KR0170079B1 - Phospholipid derivatives of acyclic nucleoside and their preparation - Google Patents

Abstract

The present invention relates to phospholipid derivatives of the novel acyclic nucleosides of formula (I) and pharmaceutically acceptable salts thereof which are effective as antiviral agents.

Wherein A is oxygen or a sulfur atom, R ₁ is a saturated or unsaturated alkyl group having 6 to 22 carbon atoms, R ₂ is a saturated or unsaturated alkyl group having 2 to 21 carbon atoms, and B is an antiviral cyclic nucleo As seeds are acyclovir, gancyclovir, and phencyclovir residues.

Description

Phospholipid derivatives of acyclic nucleosides and preparation methods thereof

1 is a graph showing the survival rate of mice compared to the control group when ACV-PCA according to the present invention after infection with herpes simplex virus type 1,

2 is a graph showing the survival rate of mice compared to the control group when ACV-PTBA according to the present invention after infection with herpes simplex virus type 1,

Figure 3 is a graph showing the change in body weight of mice when the ACV-PCA according to the present invention after infection with herpes simplex virus type 1,

4 is a graph showing the change in body weight of the mouse when the ACV-PTBA according to the present invention after infection with herpes simplex virus type 1 compared with the control.

The present invention relates to phospholipid derivatives of the novel acyclic nucleosides of formula (I) and pharmaceutically acceptable salts thereof which are effective as antiviral agents.

Where

A is oxygen or sulfur atom,

R ₁ is a saturated or unsaturated alkyl group having 6 to 22 carbon atoms,

R ₂ represents a saturated or unsaturated alkyl group having 2 to 21 carbon atoms,

B is an antiviral acyclic nucleoside represented by the following general formula;

Wherein Y is a hydrogen atom or CH ₂ OH, and Z is a carbon or oxygen atom.

Formula (I) compounds according to the invention are complexes of cyclic nucleosides with phospholipids.

Among the phospholipid derivatives of alkyl glycerol, 1-O-alkylisophospholipids and their synthetic derivatives, 1-O-alkyl-2-O-methylphosphatidyl-choline or ethanolamine, inhibit or prevent the growth and metastasis of cancer in several animals [ See: Eur. J. Cancer, 16, 1199 (1980)], immunomodulating activity; See: Springer Semin. Immunopathol., 2, 187 (1979)], and various studies have been carried out to increase antiviral or anticancer effects by combining phospholipids and nucleosides.

First, Ara-CDP-L-dipalmitin, a complex of 1,2-diacylglycerol and Ara-C (Cytarabine) [J. Med. Chem., 25, 1322 (1982); Cancer Res., 41, 2707 (1981); Biochem. Biophys. Res. Commun., 85, 714 (1978) and Ara-CDP-DL-dipalmitin [sCIENCE, 196, 303 (1977); Biochim. Biophys. Acta, 619, 604 (1980); Biochim. Biophys. Acta, 619, 619 (1980)].

In addition, British Patent No. 2,168,350 et al. Med. Chem., 29, 2038 (1986)], Ara-CDP-DL.-PBA and Ara, a complex of 1-O-alkyl-2-O-acylglycerol with Ara-C (Cytarabine) and Ara-A (Vidarabie). A method of manufacturing -CDP-DL-PCA, Ara-ADP-DL-PCA, and the like are disclosed. The complex exhibits anticancer activity against L1210 lymphatic leukemia. Among them, Ara-CDP-DL-PCA is 5 times more anti-cancer than Ara-C even with a third molar dose of Ara-C. It is. Ara-CDP-DL-PCA is hydrolyzed into Ara-CMP and phospholipids intracellularly, and the resulting 1-O-alkyl-2-O-acylglycero-3-phosphate phospholipids are subjected to 1-O- after biochemical reactions. It converts to alkyl-2-lysophosphatidyl-choline or ethanolamine, and this compound also inhibits the growth and metastasis of cancer cells and regulates immunity.

United States Patent No. 4,622,392 et al. [J. MED CHEM., 33, 1380 (1990) describe the preparation and anticancer effects of the complex of 1-S-alkyl-2-O-acylglycerol and Ara-C, which is more effective than ara-C alone. It is reported to show better anticancer effects.

