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Five 1-beta-D-arabinofuranosylcytosine conjugates and two cytidine conjugates of thioether lipids (1-S-alkylthioglycerols) linked by a pyrophosphate diester bond have been prepared and their antitumor activity against an ara-C2 sensitive (L1210/0) and two ara-C resistant L1210 lymphoid leukemia sublines in mice were evaluated. These prodrugs of ara-C include ara-CDP-rac-1-S-hexadecyl-2-O-palmitoyl-1-thioglycerol (8a), ara-CDP-rac-1-S-octadecyl-2-O-palmitoylthioglycerol (8b), and ara-CDP-rac-1-S-octadecyl-2-O-methyl(or -ethyl, -hexadecyl)thioglycerols (8c-e). The cytidine conjugates include CDP-rac-1-S-octadecyl-2-O-palmitoyl(or -methyl)- 1-thioglycerols (9a and 9b). Sonicated solutions of the conjugates existed in the form of micellar disks (size 0.01-0.04 microns). Single doses (200-400 mg/kg) of 8a and 8b produced significant increase in life span (257-371%) in mice bearing ip implanted L1210/0 leukemia. In contrast, conjugates 8c-e were less effective (ILS 19-75%) and cytidine conjugates (9a and 9b) were ineffective. Even though 8a and 8b were found to be curative in a high percentage of mice bearing ip implanted partially ara-C resistant L1210 subline [L1210/ara-C(I)], they were completely ineffective against deoxycytidine kinase deficient ara-C resistant L1210 subline [L1210/ara-C(II)]. However, the present results, together with the previous, demonstrate that 8a and 8b are promising new prodrugs of ara-C with improved efficacy.

Eight 5'-(steroid-21-phosphoryl)-9-(beta-D-arabinofuranosyl)-adenines (IV-XI) have been prepared and evaluated against L1210 lymphoid leukemia in culture. These include the 9-(beta-D-arabinofuranosyl)adenine conjugates of hydrocortisone (IV), cortisone (V), corticosterone (VI), cortexolone (VII), 11-deoxycorticosterone (VIII), prednisolone (IX), prednisone (X), and dexamethasone (XI). Conjugates IV, IX, X, and XI inhibited the in vitro growth of L1210 lymphoid leukemia cells by 50% (ED50) at a concentration of 2.3-7.8 microM, while 9-(beta-D-arabinofuranosyl)adenine (vidarabine, I) and its 5'-monophosphate (II) each showed ED50 value of 30 microM. All of the conjugates were enzymatically hydrolyzed to the corresponding steroid and II, the latter undergoing further hydrolysis to I, by phosphodiesterase I, 5'-nucleotidase, and acid phosphatase. However, these conjugates were resistant to hydrolysis by alkaline phosphatase and adenosine deaminase.

Three 1-beta-D-arabinofuranosylcytosine (ara-C) conjugates of 1-S-alkyl-phospholipids (thioether phospholipids) were tested for their antitumor efficacies against L1210 and P388 leukemia in mice. These include 1-beta-D-arabinofuranosylcytosine 5'-diphosphate-rac-1-S-hexadecyl-2-0-palmitoyl-1-thioglycerol (ara-CDP-beta-palmitoyl-DL-thiochimyl alcohol, I), ara-CDP-rac-1-S-octadecyl-2-0-palmitoyl-1-thioglycerol (ara-CDP-beta-palmitoyl-DL-thiobatyl alcohol, II), and ara-CDP-rac-1-S-octadecyl-2-0-hexadecyl-1-thioglycerol (ara-CDP-beta-cetyl-DL-thiobatyl alcohol, III). Conjugates I and II produced significant increase in life span (293-379%) and longterm survivors among mice bearing i.p. implanted L1210 lymphoid leukemia at a total dose of 400 mg (389-400 mumol)/kg. Conjugate II also displayed a strong antitumor activity against i.c. implanted L1210 leukemia in mice with an ILS range of 160-200% at a total dose of 300-450 mg (292-438 mumol/kg. Significant schedule dependency was not observed when the conjugates were administered i.p. once daily with following schedules: qd l; 1,5, 9; 1-5; and 1-9, but single doses typically produced the best effects. The i.p. administration of conjugate II gave the best results on survival of i.p. inoculated L1210 leukemic mice, then followed by the s.c. and i.m. treatments. The i.v. treatment produced a lower activity than the others. Conjugate II also exhibited a strong antitumor activity against i.p. implanted P388 leukemia in mice with ILS values of greater than 255-greater than 329% with 3-5 45-day survivors at a total dose of 300-500 mg (292-486 mumol)/kg (qd 1 or 1-5). The new conjugates I and II displayed a comparable or somewhat higher activity than the previous diacyl and 1-0-alkyl analogs.

