JPS6338360B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides novel nucleoside derivatives and formula (wherein B is adenine, cytosine, 5-fluorouracil, 5-azacytosine, 6-mercaptopurine or 7-deazaadenine, A and C are each hydrogen or hydroxy group, and W is 8-20 carbon atoms. a saturated or unsaturated alkyl group or a 2- or 3-alkoxyalkyl group having 7 to 19 carbon atoms), and W′ is a saturated or unsaturated alkyl group having 7 to 19 carbon atoms. This invention relates to novel nucleoside derivatives and methods for producing their salts. In formula (), phospholipid encompasses optical isomers LD and DL types, and representative nucleosides include:
9-β-D-arabinofuranosyl adenine (hereinafter referred to as ara-A), 1-β-D-arabinofuranosylcytosine (hereinafter referred to as ara-A),
are-C), 5-fluoro-2'-deoxyuridine or other nucleosides that can be used as anticancer and antiviral agents. The present invention relates to a novel method for producing conjugates of 1-O-alkyl phospholipids and nucleosides. A new compound with formula () was first synthesized by the inventor. The inventors [Journal of Medical
Chemistry (J.of Medical Chemistry) 25,
1322 (1982), Biochemical and Biophysical Research Communication.
Communication) 85, 715 (1978)] and other researchers [Biochimica et Biophysica Acta 69,
604 (1980)] disclosed 1,2-diacylglyceronucleoside conjugates of similar compounds. In this prior art, nucleoside-5'-monophosphomorpholinate was reacted with 1,2-diacylglycero-3-phosphate to obtain this analogous compound. However, the phospholipid moiety of the present invention consists of 1-O-alkyl-2-O-acylglycero-3-phosphate, and no novel conjugates with nucleosides have been reported in the prior art, and the present invention It was manufactured for the first time. The invention will be explained in detail below. The aim of the present invention is to provide novel anticancer and antiviral agents with unique molecular structure and physicochemical properties. Another object of the present invention is to provide a new and high-yield method for producing the above-mentioned novel anticancer and antiviral agents with unique molecular structure and physicochemical properties. Yet another object of the present invention is to provide a method for producing a new class of phospholipid conjugates as anticancer and antiviral agents. Another object of the present invention is to provide lipid vehicles ( An object of the present invention is to provide a new class of liponucleoside compounds that form lysosomes. After the anticancer agent of the present invention penetrates into tumor cells, it is separated into anticancer and antiviral nucleosides or nucleotides by a phospholipid-enzyme specific reaction or a non-specific mechanism, and the cells are treated with phospholipid-specific nucleosides or nucleotides. If it has a binding site, it becomes a specific target compound. Additionally, for nucleosides that require phosphorylation for effective activity, the conjugate provides such functionality, resulting in superior therapeutic efficacy against resisting cells lacking nucleoside kinases. Furthermore, this 1-O
- Alkylphospholipids themselves have pharmaceutical effects, especially anticancer and immunomodulating effects.
The combination of nucleosides and 1-O-alkylphospholipids produces advantageous additive or synergistic effects. In other words, 1-O-
Alkylphospholipid, 1-O-alkyl-2-
Among lysophospholipids and their derivatives, 1-
O-alkyl-2-O-methylphosphatidyl-
It is disclosed that choline or ethanolamine has suppressive and preventive effects and immune transforming effects on cancer in various animals [Anticancer Research 1, 135 and 345 (1981); Seminar in Immunopathology. (Seminar in Immunopathology) 3rd
Volume, 187-203 [1979)]. As long as it remains a conjugate,
The amino group of ara-C or ara-A is prevented from being deaminated by cytidine deaminase or adenosine deaminase. Since the conjugate itself is lipophilic, it acts as a type of sustained release prodrug, reaching the target cell in which it hydrolyzes the conjugate into phospholipids and nucleosides. Since this is the same result as administering two types of pharmaceuticals, it is possible to expect the pharmaceutical effects of both to increase the therapeutic index of anticancer drugs. The method for producing the compound of formula () is schematically shown in the reaction process diagram below. Nucleotides () are condensed to give morpholinate () or ^P1 -nucleoside-5'- ^P2 -diphenylpyrophosphate (), followed by 1-O-alkyl-2-O-acylglycero-phosphate (). Conjugate (b) is obtained from conjugate (a) by reacting with (reaction process diagram, method A or method B). Another route involves converting the phospholipid () to its morpholinate () or ^P1 -glycero-5'- ^P2 -diphenylpyrophosphate (), which is then reacted with a nucleotide () to form the conjugate (c). (d) is obtained from (reaction scheme, method C and method D). The compounds of the invention and their salts can be used in admixture with conventional pharmaceutically acceptable organic and inorganic carrier materials. The compounds of the invention may be in one of the usual acceptable forms such as emulsions, suspensions, ampoules, powders, granules, capsules, tablets, and the like. It may also contain the usual fillers, preservatives, stabilizers, dispersants, fumigants, buffers and colorants.

