CA1313498C - 3'-azido-2', 3'-dideoxyuridine anti-retroviral composition - Google Patents

Abstract

3'-AZIDO-2',3'-DIDEOXYURIDINE ANTI-RETROVIRAL
COMPOSITION
Abstract Compositions for the treatment of AIDS and ARC
having the following compound as an active ingredient:
where R1 is OH, monophosphate, diphosphate, or triphosphate; or a pharmacologically acceptable salt thereof.
The primary advantage of this compound is its highly selective anti-retroviral activity, i.e., it significantly decreases viral replication as measured as reverse transcriptase activity while demonstrating orders of magnitude less cytotoxicity than other anti-viral compounds such as AZT.
In a preferred embodiment, the compound is present in an amount sufficient to inhibit the HIV
reverse transcriptase activity but not significantly inhibit human DNA polymerase activity. Also included within the scope of this invention are 3'-azido-2',3'-dideoxyuridine 5'-mono-, di-, and triphosphate and compositions containing these compounds as the active agent.

Description

1 3 1 3 ~q~8 12 BAC~GROUND OF T~E INVENTION

13 The present invention relates to 3'-azido-2',3'-14 dideoxyuridine (referred to a~ CS-87 herein) and composition~ th~reof as agents for the prevention and 16 treatment of retroviral disease~, particularly 17 acquired human immunodeficien~y (~IDS) viru~ (HIV-1 18 al50 known as HTL~-III/LAV), which cause~ ac~uired 19 immunode~iciency syndr~me (AIDS).
~o ~
21 AIDS was recognized a~ early as 197g. Th~
22 number of case reported to the Center~ for Di~ea~e 23 ~ontrol (CDC) has increa~ed dramatically each y~ar 24 since then, and in 1982 the C~C declared AIDS a new epidemic. AIDS is generally ~ccepted at ~his ti~e to 26 be a consequen~e of i~ection wi~h the retroviru~
27 variou~lr termed human T-lymphotropic virus ty~e III
28 (~T~V~ , lymphadenopathy-as~ociated virus (LAV), 29 AIDS a~sociated retroviru~ ~ARV), or human immunodeficiency viru~ ~HIV-1). An~ibodieq ~o these 31 viruse~ are pre~ent in ovsr 80% Qf patient~ dia~no~ed 32 a~ havi~ AIDS or ~re-A~DS syndrome, and have b~en 33 found with high frequency in the identified risk 34 ~roups.
~.
` 't ,~'..' 1313~9~

1 There is corl~ide~able difficulty in diagnosing 2 the risk oE development of AIDS. AIDS is known to 3 develop in at leaSt 10% of t~e indiYiduals in~ec~ed 4 with HIV, although this percentage i~; ~uspected to be much higher.
6 A patient is generall~ diagno~3ed as havin~ AIDS
7 when a previously healthy ~dult ~ith ~n intact im~UnQ
~ system acquires i~paired T=cell immunity. The 9 impaired immunity u~ually appear~ over a period of eighteen month~ to three years. As a result of this 11 impaired immunity, the patient becomes susceptible to 12 opportuni~tic in~ections, various types of cancer ~uch 13 a~ Raposi's sarcoma, and other disorders associated 14 with reduced functioning of the immune ~ystem.
Another condition a~ociated with HIV is AIDS-16 related complex, or ARC. This condition is thou~ht to 17 lead eventua}ly to AIDS.
18 No treatment capable of preventing or reversin~
19 the immunodeficiency of AIDS or ARC i~ currently available. A11 patients with opportunistic infections 21 and approximately half of ~ll patients with ~aposi's 22 sarcoma have died within two years of diagnosis.
23 Attempts at reviving t}e immune systems in patient~
24 with AIDS have been unsucce~ful.
A number of compounds have de~onstrated ~6 antiviral activity against this virus which include 27 HPA-23, interfexons, ribavirin, phosphonofor~ate, 28 ansamycin, ~uramin, imuthiol, penicillamine, 29 rifabutin, A~-721, 3'-~zido-3'-deoxythymidine (~ZT~, and other 2',3'- dideoxynucleosides.
31 AZT appears to be the drug of choice at thi~
32 time. Howe~er, preliminary re~ult indicate that AZT
33 exhibit~ toxicity i~ a ~linical ~etting. See Yar~hoa~
34 et al., ~ancet 575-580 (1986). AZT was ori~inally 1 31 34qP~

1 ~ynthesized by Horwitz et al., J. Orq. Chem. 29, 2076-2 2078, 1974. Its activity ~ain~t Friend leukemia 3 virus la retrovirus) was ~e~orted as ~arly as 1973 4 ~ee Ostertag et al., Proc. Natl. Acad. S~i. USA 71, 4980-4985 !1974); Rrie~ et al., ~ptl. Cell. Res. 116, 6 21-29, 1978 and refere~es cited therein).
7 In general, inhibit~rs of ~ellular processes 8 will o~ten limit vir~l repli~ation, but ~uch a~ent~
9 are usually quite toxic for the host as ~ell. ~o~t of the antiviral drugs that have been discovered ~o far 11 cannot be prescribed for a prolonged period of time 12 becau~e of their toxicity. For example, a compound 13 structurally related to the compounds of the present 14 invention, idoxuridine, i~ limited in clillical usefulness to topical application in ophthalmic 16 ~olutions for treatment o~ herpetic keratitis because 17 of its toxicity to normal cells. Clearly, there is a 18 ~trong demand for new antiviral agents of low 19 toxicity.
CS-87 is a known compound. See, for example, 21 Lin et al., J. Med. Chem. 26, 1691-1696 (1983), ~in 22 and Mancini, J. ~ed. Chem. 26, 544-548, Colla et al., 23 ur. J. Med. Chem. - Chim. ~he~. 295-301 ~1985~.
24 Lin et al. te~ed the activity of both CS-87 and 25 DDC against L1210 a~d sarcoma 180 cells in vitro and 26 ~ound that both of these compounds are inactive 27 against both cell line~. ~in et al. al~o report that 28 ~oth CS-87 and DDC exhibit only marginal inhibitory 29 activity towards two particular enzyme~ isolated from L1210 cell~. Lin et al. do not di~lose a composition 31 ~ontaining ~he~e compounds in a low ~on~entration 32 ~uf~icient to inhibit replication of HIV or even that 33 these compound~ could be used to treat HIV.

