2 ' Substituted Arabinofuranosyl Nucleosides And Nucleotides Intermediates, Preparation And Use Thereof;

Technical Field

This invention concerns novel arabinofuranosyl nucleo¬ sides and nucleotides which have useful antitumor, antiviral, and antimicrobial activities, processes of preparing these nu¬ cleosides and nucleotides, and pharmaceutical compositions con¬ taining them. More particularly, the invention is concerned with 2'-deoxyarabinofuranosyl nucleosides and nucleotides hav¬ ing an azido, amino, or hydrocarbylamino group on the 2' carbon atom.

Background Art

_*

Various nucleic acid derivatives have been found to possess antitumor activity. Frequently, however, they are sus¬ ceptible to deamination (and, therefore, deactivation) by dea- inase enzymes found in mammals. This limits their effective¬ ness in antitumor therapy, for example requiring frequent, re¬ peated administrations by .injection, e.g., intravenous infu¬ sion, and/or administration in combination with a compatible inhibitor which is active against deaminase enzymes. The need for an effective, deaminase resistant antitumor agent is gener¬ ally recognized.

Disclosure of Invention

In searching for such an agent we have developed a new family of arabinofuranosyl nucleosides and nucleotides which exhibit useful antitumor, antiviral, and antimicrobial properties, and which come within the formula

wherein Z is a pyrimidinyl-1, purinyl-9, or l,3-oxazinyl-3 moiety and X is selected from the group consisting of amino, azido, hydrocarbylamino (e.g., alkylamino) of 1 to 7 carbon

0 0 0 atoms, BLN-S- , HO-P-, and HOP-. In addition, some of the * * i i

0 ■ OH H members having antitumor potency exhibit the desired resistan to enzymatic deamination.

Preparation of the 2'-azido nucleosides of the pres ent invention may be by

(a) blocking the labile hydrogen sites on a pyrimi dine, purine, or 1,3-oxazine base by silylation or alkoxylati and

(b) condensing the blocked base with a 2-azido-2- deoxyarabinofuranosyl halide of the formula

wherein each of R 3L and R 3 is hydrocarbylcarbonyl of 2 to 12 or 20 carbon atoms and Y is chloro, bromo, alkanoyl, or acyloxy, to obtain a nucleoside of the formula

wherein Z is a pyrimidinyl-1, purinyl-9, or l,3-oxazinyl-3 moiety.

The pyrimidine, purine, or 1,3-oxazine base can be substituted or unsubstituted and may be acylated with hydroly- zable acyl groups.

A preferred group of pyrimidine bases are those corre¬ sponding to the formula

a. wherein R is amino, hydroxy, thio, hydroxyla ino, alkylammo, arylamino, or aralkylamino, and R is hydrogen, fluoro, bromo, chloro, iodo, ercapto, nitro, nitrilo, thiocyanato, alkyl, alkenyl, or alkynyl.

A preferred group of purine bases are those corre¬ sponding to the formula

wherein R ^e is amino, hydrogen, hydroxylamino, thio, chloro, alkylamino, arylamino, or aralk lammo, and R is hydrogen, oxo, chloro, fluoro, amino, nitro, thio, or hydroxyalkyl.

Suitable examples of pyrimidine bases include cyto sine, uracil, thymine, 5-flourouracil, 5-azauracil, 5-azacy- tosine, dihydro-5-azauracil, dihydro-5-azacytosine, 6-azaura 6-azacytosine, 3-deazauracil, and 3-deazacytosine. Examples suitable purine bases include adenine, guanine, 6-chloropuri hypoxanthine, and xanthine, as well as the 1-deaza, 2-aza, 3 deaza, 7-deaza, 8-aza, 2,8-diaza, 7-deaza-8-aza, and 9-deaza derivatives of those compounds.

Hydrolyzable acyl groups which may be present on t heterocyclic base include acetyl, propionyl, butyryl, valeryl isovaleryl, hexanoyl, heptanoyl, octanoyl, nonanoyl, undecano lauroyl, benzoyl, phenylacetyl, phenylpropionyl, o- , m- , an p-methylbenzoyl, 3-cyclopentylpropionyl, dihydrocinnamoyl, an the like.