【table】

[Table] The following examples are for illustrative purposes only;
It is not intended to limit the invention. Example 1 1-β-D-arabinofuranosylcytosine-
5'-diphosphate-1-O-octadecyl-
2-O-palmitoyl-sn-glycerol or 1-β-D-arabinofuranosylcytosine-
5'-diphosphate-β-palmitoyl-L-
Batyl alcohol (a, ara-CDP-L-
PBA) 1.7 g (2.6 mmol) of 1-O-octadecyl-2-O-palmitoyl-sn-glycero-3-phosphate (, n=17, m=14, L-isomer)
After co-evaporation with pyridine to dryness, 1.8
g (2.6 mmol) of ara-CMP morpholinate 4-morpholine-N,N'-dicyclohexylcarboxamidinium salt (B=cytosine). The mixture was dissolved in 150 ml of dry pyridine and stirred for 5 days at room temperature under anhydrous conditions. The pyridine was then removed under reduced pressure and traces of pyridine were removed by co-evaporation of toluene. 15ml of residue
of dry acetic acid and 150 ml of chloroform -CH ₃ OH-
It was dissolved in water (2:3:1) and stirred at room temperature for 1 hour, and 250 ml of chloroform was added to this solution.
The organic solvent layer was separated and concentrated under reduced pressure, and traces of residual acetic acid were removed by coevaporation with 10 ml of toluene in three steps. 100ml of residue
Dissolved in CMW solvent, DE-52 (acetate) cellulose column (2.5 x 50 cm, with jacket, 5
After adsorption to ℃), column 0-0.15M
Elute with _NH4OAc linear gradient solvent CMW (1500 ml each). Collect 900−1500 ml of eluate and evaporate under reduced pressure.
Evaporation at temperatures below 30°C produced white crystals. The white crystalline powder was washed with water, filtered and then dissolved in CMW for conversion to sodium salt, then Amberlite CG-
50 (Na+) column (2.5 x 15 cm), the eluate was collected and evaporated under reduced pressure. Crystallization of the residue from chloroform and acetone yields 820 mg
(31.2%) of the white target compound was obtained. (1) Melting point: 199-202℃ (2) [α] ²⁵ _D = +33.5° (c = 0.23, chloroform-
Methanol-water 2:3:1) (3) NMR (90MHz): Solvent ( _CDCl3 - _CD3OD-
D ₂ O, 2:3:1): δppm0.95 (6, t,
2CH ₃ ), 1.14−1.87 (58, m, 29CH ₂ ), 2.29
(2, t, _CH2 -C=O), 3.27-4.42 (11, m,
H2′, H3′, H4′, H5′, CH ₂ −O−CH ₂ and
CH ₂ -O-), 515 (1, m, glycerol CH),
5.94 (1, d, J=7Hz, cytosine H5), 6.18
(1, d, J=5Hz, H1), 7.80 (1, d, J=
7Hz, cytosine H6) (4) Elemental analysis: C ₄₆ H ₈₇ N ₃ O ₁₄ P ₂・1.5 (CH ₃ ) ₂ CO.
