KR20020037242A - Synthesis and characterization of new antifolate having a furo[2,3-d]pyrimidine derivatives - Google Patents

Abstract

PURPOSE: Provided is a novel derivative of aminopterin methotrexate(amethopterin:MTX) and homofolic acid which are antimetabolites and act as antagonist of folic acid. Also, its manufacturing method is provided. CONSTITUTION: Aminopteridin derivative compound is represented by the formula(I), wherein R is hydrogen or methyl; and R1 and R2 are respectively hydrogen, Na, methyl ethyl or isopropyl. Its manufacturing method comprises the steps of: introducing Sandmeyer reaction to obtain a compound of the formula(3); reducing the compound of the formula(3) to NaBH4 in ethanol; adding the resultant compound furopyrimidine derivative of the formula(2) to obtain pteroic acid derivative of the formula(4); reacting the pteroic acid derivative of the formula(4) with diethyl L-glutamate in the presence of DMF(dimethylformamide) using DDC or DEPC(diethylphosphoro-cyanidate) to obtain a compound of the formula(5); and reacting the resultant compound with =S¬+-CH3, if necessary hydrolyzing the resultant compound, to obtain a compound of the formula(I).

Description

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

The present invention is a novel amino puteridine thiobenzoic acid derivative of the following structural formula [I] as a derivative of homofolic acid with aminopterin methotrexate (amethopterin: MTX), which acts as an antagonist of folic acid as a compound which is an anti-metabolant, and a method for preparing the same. It is about.

Wherein R ¹ and R ² are phil or isopropyl. It has a function as an anticancer agent which is a malignant tumor therapeutic agent similar to the compound of formula (I), and a similar known compound is mesotrisate (methotrexate, MTX: see The Merk Index 11, 5908, 1989). Has

The mesotrexate compound has a number of side effects such as inhibition of bone marrow function, reduction of white blood cells, ulceration of small vesicles, impairment of liver and kidney function, and toxicity (LD50 iv in rats: 14 mg / kg, scherf) as a side effect. It is true that it is serious and requires a lot of caution when putting it in. Therefore, the present inventors, while studying a cancer drug having a safer treatment of malignant tumors, introduces CH ₃ and H instead of the amino (NH ₂ ) group having a puteridine nucleus and the C ₂ position; And diethylglutamate were reacted with pteroic acid, an intermediate synthesized by S (thio) group instead of N-CH _{3 at} position C ₁₀ , to obtain a new stable anticancer agent. And filed as a patent.

Compounds with Pteridine nuclei have been reported for anti-cancer and anti-malaria effects. Among them, aminopteridin and amethopteridin (methotrexate: MTX), whose chemical structural formula was identified in 1940 as an antagonist of folic acid, were known for about 40 years. Reign is the most representative anticancer agent. MTX, an anticancer agent, has 7,8-dihydrofolate, which is known as one carbon transfering agent, by dihydrofolate reductase (DHFR), which is reduced by DHFR. , 8-tetrahydrofolate is formed. The compound has a 2,4-diaminopyrimidine structure similar in structure to 2-amino-4-oxo ( 3H ) pyrimidine, known as the enzyme binding site, which competitively binds to the enzyme binding active site and forms folic acid. Inhibition of acid activity has been shown to have anti-cancer effects. As such, the mechanisms for cell division and growth of plants and animals obtained by the administration of folic acid have been revealed. It suppresses the growth division of normal cells as well as cancer cells. The MTX was developed by Seeger and Montogomery and is still widely used 50 years after it started to be 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. So recently the C-2 amino group of the pteridine nucleus was substituted with CH ₃ , H and other alkyl groups and the pyrazine moiety in C-5, C-6, C-7, C-8 And other N ¹⁰ -position substitutions to develop new derivatives. As a typical example quinazoline (the antifolate N ¹⁰ -propagyl-5,8-dideazafolic acid is C-2 amino (amino) instead of Pro and the substituted N ¹⁰ position, such as CH ₃ and H pajil (propagyl) A to the quinazolineO mohaek Now, it has increased polar solubility compared to general pteridine anticancer drugs and shows almost similar activity to MTX in inhibition of thimidylate synthetase (TS) and growth inhibition of tumor cell culture. The present inventors have left the pyrimidine position enzyme binding site, which is part of the phthalinine nucleus, and replaces the pyrazine moiety with the furan form, which is generally antibacterial and has good polar solubility. The anti-cancer effect of methoktrixate (methotrexate) is higher than that of N ¹⁰ -CH ₃ . An amino group was substituted with S (thio), that is, substituted with S-methyl (= S ⁺ -CH ₃ ) instead of N-methyl to synthesize a new derivative having a nearly similar molecular weight and molecular formula was proposed as a novel material.