, 1313~

1 Lin and ~ancini report that CS-87 and DDC are 2 both inactive against hl210 cells~ No other activity 3 for these compounds i~ reported.
4 -Colla et ~l. report ~hat CS-87 i9 inacti~e agai~st a v~riety o~ viruse~. In ~arti~ular, Colla ~t 6 al. report that GS-87 is inactive against Coxsa~kie 7 virus B4, polio viru~-l, reovirus-l, parain~luenza 8 virus-3, Sindbi~ virus and mea~le~. Colla et ~1. thu~
9 con~lude that azido derivatives ~uch as CS-8~ do not have si~nificant antiviral activity.
11 In li~ht of the state of the art, it is clear 12 that there remains a ~txong need for new ~ntiviral 13 agents, especially tho~e with low toxicity to normal ~4 cells. More particularly, because of the high mortality of AIDS and the lack o~ an effective 16 treatment for this di~ease, there remains a ~reat need 17 for development of new low toxicity agents ~or ~uch 18 treatment because AIDS patients re~uire a long term 19 therapy, possibly an entire life ~pan. It ~as in thi~
context that the present invention was achieved.
21 It is therefore an object of the present ~2 invention to provide new antiviral compo~itions having 23 low toxicity towards unin~ected cells.
24 It i~ a further object of this invention to provide compo~ition~ for inhibiting the growth of ~IV.
26 It i~ yet another object of the preaent 27 invention to provide a method for the prevention and 28 treatment of infection by HIV.
ag UM~ARY OF_THE INVENTION
The~e and other obje~t~ of the inventio~, ~hich 31 will hereinafter become more rea~ily apparent, have 32 been obtained by providing compositiono havin~ the 33 followin~ compound a~ an active ingredient:

j, ---` 1 31 3~9~

" ,l`
H-N
N

~'~~1 : ~3 1 where R1 is OH, monophosphate, diphosphate, or 2 triphosphate; or a pharmacologically acceptable Ralt 3 thereof~
4 The primary advantage of this compound is it~
highly selective anti-retroviral activity, i.e., i~
6 si~nificantly decreases viral replication as mea~ured 7 as reverse transcriptase activity while demonstrating 8 orders of magnitude less cytotoxicity than other anti-9 viral compounds such as AZT.
This compound i~ provided as an active `: 11 ingredient in compositions suitable for administration 12 to a patient and are contained therein in an amount 13 ~ufficient to exhibit in vitro or i _ vi~ a~tivity 14 against ~IV. In a preferred embodiment, the compound 15 i8 present in an amount 5uf ficien~ to inhibit the HIV
16 reverse transcriptase a~tiv:ity ~ut not significantly 17 inhibit human DNA polymeraqe activity. Also included 18 within tbe scope of thi~ invsntion are 3'-azido-2',3'-19 dideoxyuridine mono-, di-, and triphosphate and ~ompositions containing these compounds as the active 21 agent.
~2 Also enco~passed by the preRent in~ention i~ a 23 method of ~revention or treatment of AIDS or ARC, 2~ which involves administeri~g a composition containing -- 1 3 1 349~

1 the above compound to a per on in~ected ~ith H~V or at 2 risk of acquiring the virus. Administration of the 3 drug may be accomplished orally, in a control~ed 4 release device or in combin tion with a liposome delivery system, by i~jectio~, ~r other ~eans kno~n to 6 those in the art, alone or in combination ~ith other 7 active a~ents.
8 BRIEF DESCRIPTIO~ OF TH~ DRA~INGS
9 Fi~ure 1 i~ a graph ~howin~ the relati~e effects o~ AZT, CS-85 (3'-azido-2',3'-dideoxy-5-ethyl-uridine) 11 and CS-87 on colony formation of human granulocytye-~2 macrophage pre~ursor cells.
13 Fi~ure 2 ~hows the effects of CS-87 on the 14 growth of (a) Vero cells and (b) human blood 15 peripheral mononuclear (PBM) cell~.
16 Figure 3 i9 a graph showing the ef ~ect of CS-85 17 and C5-87 on the weight o~ BALB/c mice.
18 Figure 4 shows the effects of CS-85 and CS-87 on 19 liver enzyme~ (a) SGPT and (b) SGOT in r~}eslls monkey3.
Fi~ure 5 compares the e~fect of C~-87, AZT an~
21 d2C (2',3'-dideoxycytidine) against HIV-1 (Strain 22 RFII) i~ HeLa-T4 cells, percent inhibitiDn versus 23 co~centration.
24 Figure 6 is a graph of the a~tivity of several nucleosides analogues a~ainst HIY-1 an~ simian 26 immunode~iciency virus (SIV) in human PBM cells as a 27 function of median effective concentration.
28 Figure 7a, b, and c are graphs of the ef~Pct o~
29 del~ye~ treatment with AZ~ ~a~, CS-87 (b), an~ CS-91 (3' azido 2',3'-dideoxycytidine) (c) in human ~M
31 cell~ as percent i~hibition ver~us day drug 32 administered.

