Synthesis and Evaluation of a Novel Synthetic Phosphocholine Lipid-AZT Conjugate that Double-Targets Wild-Type and Drug Resistant Variants of HIV

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Synthesis and Evaluation of a Novel Synthetic

Phosphocholine Lipid‐AZT Conjugate That

Double‐Targets Wild‐Type and Drug Resistant Variants

of HIV

Louis S. Kucera ^a, Susan L. Morris‐Natschke ^b, Khalid S. Ishaq ^b, Jan Hes ^c, Nathan Iyer

^a, Phillip A. Furman ^c& Ronald A. Fleming ^c

^aDepartment of Microbiology and Immunology, Wake Forest University School of

Medicine, Medical Center Blvd., Winston‐Salem, North Carolina, 27157‐1064, USA

^bUniversity of North Carolina School of Pharmacy, Chapel Hill, North Carolina, 27599,

USA

^cKucera Pharmaceutical Company, Winston‐Salem, North Carolina, 27101, USA

Version of record first published: 01 Jun 2007

To cite this article: Louis S. Kucera, Susan L. Morris‐Natschke, Khalid S. Ishaq, Jan Hes, Nathan Iyer, Phillip A. Furman

& Ronald A. Fleming (2004): Synthesis and Evaluation of a Novel Synthetic Phosphocholine Lipid‐AZT Conjugate That

Double‐Targets Wild‐Type and Drug Resistant Variants of HIV , Nucleosides, Nucleotides and Nucleic Acids, 23:1-2, 385-399

To link to this article: http://dx.doi.org/10.1081/NCN-120028335

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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS

Vol. 23, Nos. 1 & 2, pp. 385–399, 2004

Synthesis and Evaluation of a Novel Synthetic

Phosphocholine Lipid-AZT Conjugate That

Double-Targets Wild-Type and Drug

Resistant Variants of HIV^y

2

Louis S. Kucera,^1,Susan L. Morris-Natschke, Khalid S. Ishaq,

*

Jan Hes,³Nathan Iyer,¹Phillip A. Furman,³and Ronald A. Fleming³

¹Wake Forest University School of Medicine, Winston-Salem, North Carolina, USA

²University of North Carolina School of Pharmacy, Chapel Hill,

North Carolina, USA

³Kucera Pharmaceutical Company, Winston-Salem, North Carolina, USA

ABSTRACT

INK-20, a synthetic phosphocholine lipid-AZT conjugate, was evaluated for antiviral

activity against wild-type HIV-1, a matched pair of pre-AZT and post-AZT and multi-

drug resistant clinical isolates. In addition, it was tested for activity against viruses

resistant to nucleoside (AZT, 3TC) and nonnucleoside (nevirapine) reverse tran-

scriptase and protease (saquinavir) inhibitors using the syncytial plaque reduction

assay for infectious virus multiplication. The EC₅₀values were 0.004, and 0.005 mM

against wild-type HIV-1 for INK-20 and AZT, respectively. INK-20 showed little or

no cytotoxicity when assayed in CEM-SS cells and four other cell types including

PBMC. This resulted in a selective index of > 25,000 and > 20,000 for INK-20 and

AZT, respectively. When tested against a matched pair of pre-AZT and post-AZT

clinical isolates, the EC₅₀values were 0.01 and 0.03 mM for INK-20 and 0.0005 and

^yIn honor and celebration of the 70th birthday of Professor Leroy B. Townsend.

*

Correspondence: Louis S. Kucera, Wake Forest University School of Medicine, Department of

Microbiology and Immunology, Medical Center Blvd., Winston-Salem, NC 27157-1064, USA;

Fax: (336) 716-9928; E-mail: lkucera@wfubmc.edu.

385

DOI: 10.1081/NCN-120028335

1525-7770 (Print); 1532-2335 (Online)

www.dekker.com

Copyright D 2004 by Marcel Dekker, Inc.

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Kucera et al.

