CA2464681C - Preparation of thioarabinofuranosyl compounds and use thereof - Google Patents
Preparation of thioarabinofuranosyl compounds and use thereof Download PDFInfo
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- CA2464681C CA2464681C CA2464681A CA2464681A CA2464681C CA 2464681 C CA2464681 C CA 2464681C CA 2464681 A CA2464681 A CA 2464681A CA 2464681 A CA2464681 A CA 2464681A CA 2464681 C CA2464681 C CA 2464681C
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- arabinofuranosyl
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Abstract
A method for producing a compound of the formula I: (see formula I) comprises A) reacting a 2,3,5-tri-O-aryl or alkyl-4-xylose diaryl or dialkyl dithioacetal in the presence of a leaving group at the 4 hydroxyl position to produce the corresponding 1,4-dithio-D-arabinofuranoside; B) reacting said arabinofuranoside from step A) to acidolysis to form the corresponding O-acetyl-4-thio-D-arabinofuranose; C) reacting said O-acetyl-4--thio-D-arabinofuranose from step B) with a compound selected from the group consisting of a cytosine, a 5-aza compound, a 6-aza compound and blocked derivatives thereof to form the corresponding 4-thio-.infin., .beta.-D-arabinofuranosyl compound; D) converting said arabinofuranosyl compound from step C) by hydrolysis to the corresponding thio sugar derivative; and E) separating out the .infin. form or the anomeric mixture of step D) to thereby obtain the compound of formula I.
Description
WO 00/04866 ~ 02464681 2004-04-07 p~'/Ugg9118630 PREPARATION OF THIOARABrNOFURANOSYL, CC?1VIPOUNDS AND USE THEREOF
DESCRIPTION
a Federally Sponsored Research and Development This invention was supported by Grant CA-34200 from National Institutes of Dealth_ Technical Field The present invention is concerned with treating patients suffering horn cancer by administering to the patients cereaiti thioarabinofuranosyl compounds.
Compounds employed according to the present invention have exhibited good anticancer activity. Compounds employed accordix~ to the present invention are in the beta configuration as cozitrasted to the alpha configuration, which failed to exhibit anticancer activity. The. present invention also relates to inhibiting DNA
replication in a rnaaunalian cell by contacting with the thioarabinofuranosyl compounds. The present invention also relates to a nevv process for preparing the compounds employed according to the present invention.
Background of Invention Past amounts of research have accrued over the years related to developing treatments against cancers to inhibit and kill tumor cells. Some of this research has ~"0 resulted in achieving some success in finding clinically approved treauaents:
Nevertheless, efforts continue at an ever-increasing rate in view of the extreme difficulty in uncovering promising anticancer treatments. For example, even when VSO flfl/fl4S66 ' PCTIUS99116630 a compound is found to have cytotaxic activity, there is no predictability of it being selective against cancer cells.
One particular compound that has been used rather widespread is cytosine arablnoside, comfnonly referred to as .lira-C.
S ~ Sununary of Invention It has been found according to the present invention that certain thioarabinofuranosyl cytosine compounds are suitable as anticancer agents. The presence of the thiv sugar moiety surprisingly makes it possible to achieve good antitumor activity. More particularly, the present invention relates to treating a mammalian host in need of an anticancer treatment by administering to the host an effective anticancez amount of at lease one compound represented by the following formula 1_ RO ~ A
wherein each R individually is H ox an aliphatic or aromatic aryl group;
A is selected from the group consisting of NHa N / ~
Z
O
n WC7 00/L148Gt FCTIU$g9116b3a N N
o~N
> ~
X is selected from the group consisting of hydrogen, flaoto, alkoxy, alkyl, haloalkyl> alkenyl, haloalkenyl, aIkynyl, amino, monoalkylamina, dialkyiamino, cyano az~d nitro.
rt has also been found according to the present invention that the above-disclosed compounds of formula x can be used to inhibit DIVA replication in a IfJ manunalian cell by contacting the cell with at least one of these compounds.
The present invention is also concerned with a pzocess for preparing the above-identified compounds. The compounds employed according eo the present invention can be prepared by:
A) reacting a 2,3,5-tri-O-aryl or alkyl-4-xylose diaryl or dialkyl dithioacetal such as 2,3,5-tri-O-benzyl-L-xylose-dibenzyl dithioacetal in the presence of a leaving group at the 4 hydroxyl to produce the corresponding 1,4-dithio-D-arabinofuranoside such as benzyl 2,3,5-tri-O-benzyl-1,4-dithio-D-arabinofuranoside;
B) subjecting the product from step A) to acidolysis to form the corresponding. 0-acetyl-4-thio-D arabinofuranose such as 2,3,5-tri-O-benzyl-1-O-acetyl-4-thio-D-arabinofuranose;
C) reacting the product of step. B) with a cytosine, a 5- or 6-aza compound or a suitably blocked derivative thereof forming a corresponding 4-thin-a,13-D-arabinofuranosyl compound such as, in the case of cytosine, 1-(2,3,5-tri-O-benzyl-4-thin-a,13-D-arabinofuranosyl) cytosine;
D) converting the compound of step C) by hydrolysis to the corresponding thin sugar derivative such as 1-(4-thio-a,13-D-arabinofuranosyl) cytosine;
E) separating out the a form of the anomeric mixture of step D) to thereby obtain the desired compound of formula l, such as 1-(4-thio-13-D-arabonifuranosyl) cytosine.
In a broad aspect, then, the present invention relates to the compound represented by the formula 1:
RO A
S
RO
RO (1) wherein each R individually is H, an aliphatic acyl group or an aromatic acyl group;
NHZ
N
O N
NHz N~ N
ON
NHZ
N NH
O N
NHZ
X
/N
O N
wherein X is hydrogen, fluorine, alkoxy, alkyl, haloalkyl, alkenyl, haloalkenyl, alkynyl, amino, monoalkylamino, dialkylamino, cyano or vitro, for treating a solid. tumor in a mammalian host.
4a In another broad aspect; then, the present invention relates to the compound 1-(4-thio-13-D-arabinofuranosyl) cytosine for treating a mammalian host suffering from a cancer selected from the group consisting of melanoma, prostate cancer, mammary cancer, renal cancer, colon cancer and lung cancer.
In yet another broad aspect, then, the present invention relates to use of a compound represented by the formula 1:
RO A
S
RO
RO
wherein each R individually is H, an aliphatic acyl group or an aromatic acyl group;
A is x NFi N N
p N
4b N NH
O N
or X
N
~I IN
O N
wherein X is hydrogen, fluorine, alkoxy, alkyl, haloalkyl, alkenyl, haloalkenyl, alkynyl, amino, monoalkylamino, dialkylariiino, cyano or nitro, for treating a solid tumor in a mammalian host.
In a further broad aspect, then, the present invention relates to use of the compound represented by the formula 1:
RO A
S
RO
RO'~ ~l~
wherein each R individually is H, an aliphatic acyl group or an aromatic acyl group;
4c !~ 1S
NHZ
X
N
O N
NHZ
N N
J
O N
NHp .
N ~ NH
O N
NHz X
N
~N
O N
wherein X is hydrogen, fluorine, alkoxy, alkyl, haloalkyl, alkenyl, haloalkenyl, alkynyl, amino, monoalkylamino, dialkylamino, cyano or nitro, for treating a mammalian host suffering from a cancer selected from the group of melanoma, prostate cancer, mammary cancer, renal cancer, colon cancer or lung cancer.
4d In a still further broad aspect, then, the present invention relates to use of the compound represented by the formula 1:
RO A
S
RO
RO (1) wherein each R individually is H, an aliphatic acyl group or an aromatic acyl group;
A is X
N
i O N
NHp N~ N
~J
O N
J
NHZ
N NH
O N
or 4e NHZ
N
~N
O N
wherein ~ is hydrogen, fluorine, alkoxy, alkyl; haloalkyl, alkenyl, haloalkenyl, alkynyl, amino, monoalkylamino, dialkylamino, cyano or nitro for inhibiting DNA
replication in a mammalian cell.
In another broad aspect, then, the present invention relates to use of I-(4-thin-13-D-arabinofuranosyl) cytosine for treating a mammalian host suffering from a cancer selected from melanoma, prostate cancer, mammary cancer, renal cancer, colon cancer or lung cancer.