13~3~

1 DESCRIPTION OF THE ~REF~RR~D ~,MBODI~NTS
2 The present invention i~ based on the discovery 3 that 3'-azido-2',3'-dideoxyuridi~e (CS-87) and 4 acylated and phosphorylated derivatives thereof exert a hi~hly ~elective mntiviral act:ivity against ~IV
6 while, at the same time, e~hibiting remarkably low 7 toxicity towards normal cell~. Alth~ugh CS-87 is a 8 known compound per se, it h~ ~ot hitherto been kno~n 9 that this ~ompound could exert potent anti~iral activity ayainst HIV, and, accordingly, compositions 11 containing this compound in the low concentration~
12 su~ficient to exert such activity again~t HIV ~ith 13 minimal side ef~ects have been unknown. Previou~ly, 14 various workers have reported that CS-87 ha~ very little or no activity a~ainst both a wide variety of 16 viru~es and again~t certain tumor cell~. Due to the 17 low or non-existent activi~y, relativ~ly concentrated 18 solutions of this compound were utilized, but even 19 these compo~itions did not result in significant activity.
21 It h~s now been discovered that the SO%
22 effective do~e (~C00~ in cell culture of CS-87 again~t 23 HIV is less than 1 micromolar, more precisely, aoo 24 nanomolar, even taking into con~ider~tion that the effective dose and relative ~oxicity of the compound 26 vary according to the cell type. The relative non-27 toxicity of the compound has been demonstrated both in 28 cell culture and in animal~, includin~ mi~e and ag monkeys. Although C~-87 and related compounds exhibit reduced toxi~i~y to normal cell~ (see Fi~ure~ and 31 Biological Data Section below), adD~ tration of a 32 hi0h concentration o~ ~uch a drug ~ould nevertheless 33 produce some adverse side e~fects. By high 34 concentration i~ meant a dosage whi~h ~ould result in 1 31 3~9~

1 a blood serum concentration of approximately 100 ~M or 2 hi~her. Thu~, compositions having a hi~h 3 concentration of the actiYe ingredient are not 4 considered to be therapeutically effective.
Althou~h AZT i8 ~omewhat mor~ active than CS-87 6 against HIV, CS-87 has a ~imilar therapeuti~ i~dex 7 when ~e~ted in the same type of cell culture~. The 8 therapeutic i~dex of a compound i8 deter~i~ed by 9 dividin~ the inhibitory or lethal dose for 50~ o~ the population ~IC30 or LD~o) by the effective dose ~or 11 50% if the population (EC~o).
12 The discovery that the present compounds are 13 active against ~IIV at low concentrations and at the 14 same time quite low in toxicity to normal host cells at the lo~er concentration wa~ surpri~ing, since a 16 known compound of close structural similarity which is 17 pre~ently in clinical trials, AZT, exhibit~ ~ much 18 greater toxicity as mea~ur~d by various experimentsr 19 The results reported in Figure 1 clearly show a ~ignificant difference in the e~fect of CS-87 on 21 colony formation of human granulocytes-~acroph~e 22 precursor cells in comparison to Az~. It should be 23 noted that CS-87 appears to exert even lower toxi~ity 24 toward~ these cell3 than CS-85, ~hich i~ the subject 25 o~ patent application U.S. ~rial No. 857,947, filed 26 Hay 1, 1986 by the ~ame inventors, no~ U.S. Patent No~
27 4,681,933, 2~ .
29 A~ used in this invention, antiviral a~tivity refer~ to the ability of a co~po~ition to inhibit th~
31 ~rowth of HIV. The claimed compo3ition al~o exhib~t~
32 antiviral activity to~ard~ other retroviru~es.
33 The ability of the presen~ compo~i~ions to 34 inhibit HIV may be measured by variou~ expsrimental 131349~

_9_ 1 techniques. One ~uch tech~ique involv~s the 2 inhibition of viral replication in human peripheral 3 blood mononuclear cell~. The amount o~ viru~ produced 4 is determined by measuring the virus-coded reverse transcriptase ~an enzyme ~ou~d in retroviru~s~.
6 Results with this assay are illustrated in Table 3 7 ~ee Biolo~ical Data ~ection herein) and described 8 ~urther in the experimental examples below. Other 9 assay are described as follows.
NETHODS
11 Antiviral Assay~.
12 Evaluation in _ l~hYtohema~glutinin (PHA)-stimulated 13 human ~ri~heral blood _ mononuclear ~ cells 14 in~ected with HIV~ train LAV).
A. PBM (2 x 10B cells~ml; volume 5 ml) from a 16 healthy individual that have been cultured for 3-4 17 days after PHA stimulation are placed in a 25 ~mZ
18 flask in duplicate.
19 B. The medium, with the dru~ (2 times the final concentration) or without drug i~ then added to the 21 flasks (5 ml final volume 10 ml). AZT is included as 22 a positive control.
23 C. ~he ~ells are exposed to the virus (about 24 10,000 count~tml, a determined by reverse transcriptase assay~ and are then placed in a COz 26 incubator. HIV-1 (strain hAV) i~ obtained ~rom the 27 Centers ~or Disease Control, Atlanta, Georgia. The RT
28 levels of stock Yirus is usually o~er 106 spm RT~ml.
29 Similar results are obtained when Step C is performed before ~tep B.
31 D. ~n day 5, the cells and supernatant are 32 tr~nsferred to ~ 15 ml tube and centrifuged ~t about 33 900 g for 10 minutes. Five ml of ~upernatant are 34 removed and the ~irus is concentrated by .

