KR970008655B1 - New anticancer platinum (II) complex and preparation method thereof - Google Patents

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New anticancer platinum (II) complex and preparation method thereof

The present invention relates to a novel platinum (II) complex anticancer substance exhibiting excellent antitumor activity and mild toxicity, and a method for producing the same. Platinum complex anticancer agents are known to inhibit the growth and replication of malignant central cells by acting on nucleic acids, due to the shape of inorganic compounds containing platinum in the presence of ammonium and chlorine [B. Rosenberg et al., Platinum Compounds: a new class of poent antitumor agents, Nature, 223 (1969). Among these platinum complex anticancer substances, cis-dichlorodiamine platinum (common name cisplatin cisplatin) was found to be the most active drug in experimental tumor experiments, and excellent effects were recognized in clinical trials, mainly bladder cancer, testicular cancer, ovarian cancer and prostate cancer. And it has been used so far as a therapeutic agent for head and neck cancer, but has the disadvantage of showing side effects such as severe nephrotoxicity and nausea, vomiting and inner ear neurotoxicity. [A. See Ripman et al., Clinical trials of cis-diammined dichloroplatinium (NSC-119875), Cancer Chemother, Rep., 57, 191 (1973)]. And carboplatin, iproplatin, oxaliplatin and malonatoplatinum as second-generation anticancer platinum complexes for the purpose of water solubility and the like, which have a broad anti-corrosion spectrum and alleviate side effects. Malonatplatinum) has been developed.

Carboplatin is registered under the name 'Paraplatin' by Bristol Myers and is currently used in the clinic. It is a derivative of cisplatin and its anticancer spectrum and anticancer activity are similar, solubility is better than cisplatin, and renal toxicity, nausea and vomiting are reduced. Iproplatin was also completed as a second-generation platinum (IV) complex from Bristol Myers. Its nephrotoxicity is equivalent to carboplatin but is highly water soluble and exhibits various antitumor activities. [V. Bramwell et al .: Activity of JMP (CHIP) in advanced ovarian cancer: Cancer Treat, Rep., 69, 409 (1985)] Also, oxaliplatin is a stable second-generation platinum deposit studied by Nagano Nagano, Japan. More excellent effects have been recognized in metastatic breast cancer, liver cancer, melanoma and small intestine cancer, and have no cross resistance with cisplatin, and anticancer activity is also estimated to exceed cisplatin. It is also expected to be an excellent anti-cancer platinum complex that does not require hydration because its solubility is almost 8 times higher than cisplatin, nausea and vomiting. [T. Dashiro et al .: Antitumor activity of a new platinum complex: Biomed. & Pharmacother., 43, 251 (1989)] Maltonatoplatinium is a platinum deposit containing ethylenediamine and is being tested by Bristol Myers.

On the other hand, as a representative anti-cancer material in the patent literature, compounds of European Patent Publication No. 0222522 and US Patent No. 4,783,452 such as AH Heinness are disclosed, and Korea Patent Publication No. 92-18065 in the following general formula Complex anticancer substances are disclosed,

[Wherein R ₁ and R ₂ are the same or different and each is hydrogen or a lower alkyl group having 1-4 carbon atoms.]

In addition, the Republic of Korea Patent Publication No. 92-21569

[Wherein R ₁ and R ₂ are the same or different and each is hydrogen or a lower alkyl group having 1-4 carbon atoms.]

Platinum complex anticancer substances are disclosed.

However, these platinum (II) complexes have many limitations due to side effects such as nephrotoxicity, nausea, vomiting, and hearing loss despite their excellent antitumor activity. In particular, cisplatin is known to be caused by necrosis of the tubules, which increases with increasing dose, and is known to cause severe damage to the distal tubules and the collecting duct as well as the proximal tubules.

An object of the present invention is to provide a water-soluble new platinum (II) complex which is superior to the conventional platinum (II) complex and the like, and has significantly reduced side effects of nephrotoxicity. It also provides a method.

The platinum (II) complex of the present invention can be represented by the compounds of the following general formulas (I) and (I ').

Wherein R ₁ and R ₂ are the same or different and each is phenyl, pyridine, R is one or two substituted phenyl and R can be selected from methyl, ethyl, methoxy, ethoxy and the like.

R ₁ and R ₂ are preferably both phenyl.

n is an integer from 1 to 4, preferably 2 or 3.

In the above formula, 1,2-cyclohexanediamine, which acts as a carrier ligand to the platinum (II) complex, has optical activity and is limited to only cis in the isomer mixture in the present invention. That is, only cis-1,2-cyclohexanediamine was used in the present invention.