4f Summary of Drawings .
Figs. la and 1b are graphs showing metabolism of 2-devxycytidiae, 2'-deoxythiocytidine, ara c and thio ara-c to their respective triphosphates.
Fig. 2 is a graph showing the retention of ara c tiiphosphate and thin ara c triphosphate in CEM cells.
Best and Various Modes for Carrying ~ut Invention The present invention is related to treating a mammalian host in need of an anticancer ra~eacmenc, which comprises administering to the host an effective anticalzcer amount of at .least compound represented by the formula 1:
RO ~ A .
1.o 0 g Each R in formula 1 individually is preferably H or an aliphatic or aromatic acyl Eroup. Typical aliphatic acyl groups contain from 1 to 6 carbon acorns and include formyl, acetyl, and propionyl. Typical aromatic acyl groups include unsubstituted and alkyl substituted aromatic ,groups containdng 7-10 carbon atoms in.
1$ the arc~mauc group. When substituted, tl3e alkyl group typically contains 1-6 carbon atoms. Typical aromat5c acyl groups include benzoyl and para-tol.oyI.
A in formula l is preferably WD 00104866 ~ 02464681 2004-04-07 PCTlLyS99f 16630 N N
O%'N/
NHa N! 'NH
and .
o a o~N~
N
Suitable monoalkylamino groups for X ~contsin 1-6 carbon atoms and intclude monomethylamino, monoethylamino, mono-isopropylamino, mono-n-propylamino;
mono-isoburyl-amino, mono-n-butylamino and mono-n-hexylamino. The all~yrl moiety can .be stzaight or branched chain.
Suitable dialkylamino groups for Y and X contain 1-6 carbon atoms in each Itl alkyl ~roup. The alkyl gzoups can be the same or different and can be straight or branched chain. Examples of some suitable gmups are dimethyIamino, diethylamino, ethylmethylamino, dipropylamina; dibutylamino, dipentylamino, dihexylamino, methylpentylamino, ethylpropylamino and ethylhexylamino.
Suitable halogen groups for X include GI, Br and F.
Suitable alkyl groups far X typically contain 1--6 carbon atoms and can be straight or branched chain. Some examples are methyl, ethyl, i-propyl, n-propyl, i-butyl, n-butyl, pentyl and hexyl_ Suitable haloalkyl groups typically contain I-6 carbon atoms and can be straight Qr branched chain and include Cl, Br ar >substituted alkyl groups including LQ the above specifically disclosed alkyl groups.
5uitabie alkoxy groups typically contain 1-6 carbon atoms and include rnerhaxy, ethoxy, pxopoxy and butoxy, Suitable alkenyl groups typically contain 2-6 carbon atoms and include ethenyl and propenyl_ Suitable halaalkenyl groups typically contain 1-6 carbon atoms and include Cl.. Br or F substituted alltenyl groups including the above specifically disclosed alkenyl groups.
Suitable alkynyl groups typically contain 1-6 carbon atoms and include ethynyl and propynyt. , 24 The preferred compound employed according to the process of the present invention is 1-(4-thin-!3-D-arabinofuranosyl} cytosine.
WO 00104866 pCTlUS991t6630 The present invention is suitable for treating mammalian hosts including humans suffering from cancer including melanoma, prostate cancer, mammary cancer, renal cancer, colon cancer, sung cancer, lettkemias and lymphomas.
The compounds employed according to the present invention can be prepared by reacting a 2,3,5-tri-O-aryl or alkyl-4-xylose diaryl or dialkyl dithioacetal such as ~.3.~-tri-O-benzyl-I:-xylose-dibenzyl dithioacetal in the presence of a bearing gmup at the 4 hydroxyl position to produce the corresponding 1,4-dithio-D-arabinofuranoside such as Isenzyl 2,3,5-tri-O benzyl-l,4rdithio-D-arabinofuranoside.
This step can he carried aut using phosphine, iodine and imida2ole. The product from the above step is subjected eo acidolysis to form the corresponding 4-acetyl-4 thin-D arabinofuranose such as 2,3,5-tri-O-benzyl-1-~-acetyl-4-chio-D
arabinofuranose. For instance, acetic acid in the presence of rncrcuric acetate can be employed.
The product of the above step is reacted, with a cytosine, a 5- ar 6-aza 1.5 compound or a suitably blocked derivative thereof forming a corresponding 4-thio-«,13-D-arabinofuranosyl compound such as, in the case of cytosine, 1-(2,3;5-tri-O-benzyl-4-thio-cz,t3-D-arabinofuranosyl) cytosine. Suitable blocked derivatives include acyl and tximethylsilylated derivatives, The compound of the above step is converted by hydrolysis to the coaesponding thio sugar derivative such as 1-(4-thio-a,li-D-arabinofuranasyl) cytosine.
The cx form of the anomeric mixture of the above step is separated out to thereby obtain the desired compound of formula l, such as 1-(~i-thio-!3-D-arabinofuranosyl) cytosine.
Compounds according to the present invention can be prepared by the 2a process sequence shoawn in scheme I and examples 1 and 2 described hereinbelour, wlaerein the preferred compound, 1.-(4-thio~B-D-arabinofuranosyI) cytosine is illustrated for purposes of facilitating an understanding of the process sequence.
The precursor emgloyed, 2,3,5-thin-O-benzyl-L-xylose dibez~yl dithioacetal, can be praduced by the process described by Secrist, 1II et al. "'The Synthesis and f wo oo~oa8ss PCTlUSS91166~0 Biological Activity of Certain 4'-Thionucleosides, Nucleosides & Nucleotides, 14 (3-5), 675-686 (1995), disclosure of which is incorporated herein by reference.
The process of the present invention provides for a relatively efficacious meEhod far producing signifzcant quantities of the desired compowtd. Prior arc techniques are exuemely complicated and not readily suitable for producing desired amounts of the a .
compounds_ wo oo~o~sss pcr~~99nss~o c~sHn~ ~
OCFI3 En H ~ S8n Lxylo.~ --~- RO~ ~~.----,. H Bn -----s RO HO Ii Hn0 t~OBn 2: R H
1 3: it~Hn 4 s NF3z $n g N r en OAe O~N
$n0 H
6 XsH ? x~H
X~P 7n X~F
~a X
... ,~:, ' BQ O g N
so HO
is X~H
8 X~H 9 10a X=F
8a X-F
1~
6U8STiME SHEET (RULE 26) Wt) QO/U4866 . YCTIUS991i563o The pharmaceutically acceptable effective dosage of the active compound of the present invention to be administered is dependent on the species of the warm-blooded animal (mammal), the body weight, age and individual condition, and on the form of administration.
S The pha~cmaeeutical composition may be oral, parenteral, suppository or other form which delivers the compounds used in the present invention into the bloodstream of a mammal to be treated.
The compounds of the presern invention can be administered by any conventional means available for use in conjunction with pharmaceuticals;
either as 30 individual therapeutic agents or m a combination of therapeutic agents.
They can be administered alone, but generally administered with a pharmaceutical cattier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
The dosage administered will, of course; vary depending upon lanovvn IS factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the age; health and weight of the recipient;
the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; and the effect desired. A daily dosage of active ingredient can be expected to be about 0:001 to y000 milligram (mg) per kilogram (kg) of body 20 weight, with the preferred dose being 0.1 to about 30 mg/kg.
Dosage forms (compositions suitable for administration) typically contain from about 1 mg to abort 100 mg of active ingredient pet unit. In these pharmaceutics! compositions, the active ingredient will ordinarily be present in an amount of about 0.5-95 % by weight based on the total weight of the composition.
s The active ingredient can be administered orally in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage farms, such as elixirs, syrups, and suspensions. it can also be administered parenterally, in sterile liquid dosage forms. The acEive ingredient can also be administered intranasally (nose drops) or by inhalation. Other dosage forms are potentially possible such as administration transdermally, via a patch mechanism or ointment:
Gelatin capsules contain the active ingredient grad powdered carriers, such as lactose, starch, cellulose derivatives, magnesiutn stearate, stearic acid, and the like.
Similar diluents can be used to make compressed tablets. Both tablets and capsules ltl can be manufactured as sustained release products to provide far continuous release of medication over a period of hours. CompresseB tablets can be sugar-gated or film-coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.