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1 ~entrifugation at 40,000 rpm for 30 minutes ~Bec~man 2 70.1 Ti xotor~ The solubilized virus pellet i8 3 proces~ed for determination of the levels of re~erse 4 transcriptase. Results are ~xpres,sed in dpm/ml of ~ampled ~upernatant.
6 This experiment shows that CS-87 has ~ignifica~t 7 activity in inhibitin~ replication of HIY in vi~ro.
8 Evaluation in ATH-8 ~ells infected with NI~-1 (Strain 9 HTLV-III).
CS-87 was ~creened for its antiviral activity on 11 ~TH-8 cells ~S. Broder et al, NCI/NIH, ~ethe~da, 12 Maryland). ATH-8 cells are first treated with 13 polybrene ~2 ~/ml in growth medium) for 30 minute~ at 14 37C. The cells are then collected by gentle centrifugatlon (40 ~ for 15 minutes at 4C) and 16 resu~pended in clarified (8,000g for 15 minutes at 17 4C) two day old supernatant obtained fro~ ~9/HT~VIIIs 18 infected cells. Following a 60 minute adsorption 19 period at 37C, the cells are dispensed into the U-bottom wells of 96-well trays (2 x 104 cells in 0.1 ml 21 per well). AII equal volume (0.1 ml) of ~upplemented 22 RPMI 1640 medium containing test compound and t~ice 23 the normal concentration of interleukin-2 is then 24 added to each well.
Test compounds are evaluated at seven half-log ~6 dilutions, ranging ~rom 100 to 0.1 ~g/ml. Triplicate 27 virus-infected cultures and one u~infected compound 28 cytotoxicity control culture are included ~t each 29 dosage level. Cultures are incubated at 37C in a humidified atmo~phere of 5% C0~-95% air incubator.
31 The size~ of the cell pellets in the test compound 32 wells are compared to the pellet ~ize~ of infected and 33 uninifected ~ell control wells daily for 10 days. On 34 day 10 after infection ali~uots are taken ~rom "` 1 31 34q~

i~dividual ~ells ~rld the total ~ell number and cell 2 viability (based on trypa~-blue dye ex~lusion~ ~re 3 determined~ 2' ,3' Dideoxy~ytidi.ne and AZT are 4 in~luded as positive controls.

5 valuation in ETeLa-T4 cell~ in~e!~ted ~ith ~Itl-l 6 (Strain ~F-II).

7 HeLa-T4 cells (described in C'ell 47:333, 1986~

8 are maintained in Dulbecco'~ ~odified a~le medium 9 containin~ 10~ inactivated fetal calf ~ru~, p~nicillin (100 U~ml) and ~treptomycin (100 ~/ml), 11 and the antibiotic G418 (1 mg/ml). The cells are 12 ~eeded onto a 96-well plate (2 x 10~ cells/~ell per 13 0.1 ml), and t~o day~ later, the supernatant is 14 removed and the virus added. After ~dsorption for 45 minutes, the inoculum i8 removed and ~he cells wa~hed 16 with phosphate-buf~ered saline ~p~ 7.2). The 17 compounds at different concentration are then added in 18 maintenance medium (containing 2% Berum). The plates 19 are incubated in a 5% COs-95% air incubator (humidifiedJ and the ~yncytia allowed to develop for Zl 48 hours be~ore fixation. The cells ~re fixed with a 22 mixture o~ 5% glacial acetic a~id~95~ Ethanol ~or 15 23 minutes, and then stained with Giemsa (20% in P~S) for 24 4 hours. The plates are then washed and dried. The fo~i are then enumerated wi~h a dissectin~
26 ~tereomicro~cope.
27 P24 RIA ~rocedure.
28 Cell-free culture ~upernatant is adiusted to 0.5%
29 Triton*X-100 prior to determination of the HIV-l p24 levels using a DuPont RIA kit. A ~tandard curve i~
~1 generated over the range of 0.625 to 20 ngiml p24 32 u~in~ par~ially purified inactivated viral lysate 33 (calibrated against puri~ied p24). The lo~er limit of 34 sensitivity for this assay is 30 pg/ml o~ ~24.

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1 31 3~9~

.

1 WeBt~rn blot analys2s.
2 The viral pellet i5 dissolved in 2.5 mM Tri 3 buffer (pH 8.0) containing 1% 5DS, 50 mM DTT, and 4 tracking dye and ~ubjected to a ~tern blot a~alysi~
by a technique ~imilar to that ~escribed by Burnette 6 in Anal. Biochem. 112,195-203 ~19811. A ~ell defined 7 antiserum obtained from an AID8 patient i8 u~ed to 8 detect the separated proteinR. ~he int~sity of th~
9 bands is determined ~emiquantitatively (visually) or with a la~er densitometer.
11 The compounds of this in~ention have the 12 following structure:

H-N

O~N /
~ K~~l - . .

13 ~here R1 i5 OH, monophosphate, diphos~hate~ or 14 triphosphate; or a pharmacologically acceptable salt thereof, in as~ociation ~ith a pharmaceutically 16 acceptable carrier.
17 '~he compounds included in the present invention 18 may al~o be in the form of 6alts such as, ~or example, 19 potassium, sodium, and quaternary amine salts, etc.

1 31 34q~

1 Lyxo analo~s of the prese~t compvunds are also 2 encompa~ed within the ~cope o~ thi.s invention. For 3 example, the 3' substituent may have the opposite 4 configuration Prom that ~ho~n in the fîgure.
The compound~ of this ~L~vention may be 6 synthesized by method~ Xnown in the art. Lin et al, 7 Colla ~t al, and Lin and ~ancini, dis~ussed above, 8 each provide ~yntheti~ procedures ~hich may be u~ed to 9 prepare these compounds. A ~pecific method of ~ynthesis leading to CS-87 is a~ follow~.
11 5'-0-Trityl-2'deoxy~__dine(2) 12 A solution of (50 g, Q.~2 mole) of 2'-13 deoxyuridine and 62 g (0. a2 mole) of trityl chloride 14 in 350 ml of dry pyridine is placed in a preheated (100C) flask and ~tirred at 100C under air condenser 16 for two hours. The reaction mixture was cooled to 17 room temperature and slowly poured into 4 L of 18 vigorou~ly stirred ice-water. The ~olid obtained was 19 filtered, washed with water until free from pyridine, and dissolved in chloroform and dried (Na2 S04 or 21 M~S04 ) . Filtration and evaporation of chloroform 22 yielded the product as an syrup ~96 ~, g3%), ~hich was 23 used for the next reaction without further 24 purification~

3'-0-MesYl-5'-trityl-2'-deox~uridine~3) 26 To an ice cooled solution o~ 2 (96 ~, 0.2 mol) 27 in 350 ml of dry pyridine added dropwise 70 ml ~
28 mesyl chloride ~98%, sp. yr. 1480). The mixture ~as 29 stirred in ice-~ater bath for 3 hour and poured 30 ~lo~ly into fi~orously stirred ice-~ater. The ~olid 31 precipitated was filtered, wa~hed with wat0r and dried 3~ ~101 g, 94%).

