JPH04210993A - New phospholipid-nucleoside derivative - Google Patents

Abstract

PURPOSE:To obtain the title compound useful as an antitumor agent, having antitumor action, inhibitory action on tumor metastasis, excellent durability in vivo and low toxicity. CONSTITUTION:A compound shown by formula I (R1 and R2 are long-chain fatty acid residue; Ns is 5-fluoro-2'-deoxyuridin-5'-yl, bredinin-5'-yl, tubercidin-5'- yl, neplanocin-A-6'-yl or 5-fluorocytidin-5'-yl). The compound is obtained by reacting a phosphatidylcholine-based glycerophospholipid shown by formula II (R9 is choline residue) with nucleoside optionally in the presence of a metal ion by using phospholipase D in a solvent.

Description

[Detailed description of the invention]

[00011

[Industrial Application Field] The present invention relates to novel phospholipid/nucleoside derivatives. More specifically, the present invention is based on the following general formula [■]

[Formula 1] (wherein, R1 and R2 represent long-chain fatty acid residues, and Ns is a 5-fluoro-2"-deoxyuridin-5"-yl group, a predainin-5"-yl group, or a tubercidin-5' -yl group, neplanocin A-6'-yl group, and 5-fluorocytidin-5°-yl group), or a salt thereof. Regarding. [0002]

BACKGROUND OF THE INVENTION Nucleoside antitumor agents have been widely used clinically as agents useful in chemotherapy for various types of tumor cells. However, several problems have been pointed out in its application as a chemotherapeutic agent. In other words, the mechanism of action of these nucleoside antitumor agents is that they are 5''-phosphorylated to express their activity, and when inactivated by phosphorylation, deamination, etc., they rapidly become inactive. They have various drawbacks, such as being easily broken down into substances, causing tumor cells to become resistant to these antitumor drugs, and being toxic to rapidly dividing normal cells. Since ribonucleosides play an important role in the biosynthesis of intracellular glycerophospholipids and the structure of membranes, liponucleosides containing nucleosides have been chemically synthesized. [0003] On the other hand, the nucleoside-based antitumor Various compounds have been chemically synthesized as prodrugs to improve the toxicity and other shortcomings of drugs.From this background, attempts were made to synthesize ribonucleosides with antitumor activity (cytotoxicity). Arabinoside (a r a
Liponucleosides containing C) have been synthesized and have been shown to be effective to some extent (Biochimica et al.
Biophysica Acta, 619 (1980)
619-631. J.Med.Chem., 1982,
25.1322-1329). [0004]

[Problem to be solved by the invention] As mentioned above, since ponucleosides are synthesized by chemical synthesis,
Its synthesis required a multi-step reaction process, resulting in low yield and complicated steps. Moreover, for this reason, there is only an example of cytosine arabinoside as a nucleoside residue component of phospholipid/nucleoside derivatives, and therefore, its effect as an antitumor agent is ultimately limited to cytosine arabinoside (ara-C:1- (β-arabinofuranosylcytosine), and therefore the drawbacks associated with cytosine arabinoside, such as toxicity, were not improved. [0005]

[Means for solving the problem] One way to solve these drawbacks is to use nucleoside compounds other than cytosine arabinoside, but chemically converting these phospholipid/nucleoside derivatives It required a multi-step synthesis process, and the reaction conditions were difficult to set, making the synthesis practically difficult. [0006] The present inventors have conducted repeated research in an attempt to improve the synthetic method having such drawbacks, synthesize new phospholipid/nucleoside derivatives, and obtain substances superior to the above-mentioned known antitumor agents. As a result, by reacting glycerophospholipid and nucleoside in the presence of phospholipase D, the primary alcohol group of nucleoside and glycerophospholipid easily react, resulting in a novel phospholipid/nucleoside derivative represented by the general formula [■] This is what I got. [0007] The present invention was completed based on the above findings, and includes the following general formula CI)

It is a phospholipid/nucleoside derivative or a salt thereof represented by the following formula (wherein R1, R2, and Ns represent the same groups as above). [0008] First, as the glycerophospholipid used to obtain the phospholipid/nucleoside derivative represented by the general formula [■] of the present invention, for example, a phosphatidylcholine glycerophospholipid represented by the following general formula [■■] is used. Can be mentioned.