Liquid dosage forms for oral administration can contain coloring and 15 flavoring to increase patient acceptance.
In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glyeols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenterai administration preferably contain a water-soluble salt of the active ingredient; suitable stabilizing 2~ ~ agents, and, if necessary, buffer substances. Andoxidi2ing agents such as sodium bisulfate, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium EDTA.
In addition, parenteral solutions can contain preservatives, such as benzalkonium chloride. methyl- or propylgaraben, and chlombutanol.
25 Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences. Mack Publishing Company, a standard reference text in this field.
wo ov~o4ass FC'rluS99ll~s~o Useful pharmaceutical dosage forms' for adminisaation of the compounds according to the present invention can be illustrated as follows:
' Ca es A large number of unit capsules are prepared by fillitlg standard two-piece hard gelatin capsules- each with 1~ mg of powdered active ingredient, 150 nitg of lactose, 50 mg of cellulose, and 6 mg of magnesium stearate.
Soft GeEati~n Ca~,,~~ules A mixture of active ingredient in a digestible oil such as soybean oil, cottonseed oil, or olive oil is prepared and injected by means of a positive I(3 displacement pump into gelatin to form soft gelatin capsules containing I00 imu of the active ingredient., The capsules are washed and dried.
A large number of tablets axe prepared by conventional procedures so that the dosage unit was 100 mg of active ingredient, 0.2 mg of eolloidal silicon dioxide, 5 mg of magnesium stearate, 275 mg of microcryscalline cellulose, I 1 mg of starch, and 98_8 mg of lactose. Apgropriate coatirsgs may be applied to increase palatability or delay absorption.
'~Tarious modif:cations of the invention in additaon to those shown and described herezrr will be apparent to thase skilled in the art from the foregoing 2U description. Such modifications are alSO intended to fall within the scope of the appended claims.
WO 0010486d PCTJUS99116630 Tlte foregoing disclosure includes all the information deemed essential to enable those Skilled in the arc to practice the claimed invention_ Hecause the cited applications may provide further useful infarmatian, these cited materials are hereby incorporated by reference in their entirery.
The following non-lirtiitiag examples are presented to further illustrate the present invention.
WO ~O1o4866 PCTIUS991I663o Txample 1 Preparation of 1-(4-Thic~-~-n-Arabinofaranosyi) Cytosine Z,3,a-Trl-O-benzyl-t~-xylose DibenzYl Dithioaeetal (4). L-Xylose (x; 25g, 16'7 inmol) was stirred for 5 hours in 0.3°lo hydrogen chloride in methanol (675 mL) at room temperature and then neutralised with Amberlite IRA-400 OH anion exchange resin.
Tide filtrate and washings were combined and evaporated to dryness and W a crude product was purified by silica gel chromatography (CHC13I MeOH, 92:8) to afford 26.2 g of methyl L-xylofuranoside (2, 9S% yield) as an a and ~3 ( 1:1 ) mixture. MS
I 64 , (M)-. 1 GS (M+H)+, 133 (M-OCH3)".
To an ice-cold solution of 2 (14 g, 60.9 mmol) in dry tetrahydrofuran (350 rnL) was added sodium hydride (60% dispersion in mineral oil, 14.8 g, 370 mmol) and the reaction mixture was stirred for 15 min under N~. To this reaction mixtwre was added solid tetrabutylammonium iodide (0.36 g, 0.9& mmol) followed by a dropwise addition of bemyl bromide (36.6 g; 214 mrnol). The reaction mixture was stirred for 3 days at roo~o zernperature. After the addition of methanol (25 mL) the solution was evaporated.
m3der reduced pressure, and the crude product was purified by silica. gel chromatography (cyclolaexanelEtOAc, 9:1 ) to afFord pure methyl 2,3,5-tri-O-benzyl-L-xylofuxanoside (3, 2. g. 87% yield). MS 435 (1V1+H)'', 433 (M-H)+, 443 (M-OCH;)'; 'H NMIt (CD~CI;) b 7.38-7.25 (m, 30H, aromatic H--s), 4.94 (d, JH, H-la, J,~ - 4.3 Hz), 4.87 (d. IH, H-1 Vii. ,1 ~y = 0.9 Hz), 4.d4-4.45 (m, I2H, PhCH2 s), 4.37 (m. IH, H-4a.), 4..27 (dt. 1 H. H-4(3, J,,.SO = 3,7 Hz, Ja,~b = 6.5 Hz, J34 = 6.2-Hz), 4.17 (t. 1H~, H-3a, J3.~
= 6:9 Hz. J.,., =
5.6 Hz), 4.a7 (dd, 1 H, H-3~, J3,a IS
suesrm~rE sHE~r t~uu~ ash = wo aa~aasss PCTNS9911b630 = 6.2 Hz, 3,,3 - ?.5 Hz), 4.00 (dd, 1H, H-2a, J~,3 ~ 5-6 Hz?, 3.95 (t, 1H. H-2(3, J.,.3 =2.5 Hz), 3.70 (dd, IH, H-5aa, J4,jp ~ 4.5 Hz, Jse,55 = 10.4 Hz), 3.66 (dd.
1H. H-Sa(3,~, J,~,~~ = 3.7 Hz, Js~~, =10.T Hz), 3.54 {;dd, IH, H-Sba, J~.S~ = 7.5 Hz), 3.49 (dd, 1H. h1-R
Sb(3_ Ja.sh = 6.5 Hz).
To a solution of 3 (42 g, 97 mrnol) in dichlorometfiane (1000 mL) were added L~enzyl inercaptan (49.6 g, 400 xnrr3ol) and stannic chloride (4.93 g, 18_9 rnrnol), and the reaction mixture was stirred at room temperature ouerni~ht. After neutralization with ~% aqueous NaHCO~ (750mL), the organic layer was separated and the aqueous layer was exn~acted with dichloromethane (500 mL). The combined organic layers were . evaporated, and crude 4 was purified by silica gel chromatography (cyclohexauelEtpAc, 99:1 ) to afford 4 (8.53 g, 57%) of sufficient purity tv carry forward. MS 657 (M-t-t,i)'';
'H NlvIR (CDC13) 8 7.35-7.29 {m, 19H, aromatic H=s), 7.19-7_13 (m, 4H_ aromatic H=s). 7.01-6.96 (m, 2H azomatic I~'=s), 4.86.(d, 1H, PhCHX. J = 11.1 Hz), 4.'70 (huo overlapping d=s, 2H, PhCHH, PhCHH, J = I 1.1 Hz, J = 1 I.2 ITz), 4.43 (d, 1H.
PhC~'Fi, IS 11.2 Hz), 4.40 (d, 1H, PhCHH, 3 = I1.9 Hz), 4.36 (d, 1H, PhCHH, x = r L.9 Hz), 4.0f (dd, 1H. H-2, 3~ , = 3.0 Hz, 3~,~ = ?.5 Hz), 3.75-3.67 (m, 4H, two PhC~T==s), 3.68 (d, 1H. H-i. 3,~ ~ 3.0 Hz), 3_36-3.25 (m, 2~i, H-4, H-Sa), 3.15-.3.12 (m, IH, H-Sb), 2,22 (d. 1 H. 4~OH; ~ = 6.2 Hz).
W
S1188Tfft,JT~ SHEEP (RULE 26) WO 00!0d855 PCT/US99115630 2,3,5-Tri-O-benz~-1-1-~-acetyl-4-thin-n-arabinofuranose (6). To a solution of 4 (13.0 ~.. 20 rnmol) in dry 2:1 tolucnelacetonitriie {200 mL) were added triphenylphosphine (15.7 g. GO ramoi), iodine (12.? g, 50 mmol) and imidazole (5.44 g, 80 rnmol).
The ' reacuon mixture ~~-as stirred at 90 1C for 24 ti after which time the solution zvas S evaporated to di~mess. The crude product was purified by silica geI
chromatography (cyclohexane/EtOA.c. 4:1) to afford benzyl 2,;,5-tri-O-benzyl-t,4-ditJnio-D-arabinofuranoside as a syrup (5, 9.0 g, 83°/a). MS 543 (M+Hf; 'H NMR
(CDCI,) ~
7.40-7.~0 (1n, 20H. aromatic H=s), 4.69-4.42 (m, 6H, three PhCH=O=s), 4.37 (m, 1H, H-1 ): 4.?0 (m, 2H. H-2, H-3), 3.87 (s, 2H, PhCH3S-), 3.80 (dd, 1 H, H-Sa. 3a 5, = 7.4 Hz. JS,~~~, = 9.3 .Hz). 3.55 (dd, IH, H-Sb, Jd.~, = 7.i Hz), 3.47 (m, 1H, H-4). Anal.