1313~q~

1 2,3'-AnhYdro-5'-0-trit l-2'-de xyuridine(4) 2 3'-0-Mesyl 5'-0-trityl-2'-deoxyuridine (101 ~, 3 0.19 mol) WdS suspended in 350 ml of ethanol (95%i ~nd 4 the mixture ~as heat~d to reflux. To the refluxi~g mi~ture a~ed dropwise 125 ml o~ an ~queous solution 6 o~ sodium hydroxide (2 N). The reactivn mixture ~a9 7 conce~trated under vacuum. The syrupy residue wa~
8 purified by flash vacuum ~hromatography o~er a silica 9 ~el column eluting ~equentially with chloroform, chloroform-methanol (50:1) and finally Nith 11 chloroform-methanol (30~ Evaporation of the pure 12 fractions yielded 72 ~ (88%) of ~hite powder.

13 3'-Azido-5'-0-trityl-2',3'-dideoxyuridine(5) 14 A mixture of 4 ~72 g, 0.165 mol) and 50 g of lithium azide in 250 ml of dry dimethylformamide was 16 heated at 110-120~C for 12 hour~. The reaction 17 mixture was cooled and slowly poured into 4 L of ice-18 ~ater. The solid obtained was filtered, ~ashed with 19 water, and dissolved in chloro~orm and dried (MgSO4~.
Filtration and evaporation o~ chloroform yielded 63 g 21 ~80%) of the product as a syrup.

22 3'-Azido-2', 3'-dideoxyuridine (CS-87)(6) 23 A mixture of 3'-azido-5'-0-trityl-2',3'-24 dideoxyurîdine (5) (63 g, 0.132 mol) and 300 ml of acetic acid ~80%) ~as heated at 95-100C ~or two 26 hours. The reaction mixture was cooled in an ice-bath 27 ~nd the solid s2parated was filtered off. The 28 filtrate was e~aporated to dryness. The residue ~as 29 dissol~ed in a methanol-~hloro~orm mixture and 30 concentrate~ to a ~rup. The purî~ica~ion o~ the 31 residue ~y flash vacuum chromatography over a silica 32 gel column eluting sequentially with chloroform-1 ~1 34q8 1 methanol (70~ hloroform-methanol ~50:1) and 2 ~inally chloro~orm-metha~ol (30~yielded 23 g (70%) 3 of ~olorless product.
4 S'fNT}lESI ~;

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1 ~umans sufferin~ from di~eases clused by HIV ~an 2 be treated by administering to the patient a 3 pharmaceutically e~fective amount of CS-87 in the - 4 presence of a pharmaceutically acceptable carrier or diluent. '' ~ preferred carrier/diluent ~or oral 6 administration is water, especially sterilized water.
7 If ~dmini~tered intravenously, preferred 8 carrier/diluents are physiological saline or phosphate 9 buffered saline ~PBS). ~he compounds according to the present invention are included in the pharmaceutically 11 acceptable c~rrier or diluent in an amount sufficient 12 to exert a therapeuti~ally useful inhibitory effect on 13 ~IV in vivo without exhibiting adverse toxic effects 14 on the patient treated. By "HIV i~hibitory amount" i8 meant an amount of active ingredient suf~icient to 16 exert an HIV inhibitory effect as measured by, for 17 example, an a~say ~uch as the ones de~cribed herein.
18 There may also be included a~ part of the 19 composition pharmaceutically compatible binding agent~, and/or adjuvant materials. The active 21 materials can also be mixed with other active 22 materials which ~o not impair the desired action 23 and/or supplement the de~ired ac~ion. The active 24 materials according to the present invention can be admini~tered by any route, for example, orally, 26 parenterally, intravenously, intradermally, 27 subcutaneously, or topically, in liquid or solid ~orm.
28 A preferred mode of administratio~ of the 29 compounds of this invention is oral. Oral compositions will ~enerally include an inert dilue~t 31 or an edible carrier. They may be enclosed in gelatin 32 capsules or compressed into tablets. For the purpo~e 33 of oral therapeutic administration, the aForesaid 34 compounds may be incorporated with exipients and used 1 3 1 34q~

1 in the f~rm of tablets, troches, capsules, elixirs, 2 ~u-~pen~ions, ~yrups, wafers, che~ing gums and the 3 like. These preparation~ should produce a serum 4 concentration of active ingredient of from about 0.2 5 to 40 ~M. A ~ra~erre~ concentration ran~e is ~rom 0. a 6 to 20 ~M and most preferably about 1 to 10 ~M.
7 However, the concentration of active in~redient in the 8 drug composition it~elf ~ill depend on bioavailability 9 of the dru~ and other factors kno~n to those of skill in the art.
11 It is to be noted that ~osage values will also 12 vary with the specific severity of the ~i~ea~e 13 condition to be alleviated. It is to be ~urther 14 understood that for any particular subject, specific dosage regimens should be adjusted to the individual 16 need and the proPessional judgment of the person 17 administering or supervising the administration of the 18 a~oresaid compositions. It i3 to be further 19 understood that the concentration ranges set forth herein are exemplary only and they do not limit the 21 scope or practice of the invention. The acti~e 22 ingredient may be admini~tered at once, or may be 23 divided into a number of ~ma~ler dosf!s to be 24 admini~tered at varying intervals o~ time.
The tablets, pills, cap~ules, troches and the 26 like may ~ontain the followin~ in~redients: a binder 27 such a~ microcrystalline cellulose, gum tragacanth or 28 ~elatin; an excipient ~uch a~ ~tarch or lactose, a 29 disinte~rating agent such as alginic acid, Pri~ogel, corn starch and the like; a lubricant such as 31 ~agnesium stearate or Sterotes; a glidant such a~
32 colloidal ~ on dioxide; and a sweetening agent such 33 as ~ucrose or ~accharin or ~lavoring agent ~uch as 34 peppermint, methyl salicylate, or orange flavoring may I