0.33 mM for AZT, respectively. INK-20 had moderate to good activity against two

other AZT resistant variants and very good activity against a multi-drug resistant

clinical isolate compared to marked resistance of these viruses to AZT alone. INK-20

retained significant activity against viruses resistant to 3TC, nevirapine, and

saquinavir. The synthetic phosphocholine lipid-AZT conjugate INK-20 represents a

novel class of anti-HIV compounds, which may provide new strategies for the

treatment of HIV drug-resistant variants.

Key Words: Phosphocholine lipid analog conjugate; Drug resistant HIV; Anti-

retrovirus drug conjugates.

INTRODUCTION

Current treatment of HIV/AIDS involves the use of physical combination therapies

that include nucleoside and nonnucleoside reverse transcriptase inhibitors, protease

inhibitors, and T-20, the newest class of entry/fusion inhibitor, to produce a regimen of

‘‘highly active antiretroviral therapy’’ (HAART).^[1,2]Despite the potent antiretroviral

activity of HAART, major problems have been associated with each of the currently

approved classes of drugs including toxic side effects associated with long-term

HAART treatment^[3]coupled with human cases of HIV variants containing mutations

that cause resistance to one or more drugs,^[4]transmission of drug-resistant virus in

humans^[5]and poor pharmacokinetic properties. Better-tolerated drug combinations

with novel mechanisms of action and activity against drug-resistant variants are

urgently needed for the treatment of HIV/AIDS.

Initial studies with anti-HIV synthetic phosphocholine lipids alone were strongly

encouraging and indicated potent activity against wild-type HIV with little or no

cytotoxicity compared to AZT.^[6,7]These lipid molecules alone displayed a dose-

dependent inhibition of infectious virus production that was the likely result of inhibiting

a late stage in virus replication, possibly at the level of assembly.^[7–9]The molecules did

not directly inactivate preformed extracellular virus indicating that their activity was not

the result of a detergent-like effect on HIV infectivity.^[7]Subsequently, it was observed

that combinations of phosphocholine lipids and AZT resulted in an apparent synergistic

or additive effect.^[6]Recently, chemical combinations of a phosphocholine lipid (e.g.,

INK-18) with a nucleoside analog (e.g., AZT) resulted in a conjugate compound (e.g.,

INK-20) (Figure 1) that could ensure simultaneous delivery of two therapeutic agents

(phosphocholine lipid plus nucleoside analog) to infected cells and double-target the HIV

life cycle at different sites. Results presented in this paper indicate that the

phosphocholine lipid-AZT conjugate (INK-20) has significant and selective antiviral

activity against wild-type and several classes of drug-resistant HIV-1 variants.

RESULTS

Synthesis

The dialkyl phosphocholine lipid (7) shown in Scheme 1 was prepared using

previously established procedures.^[10–15]In brief, 1-thioglycerol (1) was alkylated with

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387

Figure 1. Structures of INK-20, INK-18, and AZT.

Scheme 1. Synthesis of dialkyl phosphocholine lipid backbone.

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Scheme 2. Synthesis of phosphocholine lipid-AZT conjugate (INK-20).

1-dodecyl bromide in alcoholic KOH to give compound 2. The primary hydroxyl of

2 was protected as the triphenylmethyl (trityl) ether using trityl chloride. The

resulting compound 3 was then alkylated at the secondary hydroxyl using sodium

hydride and 1-bromo-8-benzyloxyoctane (4). Compound 4 was prepared by reacting

1,8-dibromooctane with benzyl alcohol in the presence of NaOH and tetrabutylammo-

nium hydrogen sulfate.^[16]The protecting trityl ether of compound 5 was removed by

acid hydrolysis using p-toluenesulfonic acid in MeOH/CHCl₃(Scheme 1) to give the

substituted thioglycerol lipid (6). The phosphocholine 7 was formed in a two-step

sequence: reaction of the lipid primary hydroxyl with bromoethyl dichlorophosphate^[17]

followed by aqueous trimethylamine. The benzyl ether of 7 was removed by

hydrogenolysis over Pd/C giving INK-18. Finally, the terminal hydroxyl was coupled

with AZT-malonic acid (AZT-MA, 8) in the presence of DCC and DMAP/pyridine to

give INK-20 (Scheme 2). AZT-MA (8) was prepared by reaction of AZT with malonyl

dichloride (Scheme 3).

