Synthesis, and some properties of, amphiphilic ...

0 downloads 0 Views 2MB Size Report
dinucleoside phosphate derivatives of 31-azido-21,31 ... masked with lipophilic deoxycytidine residues of .... from lipophilic deoxycytidine derivatives and AZT.
Antiviral Chemistry & Chemotherapy (1994) 5(6), 387-394

Synthesis, and some properties of, amphiphilic dinucleoside phosphate derivatives of 3 dideoxythymidine (AZT)

1 - a z i d o - 21,3 1 -

H. schott,':" M. P. Haussler," P. Gowland,2 D. H. Herber" and R. A. schwendener" 'Institute of Organic Chemistry, University of TObingen, Auf der Morgenstelle 18, D-72076 Tiibinqen, Germany. 2Division of Infectious Diseases, Department of Internal Medicine, University Hospital, CH-8091 ZOrich, Switzerland. 3Division of Oncology, Department of Internal Medicine, University Hospital, CH-8091 ZOrich, Switzerland. Summary N4-hexadecyl-S'-O-(4-monomethoxytrityl)-2'-deoxycyti- . dine-3'-hydrogenphosphate was reacted with 3'-azido2',3'-dideoxythymidine (AZT) according to the hydrogenphosphate method to yield N4-hexadecyl-2'deoxycytidylyl-(3'-S')-3'-azido-2',3'-dideoxythymidi ne. N4-palmitoyl-S'-O-(4-monomethoxytrityl)-2'-deoxycytidine-3'-(2-chlorophenyl)-phosphate was condensed to AZT using the triester method to give N4-palmitoyl2'-deoxycytidylyl-(3'-S')-3'-azido-2',3'-dideoxythymidine. Both dinucleosidephosphates have amphiphilic properties and represent a new class of AZT derivatives in which the polar AZT-S'-monophosphate is masked with lipophilic deoxycytidine residues of variable stability. The AZT derivatives are water soluble, by forming micelles, and as a result of their amphiphilic nature, they can be incorporated into the llpld membranes of Iiposomes. In contrast to the micellar drug preparations, the Iiposomal formulations were shown to exert no lytic activity on human erythrocytes. Both AZT derivatives have anti HIV-1 activity in vitro. Introduction The antiretroviral activity of 3'-azido-2',3'-dideoxythymidine (AZT), which is used in the treatment of AIDS, is based on the intracellular phosphorylation of AZT to AZT5'-monophosphate. After anabolic conversion to the Received 10 March, 1994; revised 7 April, 1994; accepted 14June, 1994. *For correspondence. Tel. 0049 7071 296220; Fax. 0049 7071 295246.

triphosphate by host enzymes, AZT-5'-triphosphate interferes with the reverse transcription of viral DNA (St Clair et el., 1987; Yarchoan et al., 1989; Schinazi et al., 1992). A direct therapy with AZT-5'-phosphate is impeded by the fast dephosphorylation in serum and by the poor cellular uptake of phosphorylated nucleotides. A possible way of employing phosphorylated AZT for the therapy of HIV infections is to use masked AZT-5'-phosphate as a prodrug. Various concepts have been proposed for the masking of the 5'-phosphate group. For example, compounds have been synthesized in which the 5'-hydroxy group of AZT is esterified with phosphonoformic acid (Rosowsky et al., 1990). Similarly, prodrugs of AZT were prepared by esterifying the 5'-hydroxyl group of AZT with steroidal 17B-carboxylic acids (Sharma et al., 1993). Dinucleoside phosphates have been prepared in which AZT is bound covalently via a phosphodiester linkage to another anti-HIV active nucleoside analogue. These molecules can be cleaved intracellularly by phosphodiesterases into a nucleotide and a nucleoside, whereby both AZT-5'-monophosphate and AZT can be formed (Busso et al., 1988; Schinazi et al., 1990). For all these compounds, the masking of AZT-5'-monophosphate can only be successful if these prodrugs are taken up by cells before AZT-5'-monophosphate is released. In addition, the high polarity of these compounds further reduces cell uptake. Phosphotriester derivatives, such as O-alkyl-5'-5'dinucleotide phosphates, in which AZT is linked to cordycepin (Meier and Huynh Dinh, 1991) are less polar as are phosphotriesters of AZT with lactyl or glycolyl phosphates (McGuigan et al., 1992). Even if the cellular uptake of these prodrugs is more efficient than that of AZT-5'" monophosphate, the enzymatic release of the active moiety of the prodrug is probably inhibited by the unnatural triester linkage. Conjugates, in which AZT is linked by its 5'-hydroxy group via a natural phosphodiester bridge (Hostetler et al., 1990; Steim et al., 1990; Piantadosi et al., 1991) or via diphosphates (Van Wijk et al., 1992a,b) to phospholipids, should offer more efficient cellular uptake and enzymatic release of the active AZT-5'-monophosphate. An indispensable advantage of the phospholipid-AZT conjugates is their stable incorporation into liposomes. Cells of the

388

H. Schott et al.

macrophage lineage are known to take up large amounts of parenterally administered Iiposomes. This property makes Iiposomal phospholipid-AZT conjugates promising candidates for the targeting of AlT to the macrophage reservoir of HIV infection, thereby reducing the toxicity of the drug to other cells. To what extent the described derivatives may increase the anti-HIV activity of AZT and concomitantly reduce toxicity is not known. We present a new concept for the protection of AZT-5'-monophosphate which does not require phospholipids as lipophilic moiety and for which the syntheses are considerably less complicated and expensive.