(C~aH=~O,Sz ~ 0.?5 1-1~0) C, H.
Td a suspension of mercuric acetate (x.29 g, 22.9 mmol) in acetic acid (96 g) was added 5 (5_4? g, 10 mmol), and the resulting mixture w$s stirred at roonri temperature for 21Z. The reaction mixture Was diluted with dichloromethane (200 mL) 1$ and washed successively with water, saturated aqueous NaHC03 and 5% aqueous KGN -soltttion. The organic layer was dried over Na2SOQ and concentrated.
Chromatography o f the crude product using cyclohexaue:ethylacetate (98:2) as etuent gave a mixture of a and (3 ( 1:1 ) anon~ers of 6 (3.73 g, 78°/a) as a colorless syrup. MS
479 (M+H)''; ~ H
NMR (CDCI;) S 7.35-?.23 (m, 1SH, aromatic H=s), 6.07 (d; 0.25H, H-1(3, 3,, =
4:0 Hz), 5.98 (d, 0.75H. H-1 ce. Js~ --- 2.S Hz), 4.83-4.45 (ra, 6H, PhCH,'s), 4.26 (dd, 0.?5H, .
H-2a. J,., -- 5.4 Hz). 4.17-4.1 I (m, O.SH, H-Z(3, H-3(3), 4.03 {t; 0.75H, H
tea. J;.~ = 6 Hz). 3.80-3.67 (m. 1.25H, H-4a, H-5aa, H-Sa[3), 3.53 x_39 (m, I.75.H, H-Sbcx.
H-4(i, H-Sb(i), ?.0G (s, 3H. CI33-a and CH3-Vii). Anal. (Cz8H3aO5S ~ Q.75 H=O) C. H.
SUBS'CITUTE_SHEEC (RULE 26) WU 00/04866 ~ 02464681 2004-04-07 i~C?IUS~9/16630 I-(2,3,5-Tri-O-benzyl-4-thio-~x,~~n-arabinofuranosyl) cytosine ('7). To a suspension of 1-n-acetyl 2.3.5-tri-C7-benzyl-4-thio-n-arabinofuranose (478 mg, 1 mmol) and cytosine ( I 11.0 m~, 1 nnrial) in anhydrosis acetonitrile (25 rr~mol) were added consecutively hexamethyld'iszla2ane (HMDS.162rng,1 tnmoi) and chlorotrirnethylsilane a (TMSCI, 4;4 mg, 4 mmol), and the mixture was stirred at room temperature for Q.5 , hours. This solution was cooled to -78 ° C.
Trimethylsilyltrifluoromethane sulfonate (267 nag, I .2 mmol) was added and the resulting solution was stirred at -78 ° C for another 2.5 h, after which time the reaction was essentially complete. The mixture was warmed to room temperature, concentrated to a small volume (5 mL), diluted v~rith methylene chloride (50 mL) and then washed with water (20 rnL) followed by saturated sodium bicarbonate and water. The organic layer was dried over MgSO:, and evaporated to dryness. The residue was purified by chromatography over silica gel (SO
a. elution with CHCI3/MEOH 98:2) to afford 7 (4I2 mg 77.5%) as a coloriess syrup TLC (95:5 CHC13IME(.7lEi) R,t. 0.65; MS zle 536 (M+Li)t.
'H NIV1TZ (CDCI3) ~ $.22 (d, 1, H-dp, 3 ° 7.~6); (d; 1, H-6a; J5,6 =
7.5 I32); x.38-?.09 (m, 30, aromatic H's); 5.65 (d, 1, H-1'~, 3~.,~. = 5.7 I3.z); 6.36 (d, 1, H-I'a, Jt.,. _ 1_2 Hz);
5.44 (d. 1. H-SQ); 5.26 (d, 1, H-Sp); 4.97-4.33 (averlapgirtg multiplets, 12;
CaH3CH,), 4.26 (dd. I H-2'p, J,..s- = G.S Hz); 4.22 (m, 1, H-3'~, J3~.4. = 1 Hz); 4.1b (dd; 1, H-3'~, J;..,. = 6.4 I-iz); 4.13 (m, I. H-Z'a, J2._3. = 1.7 Hz); 3.91 (m, 1_ ~I-4'~);
3.78 (m, 1. H
5~.,); 3.73-3.63 (m. 2. H-S'e); 3.55 (tn, I, H-5'a); 3.46 (in, 1. H-4'~.
SUBS'TIfliT6 SH~61" (RLSt~ 26) WO 00104866 PC'TItJS99116b30 I-(4-'JChio-a, ~-u-arabiu~oturanosyl) cytosine (8). To a solution of boron irichloride ( 1 M solution) in dry dichloromethane (7mL, 7mmo1) cooled to -78 ~ C, -was added dropwise over a period of 30 min, a solution of compound 7 (26S mg, 0.5 mmol) in clxy diclUoromethane (10 mL). Stinting was continued overnight at -20 ~C. The solvent was removed in vacuo and the residue was coevagorated with dichloromethane (4 x ?0 mL). The residue was neutralized with saturated NaHCOs (25 tnL) and washed with chloraforn~ (IS mL). The aqueous iayer'was applied to a canon exchange (H') column and eluted with water to remove salts and tlxen eluted with 1N NHdOH to obtain the desired compound 8 (11U rng, 85°~°0) MS z!e 260(M+I~i)".
~H NMR (Me-,SO-ab) 8 7.9~ (d, .67, H-6-ø, J5.6 = 7.5 Hz); 7.9a (d. 4.33, H-6a, JS_6 --7.5 Hz); 7.17-7.03 (overlapping bs, 2, NH's); 6.33 (d, 4.67, H-i' ø, d, :,. =
4.6 Hz); 5.86 (d. 0.33, ~-I-1'oc, J,.,- = 7.3 Hz); 5.77 (d, 0.33, H-Stx): 5.70 (d, 0.67, H-5 j3): 5.61 arid 5.59 (overlapping doubiets, 1, 2'-OHø, J.,.~.,~ = 5.1 Hz, 2'-OHa. J,.,-_oH =
5.9 Hz);
5.47 (d, 0.33, 3'-OHa, J3. ~,~H = 5.1 Hz); 5.38 (d, 0.67. 3'-fJHø, J 3'.3'-(3H
= 4.2 Hz);
5.08 (t. 0.67. 5'-f)H~3; Jg~_S'.OH ~ 5.4 Hz); 4,90 (t, 0.33, S"-UHa, JS~,~,_oH
~ 5:2 Hz); 4.00-3.93 {m. 1.&7, H-2'Q, H-2's, H-3'~; 3.$6-3.76 (tn, l, IH S'« and H-5'p~; 3.67-3.55 (xn;
1. H-5' ~, + H-3' «); 3.49-3 .3 3 (m, 0.67; H ~' «, H-5' «); 3 . L7 (m. 0.67, H-4' ~).
SU86'i'ITUi"E SHEET (RULE 26~
WO OO/Od866 ~ 02464681 2004-04-07 pCTNS99/16630 :y 1-(S-D-dimethoxytrityl-4-than-Q-n-strabinofuranosyl) cytosine (9). To a solution of compound 8 (I00 mg, 0.38 mmoi) in dry pyridine (10 mL) was added 4,4'-~
Dimeth.oxytritylchloride (135 mg, 0.6 mtnol) and the solution was stirred for 2 It at room temperatuze_ The reaction mixture was evaporated to dryness and the crude visas dissolved in ethyl acetate (Z0 mL) and washed with water and evaporated to dryness to afford a solid which was purified on silica geI column (CHCI3/MeOH 98:2) to obtain pure compound 9 (96 mg, 90% based on 1:1 mixhire of oc, (3-mixture of 8). MS
~Je SG8.3 (M+Li)':
'H NMR (Me,SO-db) S 7.77 (d, 1, H-6, Jss ~ 7.5 Hz); 7.42-?.23 (m, 9, aromatic H's);
?.17 (bs, I, NH); 7,05 (bs, I; NH); 6.91-6.88 (rn, 4, aromatic H's); 6.36 (d, 1, H-1', .1,. ,. = 4.8 Ha); 5.65 (d, I, H-S); 5.76 (d, 1, 2'-OH, J2.,i..os, = 4.6 Hz);
5.43 (d, 1, 3'-OH, ~Ta-.:--cm ' 3.3 Hz); 3.98-3:9I (m, Z, H-2'; H-3'); 3.75 (s, G. OCIri;); 3.39-3.25 (m, B, H' 4'. H-~').