Biological Evaluation

Determination of Cytotoxicity in Uninfected and Antiviral Activity in

Wild-Type HIV-1 Infected CEM-SS Cells

Initial experiments were done to determine the toxic concentration 50 % (TC₅₀) in

uninfected CEM-SS cells. Results indicated that the TC₅₀in CEM-SS cells was > 100

Scheme 3. Synthesis of AZT-malonic acid (AZT-MA).

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389

Table 1. Determination of TC₅₀, EC₅₀and selective index (SI) for INK-18, INK-20 and AZT in

uninfected or HIV-1 infected CEM-SS cells.^a

Compound

TC₅₀(mM)^b

EC₅₀(mM)

SI^c

INK-18

INK-20

AZT

>100

0.61

0.004

0.005

>163

>25,000

>20,000

^aA syncytial plaque reduction assay was used to determine the EC₅₀. Five serial five-fold dilutions

of compound starting at 10 mM were tested in triplicate to obtain an average % inhibition of HIV-1

(strain IIIB) syncytial plaques. The % inhibition at each concentration was used to calculate the

EC₅₀. Results are representative of a typical experiment repeated at least once with similar results.

^bThe TC₅₀was determined using five serial five-fold dilutions of compound starting at 100 mM in

triplicate. After 6 days treatment at 37°C, the cells in suspension culture were counted using Trypan

blue exclusion and the % dead cells was measured to calculate the TC₅₀.

^cSI = TC₅₀Ä EC₅₀.

mM (100 mM was the highest concentration tested) for INK-18, INK-20, and AZT

(Table 1). Similarly, the TC₅₀of the compounds was >100 mM in four other cell

culture systems including human PBMC (Table 2). The anti-HIV activity of INK-18,

INK-20 and AZT was determined in wild-type (IIIB) HIV-1 infected CEM-SS cells.

The effective concentration 50% (EC₅₀) values were 0.61, 0.004, and 0.005 mM for

INK-18, INK-20, and AZT, respectively, (Table 1). The phosphocholine lipid-AZT

conjugate INK-20 had the best selective index (SI = >25,000) followed by >20,000 for

AZT and >163 for INK-18. These results indicate that the phosphocholine lipid-AZT

conjugate (INK-20) is well tolerated by human tissue culture cells and it has potent

anti-HIV-1 activity.

Anti-HIV Activity Against Matched Pairs of Pre-AZT and Post-AZT

Clinical Isolates

Since phosphocholine lipids alone are not inhibitors of reverse transcriptase but

target infectious HIV-1 replication at a late stage in the virus life cycle, possibly

Table 2. Determination of cell cytotoxicity in different cell cultures.

Cell cultures (TC₅₀, mM)

Compound

PBMC^a

MOLT-4^a

MRC-5^b

MRC-9^b

INK-18

INK-20

AZT

> 100

^aTC₅₀was determined by Trypan blue exclusion after 4 days incubation with compound.

^bTC₅₀was determined by neutral red assay after 3 days incubation with compounds. Results are

representative of a typical experiment repeated at least once with similar results.

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Kucera et al.

Table 3. Evaluation of compounds against a matched pair of HIV-1 pre-AZT and post-AZT

clinical isolates.^a

EC₅₀, mM

Compound

Pre-AZT H112-2

Post-AZT G910-6

Fold-increase

INK-18

INK-20

AZT

0.21

0.01

1.2

6

3

0.03

0.33

0.0005

660

^aA syncytial plaque reduction assay with CEM-SS cells was used to determine the EC₅₀. Five serial

five-fold concentrations of compound starting at 10 mM were tested in triplicate to obtain an

average % inhibition of HIV-1 syncytial plaques. The % inhibition at each concentration was used

to calculate the EC₅₀. Results are representative of a typical experiment repeated at least once with

similar results.

