1 below. The percent increase in survival is calculated by the following equation.

% Increase in survival = D / D ₀ X 100

D: Mean survival time in untreated group (days)

D: Mean survival time in days treated group

Table 1 (Effects of Prolonging Survival in Lemur Leukemia)

2) Acute Toxicity Test

A male toxicity of ddy strain (weight 25) was used for acute toxicity testing of the compounds of the formulas (1-1), (1-2), (1-3) and (1-4) according to the present invention. . Each of the four compounds is suspended in 0.2% Tween 80 solution in physiological saline and administered intraperitoneally to birds. The LD ₅₀ value was measured by an up and down mehtod, and the results are shown in Table 2 below.

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Table 2.LD ₅₀ (mg / Kg)

The preparation example of the compound of this invention is demonstrated.

Formulation Example 1

4 mg of compound (I-1)

Lactose 43mg

Cornstarch 50mg

50mg of crystalline cellulose

15 mg of hydroxypropyl cellulose

Talc 2mg

Magnesium Stearate 2mg

Ethyl Cellulose 30mg

Fatty Acid Glycer Ester 2mg

Titanium Dioxide 2mg

200 mg per tablet

In addition, the amino pteridine thiobenzoic acid compound of formula (I) according to the present invention may be mixed with a conventional pharmaceutical carrier to prepare granules, fine granules, capsules, injections, suppositories, and the like.

The compound according to the present invention can be used as an anticancer agent, an antimalarial agent, an agent for treating other skin diseases, a contraceptive agent, or the like for injection or oral administration for therapeutic purposes.

Claims (7)

Amino Pteridine Thiobenzoic Acid Compounds of Formula (I)

Where R is hydrogen, methyl, -Et or -CH ₂ CH ₂ CH ₃ , R ¹ and R ² are each hydrogen, Na, methyl, ethyl, propyl or isopropyl. The compound of claim 1, wherein R is hydrogen, methyl, —Et or —CH ₂ CH ₂ CH ₃ , and R ¹ and R ² are each hydrogen or —CH ₂ CH ₃ . As shown in the following reaction scheme, the dithiobisbenzoic acid of the following structural formula (3) is reacted with the purpyrimidine compound of the following structural formula (2) in ethanol in the presence of NaBH ₄ , to form a pteroic acid compound of the following general formula (4) Was synthesized and reacted with diethyl L-glutamate by addition of DCC or DEPC (diethylphosphoro-cyanidate) in the presence of DMF (dimethylformamide) to give the ester of Method for preparing the compound. Reaction Scheme

A process for preparing an amino pteridine thiobenzophosphate compound of formula (I) by reacting an ester type compound of formula (5) in the presence of CH ₃ I and AgClO ₄ .

Where R is hydrogen, methyl, -Et or -CH ₂ CH ₂ CH ₃ , R ¹ and R ² are each hydrogen, Na, methyl, ethyl, propyl or isopropyl. delete A pharmaceutical composition for injection or oral administration having an anticancer activity containing the amino pteridine thiobenzophosphate compound of formula (I) as defined in claim 1 together with a conventional pharmaceutical carrier. delete