1 shows the effect of prolonging survival in mouse leukemia.

Figure 2 shows the acute toxicity test.

The compound of formula (1) of the present invention is prepared by the following two routes.

1) Method 1

To replace the p -amino group of para-aminobenzoic acid with thio (S) group, Sand mayer reaction was introduced to obtain compound (3), which was then reduced to NaBH ₄ in ethanol, followed by addition of purpyrimidine derivative (2). An ester compound of formula (5) is reacted with diethyl L-gulutamate using DCC or DEPC (diethylphosphorocyanidate) in the presence of a novel compound, an intermediate, a puteroic acid derivative (4) and DMF (dimethylformamide). The compound of is obtained, followed by the = S-CH3 reaction and hydrolysis if necessary to obtain the compound in the form of the free acid of the formula [I].

Schematic of this is as follows.

2) Method 2

Alternatively, to synthesize aminoputin derivatives, compound (2) and dietyl L-glutamate were synthesized as bis sulfide compound (6) using DCC (dicyclohexylcarbodiimide) and NaBH ₄ in EtOH. After reduction to, the addition of compound (2) at room temperature yields the MTX derivative (5). After the reaction with S-methylation, alkali hydrolysis is carried out to obtain a free acid compound of Structural Formula [I].

Schematic of this is as follows.

R, R1 and R2 are as defined above.

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

Preferred compounds of the above formula (1) include the following compounds.

1) Equipment

The instrument used to identify the synthesized compound was an infrared spectrometer using Perkin-Elmer 599B and a nuclear magnetic resonance spectrometer using Varian Model T60 and Bruker FT300 MHz. The mass spectra was measured by Japan's MS25 RTA GC-MS and For analysis, Carlo Erba Elemenetal analyzer CHNS-OEA 1108 from Italy was used. The melting point was Thomas Hoover model and no temperature compensation was used. T.L.C plate was used Merck DC-Fertig platten kieselgel 60F 254.

2) reagents

p- Aminobenzoic acid and diethylcyanophosphonate were used by Aldrich, Inc., and acetamidine hydrochloride, formamidine hydrochloride and sodium ethoxide and dimethyl formamide were used by Tokyo Kasai and Aldrich.

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 Flask, and 1.26 g (10 mmol) of 2,4-diamino-4-hydroxy pyrimidine compound was added to 20 ml of DMSO and stirred at room temperature for 12 hours. After concentration 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 crystal compound showing a purple color. Yield: 68%

mp; 179-183 ° C., TLC Rf . 0.3 (Chloroform: Methanol = 90: 10)

¹ H-NMR (DMSO-d 6) δ : 4.9 (s, 2H, 8- CH ₂ ), 6.1 (s, 2H, 4-NH 2),

6.57 (s, 2H, 2-NH2), 7.45 (s, 1H, 6-CH)

Example 2

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

90 ml of distilled water was added to the flask and dissolved by adding 26 g (0.11 mol) of Na ₂ S) 9H ₂ O. Then, 3.4 g (0.05 mol) of S ₂ was added in small portions, stirred for 30 minutes, and dissolved in an ice-bath. Allow to cool. Separately, 13.7 g (0.1 mol) of P -amino benzoic acid was added to 50 ml of distilled water, and then cooled to -10 ℃ by ice-bathing. Then, 26 ml of C-HCl was added for 10 minutes to cool to 0 ℃, and NaNO ₂ A small amount of 6.9 g (0.1 mol) dissolved in 30 ml of distilled water was added in small portions for 10 minutes, mixed with the solution left at the same temperature for 30 minutes while stirring, and stirred for 30 minutes again, the mixture was filtered, and the filtrate was adjusted to pH 3 The resulting crystal compound was filtered and dried to obtain 18.0 g of the above compound.