H ₂ O C H N P Calculated value 56.51 9.20 3.92 5.77 Actual value 56.81 9.27 3.69 5.87 Example 2 1-β-D-arabinofuranosylcytosine-
5'-Diphosphate-rac-1-O-octadecyl-2-O-palmitoylglycerol (
b.ara-CDP-DL-PBA) (1) Method A (reaction process diagram, method A) The indicated compound is rac-1-O-octadecyl-2-O-palmitoylglycero-3-phosphate (, n = 17 , m=14, DL-mixture)
was prepared by condensation of ara-CMP-morpholidate (, B=cytosine) and then separated as described above to give the target compound in 35% yield. Mobility in chromatography and
NMR data agreed with theoretical values. (2) Method B (Reaction Process Diagram, Method B) The title compound was prepared by the following method. 323 mg (1 mmol) of ara-CMP, (,
B = cytosine) and 371 mg (1 mmol) of tri-n-octylamine were dissolved in 7 ml of hot methanol, then the solvent was evaporated under reduced pressure and the residue was redissolved in N,N'-dimethylformamide (DMF). and evaporated under reduced pressure to remove traces of water remaining in the residue. The dry ara-CMP-tri-O-octylammonium salt thus obtained was mixed with 10 ml of dioxane and 5 ml of dioxane.
Dissolved in DMF and added to this solution 0.3 ml diphenylphosphochloridate and 0.45 ml tri-n-
Butylamine was added and the mixture was allowed to react under anhydrous conditions at room temperature for 2 to 3 hours. After distilling off the solvent under reduced pressure, 50 ml of ether was added and P ¹ −
(1-β-D-arabinofuranosylcytosine-
5′-yl)-P2-diphenylpyrophosphate (,B=cytosine) was precipitated and incubated at 0°C for 30-
After holding for 60 minutes, the ether was removed. The precipitate was dissolved in 2 ml of dioxane, and traces of water in the precipitate were distilled off under reduced pressure. _663mg on _P2O5
(1 mmol) of rac-1-O-octadecyl-
2-O-palmitoglycero-3-phosphate (, n = 17, m = 14, DL-mixture) was dried overnight and then dissolved in 1 ml of anhydrous pyridine, and the above pyrophosphate () was dissolved in 0.5 ml of dioxane. The reaction was carried out under anhydrous conditions at room temperature for one day. After the reaction, the solvent was distilled off under reduced pressure, and 25 ml of ether was added to the thus obtained residue to precipitate the target compound. 100ml of the precipitate thus obtained
Dissolve in CMW, DE-52 (acetate) cellulose column (2.5 x 50 cm, with jacket,
After adsorption at 5°C), elution and purification as described above gave the target compound in 30% yield. Mobility in chromatography and NMR
The data agreed with the theoretical values. Example 3 1-β-D-arabinofuranosylcytosine-
5'-Diphosphate-rac-1-O-hexadecyl-2-O-palmitoylglycerol or 1-β-D-arabinofuranosylcytosine-
5'-Diphosphate-β-palmitoyl-DL
-thymyl alcohol (c, ara-CDP-L-
PCA) (1) Method A (Reaction Scheme, Method A) Basically, this compound is prepared in the same manner as described in Example 1. 4.19 g (6.6 mmol) rac-1-O coevaporated with pyridine
-hexadecyl-2-O-palmitoylglycero-3-phosphate (, n=15, m=14,
DL-mixture) in 300 ml of anhydrous pyridine.
It was reacted with 3.43 g (5 mmol) of ara-CMP morpholidate (B=cytosine) for 5 days at room temperature under anhydrous conditions. The pyridine is evaporated under reduced pressure and the residue is treated as described in Example 1,
DE-52 (acetate) cellulose column (2.5
x 50 cm, with jacket, at 5 °C), followed by linear gradient CMW containing 0-0.15 M NH ₄ Ac.