Meanwhile, European Patent No. 350,287 (VICAL INC., USA) and International Patent Application (PCT) WO 91/18914 (VICAL INC., USA) disclose examples of preparing complexes by binding phospholipids to acyclic nucleoside compounds. have. However, in this document, ACV-DP-DPG and ACV-DP-DMG and acyclovir-diphosphate- (1-O-octadecyl) glycerols using 1,2-diacylglycerol and 1-O-alkylglycerols as lipids Only examples of synthesizing acyclovir-diphosphate- (1-O-hexadecyl) glycerol are shown. In particular, the results of pharmacological tests on in vitro WI-38 cells described in European Patent No. 350,287 indicate that the phosphatidyl acyclovir developed as a complex is similar to the acyclovir, which is a reference drug in HSV-1 and HSV-2. Only the effect of the degree which shows was shown.

The present inventors have studied to find a complex exhibiting a synergistic antiviral effect superior to that of acyclovir by binding new phospholipids to acyclic nucleosides. O-acylglycero-3-phosphate (A is an oxygen or sulfur atom), and the complex of phospholipids with cyclic nucleosides, wherein the acyclic nucleoside moiety consists of acyclovir, gancyclovir or phencyclovir moieties The present invention was completed by confirming the fact that it has an excellent antiviral effect.

It is therefore an object of the present invention to provide phospholipid complexes of acyclic nucleosides which exhibit superior antiviral effects than existing acyclic nucleoside compounds.

To this end, the present invention provides phospholipid derivatives of the novel acyclic nucleosides of the general formula (I) and pharmaceutically acceptable salts thereof.

(Wherein A, R ₁ , R ₂ and B are as defined above).

Hereinafter, the content of the present invention will be described in detail.

The phospholipid portion of the phospholipid complex with an cyclic nucleoside according to the invention is 1-A-alkyl-2-O-acylglycerol, and A is selected from oxygen or sulfur atoms. The acyclic nucleoside moiety complexes with phospholipids in the case of acyclovir, gancyclovir, or phencyclovir, resulting in excellent antiviral effects.

In particular, acyclovir-5'-diphosphate-rac-1-O-hexadecyl-2-O-palmitoylglycerol (ACVDP-DL-PCA), acyclovir-5'-diphosphate-rac-1-S-octadecyl 2-O-palmitoyl-1-thioglycerol (ACVDP-DL-PTBA), acyclovir-5'-diphosphate-rac-1-O-octadecyl-2-O-palmitoylglycerol (ACVDP-DL-PBA) ), Acyclovir-5'-diphosphate-rac-1-S-hexadecyl-2-O-palmitoyl-1-thioglycerol (ACVDP-DL-PTCA), or pharmaceutically acceptable salts thereof are preferred. .

In the complex according to the present invention, acyclovir, livercyclovir, or phencyclovir inhibit the replication of the virus in infected cells with the medicinal properties and phospholipids themselves represented by conventional antiviral agents. AIDS Research and Human Retroviruses, 6, 491 1990) and cancer growth, metastasis inhibitory or prophylactic effects and immunomodulatory action {see Eur. J. Cancer, 16, 1199 (1980); Springer Semin. Immunopathol., 2, 187 (1979); Cancer Research, 47, 2599 (1987)] is expected to appear either additively or synergistically, and the complex reaches the target cell as a sustained prodrug and is broken down into phospholipids and nucleosides intracellularly. It can have the same pharmacological and antiviral effects.

Phospholipid derivatives of the cyclic nucleosides according to the present invention can be prepared by the following two methods.

First, the cyclic nucleotides are made of morpholidate (VII) or P ¹ -nucleoside-5′-P ² -diphenylpyrophosphate (VI), followed by 1-A-alkyl-2-O-acylglycerol. The compound of formula (I) can be prepared by reaction with -3-phosphate (V) (A is oxygen or sulfur atom). In addition, the phospholipid is made of morpholidate (IV) or P ¹ -glycerol-5'-P ² -diphenylpyrophosphate (III), and then reacted with an cyclic nucleotide (II) to give a compound of formula (I). It is also possible to prepare (see Biochim. Biophys. Acta, 91, 1-13 (1964); J. Am. Soc., 83, 649 (1961); And J. Am. Chem. Soc. 83. 659 (1961).

In the general formula, A, R ₁ , R ₂ and B have the same meaning as defined above.

Phospholipid complexes of 1-A-alkyl-2-O-acylglycerols (A is oxygen or sulfur atoms) with acyclic nucleosides provided by the present invention are the antiviral test results for HSV-1. It showed more than three times better effect when administered alone. [FIGS. 1 to 4: in vivo test; Mouse, intraperitoneal injection].