Silylation or alkoxylation of the labile hydrogen sites on the heterocyclic base can be accomplished by known methods. Silylation, for example, can be accomplished by the method described in British Patent Specification No. 1,070,41 The latter procedure generally involves reacting the labile hydrogen-containing base at about room temperature with a tri(lowe )alkyl-chlorosilane in the presence of a tertiary amine in an anhydrous organic solvent such as benzene, toluen xylene, and dioxane. Suitable tertiary amines include tri(lower)alkyl amines such as trimethylamine, triethylamine, and tripropylamine. Alternatively, silylation can be effecte by suspending the base in anhydrous hexa(lower)alkyldisilazan and heating to reflux.

Preparation of the 2-azido-2-deoxy-arabinofuranosyl halide can be by a multi-step synthesis involving

(a) acylating (e.g. benzoylating) 1,2-O-isopropyli dene-3-azido-3-deoxy-oc -D-glucofuranose (described by Meyer Reckendorf in Chemische Berichte, vol. 101, p. 3802 (1968)) t obtain 1,2-0-isopropylidene-6-0-acyl-3-azido-3-deoxy-°C-D-gluc furanose of the formula

f θCH

wherein R is hydrocarbylcarbonyl of 2 to 12 carbon atoms,

(b) hydrolyzing the product of step (a) to obtain

6-0-acyl-3-azido-3-deoxy-D-glucofuranose of the formula

( c ) oxidizing and hydrolyzing the product of step (b) to obtain 5-0-acyl-2-azido-2-deoxy-D-arabinofuranose of the formula

H

(d) acylating (e.g. acetylating) the product of step (c) to obtain l,3-di-0-a.cyl-5-0-acyl-2-azido-2-deoxyara- binofuranose of the formula

OMPI

wherein E? is hydrocarbylcarbonyl of 2 to 12 carbon atoms, an (e) halogenating the product of step (d) to obtai the 2-azido-2-deoxyarabinofuranosyl halide.

The same hydrolyzable acyl groups described earlier herein can generally be used in the above synthesis of the 2 azido-2-deoxyarabinofuranosyl halide. Steps- (a) and (d) of that synthesis can be accomplished by conventional acylation techniques. The step (b) hydrolysis can be performed by con tacting an aqueous solution of the compound with a cation ex change resin. Oxidation and hydrolysis (step (c)) can be achieved by reaction with conventional oxidizing agents such as sodium or potassium metaperiodate, followed by treatment with an alkali metal bicarbonate. The step (d) halogenation can be effected by contacting the compound with a metallic halide halogenating agent such as titanium tetrachloride, or stannic chloride, preferably at lower than room temperature, e.g., about 0 to 4 degrees C.

The condensation reaction of the silylated or alko lated heterocyclic base with the 2-azido-2-deoxy-arabinofura- nosyl halide can be conducted in a conventional manner for c densing such bases with saccharide halides, for example as di closed in British Patent Specification No. 1,070,413. Gener ly, the reaction is performed by simply mixing the two reac¬ tants in an aprotic solvent such as tetrahydrofuran, methyle chloride, 1,2-dichloroethane, benzene, and toluene. Use of a catalyst, such as tin tetrachloride, titanium tetrachloride, and mercury salts, is optional. The precise temperature and duration of the reaction are not critical and may be varied widely depending upon the reactants and solvents employed. Ho

ever, high temperatures promote decomposition of the saccharide halide and are therefore not preferred. Generally, the reac¬ tion temperature can be varied between about 10 degrees and 80 degrees C. for 1 hr. to several days or weeks, with the longer times being used at the lower temperatures and with the less reactive heterocyclic bases.

To unblock the 3' and 5' oxygens of the 2-azido-2- deoxy arabinofuranosyl nucleosides described above requires a conventional saponification treatment, for example using methanolic sodium. Conversion of the resultant 3' and 5' hydroxyls to phosphate, sulfamate, phosphonate, or acyl groups can be accomplished by simply reacting the nucleoside with phosphoric, sulfamic, or phosphorous acids, or with hydrocar¬ bon acids in the presence of condensing agents such as dicyclo- hexylcarbodii ide, or suitable derivatives of the acids such as halides or anhydrides thereof. Further, the 5'-hydroxyl group can be replaced by halogenation with fluoro, chloro, bromo, or iodo atoms, or by replacement of these with an amino group.