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Kucera et al.

the mechanisms of action of phosphocholine lipids^[7,8]and AZT^[18]in tissue culture

cells are not completely understood, both inhibitors can block virus maturation and

disrupt syncytial formation. Therefore, a mutation involving virus maturation and

syncytial formation might explain the fold-increase in EC₅₀for variants 2526 and 2529

to the phospholipid and AZT. Of great interest were results indicating that the MDR

5652 variant had only 3- and 1-fold increase to INK-18 and INK-20, respectively,

compared to 443-fold increase to AZT (Table 4). In summary, the phosphocholine

lipid-AZT conjugate (INK-20) has very good activity against several classes of drug-

resistant variants.

DISCUSSION

Results presented in this paper demonstrate that the synthetic phosphocholine lipid-

AZT conjugate INK-20 belongs to a novel class of anti-HIV compounds that in tissue

culture cells has potent and selective activity against HIV-1 (Table 1), as well as drug-

resistant variants (Tables 3 and 4) representing three major classes of drugs (nucleoside

and non-nucleoside reverse transcriptase and protease inhibitors).

INK-20 was synthesized as a model compound that embodies two molecules (a

phosphocholine lipid, e.g., INK-18, and a nucleoside analog, e.g., AZT) linked together

through a malonate diester bond (Figure 1). Other phosphocholine lipids and nucleoside

analogs could replace the lipid and nucleoside components, respectively. It is expected

that the ester linkages will be cleaved in cells to release both components and thus

target two different stages in the viral replication cycle (i.e., RT activity due to the

nucleoside analog and virus assembly with the production of defective progeny virus

due to the phosphocholine lipid).^[7–9]Esterase activity could be modulated through

alkyl or aryl substitution at the C-2 position of the malonate linker.

It is hypothesized that metabolism of the conjugate INK-20 releases the nucleoside

AZT, which after phosphorylation to AZT-triphosphate, inhibits the HIV-induced

reverse transcriptase.^[19]In contrast, the phosphocholine lipid portion (INK-18) does not

inhibit the reverse transcriptase activity. The evidence so far is that phosphocholine

lipids are involved in assembly of defective virus-like particles within intracytoplasmic

vacuoles.^[7,20]These viral progeny lack functional gp120 expression on the virus

surface and have reduced capacity to bind to CD4 + cells. This action does not appear

to involve virus induced synthesis and processing of virus structural proteins in infected

cells.^[8]The hypothesis is that synthetic phospholipids may inhibit transport of gp120

from the golgi to the plasma membrane or the binding of gp120 and/or gag viral

proteins to cell plasma membranes and lipid membrane rafts. There are accumulating

data indicating the role of host cell proteins, cell membranes and membrane rafts in the

entry and assembly of HIV-1 into cells.^[21]Synthetic phospholipids are membrane

interactive molecules that may have a profound effect on the lipid composition of

membrane rafts and thus alter the proper binding of viral structural proteins and

assembly of infectious HIV particles.

In summary, chemical combination of a synthetic phospholipid with another anti-

HIV drug could lead to distinct advantages compared with a physical combination of

several antiviral drugs (e.g. AZT and 3TC) including 1) increased half-life in vivo and

prolonged duration of biological action due to protection of AZT from rapid

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393

glucuronide formation at the 5’ position, 2) reduced toxicity and increased tolerability

in-vivo, 3) simultaneous delivery of constant amounts of multiple agents (e.g.

phosphocholine lipid and AZT) to virus infected cells, 4) uptake of drugs into the CNS

and lymphoid tissues which serve as HIV sanctuaries, 5) intracellular activation of the

conjugate (see review by Morris-Natschke et al.,^[9]and 6) activity of the phospholipid

against drug resistant variants.

EXPERIMENTAL

Solvents were purchased from Acros and purified/dried as necessary by standard

methods. Tetrahydrofuran (THF) and diethyl ether were distilled from sodium hydride

(NaH) and stored over Na ribbon, chloroform and dichloromethane were distilled from

CaCl₂and stored over molecular sieves type 4A, and pyridine was distilled from and

stored over KOH. Reagents were purchased from Sigma Aldrich and used as such.