Therefore, the phospholipid complex of the acyclic nucleoside according to the present invention can be used as an antiviral agent, and a dose of 0.001 to 5 g may be administered by oral or parenteral route, for example, by injection.

The compounds of the present invention and salts thereof can be prepared and used in the form of pharmaceutical preparations with pharmaceutically acceptable organic or inorganic carriers. The formulation form may be a conventional pharmaceutical formulation such as injections, capsules, suspensions, granules, powders and tablets.

Next, the Example of this invention is described. However, these examples are provided to facilitate the understanding of the present invention, and the scope of the present invention is not limited to these examples.

Example 1

Preparation of Acyclovir-5'-diphosphate-rac-1-O-hexadecyl-2-O-palmitoyl glycerol (ACVDP-DL-PCA)

Rac-1-O- dried by azeotropic evaporation with 4.5 g (6.7 mmol) of dried acyclovir-5'-monophosphoromorpholidate 4-morphine-N, N'-dicyclohexylcarboxamidinium salt and pyridine After adding 30 ml of pyridine to 4.5 g (7.1 mmol) of hexadecyl-2-O-palmitoylglycerol-3-phosphate, the pyridine was concentrated under reduced pressure, and the process was repeated twice.

300 ml of anhydrous pyridine was added to the residue from which moisture was removed, followed by stirring at 25-30 ° C. After pyridine was concentrated under reduced pressure, the remaining amount of pyridine was removed by adding 40 ml of toluene to azeotropic evaporation. 43 ml of 1N hydrochloric acid and chloroform in the residue; Methanol; Dissolve in 430 ml of water (2: 3: 1) (hereinafter referred to as solvent CMW), stir at 18-22 ° C. for 1 hour 30 minutes, and then add 200 ml of chloroform and stir at 18-22 ° C. for 30 minutes to dissolve clearly. I was. The dissolved reactants were adsorbed onto a DE-52 (acetate) cellulose column, washed with 500 mL of solvent CMW and then separated by eluting the adsorbed reaction with 2000 mL of solvent CMW containing 0.05-0.1 n ammonium acetate. 900-2000 mL fractions were collected, concentrated under reduced pressure until white crystals formed, and filtered. The obtained crystals were washed with acetone and dried to obtain a white crystal powder (ammonium salt of the target). The ammonium salt of the target was dissolved in 600 ml of solvent CMW, passed through an Amberlite CG-50 (Na ⁺ ) column, the eluent was collected and distilled under reduced pressure, and the residue was crystallized with acetone to give 2.6 g (40.3%) of the white target. Got it.

Example 2

Preparation of Acyclovir-5'-diphosphate-rac-1-S-hexadecyl-2-O-palmitoyl thioglycerol (ACVDP-DL-PTBA)

Rac-1- dried by azeotropic evaporation with dried acyclovir-5'-monophosphoromorpholidate 4-morpholine-N, N'-dicyclohexylcarboxamidinium salt 2.5 g (3.7 mmol) and pyridine 2.5 g (3.7 mmol) of S-octadecyl-2-O-palmitoyl-1-thioglycerol-3-phosphate was separated in the same manner as in Example 1 to obtain 35% yield.

Example 3

Preparation of Acyclovir-5'-diphosphate-rac-1-S-octadecyl-2-O-palmitoyl-1-thioglycerol (ACVDP-DL-PTBA)

679 mg (1 mmol) of rac-1-S-octadecyl-2-O-palmitoyl-1-thioglycerol-3-phosphate and 371 mg (1 mmol) of tri-n-octylamine were added to 10 ml of methane and warmed. After dissolving, the solvent was distilled under reduced pressure to remove the solvent, and the residue was dissolved in a small amount of N, N-dimethylformamide (DMF) to remove a small amount of water, followed by distillation under reduced pressure. The dried rac-1-S-octadecyl-2-O-palmitoyl-1-thioglycerol-3-phosphate tri-n-octylammonium salt was dissolved in 13 ml of dioxane and 8 ml of DMF, followed by diphenylphosphochlorine. 0.3 ml of data and 0.45 ml of tri-n-butyl amine were added, and the mixture was reacted at room temperature for 2 hours in anhydrous state. After distilling off the solvent by distillation under reduced pressure, 50 ml of ether was added to the mixture, followed by P ^1- (rac-1-S-octadecyl-2-O-palmitoyl-1-thioglycerol-3-yl) -P ² -diphenylpyrophosphate Precipitate was preserved after 30 to 60 minutes at 0 ℃, and then removed by adding ether. The precipitate was dissolved in 2 ml of dioxane and distilled under reduced pressure to remove traces of water.