Five new P1-(steroid-21-yl)-P2-(1-beta-D-arabinofuranosylcytosin-5'-yl)pyro phosphates (ara-CDP-steroids), five 1-beta-D-arabinofuranosylcytosine 5'-O-(alkyl)phosphates (ara-CMP-alkyl esters), and two P1-(alkyl)-P2-(1-beta-D-arabinofuranosylcytosin-5'-yl)pyrophosphat e (ara-CDP-alkyl esters) have been prepared and evaluated against L1210 lymphoid leukemia in culture and in mice (C3D2F1/J). These include ara-CDP-11-deoxycorticosterone (6a), ara-CDP-cortisone (6c), ara-CDP-corticosterone (6d), ara-CDP-cortexolone (6e), and ara-CDP-prednisone (6g), ara-CMP hexadecyl ester (7a), ara-CMP 1-cyclohexylmethyl ester (7b), ara-CMP 1-adamantylmethyl ester (7c), ara-CMP 2-(1-adamanthyl)ethyl ester (7d), ara-CMP 2-chloroethyl ester (7e), ara-CDP hexadecyl ester (9a), and ara-CDP 1-cyclohexylmethyl ester (9b). The in vitro antitumor results indicated that ara-CDP-steroids were as active as the previously reported ara-CMP-steroids and that ara-CMP and ara-CDP-alkyl esters were less growth inhibiting than ara-CDP-steroids and ara-C. However, the in vivo antitumor results indicated that ara-CDP-steroids were generally less effective than the previous monophosphate derivatives. Among them ara-CDP-corticosterone (6d) and the known ara-CDP-cortisol (6b) showed greater efficacy than ara-C with ILS value of 152% and 209%, respectively, at the optimal dose of 40 and 80 (mg/kg)/day for 9 days, while that of ara-C was 138% at the optimum dose of 9.2 (mg/kg)/day. Generally, ara-CMP alkyl esters (7a-e), given ip to the L1210 leukemic mice, were found to be toxic and ineffective. However, ara-CDP hexadecyl ester (9a) showed marginal activity (ILS, 38%). These preliminary results support the thesis that the ara-C conjugates of this type may require a lipophilic and naturally occurring moiety for improved efficacy.

The L-, D-, and D,L-isomers of 1-beta-D-arabinofuranosylcytosine 5'-diphosphate-1,2-dipalmitin, new prodrugs of ara-C5, have been evaluated for antitumor activity in L1210 lymphoid leukemic mice. The L-isomer produced significant increase in life span (ILS), and longterm survivors among mice bearing i.p. and i.c. implanted L1210 leukemia and the maximal ILS values found were greater than 543 and greater than 374% with five and four 45-day survivors out of six mice, respectively, at the optimal single doses of 300 mg/kg and 125 mg/kg. The D- and D,L-isomers also displayed significant in vivo antitumor activity against both i.p. and i.c. implanted L1210 leukemia in mice with ILS range of 144-293% at a total dose of 125-250 mg/kg. Significant schedule dependency was not observed when the conjugates were administered i.p. once daily for 5 days, once every 4 days, or as a single dose, but single doses typically produced the best effects. The L-isomer was found to be a more effective prodrug of ara-C than its isomers and other lipophilic prodrugs, 5'-O-palmitoyl-ara-C and N4-acyl-ara-C. Unlike the latter prodrugs, the new conjugates are water soluble by sonication method.