Yield; 49.9% MP; 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

2,4-diamino-5- [S- (phenyl-4-yl thi) methyl puro [2,3-d] pyrimidine (4) synthesis

0.147 g (0.74 mmole) of the compound (2) was dissolved in anhydrous DMF, 0.275 g (1.5 mmol) of 4-thiobenzoic acid compound (3) was added thereto, and anhydrous K 2 CO 3 was added thereto and reacted at 60 ° C. for 10 hours. 50 ml of solution was added to recover the crystalline compound, which was then filtered and dried to obtain 0.09 g of the compound. Yield: 79.6%

mp; 179-183 ° C., TLC Rf . 0.3 (Chloroform: Methanol = 90: 10)

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

Example 4

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

7.7 g (25 mmol) of 4,4'-dithiobis (benzoic acid) was added to 200 ml of acetonitrile. Then, 10.3 g (50 mmol) of diethyl L-glutamate was added thereto, followed by stirring for 10 minutes, followed by DCC 11,9 g (55 mmol). do. The compound was stirred for 1 day at room temperature under anhydrous conditions. The resulting dicyclohexylurea was filtered off, the filtrate was concentrated under reduced pressure to remove the solvent, and the resulting oilic residue was dissolved in 300 ml of CHCl ₃ solution. Again the solution was washed with 10% NaHCO ₃ (2x50 ml), 0.1N HCl (2x50 ml) and distilled water, and then separated by a funnel. The separated organic layer was dehydrated by adding anhydrous MgSO ₄ , and then 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 compound.

Yield; 46%

MP; 58-61 ℃

IR (KBr) Cm ⁻¹ ; 3350 (NH), 1720 (C = O), 1620,1510 (C = C)

¹ H-MNR (CDCl ₃ ); 1.23 (m, 6H, 2-0CH ₂ CH ₃ ) 4.08 (m, 4H, 2-0 CH ₂ CH ₃ ) 7.44 and 7.7 (aromatic quartet 4H, J = 8.3HZ)

8.9 (d, 1H, -CoNH = CH)

Example 5

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

[A] method

Futeric Acid Derivative Compound (4) 0.148 g (0.79 mmol) was added to 10 ml of DMF, followed by reduction with NaBH 4 in an alcohol solution. Diethyl [p- (thio) benzoyl] -L-gulutamate 0.252 g (0.75 mmole) is added correctly, followed by 0.207 g (1.5 mmole) of K 2 CO 3. The reaction is carried out at 50 ° C. for 6 hours, and then concentrated under reduced pressure. The resulting crystals are collected, washed with alcohol and washed again with ether. do. And dried to obtain 0.225 g of the resulting compound. Yield: 52.3%

[B] method

To 15 ml of 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 diethylphosphorcyanidate and 202 mg (2 mol) of Et ₃ N, followed by stirring at room temperature for 30 minutes. Let's do it. Diethyl L-glutamate 117 mg (0.6 mol) x 2 times a day was added to the mixture and stirred. TLC (91: 1 CHCl ₃ -MeOH) was added. The insoluble compounds were filtered off. The filtrate was concentrated under reduced pressure, distilled water was added thereto, crystallized and 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

¹ H-NMR (DMSO-d6) δ : 1.17 (m, 6H, CH 2 CH 3), 1.95-2.0 (m, 2H, Glu β -CH 2), 2.4 (t, 2H, Glu γ -CH 2), 4.0-4.18 ( m, 4H, 8- CH ₂ CH 3) , 4.4 (m, 1H, Glu α -CH), 4.63 (s, 2H, 8-CH 2), 6.06 (s, 2H, 4-NH 2), 6,43 (d , 2H, 2-NH2), 6.88 (d, 2H, 3'-, 5'-CH), 7.03 (s, 1H, 6-CH), 7.7 (d, 2H, 2 ', 6'-CH), 8.38 (d, 1H, CONH)

Example 6

Diethyl N- [4- [N-[-[(2,4-diaminopuro [2,3-d] pyrimidin-5yl) methyl] thio-methyllinium] 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). Then, 0.108 g (0.00052 mole) of AgClO4 is reacted for about 6 hours at 80 ° C, 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 resulting compound.

Yield 75%, Rf . 0.46

¹ H-NMR (DMSO-d6) δ : 1.2 (m, 6H, CH 2 CH 3), 1.9-2.0 (m, 2H, Glu β -CH 2), 2.32 (t, 2H, Glu γ -CH 2), 2.75 (s, 3H, CH3) 4.0- 4.2 (m, 4H, 8- CH ₂ CH3) , 4.42 (m, 1H, Glu α -CH), 4.7 (s, 2H, 8-CH2), 6.1 (s, 2H, 4- NH2), 6,36 (d, 2H, 2-NH2), 6.96 (d, 2H, 3 '-, 5'-CH), 7.1 (s, 1H, 6-CH), 7.72 (d, 2H, 2 ', 6'-CH), 8.5 (d, 1H, CONH)