1 3~ 3~q~

1 be added. ~hen the do~age uni~ form is a capsule, it 2 may contain, in addition to material of the above 3 type, a liquid carrier such a~ a atty oil. Other 4 do~age unit form~ may cvntain cthcr ~ariou~ material~
which modify the physical form of the do~age u~it, ~or 6 example, as coatings. ~hu~ tablet~ or pill~ may be 7 coated with sugar, shellac, or oth~r enteric coating 8 agents. A syrup may cont~in, :in ~ddition to the 9 active comp~u~ds, ~ucrose as a ~weetening agent a~
certain pre~ervative~, dye~ and ~olorin~s and ~lavors.
11 Materials u~ed in preparing these various compositions 12 should be pharmaceutically pure and non-toxic in the 13 amounts used.
14 The solutions or suspensions may also include the following components: a ~terile diluent ~uch as ~ater 16 for injection, saline ~olution, fixed oils, 17 polyethylene ~lycols, glycerine, propylene glycol or 18 other synthetic solvents; antibacterial agents such as 19 benzyl alcohol or methyl parabens; antioxidants cuch as ascorbic acid or ~odium bisulfite; chelating agents 21 ~uch as ethylenediaminetetraacetic acid: buffers such 22 as acetates, citrates or phosphates and agents for the 23 adjustment o~ tonicity such as sodium chloride cr 24 dextrose. The parental preparation can be enclosed in ampoules, dispo~able ~yringes or multiple do~e vials 26 made of glass or plastic.
27 The composition~ of the pre~ent invention are 28 prepared as ~ormulations with pharmaceu~ically 29 acceptable ~arriers. Pre~erred are tho~e ~arrier~
that will protect the active compound against rapid 31 elimination from the body, 6uch a~ a controlled 32 release formulation, includi~g implantc and 33 microencapsulated delivery systems. ~iode~radable, 34 biocompatable polymers can be used, such as 1 3 1 34q~
.~, ...

polyanhydrides, polyglycolic acid; collagen, and polylactic acid. Methods for preparation of such formulations will be -` apparent to those skilled :in the art.
Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) are also preferred as pharmaceutically accceptable carriers. These may be prepared according to methods known to those skilled in the art, for e~ample, as described in IJ.S. Patent No. 4,522,811. For example liposome formulations may be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and cholesterol) in an organic solvent that is then evaporated, leaving behind a thin film oE dried lipid on the surface of the container.
An aqueous solution of the active compound (e.g., CS-~7, CS-87 monophosphate, CS-87 diphosphate, and/or CS 87 triphosphate) is then introduced into the container. The container is then swirled by hand to free lipid material from the sides of the container and to disperse lipid aggregates, thereby forming the liposomal suspension.
The phosphate compounds of the present invention are prepared by phosphorylation of CS-87, as described below.
The monophosphate can be prepared according to the procedure of Imai et al., J. Ora. Chem., 34(6), 1547-1550 (June 196g). ~or example, about 100 mg of CS-87 and about 280 ~l of phosphoryl chloride are reacted with stirring in about 8 ml of dry ethyl acetate at about 0 C for about four hours. The 1 31 3~q8 1 reaction is quenched with ice. The aqueous ~ha~e i~
2 purified on an activated charcoal column, eluting with 3 5% ammonium hydroxide in a 1:1 mixture of ethanol and 4 water. Evaporation o~ the eluant gives 100 m~ of 3'-azido-2',3'-dideoxyuridine ~ono~hol~phate, a~ the 6 ammonium salt.
7 The diphosphate can be pre~areld accor~ing to the 8 ~rocedure of Davi~son et al., J. Or~. Chem., 52g9~, 9 1794-~801 ~1987). 3'-Azido-2',3'-dideoxyuridi~e diphosphate is prepared from the tosylate of CS-87, 11 which may be prepared, for example, by reacting CS-87 12 ~ith tosyl chloride in pyridine at room temperature 13 for about 24 hours, working up the product in the 14 u~ual manner (e.g., by washing, drying, and crystallizing it).
16 The triphosphate can be prepared according to 17 the procedure o~ Hoard et al., J. Am. Chem. Soc., 18 87(8), 1785-1788 ~1965). For example, 3'-azido-2',3'-19 dideoxyuridine monophosphate is activated (by making a imidazolide, according to methods known to tho~e 21 ~killed in the art) and treat~d with tributyl ammonium 22 pyrophosphate in DMF. The reaction gives primarily 23 3'-azido-2',3'-dideoxyuridine triphosphate, with ~ome 24 unreacted monopho~phate and some diphosphate.
Purification by anion excha~ge chromatography o~ a 26 DEA~ ~olumn i~ ~ollowed by isolation of CS-87 27 trîpho~phate, e.g., as the tetrasodium ~alt.
28 Structurally related analogues ~uch a~
29 pho~phorylated and acylate~ derivatives of CS-87, and the uridine and C-nucleo~ide derivatives thereof will 31 have eimilar activitie6 at generally the same in ~ivo 32 concentration range~.
33 The invention now being generally de~cribed, the 34 same will be better understood by reference to certain ~ 3~ 34qP~