R

Results Chemistry

AZT was linked via a natural 3'-5'-phosphodiester bridge to new N4-hexadecyl and N4-palmitoyl derivatives of 2'deoxycytidine (dC). The target of the four-step synthesis, which is summarized in Fig. 1, is the condensation of the lipophilic dC derivative as the protected 3'-phosphate or hydrogenphosphonate component to the 5'-hydroxy group of AZT. An alternative method of condensation in which AZT is coupled as 5'-phosphate Orhydrogenphosphonate to the 3'-hydroxy group of the lipophilic dC derivatives is not economic due to the low yields obtained with the phosphorylation of AZT. AZT may also be linked to the dC derivative via a 5'-5' phosphodiester condensation. However, this unnatural 5'-5' nucleotide linkage is unlikely to be cleaved efficiently in vivo. The conversion of dC into the corresponding lipophilic 3'-phosphate or hydrogenphosphonate components begins either with the alkylation with hexadecylamine or with the acylation with the palmitic anhydride of the N4 _ amino group of dC. The two dC derivatives possess comparable lipophilic properties but differ significantly in their stability against hydrolytic cleavage of the lipophilic sidechain. In contrast to the stable N4-hexadecyl compound, the N4-palmitoyl group is readily cleaved under basic conditions. Depending on whether the N4-alkylated or the N4 _ acylated dC derivative is condensed to AlT, two amphiphilic AZT derivatives are obtained, each of which contains a masked AZT-5' monophosphate and which have different susceptibilities to hydrolysis. N4-hexadecyl-5'-O-(4-monomethoxytrityl)-2'-deoxycytidine-3'-O-hydrogen phosphonate (compound 4, Fig. 1) was condensed according to the phosphonate method (Froehler and Matteucci, 1986) to AlT in the presence of pivaloyl chloride. The internucleoside linkage was then oxidized with iodine to obtain the phosphodiester. The reaction mixture was purified on a silica gel column and treated with aqueous acetic acid to remove the 4monomethoxytrityl (MeOTr) protecting groups. The product was subsequently fractionated on a silica gel column.

R

N~H

HO~~O~3~H ~ o I

4H

I

OH

5

8

HNJl.vCH3

J-...Njl 9 H

or

14

O=P-O-CH z

0

0

13 H H 10 H 1211 H 11 H

Fig. 1. Synthesis of the amphiphilic AZT derivatives 5a and 5b, starting from lipophilic deoxycytidine derivatives and AZT. The reaction steps are: A = 4-monomethoxytrityl chloride in pyridine; B = (1) triazole in triethylamine, (2) 2-chlorophenyl dichlorophosphate in dioxane; C (hydrogenphosphonate method) = (1) AZT, pivaloyl chloride in pyridine, 1.5 min, (2) iodine in THF/pyridine, 10 min, (3) 80% acetic acid, 30 min (50°C); D (triester method) = (1) AZT, 2,4,6-triisopropylbenzenesulfonyl chloride, N-methylimidazole in pyridine, 40 min, (2) tetrabutylammonium fluoride in THF/pyridine/water, 45 min, (3) 80% acetic acid, 30 min (50°C). The products 2-5 were purified after each reaction step by column chromatography, precipitation or crystallization.

Amphiphilic AZT derivatives

After crystallization from ethanol, 5a was obtained with a yield of 47% relative to AZT. To obtain the N4-acylated derivative 5b, the 3'-hydroxyl group of 2 was first esterified with 2-chlorophenyl phosphate, and the resulting N4-palmitoyl-5'-O-(4-monomethoxytrityl)-2' -deoxycytidine-3' -(2-chlorophenyl)-phosphate was precipitated from water as barium salt 3. The condensation to AZT was carried out according to the triester method (Reese, 1978), whereby 3 was coupled to AZT in the presence of 2,4,6-triisopropylbenzenesulfonicacid chloride and N-methylimidayole (Fig. 1). After column chromatography on silica gel, the 2-chlorophenyl group of the fully protected dinucleosidephosphate was removed with tertrabutylammonium fluoride. The resulting dimeric product was fractionated on a silica gel column. The MeOTr protecting group was removed by treatment with aqueous acetic acid. The resulting crude product was then chromatographed on a silica gel column, precipitated with hexane and further purified by repeated chromatography on silica gel yielding N4-palmitoyl-2'-deoxycytidylyl- (3' -5') -3' -azi~p- 2' ,3'-dideoxythymidine 5b. The yield relative to A2~!Jwas 37%, which was lower than that of 5a (47%). The yield of 5a dropped to 15% when the triester method was applied (data not shown). According to our experience, the major loss occurs during the cleavage of the 2-chlorophenyl protecting group. In spite of the lower yield, we used the triester method for the preparation of 5b because means of synthesis and purification of the N4-palmitoyl-dC derivatives, which are necessary for the phosphonate method, are not available. During the course of our work we avoided the development of a synthesis scheme for the dC derivatives, taking into account the rather low yield of the N4 _ palmitoyl-dC derivative. We monitored the course of the synthesis and purification of the AZT derivatives by thin layer chromatography (TLC). The synthesis products were purified until the compounds migrated on TLC as single spots (Rt values are given in Table 1). The molecular mass of the purified intermediate products was confirmed by FD or FAB technology before the next reaction step was carried out. Melting points (Fp) were not determined in the cases where the products either had broad melting ranges or decomposed upon heating. Elementary analyses of the intermediate products were not carried out because these products were isolated as foams from which the quantitative removal of the solvents would have been too difficult. In the IR spectra all characteristic vibrational regions of the structural features of 5a and 5b were found. The structures were verified by analysis of their NMR spectra. Since the one-dimenslonal '}! and 13C spectra of 5a and 5b produced narrow and overlapping signals which did not allow an exact assignment, we used two-dimensional 13C-correlated lHNMR spectroscopy for the elucidation