~EUBSTtTU'tE SHEEP (RULE 2~
1~V0 ~0/04866 ~ 02464681 2004-04-07 1-(4-Thio-(3-u-arabinofuranospl) cytosine (I4). Compound 9 (90 mg, 0.16 mmol) was treated with trifluoroaGetic acid (22 mg) in chlorafaxm (5 mL) at roam temperature foz I O min. The reaction mixture was neutralized with aq. NaHC03 and the aqueaits layer was applied on a catiou exchange column which was eluted first with water to remove salt and finally with IN NHdOH to afford compound 10 (35 mg, 85°/a), rnp218-Z20 °C
( li t' 3? 1 _322 °C); MS zle 26Q(M+H)+.
'H NMR (Me,SO-d6) 6 7.94 (d, 1, H-6, 3~.6= 7.5 Hz); ?.I2 (bs, 1, NH); ?.U4 (6s, 1.;
NHl'. _6.33 (d, 1. H-1', J' :,' = 4.6 H~); 5.70 (d, 1, H-5); 5.61 (bd, 1, 2'-OH, .T,.,,.ou =
3.1 Hz); 5.38 (6d. 1, 3'-OH, J3.,3,.cH '- 3:5 Hz); S.Q8 (bt, 1, 5'-OH, JS.,s..oH ~ 4.9 Hz);
4.U0-3.93 (m, 2, H-2', H-3'); 3.78 (m, I, H-S'a); 3.6I (m, 1, H-5'b); 3.16 (m, 'l, H-4').
Example 2 Preparatioct of 1-(4-Thin-t3-D-Arabinofnranosyl)~.FluoroCytosine 1-(2,3,5-Tri-D-benzyl-4-thio-a,tl-~?-arabiuofnranosyl)~-flunrocytosine ('7a).
To a suspension of 1-C?-acetyl 2,3,5-tri-O-benzyl-4-thio-D-arabinofmranose (478 m.g,1 ~nmol) and cytosine ( 129.0 mg, 1 mmol) in anhydrosis acetouitrile (25 mmol) were added consecutively I~zexamethyldisilazane (HIVIDS,162 mg, l mmol) and chlorotrimethylsilane (TMSCI. 434 mg. 4 mmol), and the mixture was stirred at room temperature for 0.5 llour5. Tlois solution was cooled to -78°C.
Trirnethylsilyltrifluoromethane sulfonate (2b'l mg, 1.2 mmol) was added and the resulting solution was stirred at -7&°C for 2fl another 2.5 h, after which time the reaction was essentially. complete.
The mixture was warnaed to room temperature, concentrated to a small volume (5 mL), diluted with ZI
~UBS~IME SHEET (RUCHE 26) .
WO 00I0~866 PCTJUS99/16630 tnethyleue clzlor~ide (SO mL) and then washed with water (20 mL) followed by~
saturated sodium bicarbonate and water. The organic layer was dried over. MgSO., and evaporated to dryness, The residue was purified by chromatography over silica gel (SO
g. elution with CHCI~IMEOH 98:2) to afford 7 as 2:1 oe.Ii mixture (425.2 m~
80.0%) as a colorless syntp TLC (95:5 CHCI3fM,EOH R,. 0.65; MS 7le S53 (IvI+Li)'.
'I-i NMR {CDCI3} 8 8.40 (d, I, H-6a,J=7.6); 8.10 (d, 1, H-6a, Jt,b ~ 7.5 Hz};
7..35-7.09 -(n3, 30. aromatic H's); 6.55 (d, 1, H-1'a, J, :~- = 5.7 Hz); 6.25 (d, 1, I~-1'n, 31. ,. = 1.2 ' Hz), 4.90 (d, 1. II-S~); 4.~8 (d, l, H-Sa}; 4.30-4.55 (overlapping multiplets.
12, C,~H;CHI), 4.15 (dd, 1 H-2'a, Ja~,~~ = 6.8 Hz); 4.i0 (m, 1. H-3'Q, 33,.x. = 'l Hz): 3.90 (dd, l, H ~'~,. J3~.,,- = 6.4 Hz); 3.75 (m, 1, H-2'a, Jz.w = 1.7 Hz); 3.70 (m, 1. H-4'~; 3.65 (m, I. H-5'~a): 3.50-3.55 (m,2,H-5'a); 3.45 (m, I, H-5'a}; 3.4D (m. 1, H-4'~, Z-(4-Trio-a-D-arabinofuranosyl)5-ifuorvrytosine (loa). To a solution of boron trichioride ( 1 M solution} in dry dichlorometbane (7 mL. 7 anmol} cooled to -78°C. ~~as added dropwise over a period of 30 min, a solution of corapound 7 (2273 mg.
4.5 IS mmol) in dry dichloromethane (10 mL).~ Stirring was continued overnight at -20°C:
tl3e solvent was removed in vacuo and the residue was coevaporated with dichloramethane (4 x 20 mL). The residue was neutralized with saturated NaHC03 (25 nL) and washed with chlorafomm (15 mL). The aqueous layer was applied to a cation exchange (H') column and eluted with ,hater to remove salts and then eluted with 1N
NH~OH to obtain the desired compound Sa as ?:l a.B mixture which upon crystallization with water gave pure IOa (32.2 mg, 25%) MS zle 278(M+H)', 'H NMR (Mt~SO-db) 8 8:15 (d, 1, H-6, Js.e = 7.5 Hz); 7.75 (bs. I, NH); 7.50 (bs. 1, NH); G ~5 (d, I, SUBSTCtUfE SHEEt (RU4E 2.6~
wo ooioa866 PCTlU59911~630 H-1', 1~ :,v= 4.t~ Hz); 5.65 (d, 1, H S); 5.60 (bd, 1,2'-OH. J=.,~-~H = 3.1 Hz); 5.X0 (bd., 1.3'-OH: J~ ,3'-OM = 3.5 Hz); 5.30 (bt, 1, 5'-OH, 3S..S..o~ = 4.9 Hz); 4.00-3.90 (in. 2. H-?', ~ .
H-3'); 3.75 (m, 1, H-S'n); 3.65 (m, l, H-S'~); 3.1S (m, I, H-4').
' Ototani, H.; Whistler, R.L. Preparation and Antitumo'~r Activity of 4'-Thio Analogs 0~' ?.?'-Anhydro-1-l3-17-arabinofuranosylcytosme. J. Med. Chem. 1974, 17, 535-537.
SUB~~ SHED' (R!!L~ ~6, WO 00!04866 ~ 02464681 2004-04-07 PCTlUS99/I6630 Example 3 The following in vitro tests were carried out.
Table 1 iCSfl Molar Values - ' ThioaraC 5-fluorothiaaraC~'ts~e arabinc~side LOX IMV 1 Melanoma 7 x 10'6 6 x 10'6 1 x 10-6 PC-3 Prostate $ x 10'6 1 x 10''~ S x IO'~
ZR-75-I Mammary 3 x 10'6 6 x 10'' 4 x 10' CAKI-I Renal 3 x 10'~ 2 x IO's 6 x 1U-' DLD-1 Colon 7 x 106 . 8 x 10's 5 x I0'~
ID ~ NC1-H23 Lung 5 x 10'G ' 3 x 10-6 3 x IO'' , SK-MEL 28 Melanoma 7 x 10'6 - 2 x IO'6 S1~TB-7 (CNS) 1 x IO'a ~t 2 x 10' The above tests were carried out with a 72 hour exposure to the compound using Natural Red assay for all cell lines except SK-MEL 28 which was done with SRB assay.
WU 00!04866 . PC'1'1US99~116630 Example 4 The fallowing in viva tests were carried out to illustrate the effectiveness of ' the compounds of the present invention.