Conversion of the 2 ¹ -azido group to an amino group can be accomplished by catalytic hydrogenation, for example using a nobel metal, such as platinum or palladium, catalyst.

Conversion of the amino group to a hydrocarbylamino group, such as an alkyla ino or dialkylamino group wherein the ' alkyl substituents are methyl, ethyl, or propyl, can be accom¬ plished by reacting the compound with a hydrocarbylhalide such as an alkylhalide, e.g., ethyl chloride, under conditions generally suitable for amine alkylation reactions. If mono- substitution is desired, it is preferred to first acylate the 2'-amino group.

Separation of the ©c and /3 anomers of the nucleosides and nucleotides of the present invention can be accomplished using conventional column chromatography and crystallization procedures.

The nucleosides and nucleotides of the present inven¬ tion form acid addition salts with both organic and inorganic acids. Preferred acid addition salts are those which are phar¬ maceutically acceptable, such as the addition salts of hydro-

OMPI . -» ^W,p O -

chloric acid, hydrobromic acid, sulfuric acid, phosphoric aci citric acid, acetic acid, succinic acid, maleic acid, methane sulfonic acid, p-toluenesulfonic acid, and the like.

To use the nucleosides and nucleotides of the prese invention, or their acid addition salts, as therapeutic agent for the treatment of mammals, it is preferred to formulate th particular compound in a dosage unit form comprising from abo 1 to about 500 milligrams of the compound per kilogram of the average body weight of the mammal, per dosage unit. For exam ple, the formulation may often contain about 100 to 2000 mill grams of the compound per dosage unit.

The active compound is preferably mixed or dissolve in a compatible pharmaceutical carrier such as, for example, water, gelatin, gum arabic, lactose, starches, magnesium stea rate, talc, vegetable oils, polyalkylene glycols, petroleum jelly, etc. Administration of the compounds can be enteral o parenteral. Accordingly, their pharmaceutical preparations c either be in solid form (e.g., as tablets, capsules, dragees, or suppositories) or in liquid form (i.e., as solutions, sus¬ pensions, or emulsions). The preparations may be sterilized and/or may contain adjuvants such as preserving, stabilizing, wetting, or emulsifying agents, salts for varying the osmotic pressure, buffers, or other therapeutic agents.

Best Mode for Carrying Out the Invention

The invention may be better understood by reference to the following, non-limiting examples.

EXAMPLE 1

Preparation of 5-O-Benzoyl-3-0-Acetyl-2 - Azido-2-Deoxy-D-Arabinofuranosyl Chloride

The following reaction sequence is accomplished in this example.

Benzoylation of l,2-0-isopropylidene-3-azido-3- deoxy-σ -D-glucofuranose with 1.05 molar equivalent of benzoyl chloride gives a greater than 90% yield of 1,2-O-isopropylidene- 6-0-benzoyl-3-azido-3-deoxy- -D-glucofuranose (1). .A sample of 1 is purified on silica gel (CHC1--ether; 3:1), NMR (CDC^ , TMS internal standard) 7.36-8.18 (2m, 5, aromatic), 5.93 (d, 1, J,^ = 3.5 Hz, H-l) , 4.67 (d, 1, J, _jJU = 3.5 Hz, H-2) , 4.25 - 4.80 (m, 5, H-3,4,5,6), 1.50,. 1.33 (2s, 6, 2CH ₃ ) . Crude 1, which contains a small amount of 5,6-di-O-benzoate and a trace of 5-O-benzoate derivatives, is hydrolyzed in dioxane-water (1:1) with Dowex 50 [H ⁺ ] ion exchange resin to give 6-O-benzoyl- 3-azido-3-deoxy-D-glucofuranose (2). Compound 2, which gives a poorly resolved NMR spectrum, is oxidized with sodium meta— periodate at 22-28 degrees C. for 3 hr. , followed by treatment with NaHCO _j (to hydrolyze the forrayl group) overnight to give 5-0-benzoyl-2-azido-2-deoxy-D-arabinofuranose (3), which is purified by silica gel chroraatography (CH^Cl^-ether 3:1).