389

of these structures (Braunschweiler and Ernst, 1983). From the chemical shifts, which are summarized in Table 2, it is clear that the 5'-hydroxyl group of the dC residue is free, whereas the 3'-hydroxyl group is linked to the 5'hydroxyl group of AZT via the phosphodiester bridge. A free hydroxyl group causes the C7 in dC to produce a signal at 62.21 ppm (5a) or 61.31 ppm (5b). The phosphorylation of a 5'-hydroxyl group induces a shift of the C14 signal of AZT to 83.13 ppm (5a) or 82.57 ppm (5b), an observation that has also been reported by other authors (Kalinowski et al., 1984). The different residues at the N4 position of dC strongly influence the chemical shifts of H1 and H2, and are therefore also detectable. The signal for H1 appears in 5a at 5.75 ppm, and in 5b at 7.21 ppm, whereas H2 is shifted from 7.66 to 8.28 ppm. The different substitution at the N4 position of dC also causes a shift of

Table 1. R t values of the nucleoside derivatives on silica gel plates Rtvalues in chloroform/methanol (v/v) Compound (see Fig. 1)

AZT 1 2 3 4 5b* 5at 5b t

90/10

80/20

70/30

0.44 0.63 0.80

0.68

0.29 0.60

60/40

0.58 0.34

0.63 0.50

0.44 0.46 0.19 0.25

0.59 0.62 0.33 0.40

0.70

5a 5b

'Protected by the 4-monomethoxytrityl- and 2-chlorophenyl group. tProtected by the 4-monomethoxytrityl group only.

Table 2. 1Hand 13Cchemical shifts for the resonance of the AZT derivatives 5a and 5b

'H

13C

Chemical shifts [ppm]

Chemical shifts [ppm]

Protonl carbon (see Fig. 1)

5a

5b

5a

5b

1 2 3 4 4 5 6 7 8 9 10 11 11 12 13 14

5.75 7.66 6.13 1.98 2.29 4.58 3.92 3.58 1.83 7.87 6.15 2.29 2.40 4.52 3.96 3.88

7.21 8.28 6.10 2.03 2.29 4.72 3.94 3.59 1.78 7.72 6.12 2.29 2.40 4.45 3.94 3.94

95.70 140.08 85.60 39.61 39.61 76.20 86.59 62.21 12.79 136.75 84.27 36.51 36.51 61.65 65.26 83.13

95.82 145.00 85.36 39.70 39.70 75.36 87.18 61.31 12.12 136.03 83.57 35.87 35.87 60.86 64.73 82.57

390

H. Schott et al.

100 90 80 70 01

60

01

>. 0

E Gl :::c ~

50 40 30 20 10 0

~---- ~---- [Jl

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Drug Concentration (mM) Fig. 2. Haemolysis of human erythrocytes of 5a and 5b in liposomal (6 and 0, respectively) and micellar (.A. and e, respectively) preparations.

the absorption maxima of the UV spectra. For Sa the Am ax was found at 269 nm, and for Sb it was found at 250 nm. The N4-hexadecyl residue in Sa causes a pH-dependent shift of the maximum. At a pH of 11.5 or 7 the maximum lies at 269 nm, whereas at a pH of 1.5 a shift to 312 nm was observed. With the derivative Sb a significant shift was recorded at basic pH values. Characteristic of Sb is a shoulder in the range of 300 nm. Further unambiguous proof of the structures of Sa and Sb is provided by the mass spectra. The measured molecular masses correspond to the calculated values. Uposome properties, haemolytic activity and anti-HIV-1 activity

The amphiphilic dinucleoside phosphate derivatives either are soluble as micelles in aqueous buffers, or can readily be incorporated into the bilayer membranes of Iiposomes. Small unilamellar liposomes containing Sa or Sb had mean diameters of 100±21 nm, and the AZT derivatives remained incorporated in the Iiposomes for >6 months at 4 DC. The stability of incorporation of Sa and Sb was monitored by repeated dialysis of the liposomes against phosphate buffer, which showed that the UV absorption of the drug-containing liposomes remained constant. Aqueous solutions of Sa and Sb produced micelles with mean diameters of 9 and 7.9 nm for Sa and Sb, respectively. The micellar solutions of Sa and Sb had

strong lytic activity on human erythrocytes. Fig. 2 shows that the micellar solutions of Sa and of Sb quantitatively lysed erythrocytes at AZT derivative concentrations above 0.5 mM. In sharp contrast, the haemolytic activity was abolished when the AZT derivatives were incorporated into Iiposomes. Liposome preparations of Sa and Sb were tested in vitro in a p24 immunoassay on HIV-1-infected H9 cells. AZT dissolved in PB was used as standard. As summarized in Table 3, both derivatives exert an anti-HIV-1 effect, but the IC5 0 concentrations of Sa and Sb, were 10fold higher than that of AZT. Possibly as a result of the prodrug nature of Sa and Sb, the in vitro antiviral effect was less pronounced than with free AZT, which is probably taken up by the cells at faster rates than are the AZT derivatives. The cytotoxic effect on uninfected H9 cells was determined using the MTT assay, which showed that Sa and Sb have a higher toxicity (CC5 0 ) than AZT. Empty liposomes were not toxic in either assay in a lipid concentration range of 0.08-8 mM SPC. The higher cytotoxicity observed for Iiposomal Sa and Sb is surprising. However, since the properties of the derivatives in vivo cannot be directly inferred from the results obtained in the in vitro experiments, it is of great importance to test the antiviral effects of these compounds in appropriate in vivo models.