It was eluted with a solvent (1500 ml each), and the fractions containing the target compound were combined and concentrated under reduced pressure at a temperature below 30°C to produce white crystals. The thus obtained crystals were treated with an Amberlite CG-50 (Na+) column to obtain the target compound with a yield of 30%. (1) Melting point: 202-205℃ (decomposition) (2) NMR (90MHz): Solvent (CDCl ₃ −CD ₃ OD−
D ₂ O, 2:3:1): δppm0.87 (6, t,
2CH ₃ ), 1.07−1.78 (54H, m, 27CH ₂ ), 2.35
(2, t, _CH2 -C=O), 3.27-4.35 (11, m,
H ₂ ′, H ₃ ′, H ₄ ′, H ₅ ′, CH ₂ −O−CH ₂ , CH ₂ −
O), 5.15 (1, m, glycerol CH), 5.92
(1, d, J=7Hz, cytosine H ₅ ), 6.14 (1,
d, J = 5 Hz, H ₁ ′), 7.88 (1, d, J = 7 Hz,
Cytosine H ₆ ) (3) Elemental analysis: C ₄₄ H ₈₁ N ₃ O ₁₄ P ₂ Na ₂・0.5H ₂ O C H N P Calculated value 53.21 8.43 4.23 6.24 Actual value 53.69 9.27 3.92 5.72 (2) Method B (reaction Process diagram, method C) 3.17 g (5 mmol) rac-1-O-hexadecyl-2-palmitoylglycero-3-phosphate (, m = 15, n = 14, DL-mixture) and 1.7 ml (20 mmol) ) of morpholine,
75 ml of 4.12 g (20 mmol) of N,N'-dicyclohexylcarbodiimide (DCC) and 50 ml of t-butyl alcohol.
A solution of t-butyl alcohol was added dropwise, and the mixture was reacted under reflux conditions for 2 hours. After the reaction mixture was stirred at room temperature overnight, 20 ml of water was added and stirred for 2 hours at room temperature to destroy the remaining DCC.
The white crystals thus formed are removed by filtration,
The filtrate was concentrated by evaporation under reduced pressure and extracted with ether. The extract was evaporated under reduced pressure and the resulting residue (, n = 15, m = 14, DLC-mixture) was co-evaporated twice with toluene to yield 2.13 g (6.6
mmol) of ara-CMP (, B = cytosine)
and 4.67 g (13.2 mmol) of tri-n-octylamine and the mixture was again diluted with pyridine.
After drying by step co-evaporation, it was dissolved in 200 ml of pyridine and reacted by stirring at room temperature for 7 days under anhydrous conditions. Pyridine is distilled off under reduced pressure,
Further, remaining traces of pyridine were co-evaporated with a small amount of toluene to completely remove them. 30 residue
It was dissolved in ml of acetic acid and 300ml of CMW solvent, stirred for 1 hour at room temperature, and then 500ml of chloroform was added. Separate the organic layer and concentrate under reduced pressure.
The remaining traces of acetic acid are mixed with 10 ml of toluene and 3
It was completely removed by step co-evaporation. Dissolve the residue in 100 ml of CMW solvent and DE
-52 (acetate) cellulose column (2.5 x 50
cm, jacketed at 5°C) and then 0-1.15M as described in Example 1.
Elute with a linear gradient CMW solvent containing _NH4OAc ,
The target compound was obtained with a yield of 30%. Chromatographic mobility and NMR data were consistent with theoretical amounts. Example 4 9-β-D-arabinofuranosyl adenine-
5'-Diphosphate-rac-1-O-hexadecyl-2-O-palmitoylglycerol or 9-β-D-arabinofuranosyladenine-
5'-Diphosphate-β-palmitoyl-DL
-thymyl alcohol (d,ara-ADP-DL
-PCA) (reaction scheme, method A) 1.90 g (3 mmol) rac-1-O-hexadecyl-2-O-palmitoylglycero-3-phosphate (, n=15, m=14, DL-mixture)
was dried by co-evaporation with pyridine in three steps to give 2.13 g (3 mmol) of ara-AMP morpholidate-4-morpholine-N,N'-dicyclohexyl-carboxamidinium salt (B = adenine). was dissolved in 200 ml of pyridine and the solution was stirred under anhydrous conditions at room temperature for 7 days. The pyridine was distilled off under reduced pressure and the residue was treated in a manner similar to that described in Example 1, adsorbed onto a DE-52 (acetate) cellulose column (2.5 x 50, jacketed, 5°C) and 0- Linear gradient containing 0.15M _NH4OAc
After elution with CMW solvent, Amberlite CG-