\7'/~ ~(?~p4~~ CA 02464681 2004-04-07 H
~$f.
m O
m ,n. v ,a a m ' G
o i.
.~
m ..
mm n.
DESCRIPTION
a Federally Sponsored Research and Development This invention was supported by Grant CA-34200 from National Institutes of Dealth_ Technical Field The present invention is concerned with treating patients suffering horn cancer by administering to the patients cereaiti thioarabinofuranosyl compounds.
Compounds employed according to the present invention have exhibited good anticancer activity. Compounds employed accordix~ to the present invention are in the beta configuration as cozitrasted to the alpha configuration, which failed to exhibit anticancer activity. The. present invention also relates to inhibiting DNA
replication in a rnaaunalian cell by contacting with the thioarabinofuranosyl compounds. The present invention also relates to a nevv process for preparing the compounds employed according to the present invention.
Background of Invention Past amounts of research have accrued over the years related to developing treatments against cancers to inhibit and kill tumor cells. Some of this research has ~"0 resulted in achieving some success in finding clinically approved treauaents:
Nevertheless, efforts continue at an ever-increasing rate in view of the extreme difficulty in uncovering promising anticancer treatments. For example, even when VSO flfl/fl4S66 ' PCTIUS99116630 a compound is found to have cytotaxic activity, there is no predictability of it being selective against cancer cells.
One particular compound that has been used rather widespread is cytosine arablnoside, comfnonly referred to as .lira-C.
S ~ Sununary of Invention It has been found according to the present invention that certain thioarabinofuranosyl cytosine compounds are suitable as anticancer agents. The presence of the thiv sugar moiety surprisingly makes it possible to achieve good antitumor activity. More particularly, the present invention relates to treating a mammalian host in need of an anticancer treatment by administering to the host an effective anticancez amount of at lease one compound represented by the following formula 1_ RO ~ A
wherein each R individually is H ox an aliphatic or aromatic aryl group;
A is selected from the group consisting of NHa N / ~
Z
O
n WC7 00/L148Gt FCTIU$g9116b3a N N
o~N
> ~
X is selected from the group consisting of hydrogen, flaoto, alkoxy, alkyl, haloalkyl> alkenyl, haloalkenyl, aIkynyl, amino, monoalkylamina, dialkyiamino, cyano az~d nitro.
rt has also been found according to the present invention that the above-disclosed compounds of formula x can be used to inhibit DIVA replication in a IfJ manunalian cell by contacting the cell with at least one of these compounds.
The present invention is also concerned with a pzocess for preparing the above-identified compounds. The compounds employed according eo the present invention can be prepared by:
A) reacting a 2,3,5-tri-O-aryl or alkyl-4-xylose diaryl or dialkyl dithioacetal such as 2,3,5-tri-O-benzyl-L-xylose-dibenzyl dithioacetal in the presence of a leaving group at the 4 hydroxyl to produce the corresponding 1,4-dithio-D-arabinofuranoside such as benzyl 2,3,5-tri-O-benzyl-1,4-dithio-D-arabinofuranoside;
B) subjecting the product from step A) to acidolysis to form the corresponding. 0-acetyl-4-thio-D arabinofuranose such as 2,3,5-tri-O-benzyl-1-O-acetyl-4-thio-D-arabinofuranose;
C) reacting the product of step. B) with a cytosine, a 5- or 6-aza compound or a suitably blocked derivative thereof forming a corresponding 4-thin-a,13-D-arabinofuranosyl compound such as, in the case of cytosine, 1-(2,3,5-tri-O-benzyl-4-thin-a,13-D-arabinofuranosyl) cytosine;
D) converting the compound of step C) by hydrolysis to the corresponding thin sugar derivative such as 1-(4-thio-a,13-D-arabinofuranosyl) cytosine;
E) separating out the a form of the anomeric mixture of step D) to thereby obtain the desired compound of formula l, such as 1-(4-thio-13-D-arabonifuranosyl) cytosine.
In a broad aspect, then, the present invention relates to the compound represented by the formula 1:
RO A
S
RO
RO (1) wherein each R individually is H, an aliphatic acyl group or an aromatic acyl group;
NHZ
N
O N
NHz N~ N
ON
NHZ
N NH
O N
NHZ
X
/N
O N
wherein X is hydrogen, fluorine, alkoxy, alkyl, haloalkyl, alkenyl, haloalkenyl, alkynyl, amino, monoalkylamino, dialkylamino, cyano or vitro, for treating a solid. tumor in a mammalian host.
4a In another broad aspect; then, the present invention relates to the compound 1-(4-thio-13-D-arabinofuranosyl) cytosine for treating a mammalian host suffering from a cancer selected from the group consisting of melanoma, prostate cancer, mammary cancer, renal cancer, colon cancer and lung cancer.
In yet another broad aspect, then, the present invention relates to use of a compound represented by the formula 1:
RO A
S
RO
RO
wherein each R individually is H, an aliphatic acyl group or an aromatic acyl group;
A is x NFi N N
p N
4b N NH
O N
or X
N
~I IN
O N
wherein X is hydrogen, fluorine, alkoxy, alkyl, haloalkyl, alkenyl, haloalkenyl, alkynyl, amino, monoalkylamino, dialkylariiino, cyano or nitro, for treating a solid tumor in a mammalian host.
In a further broad aspect, then, the present invention relates to use of the compound represented by the formula 1:
RO A
S
RO
RO'~ ~l~
wherein each R individually is H, an aliphatic acyl group or an aromatic acyl group;
4c !~ 1S
NHZ
X
N
O N
NHZ
N N
J
O N
NHp .
N ~ NH
O N
NHz X
N
~N
O N
wherein X is hydrogen, fluorine, alkoxy, alkyl, haloalkyl, alkenyl, haloalkenyl, alkynyl, amino, monoalkylamino, dialkylamino, cyano or nitro, for treating a mammalian host suffering from a cancer selected from the group of melanoma, prostate cancer, mammary cancer, renal cancer, colon cancer or lung cancer.
4d In a still further broad aspect, then, the present invention relates to use of the compound represented by the formula 1:
RO A
S
RO
RO (1) wherein each R individually is H, an aliphatic acyl group or an aromatic acyl group;
A is X
N
i O N
NHp N~ N
~J
O N
J
NHZ
N NH
O N
or 4e NHZ
N
~N
O N
wherein ~ is hydrogen, fluorine, alkoxy, alkyl; haloalkyl, alkenyl, haloalkenyl, alkynyl, amino, monoalkylamino, dialkylamino, cyano or nitro for inhibiting DNA
replication in a mammalian cell.
In another broad aspect, then, the present invention relates to use of I-(4-thin-13-D-arabinofuranosyl) cytosine for treating a mammalian host suffering from a cancer selected from melanoma, prostate cancer, mammary cancer, renal cancer, colon cancer or lung cancer.
4f Summary of Drawings .
Figs. la and 1b are graphs showing metabolism of 2-devxycytidiae, 2'-deoxythiocytidine, ara c and thio ara-c to their respective triphosphates.
Fig. 2 is a graph showing the retention of ara c tiiphosphate and thin ara c triphosphate in CEM cells.
Best and Various Modes for Carrying ~ut Invention The present invention is related to treating a mammalian host in need of an anticancer ra~eacmenc, which comprises administering to the host an effective anticalzcer amount of at .least compound represented by the formula 1:
RO ~ A .
1.o 0 g Each R in formula 1 individually is preferably H or an aliphatic or aromatic acyl Eroup. Typical aliphatic acyl groups contain from 1 to 6 carbon acorns and include formyl, acetyl, and propionyl. Typical aromatic acyl groups include unsubstituted and alkyl substituted aromatic ,groups containdng 7-10 carbon atoms in.
1$ the arc~mauc group. When substituted, tl3e alkyl group typically contains 1-6 carbon atoms. Typical aromat5c acyl groups include benzoyl and para-tol.oyI.
A in formula l is preferably WD 00104866 ~ 02464681 2004-04-07 PCTlLyS99f 16630 N N
O%'N/
NHa N! 'NH
and .
o a o~N~
N
Suitable monoalkylamino groups for X ~contsin 1-6 carbon atoms and intclude monomethylamino, monoethylamino, mono-isopropylamino, mono-n-propylamino;
mono-isoburyl-amino, mono-n-butylamino and mono-n-hexylamino. The all~yrl moiety can .be stzaight or branched chain.