Compound 3 is acetylated with pyridine-acetic anhy¬ dride to give an anomeric mixture (σc:^5— 4:1, determined by NMR spectroscopy) of l,3-di-0-acetyl-5-0-benzoyl-2-azido-2- deoxyarabinofuranose (4). For thecC-ano er of 4, NMR (CDCl. ) $ 7.42-8.36 (2m, 5 aromatic), 6.20 (s,l, H-l), 5.14 (dd, 1,

J^- _j = 1.5Hz,J ₃ ^ = Hz, H-3), 4.20 (d,l _/ J _;| , ₃ = 1.5 Hz, H-2 for the 3 -anomer of 4, S 7.42 - 8.36 (2m, 5, aromatic), 6.3 (d,l,J _{f A} = 4.5 Hz, H-l), 5.56 (dd,l,J _A3 = 8.0 Hz, _q _ ₃ = 6 Hz, H-3), 4.09 (dd,l,J _{f 3} = 4.5 Hz, J^ ₃ * 8.0 Hz, H-2).

Starting from 109 g. of l,2-0-isoptopylidene-3-azi 3-deoxy-cc-D-glucofuranose, 86.4 g. of 4 (53.3% yield) is ob tained. Compound 4 is converted to a mixture of 1-chloro de rivatives 5 and 6 (5:6 * 4:1) by treatment with TiCl^ at 0-4 degrees C. for 3 hr. Compounds 5 and 6 are separated by sil gel chromatography (toluene-ethyl acetate, 8:1). For compou 5, NMR (CDC1 ₃ ), S 7.32 - 8.20 (2ra, 5H, aromatic), 6.13 (s,l,H-l), 5.10(d,l,J= 4.5 Hz, H-3), 2.16 (S, 3H, Ac); 6 NMR (CDCl^) S 1 .22 - 8.20 (2m, 5, aromatic), 6.25 (d,l,J _{I A} = 4. Hz, H-l), 5.65 (dd,l,J _{λ 3} = 8.5,J^« = 6.0 Hz, H-3), 4.31 (dd,l,J _{l λ} = 4.5 Hz, _{Λ 3} = 8.5 Hz, H-2), 2.13 (s,3H, Ac).

EXAMPLE 2

Preparation of l-(2-Azido-2-Deoxy-3-0- Acetyl-5-O-Benzoyl-D-Arabinof ranosyl) Cytosine

To a stirred solution of 7.8 g. of 2-azido-2-deoxy O-acetyl-5-O-benzoyl-D-arabinofuranosyl chloride in 300 ml. 1,2-dichloroethane is added 6 g. of bis(triraethylsilyl) cyto sine dissolved in 200 ml. of 1-2-dichloroethane. The soluti is stirred at 60-65 degrees C. for 3 days, cooled to room te perature and washed successively with 100 ml. of a saturated NaHCO solution and 100 ml. of water. It is then dried and evaporated at reduced pressure at 45 degrees C, and the res due is dissolved in 50 ml. of chloroform. The chloroform so tion is applied to a silica-gel column and the 3 and cc anome are eluted with an acetyl mixture of chloroform and 2-ρropan (10:1). The 3 -anomer, l-(2-azido-2-deoxy-3-0-acetyl-5-0- benzoyl-B-D-arabinofuranosyl) cytosine, is eluted from the c umn first, and is obtained after evaporation of the solvent 37% yield. Theoc-anomer, l-(2-azido-2-deoxy-3-0-benzoyl- OC - arabinofuranosyl) , cytosine, is obtained in 10% yield.

/-BU

' O

Preparation of l-{2-Azido-2-Deoxy-,-S-D- Arabinofuranosyl) Cytosine ("Cytarazid" )

To a stirred solution of 14 g. of l-(2-azido-2-deoxy- 3-0-acetyl-5-0-benzoyl- 3-D-arabinof ranosyl) cytosine in 500 ml. of methanol is added a catalytic amount of sodium methoxide and the solution is stirred at room temperature overnight. The solution is evaporated to a syrup which is extracted twice with ^' 200 ml. of ether. The residue is dissolved in 100 ml. of meth¬ anol and passed through a short column of Dowex 50 [ H^ ⁺ ] ion exchange resin. The column is washed with 500-1000 ml. of methanol and the methanol solution is evaporated to a syrup. The syrup is crystallized from ethanol to give 8.2 g. (85%) of l-(2-azido-2-deoxy- 3 -D-arabinofuranosyl) cytosine, to which the trivial name cytarazid is assigned; λ _mβx CH^OH = 273 nm, m.p. 157-158 (dec), NMR ( DMSO-d _g , TMS) S 1.16 (C _g H), 7.20 (NH^), 6.17 (C . H), 5.75 (C^H), 5.86 (C H) , 5.07 (O^H).