NMR spectra were recorded on Varian 300 spectrometer and are reported in delta units

1

with TMS resonance at 0 ppm used as reference resonance for H spectra. The mass

spectra were recorded on Micromass Quattro II Triple Quadrupole mass spectrometer

equipped with Z–spray and nanospray sources. The melting points were obtained on

Thomas-Hoover melting point apparatus and are uncorrected. Silica gel plates (MK6

F₂₅₄) were used for thin layer chromatography and spots visualized by UV₂₅₄, iodine,

and NH₄SO₄charring. Chromatographic purification was performed on ISCO

CombiFlash 100c flash chromatograph using ISCO RediSep silica gel columns.

1-S-Dodecyl-3-O-trityl-rac-1-thioglycerol (3). 1-S-Dodecyl-3-O-trityl-rac-thio-

glycerol (3) was prepared by alkylation of 1-thioglycerol (1) followed by tritylation

of the resulting thioether (2) as previously described.^[10–14]Structures were confirmed

1

by H NMR.

1-Bromo-8-benzyloxyoctane (4). 1,8-Dibromooctane (272 g, 1 mol) was added

to a mixture of NaOH (41 g), water (82 ml), tetrabutylammonium hydrogen sulfate

(8.0 g) and benzyl alcohol (43 g, 0.34 mol).^[16]This heterogeneous system was stirred

vigorously for 22 h at room temperature (25°C). The reaction mixture was extracted

with 500 ml and then 200 ml of hexane. The combined hexane layers were extracted

with 100 ml of water and dried over magnesium sulfate overnight. After filtration and

solvent evaporation in vacuo, the thick, brown oil (290 g) was flash chromatographed

on silica gel using EtOAc gradient in hexane (0% ! 10% EtOAc) to afford 62.2 g

(61.2%) of product as a thick, light tan, lachrymating oil. TLC: 5% EtOAc:95%

1

hexane, R_f= 0.55. Structure was confirmed by H NMR.

1-S-Dodecyl-2-(8-benzyloxyoctyl)-3-trityl-rac-1-thioglycerol (5). Sodium hy-

dride (60%, 12 g, 0.3 mol) was added to 300 ml of dry THF and chilled to 0°C in

an ice-salt bath. The suspension was stirred 20 min and 108 g (0.208 mole) of

compound 3 in 300 ml THF were slowly added over period of 1 h. Stirring was

continued 30 min and 62.1 g (0.21 moles) of l-bromo-8-benzyloxyoctane (4) in 100 ml

THF were added. After removing the ice bath, the reaction mixture was stirred 1 h at

room temperature, then refluxed 42 h. Heating was discontinued and the reaction

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Kucera et al.

mixture cooled in an ice bath. Ice (50 g) was added slowly, solution stirred 1 h, and

saturated sodium bicarbonate (100 ml) was added followed by 350 ml of saturated

NaCl. The milky emulsion was extracted with 2 ꢀ 700 ml of CH₂Cl₂, and organic

layers combined and dried over Na₂SO₄for 20 h. Solvents were evaporated in vacuo

and residual oil (230 ml) flash chromatographed on silica gel with EtOAc gradient in

hexane (0% ! 6% ! 15% EtOAc) to give 53.8g (35.2%) of pure product (5) as light

tan oil, together with 18.6 g of impure 4, and 55.3 g of pure 3. TLC: 10% EtOAc:90%

1

hexane, R_f= 0.8. Structure was confirmed by H NMR.

1-S-Dodecyl-2-(8-benzyloxyoctyl)-rac-1-thioglycerol (6). Compound 5 (35 g,

0.048 mol) was dissolved in a mixture of 250 ml anhydrous CHCl₃and 125 ml anhydrous

MeOH, containing 1.0 g of p-toluenesulfonic acid monohydrate and stirred 3 h at 40°C, at

which time the reaction was complete. The reaction mixture was shaken repeatedly with

400 ml of saturated NaHCO₃until carbon dioxide evolution ceased. After drying over

Na₂SO₄for 10 h, the solvent was removed at reduced pressure. Hexane (100 ml) was

added and evaporated in vacuo. Hexane (25 ml) was added to the resulting oil to prevent

crystallization of methoxy triphenylmethane and this mixture was flash chromatographed

on silica gel with gradient of EtOAc in hexane (0% ! 12% EtOAc). Product was

obtained in 67% yield (15.8 g) as thick oil. TLC: 10% EtOAc:90% hexane, R_f= 0.2. The

1

structure was confirmed by H NMR.