[Image Omitted] [1] (In the formula, R1 and R2 represent the same groups as above,
(R3 represents a choline residue) [0009] In the phosphatidylcholine glycerophospholipid represented by the general formula [:II], the groups R1 and R2 represent the same or different long-chain fatty acid residues; For example, it is a long chain fatty acid residue having 16 to 20 carbon atoms, and in detail, for example, valmitoyl, stearoyl,
Long-chain saturated fatty acid residues with 16 to 20 carbon atoms such as dodecanoyl, palmitooleoyl, oleoyl, liloil,
Examples include long-chain unsaturated fatty acid residues having 16 to 20 carbon atoms and having 1 to 4 unsaturated bonds, such as lilnoyl and arachitonyl. [00101 Compounds of general formula (II) include saturated or unsaturated fatty acid residues such as dipalmitoyl phosphatidylcholine in which R1 and R2 are both valmitoyl groups, and silyloyl phosphatidylcholine in which R1 and R2 are both lyluyl groups. It may also be a natural phosphatidylcholine in which R1 and R2 are represented by a Radyl group, which is a mixture of long chain fatty acids having 16 to 20 carbon atoms. Further, the phosphatidylcholine having these R1 and R2 groups may be synthesized using a fatty acid having 16 to 20 carbon atoms as appropriate, or a commercially available one may be used. [0011] Further, as the nucleoside used in the present invention, for example, 5-fluoro-2'-deoxyuridine [:5-Fluoro-2'-deoxyuridine]
ine; hereinafter abbreviated as FUDR], predeinine (B r
e d 1nin; 4-Carbamoyl-1-β
-β-ribofuranosyl-imidazo
lium-501ate], tubercidin (Tube
rcidin; 7-Deazaadenos 1ne)
, Neplanocin A (Nep 1 anocinA;
1-β-(6-amin. 9H-9-yl)-4-hydroxymethy14
-cyclopentene-2a,3a-diol
;hereinafter abbreviated as NepA], 5-fluorocytidine [:5
-Fluorocyt 1dine; hereinafter abbreviated as FCR]. [0012] Furthermore, the phospholipid/nucleoside derivative represented by the general formula [■] can be obtained by reacting the above-mentioned glycerophospholipid and nucleoside in a solvent using phospholipase D in the presence of a metal ion as necessary. You can get it. As the phospholipase D used, for example, Streptomyces sp.
Phospholipase DP derived from @AA586; FERMP-6100 (Japanese Unexamined Patent Publication No. 152481/1981, Catalog No. P-39 manufactured by Toyo Jozo Co., Ltd.) is preferred. The amount used is 1 to 100 units of phospholipase per 1 mg of phosphatidylcholine, preferably 1 to 100 units.
It is a unit. [0013] Further examples of the solvent include a two-layer solvent consisting of an organic solvent layer such as ether, benzene or chloroform, and a buffer solution of pH 3 to 9, preferably a buffer solution of pH 4 to 6, consisting of an organic solvent layer and an aqueous layer. It will be done. Furthermore, calcium chloride is usually used as a water-soluble salt for forming metal ions, and the reaction temperature is usually 20 to 6
At 0°C, a reaction time of 30 minutes to 5 hours is sufficient. The phospholipid/nucleoside derivatives obtained in this way are
It can be purified by separation method and silica gel chromatography. [0014] The one-step synthesis method for the phospholipid/nucleoside derivative of the present invention as described above is shown as follows.