Discussion

The condensation of a lipophilic derivative of a natural nucleoside to AZT leads to amphiphilic AZT derivatives that contain masked AZT-5'-monophosphate. The syntheses start with easily accessible compounds and are practicable at preparative scales. The lipophilic nucleoside residue may exert a protective or slow-release effect on the active part of the molecule. Furthermore, it is conceivable that the lipophilic nucleoside residue favours the cellular uptake of the phosphorylated AZT derivative. It is expected that Iiposomally administered AZT derivatives should be able to reach the HIV reservoirs in the monocytic cell system to exert an antiviral effect there. The lipophilic side-chains are necessary for the stable incorporation of the derivatives into the bilayer membranes of

Table 3. In vitro anti-HIV-1 activity, ICso: inhibitory concentration (the concentration of test compound required to decrease p24 production by 50%). CC so: cytotoxic concentration (the concentration of test compound that reduces cell Viability of uninfected cells by 50%). The selectivity index is the ratio of CC so to ICso ICso

cc.,

Compound

(I.lM)

(mM)

Selectivity index

AZT

0.005 0.05 0.05

3.16 0.12 0.02

6.3 x 10s 2.4 x 103 400

5a 5b

Amphiphilic AZT derivatives liposomes. The hydrophilic AZT moiety renders the dimers soluble in aqueous media through the formation of micellar structures. However, micellar solutions of the AZT derivatives were shown to exert a strong haemolytic activity. This finding excludes the possibility of their application as aqueous solutions for parenteral applications. As shown in the experiments with virus-infected cells, liposomal preparations of the AZT derivatives exert strong antiviral effects on HIV-1 viruses residing in infected cells. Our strategy of obtaining amphiphilic dinucleoside phosphate derivatives, which are composed of a non-toxic natural nucleoside and a therapeutically active nucleoside, from therapeutically active nucleosides is not restricted to AZT. This concept can be used to develop new derivatives of known cytostatic and virostatic drugs based on nucleosides.

Materials and Experimental procedures: chemistry UV spectra were recorded using a Perkin Elmer Lambda 5 UVNIS spectrophotometer. 1H-NMR .§pectra were recorded with a Bruker AMX 400 spectromet~' and IR spectra with a Bruker lFS 48 infrared spectrophotonteter, Mass spectra (electrospray; ES) of compounds Sa and 5b were obtained using a Sciex API III with an electrospray source. TLC was performed on precoated silica gel plates 60 F254 (0.25 mm, Merck). The nucleoside derivatives were detected at a wavelength of 254 nm as black spots which turned brown after spraying with perchloric acid (60%) and heating (sugar moieties). UV-active spots that turned yellow after perchloric acid treatment indicated the presence of MeOTr protecting groups. TLC spots of nucleoside derivatives that were protected by MeOTr groups and contained a phosphate group or a phosphodiester linkage turned green after spraying with a molybdic reagent. When the MeOTr group was absent, these derivatives formed blue molybdate complexes. Nucleoside derivatives with palmitoyl or hexadecyl residues formed fluorescent spots (366 nm) after spraying with 2,7-dichlorofluoresceine (0.2%) in ethanol. Salicylic acid and its derivatives appeared as light blue spots at a wavelength of 254 nm. Impurities with amines and amide linkages were detected by treatment with chlorine gas followed by a tolidine spray reagent (Pataki, 1963). Column chromatography was carried out on silica gel 60 (0.04-0.063 nm). The follOWing substances were synthesized in our laboratory according to previously published methods: 4-monomethoxy trityl chloride (Gomberg and Buchler, 1923), 2,4,6-triisopropylbenzenesulfonyl chloride (Lohrmann and Khorana, 1966), 2chlorophenyl dichlorophosphate (Owens and Reese, 1974), salicylchlorophosphite (Anschutz and Emery, 1887), N4 _ hexadecyl-5'-0-(4-monomethoxytrityl)-2'-deoxycytidine-3'-0hydrogenphosphonate (compound 4 in Fig. 1; Schott et al., 1994).

/If-Hexadecyl-2'-deoxycytidylyl-(3'-5J-3'azido-2', 3'dideoxythymidine (Sa) Condensation and oxidation. AZT (3.5 g, 13 mmol) and N4-hexadecyl-5'-0-(4-monomethoxytritYI)-2'-deoxycytidin-3'-0-hydro-

391

genphosphonate (10.2 g, 13 mmol) were dissolved in dry pyridine (50 ml) and further dried by repeated evaporation with anhydrous pyridine (20 ml) until the solution was a syrup. Anhydrous pyridine (100 ml) and plvaloyl chloride (8 ml, 64 mmol) were added and stirred for 1.5 min before the reaction was stopped by the addition of water (5 ml). The solution was titrated with iodine (0.2 M) in THF/pyridine (18: 1, v/v) until the solution kept a dark red colour. The product was concentrated in vacuo, and after addition of an aqueous solution of NaHSO s (2%, 350 ml) the mixture was extracted with ethyl acetate (350 ml). The organic layer was washed twice with a saturated NaCI solution (100 ml), filtered through Na2S04 and concentrated in vacuo to a syrup.