In the present invention, the stereoisomer is limited only by the transmer because the chelate ring and the cyclohexane ring of the platinum complex (II) are coplanar and easily react with DNA.

In the present invention, the platinum (II) complex anticancer substance can be prepared by the following method.

In the above formula, the definitions of R ₁ and R ₂ are the same as in general formula (I), and the definition of n is the same as in general formula (I).

In addition, 1,2-cyclohexanediamine of the starting material was only cis-1-1,2-cyclohexanediamine.

In addition, the platinum (II) complex of the present invention may be formulated according to a conventional method with a pharmaceutically acceptable carrier and, if necessary, with an adjuvant, and may be formulated as an injection solution or an intravenous infusion solution.

Example 1

Preparation of # 1,3-bis (diphenylphosphino) propane (cis-1,2-cyclohexanediamine) platinum (II) nitrate- [pt (cis-1-dach) (DPPP)] · (NO ₃ ) Manufacture of

[Step 1]

2.50 g (6.02 mmol) of K ₂ PtCl ₄ (MW 415.11) was dissolved in 80 ml of distilled water and placed in a reaction vessel, and 1.13 g (6.02 mmol) of trans-1--1-ach (1,2-cyclohexanediamine) and 20 ml of distilled water The solution was slowly added to the reaction vessel, brought to pH 6.5 with 5% NaOH solution, and stirred at room temperature for 1 hour for reaction. The resulting yellow crystals were filtered through a 3G4 standard glass filter (cis-1-dach), suction filtered, lyophilized, and re-purified several times with a small amount of distilled water to obtain [Pt (cis-1-dach) Cl ₂ ] (MW 380.2) 1.92 g was obtained. This substance was insoluble in water and poorly soluble in organic solvents such as ethanol.

Yield: 83.9%

IR (KBr): 3255, 3172 (NH ₂ ) cm ^-1

[Step 2]

1 g (2.63 mmol) of [Pt (cis-1-dach) Cl ₂ ] obtained in step 1 was suspended in 150 ml of distilled water of a light shielding reaction vessel, and 0.89 g (5.26 mmol) of AgNO ₃ was dissolved in 10 ml of distilled water. The reaction mixture was added to the mixture and stirred at room temperature for 24 hours. The reactant was filtered under light shielding to remove AgCl precipitate and unreacted substance formed during the reaction, and the filtrate was concentrated under reduced pressure and lyophilized. The resulting white crystalline mole was refined with distilled water to obtain 0.91 g of [Pt (cis-1-dach) (NO ₃ ) ₂ ] (MW 433.2). This substance was soluble in water but poorly soluble in organic solvents such as ethanol.

Yield: 79.9%

IR (KBr): 3215, 3120 (NH ₂ ) 1384 (NO ₃ ) cm ^-1

[Step 3]

500 mg (1.15 mmol) of [Pt (cis-1-dach) · (NO ₃ ) ₂ ] obtained in step 2 above was dissolved in 150 ml of distilled water, and again 1,3-bis (diphenylphosphino) propane (DPPP) 480 mg (1.15 ml) was dissolved in 30 ml of acetone, and the solution was added to the solution with stirring. After reacting for 5 hours, the solvent was distilled under reduced pressure to organic solvent and lyophilized. The resulting yellowish white crystals were reconstituted with distilled water to obtain 457 mg of [Pt (cis-1-dach) (DPPP)] (NO ₃ ) ₂ .H ₂ O (MW 845.7). This material was dissolved in water and dissolved in acetone and ethanol.

Yield: 47%

IR (KBr): 3298, 3182 (NH ₂ ) 1440, 1162 (PC) 1392 (NO ₃ ) cm ^-1

C-NMR (DMSO): δ 18.0 21.8 24.1 32.4 60.4 125.8 128.8 129.1 132.0 132.2 133.1 ppm

Elemental analysis;

Calculation: H 4.77 C 46.87 N 6.62 (%)

Found: H 4.58 C 47.03 N 6.80 (%)

Example 2

Preparation of # 1,2-bis (diphenylphosphino) ethane # (cis-1,2-cyclohexanediamine) platinum (II) nitrate

Preparation of [Pt (cis-1-dach) (DPPE)] · (NO ₃ ) ₂

500 mg (1.15 mmol) of [Pt (cis-1-dach) · (NO ₃ ) ₂ ] obtained in step 2 of Example 1 above were dissolved in 150 ml of distilled water, and 460 mg (1.15 ml) of DPPE was dissolved in 30 ml of acetone. 425 mg of [Pt (cis-1-dach) (DPPE)] (NO ₃ ) ₂ .2H ₂ O (MW 869.7) was obtained in the same manner as in [Step 3] of Example 1). This material is soluble in water and in acetone and ethanol.