1-S-Dodecyl-2-O-(8-benzyloxyoctyl)-rac-thioglycero-3-phosphocholine (7). A

magnetically stirred mixture of anhydrous diethyl ether (50 ml) and THF (50 ml) was

cooled to 0°C in an ice bath under argon atmosphere and 3 ml (5.35 g, 0.022 mol)

of freshly distilled 2-bromoethyl dichlorophosphate (prepared as given in Ref. [17])

in 10 ml anhydrous pyridine were added dropwise. Compound 6 (4.93 g, 0.01 mole),

dissolved in 50 ml of anhydrous diethyl ether was added, the reaction mixture allowed

to warm to room temperature, then refluxed 4 h, and cooled to 0°C. Water (6 ml)

was added and stirring continued 30 minutes. Solvents were removed in vacuo, and

the resulting semisolid dissolved in 1:1 CHCl₃:MeOH (25 ml), mixed with 10 g of

silica gel and the solvents evaporated. The residue was packed in an injection cartridge,

then eluted onto 120 g silica gel column (ISCO RediSep) and eluted with a gradient

of MeOH in CHCl₃(0% ! 15% ! 30% ! 40% MeOH). Fractions were analyzed

on silica gel TLC developed with CHCl₃:MeOH:H₂O 75:25:4, and those containing

the R_f= 0.65 component (fractions 62–71) were combined and solvent evaporated

to afforded 3.1 g (45.5%) of chromatographically pure product as glassy light tan

1

solid. The structure was confirmed by H NMR. This intermediate (1.4 g, 0.002 mol)

was dissolved in 25 ml of CHCl₃, and 25 ml of isopropyl alcohol was added followed

by 40 ml of 40% aqueous trimethyl amine. The slightly turbid solution was

stirred vigorously and refluxed (63°C) for 20 h. The reaction mixture was allowed

to cool to room temperature (about 1 h) and 2.75 g (0.01 mole) of Ag₂CO₃was

added. Stirring and reflux were resumed and continued for 1.5 h. Therefore, heating and

stirring were stopped and most of the black sediment was filtered using a sintered glass

funnel (medium). CHCl₃(25 ml) was then added and the mixture formed two layers. The

lower (organic) layer was separated and evaporated to dryness, and the residue dissolved

in MeOH (20 ml). This solution was mixed with 10 g of silica gel, evaporated to dryness,

and packed in an injection cartridge which was then eluted on 10 g silica gel column

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395

(ISCO RediSep) and eluted with MeOH:CHCl₃gradient (100% CHCl₃followed by

linear gradient to 80% MeOH). Product (7) was obtained in 66% (0.8 g) as a light tan

glassy and extremely hygroscopic material. TLC: CHCl₃:MeOH:H₂O 75:25:4,

1

R_f= 0.3. The structure was confirmed by H NMR.^[14]

1-S-Dodecyl-2-O-(8-hydroxyoctyl)-rac-thioglycero-3-phosphocholine (INK-18).

The phosphocholine lipid 7 (0.97 g, 1.5 mmol, was dissolved in 150 ml of absolute

EtOH and transferred into a Parr hydrogenation bottle under stream of argon. 10% Pd

on activated carbon (1.0 g) was added and the bottle was placed into a Parr

hydrogenation apparatus. The system was repeatedly evacuated and flashed with

hydrogen and finally pressurized to 50 psi. Hydrogenation proceeded at room

temperature (25°C) for 32 h until a constant pressure was reached for 8 h. The system

was evacuated and flushed twice with argon. The catalyst was filtered off and washed

with 25 ml of absolute ethanol, and the solvent was evaporated in vacuo. The glassy,

hygroscopic residue (1.3 g) was dissolved in 15 ml of CHCl₃containing 1% EtOH,

injected on 10 g silica gel column (ISCO RediSep) and eluted with MeOH. Crude

product (1.04 g) was obtained as thick oil, which was dried by repeated co-evaporation

with 4% benzene in EtOH. The resulting glassy semisolid was placed in high vacuum at

40°C and dried 24 h to afford 0.81 g (95%) of chromatographically pure, hygroscopic,

1

sticky solid. The structure was confirmed by H NMR and mass spectrometry.