Detritylation. The remaining syrup was dissolved in acetic acid (80%, 200 ml) and stirred for 30 min at 50°C. The complete removal of the MeOTr group was confirmed by TLC. The dried mixture was dissolved in chloroform/methanol (7: 3, v/v; 100 ml) and chromatographed on a silica gel column using a five-step gradient [step 1: chloroform (41); steps 2-5: chloroform/methanol 9: 1, v/v (41); 17: 3 v/v (41); 4: 1,. v/v (41) and 3: 1, vtv (81), respectively]. The desired compound Sa was eluted during the fourth and fifth steps. The pooled fractions (UV-, molybdic reagent- and fluorescence-positive) were concentrated in vacuo to yield 4.8 g (47%) of a white powder which was chromotographically purified from remaining traces of impurities. Aliquots (2 g) dissolved in water (20 rrll) were chromatographed on a G10 Sephadex column (100x5cm). The isolated fractions of the main peak were concentrated to about 20 ml. Aliquots (1 ml) of the concentrated solution were then chromatographed by HPLC on a to-um Polygosil C a column (25 x 0,46 cm) at a flow-rate of 1.5 ml mln', using a mixture of A: 20% 0.1 M NH4Ac and B: 80% methanol as the eluents. The eluate between 9.0 and 10.5 min was collected and lyophilized, resulting in analytically pure Sa. UV (phosphate buffer pH 7) Amax=269 nm; IR (KBr) 3700-31 00 cm' (NH and OH ofthymidine and cytidine), 2854cm-1 (CH of the hexadecyl residues), 2110cm-1 (Ns of AZT), 1700cm-1 (C=O of thymine and cytosine). 1646cm-1 (NH of cytosine), 1270cm-1 (P=O of the phosphodiester bond) and 1059 crn" (P-O-C of the phosphodiester bond). MS (ES), m/z 779.1 (M-W), 444.1 (M-cytosine residue-W), 530.0 (M-AZT), 345.8 (AZT monophosphate), 305.3 (cytidine monophosphate). Elementary analysis: calc.: C 52.62, H 7.44, N 14.03; found: C 52.56, H 7.25, N 14.15; CssHs7Na01OP x H20.

/If -Palmitoyl-2'-deoxycytidine (1) 2'-Deoxycytidine (22.7 g, 100 mmol) dissolved in warmed H20 (64 ml) was added to palmitic anhydride (84 g, 170 mmol) dissolved in warmed dioxane (1.31). The resultant mixture was refluxed for 1 h, cooled and then concentrated in vacuo to dryness. The residue was dissolved by addition of a mixture (400 ml) of chloroform/methanol (4: 1; v/v). The solution was rapidly washed three times with 2N NaOH (150 ml each time). The addition of water (20 ml) accelerated the separation of the two phases. The organic layer was immediately filtered through Na2S04 and concentrated in vacuo to dryness, yielding 42.5 g (91%) of 1 as a colourless solid; m.p. 123-125°C. MS (FD), m/z 466.2 (M + W), 350.3 (N4-palmitoyl-cytosine + W), 256.1 (palmitoylamide + W), 228.1 (2'-deoxycytidine + W).

392

H. Schott et al.

lifI-Palmitoyl-5'-O-(4-monomethoxytrityl)-2'-deoxycytidin e (2) 4-Monomethoxytrityl chloride (17.2g, 56mmol) was added quickly to 1 (21.9 g, 47 mmol) dissolved in pyridine (80ml). The reaction mixture was stirred for 2 h at ambient temperature. The tritylation was stopped by adding methanol (10 ml), and the reaction mixture was concentrated in vacuo to an oily residue which was coevaporated twice with toluene (50 ml) in order to remove the pyridine. The remaining oil was dissolved in chloroform (100 ml) and chromatographed on a silica gel column (20 x 9 cm) using a three-step gradient [step 1: chloroform (41); steps 2-3: chloroform/methanol 97: 3, v/v (41) and 19: 1, v/v (41), respectively]. The desired 2 was eluted during the second step. The pooled fractions (UV- and trityl-positive) were concentrated in vacuo to a colourless foam yielding 30.2 g (87%) of 2. MS (FAB), m/z 738.6 (M+), 465.2 (N4-palmitoyl-z-deoxycytldlne"), 350.3 (N4-palmitoylcytosine + W), 273.0 «MeOTrt).

Barium salt of lifI-palmitoyl-5'-O-(4-monomethoxytrityl)2'-deoxycytidine-3'-(2-chlorophenyl)-phosphate (3) 1H-1,2,4-Triazole (6.7g, 97mmol) was dissolved in dry dioxane (160 ml). To this solution, triethylamine (13.4 ml, 113 mmol) and 2-chlorophenyl dichlorophosphate (9.3 ml, 53 mmol) were added. The sealed reaction mixture was stirred for 30 min at ambient temperature. The mixture containing 2chlorophenyl-bis-(1,2,4-triazol-1-yl)-phosphate and a precipitate of triethylammonium chloride was filtered into a pyridine solution (50 ml) of 2 (11.7 g, 16 mmol) and the resulting clear, pale yellow solution was stirred for 20 min at ambient temperature. TLC in chloroform/methanol (8: 2, v/v) showed that the reaction was complete with the appearance of a trityl-posilive spot near the baseline. This solution was slowly added to precooled water (500 ml, 4°C). The barium salt of the phosphorylated product was obtained as a precipitate by the dropwise addition of the aqueous solution to a vlqorously stirred and cooled aqueous solution of BaCI2 (1%, 1.51, 4 0G). The resulting suspension was stirred for 1 h and the precipitate collected by filtration. After three washings with water, the product was dried in vacuo over P4010' yielding 15.9 g (95%) of 3 as a white powder. MS (ES), m/z 982.2 (M-W), 709.2 (M-(MeoTrt), 348.2 (N4-palmitoylcytosine - W).

