Cytotoxic liponucleotide analogs

#This invention was funded in part by Grant Nos. CA 15898 and CA 13943 from the National Cancer Institute, National Institutes of Health.

#This invention relates to a new class of cytotoxic liponucleotide analogs (sometimes abbreviated herein as CLNA) drugs having useful cytotoxic activity, to methods of preparing and using such drugs and to pharmaceutical compositions containing cytotoxically effective amounts of such drugs as a primary active ingredient.

#Cancer can be considered as a group of diseases that can occur in any tissue, organ or system of the body. The causes of all cancers are not known, nor are there any reported major qualitative metabolic differences between cancer cells and host tissue cells of origin. Accordingly, cancer chemotherapy, unlike the chemotherapy of infectious diseases wherein the disease-causing organism itself offers a distinct metabolic or structural biologic target, has far more restrictive fundamental concepts on which to pattern therapeutic treatment.

#A mixture of 0.2 M acetate buffer (pH 5.6, 130 ml), 1.0 M aqueous CaCl.sub.2 solution (36 ml), and phospholipase-D (150 mg) was stirred for 30 minutes at 25.degree.. A suspension of 3.0 g of egg lecithin in 70 ml Et.sub.20 was added to the above mixture and stirred at 25.degree. for 4 hr. The progress of the reaction was checked by TLC; when the reaction was completed (4-5 hr.), the ether was removed by a N.sub.2 stream. The reaction product was extracted with 200 ml of CHCl.sub.3 /CH.sub.3 OH (1:1, v:v). The chloroform phase was separated and then again extracted with 100 ml of CHCl.sub.3. The chloroformic phases were combined, washed twice with 50 ml volumes of H.sub.2 O and the solvent was removed under reduced pressure. The residue was dissolved in Et.sub.2 O and the solution was dried over Na.sub.2 SO.sub.4 and filtered. The filtrate was concentrated to a volume of about 40 ml and phosphatidic acid, mostly as the calcium salt, was precipitated by addition (ca 150 ml) of CH.sub.3 OH. The mixture was allowed to cool (ca 5.degree.) overnight and centrifuged; the precipitate was washed with cold acetone and with 95% ethanol, then dried to yield 1.8 g of phophatidic acid, mostly as a calcium salt.

#The phosphatidic acid (500 mg), chloroform (20 ml), methanol (20 ml), and 0.1 N HCl (20 ml) was mixed well in a separator. The chloroform phase was separated and evaporated to dryness with 10 ml of benzene. The residue was lyophilized and thoroughly dried under reduced pressure (10.sup.-1 mm Hg). The dry phosphatidic acid, 470 mg (0.636 mmol), and 1-beta-D-arabinofuranosylcytosine 5'-(hydrogen morpholino phosphonate) compound with N,N'-dicyclohexyl-4-morpholine carboxamidine (1:1), 660 mg (0.802 mmol), was suspended in 20 ml of dry benzene and evaporated to dryness. The evaporation process (20 ml dry benzene) was repeated and 25 ml of dry distilled pyridine was added to the dry residue; the mixture then was stirred in a tightly stoppered flask at room temperature for four days. Pyridine was removed by a vacuum pump at 30.degree. and the residue in the flask was dried in a vacuum desiccator over H.sub.2 SO.sub.4 for 16 hr. The residue, almost free from pyridine, was shaken with 50 ml of chloroform-methanol (1:1, v:v) and 20 ml of 0.1 N ice-cold HCl. The chloroform phase was removed. The aqueous phase was once more extracted with 10 ml of CHCl.sub.3. The chloroformic layers were combined and carefully washed with 10 ml of cold H.sub.2 O to avoid excessive emulsion formation. The aqueous layer was removed and the chloroform phase was dried over Na.sub.2 SO.sub.4 and filtered; the residual Na.sub.2 SO.sub.4 and flask were washed with CHCl.sub.3, followed by filtration. The filtrates were combined and concentrated under reduced pressure. The residue was deposited on a short, wide column (2.75.times.3.0) of silica gel. The column was eluted with chloroform and then with increasing concentrations of methanol in chloroform. Most of the unreacted phosphatidic acid was eluted with 20 to 25% methanol in chloroform; the product was eluted with CHCl.sub.3 /MeOH (1:1, v:v) and had traces of impurities, mainly phosphatidic acid. Fractions containing the desired product (TLC) were combined and the pH of the eluate (pH=6.5) and was brought to 7.5 (pH meter) with methanolic NH.sub.4 OH. The solvent was evaporated and the residue was dissolved in 20 ml of chloroform/pyridine/formic acid 80% (50:30:7, v:v:v) and applied to a second silica column (2.times.25 cm) prepared in the same solvent system and eluted with 200 ml of the same mixture. The column was eluted with CHCl.sub.3 /MeOH (9:1, v:v), the methanol concentration being gradually increased to 50% to elute the desired product. The desired fractions were combined and the solvent evaporated to ca 2-3 ml to obtain a white precipitate. To this mixture cold acetone (5 ml) was added and the suspension was centrifuged. The supernatant was discarded and the residue was dissolved in a mixture of 10 ml of chloroform methanol (2:1, v:v); cold 0.1 N HCl was added to pH 2.5. The aqueous phase was discarded and washed with H.sub.2 O by centrifugation. The chloroform phase was brought to pH 7.5 with 2 N methanolic ammonium hydroxide and the solvent was evaporated under reduced pressure. The residue was thoroughly dried in a vacuum desiccator over P.sub.2 O.sub.5 to give 300 mg of a white amorphous solid: mp yellow, then brown ca 180.degree., melts (brown mass) 218.degree.-220.degree. C. dec; R.sub.f (TLC) 0.72 [silica, CHCl.sub.3 /MeOH/H.sub.2 O/glacial HOAc (25:15:4:2)]; ir (KBr) 2915, 2849, 1724, 1575 (broad), 1235 (broad), 1117, 1064 (broad), 926 (broad), 784 (broad), 712 (broad).