5’-Malonyl AZT (8). One gram of AZT was dissolved in 30 ml of anhydrous

acetonitrile and added dropwise to a solution of 630 mg of malonyl dichloride in 20 ml

of acetonitrile at 0°C. The reaction mixture was stirred for 2 h at 0°C then at 8–10°C

for 4.5 h. Thin layer chromatography showed that the reaction was complete. Water

(4 ml) was added. Solvents were removed in vacuo and the residue purified by silica

gel chromatography eluting with CHCl₃:MeOH to give pure product in a 68% yield as

1

a glassy solid. The structure was confirmed by H NMR.

1-S-Dodecyl-2-O-[8-(5’-malonyl-AZT)octyl]-rac-thioglycero-3-phosphocholine

(INK-20). Pyridine (200 ml) was freshly distilled from KOH and used in this

experiment, which was performed entirely in an argon atmosphere. 5’-Malonyl AZT (8)

(0.353 g, 0.001 mole) was dissolved in 6 ml of dry pyridine and evaporated to dryness.

This procedure was repeated twice. The dry residue was then dissolved in 10 ml of

pyridine, 0.62 g (0.003 mole) of dicyclohexylcarbodiimide was added, and the mixture

evaporated to dryness. This process (addition of pyridine followed by evaporation) was

repeated twice. p-Dimethylaminopyridine (0.12 g, 0.001 mole) in 10 ml of pyridine was

added, and the solution then evaporated to dryness. This procedure was repeated twice

with 10 ml of pyridine. Finally, 0.57 g (0.001 mole) of INK-18 in 10 ml of pyridine

were added, evaporated to dryness (repeated twice), and then redissolved in 20 ml of

pyridine. The flask was sealed and immersed in a 40°C water bath. The reaction

mixture was stirred at this temperature for 18 h. Water (1 ml) was added, and stirring

continued at 40°C for 1 h. The solvent was evaporated to dryness, the residue dissolved

in 10 ml of MeOH: chloroform (1:2), mixed with 5 g of silica gel, evaporated to

dryness and eluted onto a 35 g silica gel column (ISCO RediSep) with a gradient of

MeOH in chloroform 0–66%. Chromatography gave 0.37 g of impure product, which

was re-chromatographed on 4 g column (ISCO RediSep) with a MeOH:CHCl₃gradient

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(0% ! 80%) to yield 0.18 g (19.9%) of chromatographically pure INK-20. TLC:

1

CHCl₃:MeOH:NH₄OH 75:25:5, R_fꢁ 0.3. The structure was confirmed by H NMR

and mass spectrometry.

Virology

Virus stocks. HIV-1 (strain IIIB) was propagated in H9IIIB persistently infected

cells as previously described.^[7]HIV-1 pre-AZT (H112-2) and post-AZT (G-910-6; RT

mutant at codon 215) clinical isolates and drug-resistant variants (2948, 2970, 2526,

2529, 1392) were obtained through the AIDS Research and Reference Reagent

Program, NIH, Bethesda, MD, USA. The MDR 5652 resistant HIV-1 was received

from Dr. Mark Wainberg, Lady Davis Institute, Jewish General Hospital, Montreal,

Quebec, Canada. Stocks of drug resistant variants of HIV-1 were propagated in acutely

infected CEM-SS cells using methods described by Krugner-Higby et al.^[8]

Cell cultures. CEM-SS, PBMC and Molt-4 lymphoid cells were grown and

maintained in RPMI-1640 medium supplemented with penicillin (100 U/ml), streptomy-

cin (100 mg/ml), 2 mM ^L-glutamine and 20% fetal bovine serum (growth medium)

as previously described.^[7]MRC-5 and MRC-9 human fibroblast cells were grown

and maintained in Dulbecco’s minimal essential medium supplemented with penicillin

(100 U/ml), streptomycin (100 mg/ml), 2 mM ^L-glutamine and 10% fetal bovine serum.