lifI-Palmitoyl-2'-deoxycytidylyl-(3'-5')-3'-azido-2', 3'dideoxythymidine (5b) Condensation. For the condensation, 3 (15.0 g, 15 mmol) and AZT (4.0 g, 15 mmol) were dissolved in anhydrous pyridine (50 ml) and further dried by repeated evaporation with anhydrous pyridine (50 ml). The syrup was dissolved in anhydrous pyridine (100 ml) and 2,4,6-triisopropylbenzenesulfonyl chloride (5.9 g, 19.5 mmol) and N-methylimidazole (4.9 ml, 59 mmol) were quickly added. The reaction mixture was shaken for 40 min at ambient temperature. The condensation was monitored using TLC in chloroform/methanol (1 : 1, v/v). AZT and the fully protected condensation product migrated more or less rapidly (see Table 1), whereas 3 remained near the baseline. 5b appeared on the TLC plate as a new UV-and

trityl-positive spot. The condensation was stopped by the addition of water (5 ml) when 3 had disappeared. After concentration, the oily residue obtained was co-evaporated twice with toluene (50 ml) to remove pyridine. The residue was dissolved in chloroform (150 ml) and fractionated on a silica gel column (10 x 9 em) using a four-step gradient [step 1: hexane/chloroform 1 : 1, v/v (41); step 2: chloroform (41); steps 3 and 4: chloroform/methanol 99: 1, v/v (41) and 49: 1, v/v (41), respectively]. The fully protected condensation product was eluted during the third step. The fractions containing the dinucleoside phosphate (UV-, trityl- and fluorescence-positive) were pooled and evaporated in vacuo to a colourless foam.

Cleavage of the phosphate protecting group. The foam (11.4 g) was added to tetrabutylammonium fluoride (11.3 g, 36 mmol) which was dissolved in a mixture (700 ml) of THF/water/pyridine, 8: 1 : 1 (v/v/v). The resulting reaction mixture was stirred for 45 min and concentrated in vacuo to a syrup which was coevaporated twice with toluene (50 ml). The oily residue was dissolved in ethyl acetate (200 ml) and washed three times with water (100 ml), filtered through Na2S04 and concentrated to 40 ml. The tritylated dinucleoside phosphate was obtained as an oily residue by dropping the solution under vigorous stirring into n-hexane (600ml). The isolated precipitate was dissolved in chloroform (150 ml) and fractionated on a silica gel column (10 x 9 em) using a five-step gradient [step 1: chloroform (41); steps 2-5: chloroform/methanol 19: 1, v/v (41), 9: 1, v/v (41), 17: 3, v/v (41) and 4: 1, vlv (41), respectively]. During the third step the tritylated dinucleoside phosphate was eluted. The fractions containing the desired product (UV-, trityl-, molybdic reagent- and fluorescence-positive) were pooled and evaporated in vacuo to a white foam. Detritylation. As for the detritylation of 5a, the foam was stirred in acetic acid (80%). at 50°C for 30 min. The reaction mixture was concentrated in vacuo to an oily residue which was dissolved in a mixture of chloroform/methanol (100 ml) (4:1, v/v) and chroma,tographed on a silica gel column (20x9cm) using a three-step gradient [steps 1-3: chloroform/methanol 17: 3, v/v (41), 4: 1, v/v (81) and 3 : 1, vlv (81), respectively]; During the second step the desired product 5b was eluted. The fractions of 5b were pooled and evaporated in vacuo, yielding 4.4 g (37%) of 5b as a white powder which was chromatographically purified from remaining traces of impurities. Aliquots (1 g) dissolved in water (20ml) were chromatographed on a G10 Sephadex column (100x5cm). The isolated fractions of the main peak were concentrated to about 20 mi. Aliquots (1 ml) of the concentrate were then chromatographed on a 10-J-lm Polygosil Cs column (25x0.46cm) at a flow-rate of 1.5ml mln", The eluate between 9.5 and 11.2 min was collected and lyophilized, resulting in analytically pure 5b. UV (phosphate buffer pH 7) Amax=250 nm; IR (KBr) 3700-31 00 crn" (NH and OH of thymidine and cytidine), 2925 cm" (CH of the palmitoyl residues), 2108 om'' (N3 of AZT), 1696 cm" (C = 0 of thymine and cytosine), 1273 cm' (P = 0 of the phosphodiester bond) and 1062cm-1 (P-O-C of the phosphodiester bond). The'presence of the palmitoyl residue in 5b was demonstrated by the vibrations at 1570 cm" for COHN of palmitoyl and cytosine amide bonds which were absent in the spectrum of 5a. MS (ES), m/z 793.1 (M-W), 444.1 (M-cytosine residue-W), 345.8 (AZT monophosphate), 305.3 (cytidine monophos-

Amphiphilic AZT derivatives phate). Elementary analysis: calc.: C 51.72, H 7.07, N 13.78; found: C 52.03, H 7.29, N 13.43; C3sHssNa011PxH20.