Yield: 42.8%

IR (KBr): 3141, 3050 (NH ₂ ) 1441, 1180 (PC) 1392 (NO ₃ ) cm ^-1

C-NMR (DMSO): δ 24.1 24.3 31.6 60.8 126.3 129.4 132.2 132.4 133.1 134.6 ppm

Elemental Analysis:

Calculation: H 4.88 C 44.30 N 6.46 (%)

Found: H 4.93 C 44.84 N 6.39 (%)

Example 3

Anticancer activity test

2 × 10 L-120 and P-388 rat leukemia cancer cells were transplanted into 20 ml of RPMI medium supplemented with 10% fetal bovine serum and 100 units / L of penicillin G and streptomycin and cultured in a 37 ° C., 5% CO ₂ incubator. It was. On the other hand, murine melanoma cancer cells M-14 were cultured in DMEM medium under the same conditions as in the case of leukemia cancer cells L-1210 and P-388. On day 3 of transplantation, cells were collected by centrifugation at 1000 rpm for 5 minutes and then medium was exchanged. The medium was exchanged again on day 4, which is a cell multiplier, and diluted to 10cells / ml. The platinum (II) complex synthesized in Examples 1 to 2 was prepared at 500uM to 5uM. 0.1 ml of cell diluent and 0.1 ml of various concentration samples were added to the 96 well titer plate and incubated for 48 hours. After incubating for 4 hours by adding 0.05 ml of 5 mg / ml MTT solution, the supernatant was removed, and the precipitate was dissolved by adding 0.05 ml of DMSO, and the absorbance was measured at 630 nm with an ELISA reader. Cisplatin was used as a comparative drug, and a cell group cultured under the same conditions without a sample was used as a control, and the determination was expressed in% by the following formula.

The results are shown in Table 1-3 below.

For reference, the platinum (II) complex synthesized in Examples 1 to 2 is

Example 1

[Pt (cis-1-dach) (DPPP)] (NO ₃ ) ₂

Example 2

[Pt (cis-d-dach) (DPPE)] · (NO ₃ ) ₂

to be.

Table 1 below shows the anticancer effect of the leukemia cancer cells L-1210 of the platinum (II) complex synthesized in Examples 1 to 4. As shown in Table 1 below, the compound of the present invention showed high or similar anticancer activity to the leukemia cancer cell L-1210 at 500uM and 50uM compared to cisplatin, and at a low concentration of 5uM, the anticancer activity was more prominent than cisplatin. You can see.

TABLE 1

Anticancer Effect on Leukemia Cancer Cell L-1210

Table 2 below shows the anticancer effect of the leukemia cancer cells P-388 of the platinum (II) complex synthesized in Examples 1-2.

As shown in Table 2 below, it can be seen that the anticancer activity of the compound of the present invention on leukemia cancer cell P-388 is similar to or higher than that of cisplatin, and particularly, even at low concentrations of 50 uM and 5 uM, the anticancer activity is higher than that of cisplatin. Could.

TABLE 2

Anticancer Effect on Leukemia Cancer Cell P-388

Table 3 below shows the anticancer effects of the compounds synthesized in Examples 1 and 2 against melanoma cancer cells M-14.

Compounds of the present invention showed similar or higher anticancer activity to cisplatin against melanoma cancer cell M-14.

TABLE 3

Anticancer Effect on Melanoma Cancer Cell M-14

Example 4

Toxicity test (effect on rabbit proximal tubule epithelial cells)

According to the method of Jeong et al. About 2.0kg rabbit was killed by dislocation of the neck, kidneys were extracted, and phosphate buffer solution was injected through the renal artery and washed. After washing twice with DME / F ₁₂ medium, 0.5% iron oxide solution was injected, and only the neocortex was peeled and placed in DME / F ₁₂ medium and homogenized with Downs-homogenizer.

Homogenator was transferred to the DME / F ₁₂ medium and the tubules and glomeruli collected in the 253 um mesh filter and the 83 um mesh filter, and the glomeruli were removed using a magnetic stirrer bar. Immediately thereafter, trypsin inhibitor and collagenase were added thereto, followed by incubation at room temperature for 2 minutes, followed by DME / F ₁₂ medium containing insulin (5ug / ml), transferrin (5ug / ml), and hydrocortisone (5 × 10 ^-8 ). Inoculated in a cultivation cantilever by floating in a predetermined amount, and cultured for 2 weeks at 37 ℃ in a CO ₂ incubator and carried out a cytotoxicity test, the results are shown in Table 4 below.