TC₅₀determinations. Test compounds were prepared as stock reagents by

dissolving in absolute ethanol (INK-18, INK-20) or PBS (AZT) at a final concentration

of 10 mM and stored at ꢂ 20°C. To test for cytotoxicity, the stock solutions were

brought to room temperature (25°C) and serial dilutions were prepared in RPMI-1640

growth medium and added to 10,000 CEM-SS cells/well in triplicate wells of a 96-well

plate. A viable cell count was made prior to the test to ensure more than 90% cell

viability. Log phase cells were treated with compound at 37°C. Trypan blue cell counts

were made using a conventional light microscope to determine the viable cell count in

treated and untreated CEM-SS, PBMC or MOLT-4 cell cultures. From the total cell

counts, a TC₅₀for cytotoxicity was calculated as previously described by Chou et al.^[22]

and cited by Piantadosi et al.^[6]A neutral red test was used to determine cell cytotoxicity

in MRC-5 and MRC-9 cell monolayer cultures. The protocol was the same as described

above except that instead of using Trypan blue cell counts, the overlay medium in each

well was aspirated and each well received 200 ml of 0.01% neutral red (Sigma cat. #N-

2880) in phenol red free D-MEM medium. After 2 hrs incubation at 37°C, the cells were

washed with PBS to remove residual neutral red and then 200 ml lysis reagent containing

50% ethanol, 1% glacial acetic acid, 49% distilled water was added to each well to lyse

the cells. In addition, 12 blank wells each received 200 ml of the lysis reagent. After 30

mins incubation at 37°C on a rotator platform the OD₅₁₀was read using an automatic

ELISA plate reader. The % inhibition in OD₅₁₀in the test wells relative to the untreated

control wells and minus the OD₅₁₀from the blank wells was used to calculate the TC₅₀

by the method of Chou et al.^[22]and cited by Piantadosi et al.^[6]

EC₅₀determinations. The syncytium plaque reduction assay for infectious virus

multiplication was used in the presence or absence of the phosphocholine lipid-AZT

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397

conjugate (INK-20) or control compounds (INK-18, AZT) as previously described by

us.^[7]The syncytium plaques were counted on day 5 or 6 post-infection and treatment

and the percentage inhibition and effective concentrations 50% (EC₅₀) were calculated

by the method of Chou et al.^[22]and cited by Piantadosi et al.^[6]

CONCLUSION

Phosphocholine lipid-nucleoside conjugates offer a novel approach to designing

new strategies for double-targeting HIV infected cells simultaneously with two active

drugs and attacking drug-resistant virus variants with a new compound (INK-20) that

has a novel mechanism of action that may involve inhibition of virus structural protein

binding (e.g. gag, gp120) to cell membranes and membrane rafts during the process of

virus assembly.^[21]

ACKNOWLEDGMENTS

This manuscript is dedicated to a colleague, Dr. Leroy B. Townsend, for his

inspiration and dedicated role as a collaborator in several scientific publications jointly

made with one of us (L.S.K) over the span of several years. The present research

described in this manuscript was funded in part by NCI grant CA 12197 awarded to the

Comprehensive Cancer Center of Wake Forest University Health Sciences, the Cell

Culture and Virus Vector Core Laboratory, Kucera Pharmaceutical Company, Winston-

Salem, NC 27101 and the North Carolina Small Business & Technology Development

Center, State Headquarters, Raleigh, NC 27601.

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Received August 8, 2003

Accepted October 29, 2003

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Synthesis and Evaluation of a Novel Synthetic Phosphocholine Lipid-AZT Conjugate that Double-Targets Wild-Type and Drug Resistant Variants of HIV

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