Materials and Experimental procedures: virology and other Uposomes Small unilamellar liposomes were prepared by filter extrusion (Hope et al., 1985). The basic lipid composition was soy phosphatidylcholine (SPC), cholesterol, D,L-a.-tocopherol in molar ratios of 1:0.2:0.01. Typically, 1 ml of Iiposomes contained SPC (10 mg), cholesterol (1.5 mg), D,L-a.-tocopherol (0.1 mg) and either 5a or 5b (4 mg), corresponding to 5 mM of drug. The Iipid/AZT derivative mixtures were dissolved in methylene chloride/methanol (1:1, v/v) and, after exhaustive evaporation of the organic solvents at 40°C, aqueous suspensions were formed by the addition of phosphate buffer (PB, 67 mM, pH 7.4). The rnultllamellar liposome suspensions were filtered sequentially through Nuclepore™ filters of decreasing pore sizes (094, 0.2 and 0.1 11m) in a Lipex™ Extruder. Ailliposome preparations were sterilized by filtration (0.20 or 0.45 11m). The concentrations of the liposomal AZT proitugs were determined spectrophotometrically from liposom~""aliquots dissolved in methanol. Hydrodynamic Iiposome diameters were determined with a Nicomp 370 particle-sizer (Particle Sizing Systems, Santa Barbara, CAl.

Micellar drug solutions Micellar drug solutions were obtained by dissolving 5a or 5b (5 mM) in phosphate buffer (PB, 67 mM, pH 7.4) and sterilizing by filtration through 0.2-l1m filters. Mean micelle diameters (1mg drug mr' in 0.9% NaCI) were measured with a Nicornp 370 particle-sizer.

Haemolytic activity of the lipophilic AZT derivatives The AZT derivatives incorporated into Iiposomes or dissolved in 0.9% NaCI as micelles were incubated at different concentrations (25-160 mM)with human erythrocytes (2 ml) for 60 min at 37°C. Aliquots of the supernatants obtained after centrifugation (1000 rpm, 15 min) were diluted 1: 100 in 0.9% NaCI, and the concentration of haemoglobin was determined by calculating the difference between the absorbances at 577 and 561 nm. Total haemolysis (100%) was obtained by incubation of erythrocytes in water containing Triton X-100 (0.02%) at a ratio of 1 : 1 (v/v).

Antiviral activity in HIV-1-infected H9 cells The AZT derivatives 5a and 5b were analysed for their effect on the p24 antigen production in the supernatant of HIV-1infected cells (Chesebro and Wehrly, 1988). H9 cells (Medical Research Council, AIDS Reagent Project) were infected with HIV-1 IIiB at 0.004 50% tissue culture infective dose per cell for 1-2 h at 37°C, extensively washed to remove virus, and then distributed in 48-well plates at 1.2 x 105 cells per well before the addition of medium containing drugs or empty Iiposomes

393

(0.08-8 11M phospholipids) as control. After 3 days of incubation at 37°C and 5% CO2, the antiviral activity was measured as the reduction of p2Li- antigen in the cell-free supernatant. The p24 antigen was measured in duplicate by an enzyme-linked immunoassay according to the manufacturer's instructions (NEN Research Products, Boston, MA). Uninfected drugtreated toxicity controls were maintained and routinely analysed for cell proliferation and viability by the trypan blue exclusion method.

Cytotoxic activity in untntected cells The cytotoxic effect of the drugs on H9 cells was determined using the MTT test (Mosman, 1983). Briefly, in 96-well plates, 1.2 x 105 viable H9 cells per well were incubated in complete RPMI medium with AZT, 5a and 5b (4I1M-1 nM) for 3 days at 37°C and 5% CO 2, The plates were then centrifuged (900 g, 10 min) and serum-free RPMI medium (100 ml) was added. To the cell suspension, 25111 of a MTT (2.5 mg ml") solution was added and the plates were incubated for 3 h at 37°C, 5% CO2, The formazan product produced in viable cells was measured in an ELISA reader at 550 nm. Cytotoxic concentrations (CCso, Table 3) of the drugs were determined from qraphs of drug concentration versus cell viability.

Acknowledgements The authors thank K. Schmeer and R. Zahner for their technical assistance. This work was partly supported by the Swiss Bundesamt fOr Gesundheitswesen, grant 92-7090, the Swiss National Science Foundation, grant 32-29979.90, and the Sassella and EMDO Foundations.

References Anschutz, R., and Emery, W.O. (1887) Uber die Einwirkung von Phosphortrichlorid auf Salicylsaure und auf Phenol. Ann Chern Justus Liebigs 239: 301-313. Braunschweiler, L., and Ernst, R.R. (1983) Coherence transfer by isotropic mixing: Application to proton correlation spectroscopy. J Magn Resonance 53: 521-528. Busso, M., Mian, A.M., Hahn, E.F., and Resnick, L. (1988) Nucleotide dimers suppress HIV expression in vitro. AIDS Res Human Retroviruses 4: 449-455. Chesebro, B., and Wehrly, K. (1988) Development of a sensitive quantitative focal assay for human immunodeficiency virus infectiVity. J Viro/52: 3779-3788. Froehler, B.C., and Matteucci, M.D. (1986) Nucleoside HPhosphonates: Valuable intermediates in the synthesis of deoxyoligonucieotides. Tetrahedron Lett 27: 469-472. Gomberg., M., and Buchler, C.C. (1923) Para-Benzyloxy- and Para-Methoxy-triphenylmethyl. J Am Chern Soc 45: 217218. Hope, M.J., Bally, M.B., Webb, G., and Cullis, P.R. (1985) Production of large unilamellar vesicles by a rapid extrusion procedure. Characterization of size distribution, trapped volume and ability to maintain a membrane potential. Biochim Biophys Acta 812: 55-65. Hostetler, K.Y., Stuhmiller, L.M., Lenting, H.B., van den Bosch, H., and Richman, D.O. (1990) Synthesis and antiretroviral