[0015] The phospholipid/nucleoside derivative obtained in this way has a phosphoric acid group moiety of the phospholipid and the nucleoside position 5′ (or 6″ in the case of NepA)
The -class hydroxyl group portions (position) are bonded together through a base exchange reaction. Furthermore, this derivative can also be made into a non-toxic salt such as a sodium salt, and is generally administered orally in the form of tablets, granules, liquids, etc., and parenterally in the form of halved or suspended in distilled water for injection. It can be administered in cloudy liquid form. [0016]

Effects of the Invention: The phospholipid/nucleoside derivative of the present invention obtained in this way has greater fat solubility than the nucleoside used as the original raw material, so it remains in the body for a long time (therefore, its activity is sustained). 5”-monophosphate forms of antitumor nucleosides are resistant to inactivation such as deamination, phosphorylation, and reduction, and have increased affinity for biological membranes. The novel phospholipid/nucleoside derivative according to the present invention has advantages such as being produced within the body, having sustained and enhanced activity, and having low toxicity. in vivo
) has been shown to have prominent antitumor effects. Furthermore, anti-metastatic effects have been observed, which inhibit the metastasis of tumors generated in vivo to other sites. (00181 Regarding the phospholipid/nucleoside derivatives of the present invention)
88 carcinoma) and Ehrlich ascites carcinoma (
Ehrich ascites carcinom
The results of investigating the antitumor activity against a) are shown below. [0019] Antitumor Effect> Sample: Listed in Table 1. [00201 Animals: BDF+ or ICR mice, 5-6 weeks old, male, 5 mice per group, 7 mice in the control group (non-drug administration group). [00211 tumor cells: P-388 leukemia cells = 1 x 1
0610, 2 ml was implanted intraperitoneally into BDF1 mice. Ehrlich ascites cancer cells: 2X10610.2ml I
Transplanted intraperitoneally into CR mice. [0022] Sample Preparation Dosing Schedule: Suspend each sample in Tris-HCl buffered saline by sonication. 0.0% per 10g mouse weight. 1 ml was administered intraperitoneally. Store prepared samples at 4°C, protected from light. Administration: P-388 leukemia; administered once a day for 3 to 5 days starting the day after tumor transplantation. Ehrlich ascites carcinoma; post-tumor transplantation 2
Administer once a day for 2 to 7 days from day 1 (the day after next). Sample administration details are shown in Tables 1 and 2. [0023] The life extension rate was determined as follows. Average life expectancy (ILS) (%) for sample administration group =
Average survival days of X100 control group [0024] Observation period = 35 days (partially 30 days),
Mice that survived the final test are not included in the survival rate. [0025] Control group mean survival days: P-388 leukemia transplant group Nia, 57-7.79 days Ehrlich ascites cancer transplant group: 15.14-15.43 days [00
26]

[Table 1] [0027]

[Table 2] [0028]

[Examples] The present invention will be specifically described below with reference to Examples, but the present invention is not limited by these in any way. [0029] Example 1 2.0 g of Neplanocin A (NepA) was added to 100 mM
100mM acetate buffer containing calcium chloride (pH 5, 6)
) and stirred at 45°C for 20 minutes. In addition, phospholipase D-P (derived from Streptomyces genus,
Toyo Jojo Co., Ltd.) 10■ (specific activity = 160 units/■) and dipalmitoylphosphatidylcholine 1.5g at 30
The mixture was added as a solution in m1 chloroform (manufactured by Merck, for liquid chromatography), and stirred at 45° C. for 3 hours to react. After the reaction, the reaction solution was cooled. To this reaction solution, 20 ml of methanol was added to separate the layers, and the organic layer was collected. To the remaining aqueous layer, 30 ml of chloroform and 15 ml of methanol were added to separate the layers. Combine the organic layer and add water 2
0 ml and 20 ml of methanol were added to separate the liquids, filtered through Whatman 1-PS filter paper, and then dried under reduced pressure. After adding 30 ml of a chloroform:ethanol (1:1) mixture to the residue and drying it under reduced pressure again, the residue was dissolved in a small amount of chloroform and applied to a flash column (Merck, Silica Gel A).
Charge to rt9385, diameter 4cm x 15cm),
From chloroform, chloroform:methanol mixture (2
0: 1), (7: 1), (4: 1), (3: 1)
), (2:1) was developed and eluted in this order. The eluate was dried under reduced pressure to obtain 0.31 g (yield 17.0%) of a compound represented by the following structural formula [■b] as a white powder. [00301