1 specific examples ~hich are include~ herein ~or 2 purpose~ of illustration only and are not intended to 3 be limiting of the invention or any embodiment 4 thereof, unle~ ~pecified.
Various experiments ~ere ~onducted to ~how the 6 effects of CS-87 on the gro~th o~ certain cell~, the 7 effects of CS-87 when administerecl in vivo, and the 8 effects of CS-87 on HIV replication.
9 The Ef f ect o~ CS-87 on Cell Growth.
The effect of CS-87 on colony formation of human 11 granulocytes -macrophage precursor cells was compared 12 with the effects of AZT and CS-85 in Figure 1. The 13 effect of CS-87 on Vero cells is ~hown in Figure 2a.
14 Vero cells are very fast growing cells, and it can be seen from Figure 2a that up to a concentration o~
16 about 400 micromolar, there is relatively little 17 toxicity to these cells. P9M cells are somewhat more 18 ~ensitive to CS-87 than the Vero cell , but these 19 cells will still tolerate a concentration of CS-87 o~
up to about 200 micromolar before significant ~1 inhibition i5 noted (~ee ~igure 2b).
22 E~fect o~ CS-87 on Animals.
23 Figure 3 ~hows the effect of CS-85 and CS-87 on 24 the weight of uninfected BALB/c mice. It can be ~een that there is no signi~icant difference between CS-87, 26 CS-85 r and the control. The hematologic values of NIH
27 Swis~ mice trea~ed with AZT and CS-87 (admini~tered 28 orally at 0.5 mg/ml ad libitum for a total of 67 ~ayQ) 29 is shown in Table 1. The signi~icantly less toxicity of Cs-87 a~ compared to AZT is apparent ~rom the 31 differences in the RBC and MCV values.
32 As can be ~een in ~igure 4, there ~as a tran~ient 33 elevation in monkeys of SGPT and S~OT with intravenous 34 administration of C~-87. Ho~ever, the animals ~ere , ,., ,. - ~ , ...

~ 3~3~9~

-~2-1 back to normal after one ~eekO It can ~e seen in 2 Figure 4 that oral administration ~po) is les~ toxic 3 than intr~ven~u~ admini~tration.
4 The p~armocokinetics ~erum and brain level~) of CS~87 in adult mice is ~hown in Table 2. I~ rhesu~
6 monkey~, the pharmocoXinetic parameters a~ter an oral 7 dose of 920 mg of CS-87 are a~ ~ollo~ws:
8 elimination t~/ 2 k = 0.48 h 9 clearance = 1.74 L/hJkg AUC c 113.43 mg h/L
11 volume of distribution.(V~) = 1.2 L/kg 12 absorption rate constant (kQ) =1.73 h-13 absorption rate ti/z = 0.40 h 14 elimination rate constant, k = 1.43 h-l E~fect of CS-87 on the Replicatio of HIV in Different 16 T~pe~ of Cells.
17 CS-87 has ~elective anti-HIV-1 activity in human 18 peripheral blood mononuclear (PBM) cells, as sho~n by 19 Table 3 in combination wi h the ~oregoing data.
Since the antiviral activity o~ nucleoside 21 analogs ~ay depend on the type of assay and ~ell type 22 used, the activity o~ CS-87 ~as determined in ~ario~s 23 NIV-1 susceptible cells. In addition to PBM cells, 24 ATH-8 and the recently described HeLa-T4 cells (Cell 47:333, 19863 were usedO The m~dian effective dose 26 (~C50) or minimum inhibitory concentration ~MIC~ of 27 CS-87 relative to 3'azido-3'-deoxythymidine (AZT) and 28 2',3'-dideoxycytidine (d2C) is described in Table 4.
29 The results indicate that the potencg of CS 87 and re~ated ~ompounds varied accordin~ to the cell 31 system u~ed. Although CS-87 had comparable activity 32 to d2C in AT~-8 cells, it was less active in HeLa-T4 33 and PBM cells. CS-8~ was not toxic to the uninfected 34 cells when tested up to 200 ~M. The percent ,., ,.,, . ; .

-- ~ 31 3~q~

1 inhibition of CS-87, AZT, and d2C againct HIY-l 2 ~Strain ~FII) in HeLa-T4 ~ells as a function of 3 concentration is s~own in Fi~ure 5.
4 The_Ef~e~t o~ CS-87 on Human, Non-~luman Primate, and Murine Retroviruses.
6 A compari~on of the differenc~s in median 7 effective antiviral concentrations ~etween human, non-8 human primate, and murine retroviru~es is sho~n in 9 Table 5. CS-87 is much mOrQ ef~ective a~ain~t ~IV
~I.AV-l) and SIV (5MM) than ~gainst ~riend (13Y-10~ or 11 Mouse ecotropic (Cas-Br-M virus). A further 12 comparison of the activity of several nucleoside 13 analogues, including CS-87, against HIV-l and SIV in 14 human P~M cell~ i8 ~hown in Figure 6. The ef~ect o~
delayed treatmen~ with AZT, CS-87 or CS-91 on the 16 replication of HIV-1 and SIV in human P~M cells is 17 shown in Figure 7a, b, and c. It is apparent that the 18 time of administration i8 critical to the 19 effectiveness of the treatment.
Table 6 shows the effect of the 5'-triphosphates 21 of CS-87 and AZT on HIV reverse transcriptase and 22 alpha DNA polymerase, again demonstrating the extreme 23 selectivity of the CS-87 compound against HIV and not 24 the host ~ell ~-DNA polymerase activity).
a5 Modifications and variations of the present 26 invention, compo~ition~ for the treatment of HIV
27 including 3'-azido-2',3'-dideoxyuridine and 28 derivatives thereof, will be obvious to those skill2d 29 in the art from the ~oregoin~ detailed description of the invention. Such modifications and variations are 31 intended to come within the scope of the appended 32 claims.