50 (Na+) column, 851 mg (29%) of the sodium salt was obtained. (1) NMR (90MHz): Solvent (CDCl ₃ −CD ₃ OD−
D ₂ O, 2:3:1): δppm0.92 (6, t,
2CH ₃ ), 1.07−1.78 (54H, m, 27CH ₃ ), 2.35
(2, t, _CH2 -C=O), 3.27-4.35 (11, m,
H ₂ ′, H ₃ ′, H ₄ ′, H ₅ ′, CH ₂ −O−CH ₂ ; CHz
-O), 5.07 (1, m, glycerol CH), 6.33
(1, d, J = 4.5H ₂ , H ₁ '), 8.17 (1, s, adenine H ₂ ), 8.40 (1, s, adenine H ₈ ) Experiment 1 Intraperitoneal administration of L1210 to lymphocytic leukemia mice Antitumor activity: 1×10 ⁶ or 1×10 ⁵ in DBA/2J mice
L1210 lymphocytic leukemia cells were administered intraperitoneally, and 24 hours later, the drug was dissolved in 0.9% NaCl and injected into mice, and the survival rate was measured for 45 days. The test was conducted in accordance with the US National Cancer Institute protocol (Cancer, Chemotherapy Reports 3, 1-103, 1972). The processing plan is qd1, qd1, 5, 9
and qd1-5. The optimal dosage in Table 1 is
It is the amount that produces maximum activity. A wide dose range was tested. Activity was measured by comparing the increase in longevity. This was done by comparing the average lifespan of test and control mice. Table 1 shows ara-C and its conjugate ara
-DCP-DL-PBA (b) and ara-CDP-
The treatment results of PCA (a) are shown. The first part shows the results of ara-C sensitive L1210 lymphocytic leukemia mice. Untreated control mice died 7 or 8 days after inoculation with tumor cells. ara-C
When injected at an optimal single dose of 400 mg (1644 micromoles)/Kg, the increase in lifespan (%ILS) was 14
%, 200mg (822 micromoles) once a day
When injected for 5 days, the ILS was 129%.
However, the optimal single dose of 400 mg (395 and 407 micromoles)/Kg of the conjugates ara-CDP-DL-PBA (Ib) and ara-CDP-DL-PCA (Ic)
showed excellent ILS of 257% and 293%, respectively. Five injections at the optimal dose also produced excellent ILS of 229% and 264%, respectively. ara
Comparison of single doses of -C and its conjugates is out of scope. In the 5-dose regimen, the conjugate is ara-
A dose of 1/8 or 1/10 of the C mol dose was twice as effective, and the toxicity was significantly lower. Secondly,
Therapeutic treatment of ara-C resistant L 1210 lymphocytic leukemia mice with low abundance deoxycytidine kinase resulted in untreated control mice dying 8 to 11 days after inoculation with tumor cells. A single dose treatment with ara-C had little effect (6% ILS), and a 5-day treatment resulted in a 65% ILS.
These results indicate that ara-C contains only a minimal amount of deoxycytidine kinase. However, conjugates of ara-C, i.e. ara-CDP
−DL−PBA(Ib) and ara−CDP−DL−PCA
(Ic) has produced remarkable therapeutic results and holds great promise for fighting cancer. Optimal dosage used as a single dose or 80mg/Kg per day for 5 days
At 400 mg/Kg, ILS was 259 to >356%, 6
One to three of the mice survived for more than 45 days and were completely cured; −CDP−PBA−(Ib) is more than 290%
ara-CDP-PGA-(Ic) showed more than 374% ILS, and 3 or more mice, especially ara-
In the case of CDP-DL-PCA, 6 out of 6 mice survived for more than 45 days and were cured. For example, a dose of 1/3 mole of ara-C was 5 times more effective. The fact that the conjugate is effective in deoxynase-deficient ara-C-resistant L1210 lymphocytic leukemia mice indicates that the conjugate is introduced into the cell by endocytosis or other mechanisms and is enzymatically converted to phospholipids. The ara-CMP thus liberated is continuously phosphorylated to ara-CTP. Therefore, the deoxycytidine kinase required for phosphorylation of ara-C is not required since ara-CMP is released from the conjugate. Advantages As shown in the above studies, these conjugates exhibit greater activity than the parent drug ara-C, even at lower doses, which in turn may result in an improved therapeutic index of the parent drug. It turns out. Furthermore, ara-