Suitable dialkylamino groups for Y and X contain 1-6 carbon atoms in each Itl alkyl ~roup. The alkyl gzoups can be the same or different and can be straight or branched chain. Examples of some suitable gmups are dimethyIamino, diethylamino, ethylmethylamino, dipropylamina; dibutylamino, dipentylamino, dihexylamino, methylpentylamino, ethylpropylamino and ethylhexylamino.
Suitable halogen groups for X include GI, Br and F.
Suitable alkyl groups far X typically contain 1--6 carbon atoms and can be straight or branched chain. Some examples are methyl, ethyl, i-propyl, n-propyl, i-butyl, n-butyl, pentyl and hexyl_ Suitable haloalkyl groups typically contain I-6 carbon atoms and can be straight Qr branched chain and include Cl, Br ar >substituted alkyl groups including LQ the above specifically disclosed alkyl groups.
5uitabie alkoxy groups typically contain 1-6 carbon atoms and include rnerhaxy, ethoxy, pxopoxy and butoxy, Suitable alkenyl groups typically contain 2-6 carbon atoms and include ethenyl and propenyl_ Suitable halaalkenyl groups typically contain 1-6 carbon atoms and include Cl.. Br or F substituted alltenyl groups including the above specifically disclosed alkenyl groups.
Suitable alkynyl groups typically contain 1-6 carbon atoms and include ethynyl and propynyt. , 24 The preferred compound employed according to the process of the present invention is 1-(4-thin-!3-D-arabinofuranosyl} cytosine.
WO 00104866 pCTlUS991t6630 The present invention is suitable for treating mammalian hosts including humans suffering from cancer including melanoma, prostate cancer, mammary cancer, renal cancer, colon cancer, sung cancer, lettkemias and lymphomas.
The compounds employed according to the present invention can be prepared by reacting a 2,3,5-tri-O-aryl or alkyl-4-xylose diaryl or dialkyl dithioacetal such as ~.3.~-tri-O-benzyl-I:-xylose-dibenzyl dithioacetal in the presence of a bearing gmup at the 4 hydroxyl position to produce the corresponding 1,4-dithio-D-arabinofuranoside such as Isenzyl 2,3,5-tri-O benzyl-l,4rdithio-D-arabinofuranoside.
This step can he carried aut using phosphine, iodine and imida2ole. The product from the above step is subjected eo acidolysis to form the corresponding 4-acetyl-4 thin-D arabinofuranose such as 2,3,5-tri-O-benzyl-1-~-acetyl-4-chio-D
arabinofuranose. For instance, acetic acid in the presence of rncrcuric acetate can be employed.
The product of the above step is reacted, with a cytosine, a 5- ar 6-aza 1.5 compound or a suitably blocked derivative thereof forming a corresponding 4-thio-«,13-D-arabinofuranosyl compound such as, in the case of cytosine, 1-(2,3;5-tri-O-benzyl-4-thio-cz,t3-D-arabinofuranosyl) cytosine. Suitable blocked derivatives include acyl and tximethylsilylated derivatives, The compound of the above step is converted by hydrolysis to the coaesponding thio sugar derivative such as 1-(4-thio-a,li-D-arabinofuranasyl) cytosine.
The cx form of the anomeric mixture of the above step is separated out to thereby obtain the desired compound of formula l, such as 1-(~i-thio-!3-D-arabinofuranosyl) cytosine.
Compounds according to the present invention can be prepared by the 2a process sequence shoawn in scheme I and examples 1 and 2 described hereinbelour, wlaerein the preferred compound, 1.-(4-thio~B-D-arabinofuranosyI) cytosine is illustrated for purposes of facilitating an understanding of the process sequence.
The precursor emgloyed, 2,3,5-thin-O-benzyl-L-xylose dibez~yl dithioacetal, can be praduced by the process described by Secrist, 1II et al. "'The Synthesis and f wo oo~oa8ss PCTlUSS91166~0 Biological Activity of Certain 4'-Thionucleosides, Nucleosides & Nucleotides, 14 (3-5), 675-686 (1995), disclosure of which is incorporated herein by reference.
The process of the present invention provides for a relatively efficacious meEhod far producing signifzcant quantities of the desired compowtd. Prior arc techniques are exuemely complicated and not readily suitable for producing desired amounts of the a .
compounds_ wo oo~o~sss pcr~~99nss~o c~sHn~ ~
OCFI3 En H ~ S8n Lxylo.~ --~- RO~ ~~.----,. H Bn -----s RO HO Ii Hn0 t~OBn 2: R H
1 3: it~Hn 4 s NF3z $n g N r en OAe O~N
$n0 H
6 XsH ? x~H
X~P 7n X~F
~a X
... ,~:, ' BQ O g N
so HO
is X~H
8 X~H 9 10a X=F
8a X-F
1~
6U8STiME SHEET (RULE 26) Wt) QO/U4866 . YCTIUS991i563o The pharmaceutically acceptable effective dosage of the active compound of the present invention to be administered is dependent on the species of the warm-blooded animal (mammal), the body weight, age and individual condition, and on the form of administration.
S The pha~cmaeeutical composition may be oral, parenteral, suppository or other form which delivers the compounds used in the present invention into the bloodstream of a mammal to be treated.
The compounds of the presern invention can be administered by any conventional means available for use in conjunction with pharmaceuticals;
either as 30 individual therapeutic agents or m a combination of therapeutic agents.
They can be administered alone, but generally administered with a pharmaceutical cattier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
The dosage administered will, of course; vary depending upon lanovvn IS factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the age; health and weight of the recipient;
the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; and the effect desired. A daily dosage of active ingredient can be expected to be about 0:001 to y000 milligram (mg) per kilogram (kg) of body 20 weight, with the preferred dose being 0.1 to about 30 mg/kg.
Dosage forms (compositions suitable for administration) typically contain from about 1 mg to abort 100 mg of active ingredient pet unit. In these pharmaceutics! compositions, the active ingredient will ordinarily be present in an amount of about 0.5-95 % by weight based on the total weight of the composition.
s The active ingredient can be administered orally in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage farms, such as elixirs, syrups, and suspensions. it can also be administered parenterally, in sterile liquid dosage forms. The acEive ingredient can also be administered intranasally (nose drops) or by inhalation. Other dosage forms are potentially possible such as administration transdermally, via a patch mechanism or ointment:
Gelatin capsules contain the active ingredient grad powdered carriers, such as lactose, starch, cellulose derivatives, magnesiutn stearate, stearic acid, and the like.
Similar diluents can be used to make compressed tablets. Both tablets and capsules ltl can be manufactured as sustained release products to provide far continuous release of medication over a period of hours. CompresseB tablets can be sugar-gated or film-coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.
Liquid dosage forms for oral administration can contain coloring and 15 flavoring to increase patient acceptance.
In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glyeols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenterai administration preferably contain a water-soluble salt of the active ingredient; suitable stabilizing 2~ ~ agents, and, if necessary, buffer substances. Andoxidi2ing agents such as sodium bisulfate, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium EDTA.
In addition, parenteral solutions can contain preservatives, such as benzalkonium chloride. methyl- or propylgaraben, and chlombutanol.
25 Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences. Mack Publishing Company, a standard reference text in this field.
wo ov~o4ass FC'rluS99ll~s~o Useful pharmaceutical dosage forms' for adminisaation of the compounds according to the present invention can be illustrated as follows:
' Ca es A large number of unit capsules are prepared by fillitlg standard two-piece hard gelatin capsules- each with 1~ mg of powdered active ingredient, 150 nitg of lactose, 50 mg of cellulose, and 6 mg of magnesium stearate.
Soft GeEati~n Ca~,,~~ules A mixture of active ingredient in a digestible oil such as soybean oil, cottonseed oil, or olive oil is prepared and injected by means of a positive I(3 displacement pump into gelatin to form soft gelatin capsules containing I00 imu of the active ingredient., The capsules are washed and dried.
A large number of tablets axe prepared by conventional procedures so that the dosage unit was 100 mg of active ingredient, 0.2 mg of eolloidal silicon dioxide, 5 mg of magnesium stearate, 275 mg of microcryscalline cellulose, I 1 mg of starch, and 98_8 mg of lactose. Apgropriate coatirsgs may be applied to increase palatability or delay absorption.