1 31 34q~

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Time after treatmentSerum level~ Braln levels ~atio:
(min.) (~g/~ y,1~l) X~/S

71.3 '~.10 4.3 125.7 7.86 6.3 31 91.0 11.11 12.~
: 3~ 55.5 5.67 10.2 71 24.5 6.40 26.1 72 54.8 7.33 13.

O Mice were administered the drug at B do~e of 250 mgtkg iv.

Dlfference~ ln Median ~ffective Antlviral Concentratlons ~etween Human, Non~Hu~an Primate, and Murine Retroviruee~

~C50f~/U~
H/V S/V firi~ndl~ous~ ecofropic fLAV-1J ~S~MJ ~EY-10) tCos-~r-~lJ
AZT O.002 0.002 0.004 0.007 CS-87 0.28 0.20 > 10 9.10 CS-91 0.39 1.16 ND 77.9 HS-20 2.40 0.52 0.89 0.79 HS-21 3.20 2.30 0.70 1.16 1 31 34~8 o ~+ o o ~--~
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1 31 34~

Sl~SARY OF ANTIVIRAL ACTI-aITY AGAINST I~IV-l OF CS~37, AZT
AllD D2C I~ IOUS OELL 5YSTEMS

Tre~tment Cell ~stem ~JlM ~
PBM (EC 50) ~eI~-T4 (BC 50~ A~1-8 ~MIC) CS-87 0.18 1.2~ 0.40 AZT 0.002 0.34 0.32 d2C(ddC) 0.011 0.48 0.47 l~PFECT OF THE 5'-TRIPHOSPMATES OE' CS-87 AND AZT ON MIV Rl~V~RSE

Compound HIV RT Ho~t cell ~X-DNA pol R~tlo:
Rl, nM Rl, mM Cellular/HIV
CS-87-TP9 1. 2 133, 333 AZ$-r~40U 0.23Cl 5,750 Accordln~ to Pur~an O'C 31. Proc. Natl. Ac~d. Sci. 1986.

Claims (15)

1. A pharmaceutical composition comprising a HIV inhibitory amount of a compound having the formula:
where R1 is OH, monophosphate, diphosphate, or triphosphate; or a pharmacologically acceptable salt thereof, in association with a pharmaceutically acceptable carrier.

2. The pharmaceutical composition of Claim 1, wherein said pharmaceutically acceptable carrier protects the compound against rapid elimination from the body.

3. The pharmaceutical composition of Claim 1, wherein said pharmaceutically acceptable carrier comprises a liposomal suspension.

4. The pharmaceutical composition of Claim 3, wherein R1 is monophosphate, diphosphate, or triphosphate.

5. The pharmaceutical composition of Claim 1, wherein R1 is monophosphate, diphosphate, or triphosphate.

6. The pharmaceutical composition of Claim 1, wherein R1 is OH.

7. The pharmaceutical composition of Claim 1, comprising a HIV reverse transcriptase inhibitory amount of said compound.

8. The pharmaceutical composition of Claim 7, comprising a human DNA polymerase non-inhibitory amount of said compound.

9. A method for making a composition for the treatment of viral diseases, comprising encapsulating 3'-azido-2',3'-dideoxyuridine with a pharmaceutically acceptable carrier in an enteric coating, the resulting composition providing 3'-azido-2',3'-dideoxyuridine in a dosage resulting in a patient blood stream concentration of from about 0.2 to 40 µM.

10. A method for making a composition for the treatment of viral diseases, comprising encapsulating 3'-azido-2',3'-dideoxyuridine in a biodegradable implant, the resulting composition providing 3'-azido-2',3'-dideoxyuridine in a dosage resulting in a patient blood stream concentration of from about 0.2 to 40 µM.

11. A method for making a composition for the treatment of viral diseases, comprising providing a liposomal suspension for delivery of 3'-azido-2',3'-dideoxyuridine in a dosage resulting in a patient blood stream concentration of from about 0.2 to 40 µM.

12. A method according to claim 9, claim 10 or claim 11, wherein said 3'-azido-2',3'-dideoxyuridine is synthesized by reacting 2'-deoxyuridine with trityl chloride to produce 5'-O-trityl-2'-deoxyuridine, reacting the 5'-O-trityl-2'-deoxy-uridine with mesyl chloride to produce 3'-O-mesyl-5'-O-trityl-2'-deoxyuridine, refluxing the 3'-O-mesyl-5'-trityl-2'-deoxy-uridine under alkaline conditions to produce 2,3'-anhydro-5'-O-trityl-2'-deoxyuridine, reacting lithium azide with 2',3'-anhydro-5'-O-trityl-2'-deoxyuridine to produce 3'-azido-5'-O-trityl-2',3'-dideoxyuridine and heating the 3'-azido-5'-O-trityl-2',3'-dideoxyuridine in acetic acid.

13. A method according to claim 9, claim 10 or claim 11, wherein said 3'-azido-2',3'-dideoxyuridine is first phosphorylated to form the mono-, di-, or triphosphate.

14. The method of claim 9, claim 10 or claim 11, wherein said 3'-azido-2',3'-dideoxyuridine is first acetylated.

15. A method for making a composition for the treatment of viral diseases, comprising mixing 3'-azido-2',3'-dideoxy-uridine with compounds selected from the group consisting of water for injection, saline, oils, polyethylene glycols, glycerine, propylene glycol, antibacterial agents, anti-oxidants, chelating agents, buffers, and agents for adjusting tonicity, the resulting composition providing 3'-azido-2',3'-dideoxyuridine in a dosage resulting in a patient blood steam concentration of from about 0.2 to 40 µM.