The o -anomer, l-(2-azido-2-deoxy-3-0-acetyl-5-0- benzoyl-σc-D-arabinof ranosyl) cytosine, is deblocked in the same manner; /^ _β „ CH ₃ 0H = 273 nm, m.p. 175-176(dec) , NMR (DMS0-d ₆ , TMS) £ 7.68 (C H) , 7.23 (NH _λ ) , 5.76 (C, H) , 5.82 (C ₅ H) , 4.93 (0 ₅ . H).

EXAMPLE 4

Preparation of l-(2-Amino-2-Deoxy-/B-D- Arabinofuranosyl) Cytosine ("Cytaramin" )

To a solution of 1 g. of l-(2-azido-2-deoxy- _/! 3-D-ara- binofuranosyl) cytosine in 200 ml. of methanol is" added a cata¬ lytic amount of PtO _Λ and the mixture is hydrogenated at room temperature and atmospheric pressure for 1.5 hrs. The mixture is filtered ^' through a Celite pad and the filtrate is evaporated to a syrup which is crystallized from methanol to give 820 mg. (91%) of l-(2-amino-2-deoxy--3-D-arabinofuranosyl) cytosine, to which the trivial name cytaramin is assigned; _{r a} ( ^NH a.) ⁼ 276, m.p. 209, NMR (DMSO-dg, TMS) <$ 7.79 (CgH), 7.03 (NH^) , 5.99 (C , H) , 5.68 (C ₅ H) .

OMPI

EXAMPLES 5 - 10

Following the general condensation and deblocking procedures set forth in Examples 2 and 3 herein, but using th below-listed heterocyclic bases as reactants, in place of the silylated cytosine, the indicated arabinofuranosyl nucleoside a-re obtained:

Example No. Heterocyclic Base Arabinofuranosyl Nucleosi 5 Bis (Trimethylsilyl) 1-(2-Azido-2-Deoxy-D- Oracil Arabinofuranosyl) ϋracil

Bis (Trimethylsilyl) l-(2-Azido-2-Deoxy-D- Thy ine Arabinofuranosyl) Thymine

Bis (Trimethylsilyl) l-(2-Azido-2-Deoxy-D- -5-Fluorouracil Ara inofuranosyl)-5- Fluorouracil

Bis (Trimethy1sil 1) 9-(2-Azido-2-Deoxy-D- -N-Benzoyl Adenine Arabinofuranosyl) Adenin

Tris (Trimethylsilyl) 9-(2-Azido-2-Deoxy)-D- -N-Acetyl Guanine Arabinofuranosyl) Guanine

10 Trimethylsilyl- 9-(2-Azido-2-Deoxy-D 6-Chloropurine Arabinofuranosyl)-6-Chlor purine

EXAMPLE 11

A portion of each of the anomers of the arabinofura nosyl nucleosides prepared in Examples 5-10 is converted to i 2'-amino counterpart by the catalytic hydrogenation procedure set forth in Example 4 herein.

EXAMPLE 12 .

In Vitro Cytotoxicity Testing

Cytarazid, the /3 -anomer of the nucleoside prepared in Example 3 herein, and cytaramin, the nucleoside prepared i Example 4 herein, are evaluated _in vitro for growth inhibitin potency against mammalian cancer cells by a micro technique, using the culture conditions described by Bobek et al. in J. Med. Chem., vol. 20, p. 458 (1977), whereby 0.5 ml. aliquots

OM

compound are introduced into 16 x 125 mm. screw cap culture tubes, followed by 0.5 ml. portions of the medium containing 1 x 10 ^ff mammalian cancer cells. The cultures are incubated at 37 degrees C. for 40 hr., after which time the viable cells are counted by Trypan Blue exclusion. During this time the cell number in the controls increases about four- to nine-fold with an average cell viability of 99%. The test results are as fol¬ lows:

Concentration (Molar) for 50% Inhibition of Growth

Cell Line Cytarazid Cytaramin

-7 -s

HeLa 2 x 10 3 x 10

Molt 4F (T-type from ly phoblastic leukemia) 7 x 10 8 Not Tested

L-1210 6 x 10 ^"7 4 x lθ ^"6

When subjected to this same cytotoxicity test, the uracil derivative counterparts of cytarazid and cytaramin ex¬ hibited no growth inhibiting potency.