#2, 3-Dihexadecylglycerylphosphoric acid (103 mg, 159 umol) and 1-beta-D-arabinofuranosylcytosine 5'-(hydrogen morpholino phosphonate) compound with N,N'-dicyclohexyl-4-morpholine carboxamidine (1:1) (153 mg, 223 umol) were suspended in 10 ml of benzene and the mixture was evaporated; the evaporation process then was repeated two more times. Pyridine (15 ml) was added rapidly to the reaction mixture, which then was stirred (magnetic bar) for 70 hours at room temperature (25.degree.) in a tightly stoppered ground glass flask. Most of the solvent was removed under reduced pressure (10-15 mm/oil pump) and the product in the original reaction flask was dried in vacuo (0.5-1.0 mm) over concentrated H.sub.2 SO.sub.4 in a desiccator for one day. The product, a light-yellow-colored solid film, was dissolved in 10 ml of ice-cold water, and 0.5 N HCl was added dropwise to the solution until the pH was 2.0; the mixture appeared as a white flaky solid suspended in H.sub.2 O. To the acidified suspension was added 15 ml of CHCl.sub.3 -MeOH (1:1) and the mixture was shaken gently. An emulsion readily formed which was broken upon letting the mixture stand in the cold (ice-bath) for ca one-half hour. The chloroform layer was separated and the aqueous phase was extracted two more times with 15-ml portions of CHCl.sub.3 /MeOH (1:1). To the separated and then combined chloroform phases 10 ml of MeOH/H.sub.2 O (2:1) was added and pH of the solution was brought to 7.8 with 2 N methanolic NH.sub.4 OH. The aqueous phase was separated and the chloroform phase was repeatedly (4-5 times) extracted with MeOH/H.sub.2 O (2:1). The original aqueous phase and the combined MeOH/H.sub.2 O extracts then were placed in a beaker and the solution was concentrated to about 1 ml with a stream of N.sub.2. To the concentrated mixture 10 ml of acetone was added which resulted in further deposition of a white solid. The mixture then was kept at -40.degree. for 2-3 hours, centrifuged and the solvent removed by decantation. The white amorphous solid was washed with 2 ml of Et.sub.2 O and was dried in vacuo (0.5-1.0 mm) over P.sub.2 O.sub.5 for two days at room temperature to give 50 mg (30% yield) of an amorphorous off-white-colored solid: mp 170.degree. (softened), 190.degree. dec; R.sub.f (TLC) 0.68 [silica, CHCl.sub.3 /MeOH/H.sub.2 O/glacial HOAc (25:15:4:2)], 0.67 [n-propanol/conc NH.sub.4 OH/H.sub.2 O (20:20:3)]; ir (KBr) 3226, 2915, 2849, 1724, 1460, 1227, 1111, 1058, 943, 800 and 717; 1H NMR (pyridine-d.sub.5) 1.00 (distorted t, CH.sub.3), 1.40 (s, CH.sub.2), 2.25-3.05 (poorly resolved m, CH.sub.2 CO), 4.20-4.90 (broad unresolved m centered at 4.50, CH.sub.2 O, CHO, CHN), 5.10-6.09 (unresolved m), 7.30-7.75 (unresolved m); uv.sub.max [MeOH/H.sub.2 O(2:1)] 276 nm.