C had a short half-life and required continuous administration to be effective, whereas the conjugate has greater and better activity in a single dose. Therefore, the conjugate can be used as a sustained release drug. In particular, the fact that the conjugate is effective in ara-C-resistant L1210 lymphocytic leukemia mice is due to the fact that it hydrolyzes in cancer cells to release ara-C and phospholipids, and is highly effective in deoxycytidine kinase-deficient resistant cells. means valid. Phospholipids are 1-O-alkyl-2-O-acylglycero-3-phosphates, and after biochemical reactions, 1-O-alkyl-
Converted to 2-lysophosphatidyl-choline or -ethanolamine, these compounds themselves have growth- and transformation-inhibiting and immunotransforming effects on cancer cells, so they exhibit additive and synergistic effects. There is expected. The conjugate is simply
It is not considered to be a prodrug of ara-C, but rather a novel drug. Furthermore, compared to other lipophilic prodrugs, the present conjugates have the advantage of being transparently suspended in water by ultrasound. leukemia patient
For treatment with ara-C, it has been reported in the literature that the development of resistance to this drug is related to the relative cellular content of deoxycytidine kinase and cytidine deaminase [Annals of the New York Academy of Sciences] AoAcademy of Science)
255 247 (1975)]. On the other hand, the ara-C conjugate is resistant to ditidine deaminase and does not hydrolyze amino groups. This conjugate is considered to have great effects in the treatment of ara-C resistant leukemia patients.

【table】

【table】

Claims (1)

[Claims] 1 formula (wherein B is adenine, cytosine, 5-fluorouracil, 5-azacytosine, 6-mercaptopurine or 7-deazaadenine, A and C are each hydrogen or hydroxy group, and W is 8-20 carbon atoms. a saturated or unsaturated alkyl group or a 2- or 3-alkoxyalkyl group having W' is a saturated or unsaturated alkyl group having 7-19 carbon atoms) and pharmaceutically acceptable Non-toxic salt. 2 formulas nucleotides having the formula morpholidate or formula having the formula (wherein B is as defined above) (wherein B is as defined above)
P ¹ -nucleoside-5′-P ² -diphenylpyrophosphate and then the formula (wherein W is a saturated or unsaturated alkyl group or a 2- or 3-alkoxyalkyl group having from 8 to 20 carbon atoms, and W' is a saturated or unsaturated alkyl group having from 7 to 19 carbon atoms. ) having the formula (wherein A and C are each a hydrogen or hydroxy group) or a salt thereof. A method for producing a nucleoside conjugate having the formula (). 3 formulas (wherein W is a saturated or unsaturated alkyl group or a 2- or 3-alkoxyalkyl group having from 8 to 20 carbon atoms, and W' is a saturated or unsaturated alkyl group having from 7 to 19 carbon atoms. ) is condensed to give the formula (In the formula, W and W′ are as defined above.)
Phospholipid morpholidate or with the formula (In the formula, W and W′ are as defined above.)
P ¹ -glycero-5′-P ² -diphenylpyrophosphate with the formula (wherein B is adenine, cytosine, 5-fluorouracil, 5-azacytosine, 6-mercaptopurine or 7-deazaadenine) by reacting with a nucleotide having the formula (wherein A and C are each a hydrogen or hydroxy group) or a salt thereof. A method for producing a nucleoside conjugate having the formula (). 4. The compound according to claim 1, wherein the target compound is 1-β-D-arabinofuranosylcytosine-5'-diphosphate-1-O-octadecyl-2-O-palmitoyl-sn-glycerol. 5 The target compound is 1-β-D-arabinofuranosylcytosine-5'-diphosphate-rac-1-O
-Octadecyl-2-O-palmitoylglycerol. 6 The target compound is 1-β-D-arabinofuranosylcytosine-5'-diphosphate-rac-1-O
-hexadecyl-2-O-palmitoylglycerol. 7 The target compound is 9-β-D-arabinofuranosyl adenine-5'-diphosphate-rac-1-O
-hexadecyl-2-O-palmitoylglycerol.