394

H. Schott et al.

activity of phospholipid analogs of azidothymidine and other antiviral nucleosides. J Bioi Chem 265: 6112-6117. Kalinowski, H.-O., Berger, S., and Braun, S. (eds) (1984) 13CNMR-Spektroskopie, 1st edn. Stuttgart: Georg Thieme Verlag, p. 399-400. Lohrmann, R., and Khorana, R.G. (1966) The use of 2,4,6-triisopropylbenzenesulfonyl chloride for the synthesis of internucleotide bonds. JAm Chem Soc 88: 829-833. McGuigan, C., Nickson, C., O'Connor, T.J., and Kinchington, D. (1992) Synthesis and anti-HIV activity of some novel lactyl and glycolyl phosphate derivatives. Antiviral Res 17: 197-212. Meier, C., and Huynh Dinh, T. (1991) O-Alkyl-5',5'-dinucleoside-phosphates as combined prodrugs of antiviral and antibiotic compounds. Bioorg Med Chem Lett 1: 527-530. Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Meth 63: 55-63. Owens, G.R., and Reese, C.B. (1974) Preparation of aryl- and 2,2,2-trihaloethyl dihydrogenphosphates. Synthesis 704705. Pataki, G. (1963) Revelation von Carbobenzoxy-Aminosauren auf Dunnschicht-chromatogrammen. J Chromatogr12: 541. Piantadosi, C., Marasco, C.J.J., Morris Natschke, S.L., Meyer, K.L., Gumus, F., Surles, J.Rfii Ishaq, K.S., Kucera, L.S., Iyer, N., Wallen, C.A., Piantac!p~i, S., and Modest, E.J. (1991) Synthesis and evaluation of novel ether lipid nucleoside conjugates for anti-HIV-1 activity. J Med Chem 34: 1408-1414. Reese, C.B. (1978) The chemical synthesis of oligo- and polynucleotides by the phosphotriester approach. Tetrahedron34: 3143-3179. Rosowsky, A, Saha, J., Fazely, F., Koch, J., and Ruprecht, R.M. (1990) Inhibition of human immunodeficiency virus type 1 replication by phosphonoformate esters of 3'-azido-3'deoxythymidine. Biochem Biophys Res Commun 172: 288-294. St Clair, M.H., Richards, C.A., Spector, T., Weinhold, K.J., Miller, W.H., Longlois, AJ., and Fuhrmann, PA (1987) 3'Azido-3'deoxythymidine triphosphate as an inhibitor and

substrate of purified human immunodeficiency virus reverse transcriptase. Antimicrob Agents Chemother 31: 19721977. Schinazi, R.F., Sommadossi, J.-P., Saalmann, V., Cannon, D.L., Xie, M.Y., Hart, G.C., Smith, GA, and Hahn, E.F. (1990) Activities of 3'-azido-3'-deoxythymidine nucleotide dimers in primary lymphocytes infected with human immunodeficiency virus type 1. Antimicrob Agents Chemother 34: 1061-1067. Schinazi, R.F., Mead, J.R., and Feorino, P.M. (1992) Insights into HIV chemotherapy. AIDS Res Hum Retroviruses 8: 963-990. Schott, H., Haussler, M.P., and Schwendener, RA (1994) Synthese und Eigenschaften von N4-Hexadecyl-2'-desoxycytidylyl-(3'-5')-5-ethyl-2'-desoxyuridin und 2'-Desoxythymidylyl-(3'-5')-N 4 -hexadecyl-t-p-Dearabinofu ranosylcytosin, zwei Vertreter einer neuen Prodrug-Gruppe. Liebigs Ann Chem 277-282. Sharma, AP., Ollapally, AP., and Lee, H.J. (1993) Synthesis and anti-HIV activity of prodrugs of azidothymidine. Antiviral Chem Chemother4: 3-96. Steim, J.M., Camaioni Neto, C., Sarin, P.S., Sun, DK, Sehgal, RK, and Turcotte, J.G. (1990) Lipid conjugates of antiretroviral agents. I. Azidothymidine-monophosphate-diglyceride: anti-HIV activity, physical properties, and interaction with plasma proteins. Biochem Biophys Res Commun 171: 451-457. Van Wijk, G.M.T., Hostetler, K.Y., and van den Bosch, H. (1992a) Antiviral nucleoside disphosphate diglycerides: improved synthesis and facilitated purification. J Lipid Res 33: 1211-1219. Van Wijk, G.M.T., Hostetler, K.Y., Suurmeijer, C.N.S.P., and van den Bosch, H. (1992b) Synthesis, characterization and some properties of dideoxynucleoside analogs of cytidine diphosphate diacylglycerol. Biochim Biophys Acta 1165: 45-52. Yarchoan, R., Mitsuya, H., and Broder, S. (1989) Clinical and basic advances in the antiretroviral therapy of human immunodeficiency virus infection. Am J Med87: 191-200.