305.2 mg (1 mmol) of acyclovir-5 -'- monophosphate was dried overnight in the presence of P ₂ O ₅ , dissolved in 1 ml of anhydrous pyridine, mixed with the above-mentioned pyrophosphate dissolved in 0.5 ml of dioxane, under anhydrous conditions 1 It was allowed to react at room temperature for one day. After distilling off the solvent under reduced pressure, 30 ml of ether was added to the residue to precipitate the target substance. This was dissolved in 100 ml of solvent CMW, adsorbed onto a DE-52 (acetate) cellulose column, and eluted and purified in the same manner as above to obtain the target product in a yield of 30%.

Analysis data is the same as in Example 2.

[Test Example]

Intraperitoneal administration: Samples weighed precisely with electronic balance were suspended in physiological saline and exposed to ultrasound (20 min) for complete dissolution.

The dissolved sample was used after filtration and sterilization using a 0.02 μm membrane filter. The specimens were intraperitoneally administered with ACVDP-DL-PCA and ACVDP-DL-PTBA at 25 mg / kg, 50 mg / kg, 100 mg / kg and 200 mg / kg in mice (n = 6). In addition, 12.5mg / kg and 25mg / kg of acyclovir were administered as a positive control group and physiological saline was administered to the control group.

Infection system used for evaluation: The infection system was nasal inhalation infection of herpes simplex virus type-1 and herpes simplex virus type-1 in BALB / C mice (♂, 5 weeks old). The route of administration of the evaluation agent was intraperitoneally 0.2 ml / mouse and was administered for 5 days immediately after infection with herpes simplex virus type-1. The test period was 3 weeks after virus infection.

The drug efficacy was evaluated by comparing the weight increase and survival period between the administration groups, and as a result, the survival rate after the virus infection is shown in FIG. 3 and FIG.

Intraperitoneal administration group: In FIGS. 1 to 4, the control group administered with saline showed a sharp weight loss after 9 days and was annihilated at 11 days. Acyclovir 12.5 mg / kg group also disappeared on day 12 and 14 with rapid weight loss after day 9. In the acyclovir 25 mg / kg group, ACDP-DL-PCA, ACVDP-DL-PTBA 50 mg / kg, 100 mg / kg, 200 mg / kg group, the incidence of death was slow and the weight change was also moderate.

In the results of Figs. 1 and 2, acyclovir 25 mg / kg group compared with ACVDP-DL-PCA and ACVDP-DL-PTBA 100 mg / kg group (Acyclovir: ACVDP-DL-PCA = 1: 1 molar ratio) ACVDP-DL-PCA and ACVDP-DL-PTBA are more than three times more effective than the control drug acyclovir against -1 infection. Therefore, it can be confirmed that ACVDP-DL-PCA and ACVDP-DL-PTBA are antiviral drugs that can exert an excellent effect by intraperitoneal administration than acyclovir.

Claims (4)

Phospholipid complexes of cyclic nucleosides of formula (I) and pharmaceutically acceptable salts thereof, used as antiviral agents. Wherein A is oxygen or a sulfur atom, R ₁ is a saturated or unsaturated alkyl group having 6 to 22 carbon atoms, R ₂ is a saturated or unsaturated alkyl group having 2 to 21 carbon atoms, and B is a general formula As indicated antiviral acyclic nucleosides; Wherein Y is a hydrogen atom or CH ₂ OH and Z is a carbon or oxygen atom. A compound according to claim 1, wherein R ₁ is a saturated or unsaturated alkyl group having 12 to 20 carbon atoms. Acyclovir-5'-diphosphate-rac-1-O-hexadecyl-2-O-palmitoylglycerol (ACVDP-DL-PCA), acyclovir-5'-diphosphate-rac-1-S used as an antiviral agent -Octadecyl-2-O-palmitoyl-1-thioglycerol (ACVDP-DL-PTBA), acyclovir-5'-diphosphate-rac-1-O-octadecyl-2-O-palmitoylglycerol (ACVDP- DL-PBA), acyclovir-5'-diphosphate-rac-1-S-hexadecyl-2-O-palmitoyl-1-thioglycerol (ACVDP-DL-PTCA) or pharmaceutically acceptable salts thereof. The compound according to claim 1 or 2, wherein the salt is a sodium salt.