Example 7

Diethyl N- [4- [N-[-[(2,4-diaminopuro [2,3-d] pyrimidin-5yl) methyl] thio-methyllinium] benzoyl] -L-gluta Mic Acid Synthesis [I]

0.174 g (0.336 mmol) of the above-mentioned [I] ester compound is added to 2 ml of DmF, followed by 3 ml of acetic acid, and then slowly added an excess of 0.5 g of CH ₃ I (methyladioxide). Then, 0.108 g (0.00052 mole) of AgClO4 is reacted for about 6 hours at 80 ° C, concentrated under reduced pressure, and cooled. The resulting crystals are collected, washed with alcohol and washed again with ether. And it dried and obtained 0.09g of produced compounds.

Yield 58.1%, Rf . 0.46

¹ H-NMR (DMSO-d6) δ : 1.2 (m, 6H, CH 2 CH 3), 1.9-2.0 (m, 2H, Glu β -CH 2), 2.32 (t, 2H, Glu γ -CH 2), 2.75 (s, 3H, CH3) 4.0- 4.2 (m, 4H, 8- CH ₂ CH3) , 4.42 (m, 1H, Glu α -CH), 4.7 (s, 2H, 8-CH2), 6.1 (s, 2H, 4- NH2), 6,36 (d, 2H, 2-NH2), 6.96 (d, 2H, 3 '-, 5'-CH), 7.1 (s, 1H, 6-CH), 7.72 (d, 2H, 2 ', 6'-CH), 8.5 (d, 1H, CONH)

1) Antitumor Activity Test

Antitumor activity against the compounds of the formulas (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 with BDF1, a mouse. Each group consists of six birds. The next day, the four compounds were each suspended in 80 solutions of 0.2% physiological saline and then intraperitoneally administered to determine the number of days of survival. 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 days without treatment

D: mean survival time (days) of treatment group

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 was suspended in 80% solution of 0.2% physiological saline and administered intraperitoneally to live mice.

LD values were measured by an up and down method, and the results are shown in Table 2 below.

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

Formulation Example]

4 mg of compound (I-1)

Lactose 43mg

Cornstarch 50mg

Crystalline 50mg

Hydroxypropyl Cellulose 15mg

Talc 2mg

Magnesium Stearate 2mg

Ethyl Cellulose 30mg

Fatty Acid Glycer Ester 2mg

Titanium Dioxide 2mg

200 mg per tablet

In addition, it may be formulated in the form of granules, fine granules, capsules, injections, suppositories, and the like.

.

Claims (7)

Amino Pteridine Derivative Compounds of Formula (I) Wherein R is hydrogen or methyl and R 1 and R 2 are each hydrogen, Na, methyl ethyl or isofurophyll. In the case of claim 1, when R = H, CH3, -Et, -CH2CH2CH3 And R 1 and R 2 are hydrogen, methyl, ethyl, Na (sodium), protopill, or isopropyl, respectively. Sendaimer reaction of paraaminobenzoic acid (1) followed by reduction, followed by the action of 2,4-diamino-5-chloromethylfuro [2,3-d] pyrimidine to synthesize a derivative of phthalic acid. To prepare an aminoputidinethiobenzoic acid derivative compound by reacting with diethyl parathionyl-L-gulutamate using DCC method or dicyano phosphinocyanodate Compound (5) or (6) was synthesized by the action of 2,4-diamino-5-chloromethylpuro [2,3-d] pyrimidine and acidified using AgClO ₄ in the structural formula of each compound [I]. Method for preparing MTX derivative compound in carboxylic acid form or ester form to form = S-CH ₃ in solution Diethyl N- [4- [N-[-[(2,4-diaminopuro [2,3-d] pyrimidin-5yl) methyl] thio (S) -methyl] benzoyl] -L-gl A process for preparing a structural formula (amino puteridine thio-methyl benzoic acid derivative compound of I0 by reacting rutamic acid diethyl ester with formdiamine salt or acet amidine salt in the presence of alkali Use as an injection or oral administration for the purpose of treating anticancer drugs, antimalarial drugs, other skin disease treatments, contraceptives, etc. in the use of the above [I] chemical structural formula The paraaminobenzoic acid was subjected to sandmeier reaction, followed by reduction of sulfide with sodium borohydride, to which compound (2) was added to prepare a compound of formula (4), followed by reaction of an ester compound of glutamic acid. A method for producing the aminoputeridine methylthiobenzoic acid derivative compound of the structural formula [I].