[Chemical formula 4] (In the formula, both R1 and R2 represent a valmitoyl group) UV absorption spectrum λmax: 261n
m (methanol chloroform-20: 1) FAB mass spectrum: m/e894 (MH) Rf value: 0.
38 (chloroform: methanol: water = 65:25:3
was used as a developing solvent, an Art5715 plate manufactured by MerCk was used, and spots were detected using a UV lamp and a molybdenum blue reagent. Note that the following Rf values were measured under the same conditions. ) The antitumor activity of this compound is as shown in Table 1, and an acute toxicity test at 250 μ/kg showed no abnormalities. [0031] Examples 2 to 4 Using various nucleosides shown in Table 3 instead of NepA in Example 1, the following procedure was carried out in the same manner as in Example 1 to obtain the target phospholipid represented by the general formula CI). A nucleoside derivative was obtained. All of these exhibited useful antitumor activity. Further, as a result of acute toxicity testing of all the compounds in Table 3 at a dose of 150 mg/kg, no abnormality was observed. [0032] In Table 3, * marks indicate that 100 mM acetate buffer (pH 5, 6) containing 100 mM calcium chloride was added to
The case where 5 ml was used is shown, and the mark ** indicates that the target product was separated and recovered based on the following method. That is, 20 ml of methanol was added to the reaction-completed solution after cooling, insoluble matter was filtered off, and methanol:chloroform (1:
The organic layer was washed with brine, filtered through Whatman 1-PS filter paper, and then dried under reduced pressure. The residue was dissolved in a small amount of chloroform and charged to a flash column (Merck silica gel Art 7747, diameter 4 cm x 15 cm), and the mixture was converted from chloroform to chloroform:methanol (10:1) to (7:1).
1), (5:1), (3:1), and (2:1) were developed and eluted in this order and dried under reduced pressure to recover the desired powder. [0033]

[Table 3] [0034] Examples 5 to 8 Various nucleosides shown in Table 4 were mixed in 100 mM acetate buffer (pH 5, 6) containing 100 mM calcium chloride 20
After stirring at 45° C. for 20 minutes, 10 mg of phospholipase DP (manufactured by Toyo Jozo Co., Ltd.) and 1.5 g of phosphatidylcholine (egg yolk lecithin) were added as a chloroform solution of 30 ml. Next, the mixture was stirred and reacted at 45° C. for 3 hours, and then cooled. After the reaction, the liquid was separated in the same manner as in Example 1, and silica gel chromatography was performed to obtain the desired phospholipid/nucleoside derivative represented by the general formula [■]. All of these exhibited useful antitumor activity. Further, as a result of acute toxicity testing of all the compounds in Table 4 at a dose of 150 mg/kg, no abnormality was observed. In Table 4, *, **, and ■) have the same meanings as above. [0035]

[Table 4] [0036] Examples 9 to 13 The following procedures were carried out in the same manner as in Example 1, except that in Example 1, various nucleosides shown in Table 5 were used in place of NepA, and distearoylphosphatidylcholine was used in place of sibaytylphosphatidylcholine. The desired product, a phospholipid/nucleoside derivative represented by the general formula [■], was obtained. All of these showed useful antitumor activity. In addition, each compound shown in Table 5 was used at 150 mg/
kg was intraperitoneally administered to mice and an acute toxicity test was conducted, and no abnormalities were observed. [0037]

[0038] Furthermore, below, phospholipid/nucleoside derivatives produced in the same manner as in Example 1 by using various raw material compounds below in place of the phospholipid and nucleoside in Example 1 are listed. [0039]

[Table 6]

[0040]

[Table 7]

Claims (2)

[Claims] [Claim 1] The following general formula [■] [I] (wherein, R1 and R2 represent long-chain fatty acid residues, and Ns is 5-fluoro-2'-deoxyuridine 5''-
yl group, predeicon-5'-yl group, tubercidin-
5"-yl group, neplanocin A-6"-yl group and 5
A novel phospholipid/nucleoside derivative or a salt thereof, represented by (representing a nucleoside residue selected from the group consisting of -fluorocytidine-5"-yl group). Claim 2: In the general formula CI), R1 and R2
The novel phospholipid/nucleoside derivative or salt thereof according to claim 1, wherein is a valmitoyl group and Ns is a neplanocin A-6'-yl group.