EXAMPLE 13 In Vivo Cytotoxicity Testing

Cytarazid and cytaramin are evaluated in vivo for growth inhibitory potency against leukemic cells by intra- peritoneally inoculating DBA/2 HaDD mice with an IP-PBS saline suspension of 1 x 10 ^s L-1210 leukemic cells, waiting 24 hrs., and then administering the test compound intraperitoneally in 0.2 ml. of saline-phospate buffer solution (pH 7.0).

Survival

Compound Dosage No. of Mice Time ( days Control 4 groups 8.7

6 mice/group

Cytarazid 40 mg/kg administered 4 groups twice per day 8 hrs. 6 mice/group 120 apart for 2 day's begin¬ ning 24 hrs. after tumor inoculation

Cytaramin 75 mg/kg administered 4 groups twice per day 8 hrs. 6 mice/group 120 apart for 2 days begin¬ ning 24 hrs. after tumor inoculation

EXAMPLE 14 Enzymatic Deamination Resistance Testing

Partially purified CR-CdR deaminase is prepared fro two different sources: 1) human liver, following the procedur of Wentworth and Wolfenden, Biochemistry, vol. 14, p. 5099 (1975), and 2) blast cells of patients with acute myelocytic leukemia, using ammonium sulfate fractionation and DEAE colum chromatograph . The assay procedure is described by Wentwort and Wolfenden, ibid. Under these conditions 50% of the comme cially available anti-tumor agent, cytarabine, is deaminated 45 minutes, whereas no significant deamination of cytaramin o cytarazid is detected in 8 hrs.

EXAMPLE 15

In Vitro Antimicrobial Testing

Cytarazid and cytaramin both prove effective to pre vent the growth of E. coli and S. faecium at concentrations o about 0.08 to 1.5 zM, when evaluated by the following assay procedure, which is described in greater detail by Bobek et a in J. Med. Chem. , vol. 13, p. 411 (1970):

S. faecalis is grown in the medium of Flynn et al . (1951) from which uracil and the purines are omitted, and to

which 1 imug/ml of folic acid is added. E. coli is grown in the synthetic medium described by Gray and Tatu (1944). The as¬ says are carried out by placing 1 ml. portions of the media into 13 x 100 mm Pyrex culture tubes and adding 1 ml of water or of the solution containing the test compound. Steriliza¬ tion is carried out by autoclaving or filtration.

The inocula are prepared from cultures of the test organisms grown in 5 ml of the basal medium for 20 hr. at 37 degrees C. Following centrifugation and washing twice with iso- tonic saline, the cells are resuspended in enough saline to yield an optical density of 0.30 at 470 mix as measured in a Beckman Model B spectrophotometer. A 1 ml. portion of this suspension containing approximately 1.5 x 10 cells is diluted tenfold in saline, and 1 drop of this final dilution is placed in each assay tube. Incubation proceeds for 20 hours at 37 de¬ grees C. All E. coli assays are carried out by shaking the cultures during incubation. The extent of growth is determined by means of a Klett-Summerson photoelectric colorimeter using a red filter (640-700 nut ) .

EXAMPLE 16

In Vitro Antiviral Testing

Cytarazid and cytaramin, when tested _iιι vitro against Herpes Simplex types I and II viruses, utilizing the plaque re¬ duction technique described by Dulbecco, Proceedings National Academy of Sciences, vol. 38, p. 747, exhibit significant antiviral properties. Cytarazid, for example, when used at 50^*M concentration against Herpes Simplex type I, inhibited in. excess of 99.9% (3 log) of the virus and when used against type II inhibited greater than 99% (2 log).