TABLE 4

Toxicity to Proximal Tubular Epithelial Cells in Rabbits

As shown in Table 4, the compounds of the present invention can be seen that the toxicity to rabbit proximal tubule epithelial cells significantly lower than the cisplatin at the experimental concentration of 500uM, 50uM and 5uM.

Example 5

Toxicity test (effect on normal human kidney cells)

Kidneys were removed from a cancer patient who had undergone resection of the renal cancer and the normal tissue site was taken, washed several times in DME / F ₁₂ medium containing penicillin G and streptomycin, and then the renal capsule was removed and the scalp was thinned using a scalpel. After cutting, the cells were homogenized under sterile state and suspended in a certain amount of DME / F ₁₂ medium.

0.2 ml of inhibitor and collagenase (10 mg / ml) were added and incubated at room temperature for 2 minutes, then suspended in DME / F ₁₂ medium containing 1% fetal bovine serum and inoculated in a culture dish. 37 ° C. and 5% CO ₂ Incubated for 2 weeks in the incubator. The primary cultured cells obtained above were subjected to cytotoxicity experiments according to the same method as the MTT assay using rabbit proximal tubular epithelial cells, and the results are shown in Table 5 below.

TABLE 5

Toxicity to normal kidney cells in the human body

As shown in Table 5, the compound of the present invention was found to significantly lower toxicity compared to cisplatin even at a high concentration of 500uM with respect to normal human kidney cells.

Example 6

Toxicity test (thymidine intake test)

Primary cultures were inoculated with the proximal tubular epithelial cells of rabbit kidney and the renal cortical cells of human normal kidney, which were 7 to 10 days old, inoculated in 10 wells per 24 well plate and incubated for 1 hour. The platinum compound was added to 50uM per well and incubated for 48 hours at 37 ° C. in a 5% CO ₂ incubator.

H-thymidine at a concentration of 1uci / ml was added, followed by incubation for 24 hours, and the cells collected by trypsin treatment were washed with 10% TCA (trichloroacetic acid) and phosphate buffer solution, and 0.5M-NaOH was added thereto at 37 ° C. After dissolving at 2 hours and neutralized with 0.5M-HCL, 0.1ml each of the skintilization vials contained in 10ml of the skintilation cocktail was measured by beta-counter. As a comparative drug, cisplatin was used, and the cells cultured under the same conditions without sample were used as a control group to obtain 100% thymidine intake rate, and the cell survival rate was calculated from the thymidine intake rate according to each sample solution. It is shown to 6-7.

TABLE 6

Thymidine Intake on Proximal Tubular Epithelial Cells in Rabbits

* Concentration of compound: 50 uM

Cell concentration: 1 × 10 ⁵

As shown in Table 6, the compound of the present invention was found to be significantly higher thymidine uptake rate in the rabbit proximal tubule epithelial cells than cisplatin, significantly reduced toxicity to rabbit kidney cells.

TABLE 7

Ingestion of thymidine for normal kidney cells in the human body

* Concentration of compound: 50 uM

Cell concentration: 1 × 10 ⁵

As shown in Table 7, the compound of the present invention showed a significantly higher thymidine uptake rate than cisplatin for normal kidney cells of the human body, indicating that the toxicity to human kidney cells was significantly reduced.

Claims (7)

Anticancer platinum (II) complex represented by the following general formula (1) and its nitrate Wherein R ₂ and R ₂ are the same or different and are each phenyl, pyridine, phenyl substituted with one or two, and R may be selected from methyl, ethyl, methoxy, ethoxy and the like. n is an integer from 1 to 4, preferably 2 or 3. The anticancer platinum (II) complex according to claim 1, wherein both R ₁ and R ₂ are phenyl. '1,3-bis (diphenylphosphino) propane (cis-1,2-cyclohexanediamine) platinum (II) nitrorate. ｛1,2-bis (diphenylphosphino) ethane｝ (cis-1,2-cyclohexanediamine) platinum (II) nitrorate. The anticancer platinum (II) complex represented by the following general formula (I) and a method for producing the nitrate thereof. In the general formula (I), R ₁ and R ₂ are the same or different, and each of phenyl, pyridine, R is one or two substituted phenyl and R may be selected from methyl, ethyl, methoxy ethoxy and the like. n is an integer from 1 to 4, preferably 2 or 3. Starting material 1,2-cyclohexanediamine is cis-1,2-cyclohexanediamine. The method for producing an anticancer platinum (II) complex according to claim 5, wherein both R ₁ and R ₂ are phenyl. An anticancer composition comprising the platinum (II) complex according to claim 1 as an active ingredient.