Three 1-beta-D-arabinofuranosylcytosine 5'-diphosphate-1,2-dipalmitins from L-, D-, and DL-alpha-dipalmitoylphosphatidic acids have been synthesized and their antitumor activity against two ara-C2 resistant L1210 lymphoid leukemia sublines in mice were evaluated. These new prodrugs of ara-C include ara-CDP-L-dipalmitin, ara-CDP-D-dipalmitin, and ara-CDP-DL-dipalmitin. The L and DL isomers produced significant increase in life span (greater than 400%) and four to five long-term survivors (greater than 45 days) out of six animals bearing ip implanted partially ara-C resistant L1210 subline [L1210/ara-C (I)], while the D isomer displayed a marginal activity (ILS 100-121%). In contrast, the L isomer was completely ineffective against deoxycytidine kinase deficient ara-C resistant L1210 subline [L1210/ara-C (II)]. However, the results demonstrate that the L and DL isomers of ara-CDP-dipalmitin are promising new prodrugs of ara-C with improved efficacy.

Three new 1-beta-D-arabinofuranosylcytosine conjugates of ether lipids (alkyl glycerols) linked by a pyrophosphate diester bond have been prepared and evaluated against mouse leukemia L1210 and P388. These include ara-CDP-rac-1-O-hexadecyl-2-O-palmitoylglycerol (9a) (ara-CDP = 1-beta-D-arabinofuranosylcytosine 5'-diphosphate), ara-CDP-rac-1-O-octadecyl-2-O-palmitoylglycerol (9b), and ara-CDP-rac-1-O-octadecyl-2-O-methylglycerol (9c). Among them, conjugate 9a produced significant increase in life span (200-293%) in mice bearing ip and ic implanted L1210 lymphoic leukemia at a total dose of 400-500 mg (406-508 mumol/kg). Significant schedule dependence was not observed when the conjugate was given ip once daily on day 1, days 1, 5, and 9, and days 1-5. The new conjugates are water soluble by sonication.

Six 5'-(steroid-21-phosphoryl)-1-(beta-D-arabinofuranosyl)cytosines have been prepared and evaluated against L1210 lymphoid leukemia in culture and in mice (C3D2F1/J). These include the ara-C conjugates of 11-deoxycorticosterone (5a), corticosterone (5b), cortexolone (5c), fludrocortisone (5d), 6 alpha-methylprednisolone (5e), and dexamethasone (5f). When the optimum dosage of ara-C [38 (mumol/kg)/day X 5] was given to mice bearing L1210, the ILS value found was 89%. A simple mixture of each steroid and ara-C gave ILS values that were on the whole significantly less than that of the parent nucleoside. However, of six conjugates, all but two (5d and 5f) were more active than ara-C at their optimal doses. Both corticosterone- (5b) and cortexolone-p-ara-C (5c) were especially effective at the respective optimal doses of 76.7 and 115 (mumol/kg)/day X 5. These gave ILS values of 200% each. All of the conjugates were demonstrated to be enzymatically hydrolyzed to the corresponding steroid and ara-CMP, and the latter was further shown to be hydrolyzed to ara-C by phosphodiesterase I, 5'-nucleotidase, and acid phosphatase. However, they were shown to be resistant to hydrolysis by alkaline phosphatase.

The antitumor activity and toxicity of two new 1-beta-D-arabinofuranosyl-cytosine (ara-C) conjugates of cortisol and corticosterone linked through a phosphodiester bond between the 5' and 21 positions of the respective moieties (cortisol- and corticosterone-p-ara-C) were investigated in L1210 lymphoid leukemia cells in mice. They are highly active against both i.p.- and i.c.-implanted ara-C-sensitive lymphoid leukemia in mice, exceeding the activity produced by the parent drug, ara-C. For example, corticosterone-p-ara-C exhibited the respective ILS values of 306% at 50 mg/kg/day X 9 and 294% at 75 mg/kg/day X 9 on survivals of i.p.- and i.c.-inoculated L1210 leukemic mice. The effectiveness of the conjugates seems to depend on schedules of the treatments. The 9-day continuous treatments showed a better therapeutic effectiveness than those with either a 5-day, a single or a widely spaced (q 4d., 1, 5, 9) treatment. However, they were found to be marginally effective against i.p.-implanted ara-C-resistant L1210 leukemia in mice. They were also inhibitory against proliferation of human leukemia-lymphoid cells in culture. Their superior antitumor activity and resistance to cytidine deaminase suggests that they serve as a prodrug form of ara-C or ara-CMP.