'~Tarious modif:cations of the invention in additaon to those shown and described herezrr will be apparent to thase skilled in the art from the foregoing 2U description. Such modifications are alSO intended to fall within the scope of the appended claims.
WO 0010486d PCTJUS99116630 Tlte foregoing disclosure includes all the information deemed essential to enable those Skilled in the arc to practice the claimed invention_ Hecause the cited applications may provide further useful infarmatian, these cited materials are hereby incorporated by reference in their entirery.
The following non-lirtiitiag examples are presented to further illustrate the present invention.
WO ~O1o4866 PCTIUS991I663o Txample 1 Preparation of 1-(4-Thic~-~-n-Arabinofaranosyi) Cytosine Z,3,a-Trl-O-benzyl-t~-xylose DibenzYl Dithioaeetal (4). L-Xylose (x; 25g, 16'7 inmol) was stirred for 5 hours in 0.3°lo hydrogen chloride in methanol (675 mL) at room temperature and then neutralised with Amberlite IRA-400 OH anion exchange resin.
Tide filtrate and washings were combined and evaporated to dryness and W a crude product was purified by silica gel chromatography (CHC13I MeOH, 92:8) to afford 26.2 g of methyl L-xylofuranoside (2, 9S% yield) as an a and ~3 ( 1:1 ) mixture. MS
I 64 , (M)-. 1 GS (M+H)+, 133 (M-OCH3)".
To an ice-cold solution of 2 (14 g, 60.9 mmol) in dry tetrahydrofuran (350 rnL) was added sodium hydride (60% dispersion in mineral oil, 14.8 g, 370 mmol) and the reaction mixture was stirred for 15 min under N~. To this reaction mixtwre was added solid tetrabutylammonium iodide (0.36 g, 0.9& mmol) followed by a dropwise addition of bemyl bromide (36.6 g; 214 mrnol). The reaction mixture was stirred for 3 days at roo~o zernperature. After the addition of methanol (25 mL) the solution was evaporated.
m3der reduced pressure, and the crude product was purified by silica. gel chromatography (cyclolaexanelEtOAc, 9:1 ) to afFord pure methyl 2,3,5-tri-O-benzyl-L-xylofuxanoside (3, 2. g. 87% yield). MS 435 (1V1+H)'', 433 (M-H)+, 443 (M-OCH;)'; 'H NMIt (CD~CI;) b 7.38-7.25 (m, 30H, aromatic H--s), 4.94 (d, JH, H-la, J,~ - 4.3 Hz), 4.87 (d. IH, H-1 Vii. ,1 ~y = 0.9 Hz), 4.d4-4.45 (m, I2H, PhCH2 s), 4.37 (m. IH, H-4a.), 4..27 (dt. 1 H. H-4(3, J,,.SO = 3,7 Hz, Ja,~b = 6.5 Hz, J34 = 6.2-Hz), 4.17 (t. 1H~, H-3a, J3.~
= 6:9 Hz. J.,., =
5.6 Hz), 4.a7 (dd, 1 H, H-3~, J3,a IS
suesrm~rE sHE~r t~uu~ ash = wo aa~aasss PCTNS9911b630 = 6.2 Hz, 3,,3 - ?.5 Hz), 4.00 (dd, 1H, H-2a, J~,3 ~ 5-6 Hz?, 3.95 (t, 1H. H-2(3, J.,.3 =2.5 Hz), 3.70 (dd, IH, H-5aa, J4,jp ~ 4.5 Hz, Jse,55 = 10.4 Hz), 3.66 (dd.
1H. H-Sa(3,~, J,~,~~ = 3.7 Hz, Js~~, =10.T Hz), 3.54 {;dd, IH, H-Sba, J~.S~ = 7.5 Hz), 3.49 (dd, 1H. h1-R
Sb(3_ Ja.sh = 6.5 Hz).
To a solution of 3 (42 g, 97 mrnol) in dichlorometfiane (1000 mL) were added L~enzyl inercaptan (49.6 g, 400 xnrr3ol) and stannic chloride (4.93 g, 18_9 rnrnol), and the reaction mixture was stirred at room temperature ouerni~ht. After neutralization with ~% aqueous NaHCO~ (750mL), the organic layer was separated and the aqueous layer was exn~acted with dichloromethane (500 mL). The combined organic layers were . evaporated, and crude 4 was purified by silica gel chromatography (cyclohexauelEtpAc, 99:1 ) to afford 4 (8.53 g, 57%) of sufficient purity tv carry forward. MS 657 (M-t-t,i)'';
'H NlvIR (CDC13) 8 7.35-7.29 {m, 19H, aromatic H=s), 7.19-7_13 (m, 4H_ aromatic H=s). 7.01-6.96 (m, 2H azomatic I~'=s), 4.86.(d, 1H, PhCHX. J = 11.1 Hz), 4.'70 (huo overlapping d=s, 2H, PhCHH, PhCHH, J = I 1.1 Hz, J = 1 I.2 ITz), 4.43 (d, 1H.
PhC~'Fi, IS 11.2 Hz), 4.40 (d, 1H, PhCHH, 3 = I1.9 Hz), 4.36 (d, 1H, PhCHH, x = r L.9 Hz), 4.0f (dd, 1H. H-2, 3~ , = 3.0 Hz, 3~,~ = ?.5 Hz), 3.75-3.67 (m, 4H, two PhC~T==s), 3.68 (d, 1H. H-i. 3,~ ~ 3.0 Hz), 3_36-3.25 (m, 2~i, H-4, H-Sa), 3.15-.3.12 (m, IH, H-Sb), 2,22 (d. 1 H. 4~OH; ~ = 6.2 Hz).
W
S1188Tfft,JT~ SHEEP (RULE 26) WO 00!0d855 PCT/US99115630 2,3,5-Tri-O-benz~-1-1-~-acetyl-4-thin-n-arabinofuranose (6). To a solution of 4 (13.0 ~.. 20 rnmol) in dry 2:1 tolucnelacetonitriie {200 mL) were added triphenylphosphine (15.7 g. GO ramoi), iodine (12.? g, 50 mmol) and imidazole (5.44 g, 80 rnmol).
The ' reacuon mixture ~~-as stirred at 90 1C for 24 ti after which time the solution zvas S evaporated to di~mess. The crude product was purified by silica geI
chromatography (cyclohexane/EtOA.c. 4:1) to afford benzyl 2,;,5-tri-O-benzyl-t,4-ditJnio-D-arabinofuranoside as a syrup (5, 9.0 g, 83°/a). MS 543 (M+Hf; 'H NMR
(CDCI,) ~
7.40-7.~0 (1n, 20H. aromatic H=s), 4.69-4.42 (m, 6H, three PhCH=O=s), 4.37 (m, 1H, H-1 ): 4.?0 (m, 2H. H-2, H-3), 3.87 (s, 2H, PhCH3S-), 3.80 (dd, 1 H, H-Sa. 3a 5, = 7.4 Hz. JS,~~~, = 9.3 .Hz). 3.55 (dd, IH, H-Sb, Jd.~, = 7.i Hz), 3.47 (m, 1H, H-4). Anal.
(C~aH=~O,Sz ~ 0.?5 1-1~0) C, H.
Td a suspension of mercuric acetate (x.29 g, 22.9 mmol) in acetic acid (96 g) was added 5 (5_4? g, 10 mmol), and the resulting mixture w$s stirred at roonri temperature for 21Z. The reaction mixture Was diluted with dichloromethane (200 mL) 1$ and washed successively with water, saturated aqueous NaHC03 and 5% aqueous KGN -soltttion. The organic layer was dried over Na2SOQ and concentrated.
Chromatography o f the crude product using cyclohexaue:ethylacetate (98:2) as etuent gave a mixture of a and (3 ( 1:1 ) anon~ers of 6 (3.73 g, 78°/a) as a colorless syrup. MS
479 (M+H)''; ~ H
NMR (CDCI;) S 7.35-?.23 (m, 1SH, aromatic H=s), 6.07 (d; 0.25H, H-1(3, 3,, =
4:0 Hz), 5.98 (d, 0.75H. H-1 ce. Js~ --- 2.S Hz), 4.83-4.45 (ra, 6H, PhCH,'s), 4.26 (dd, 0.?5H, .
H-2a. J,., -- 5.4 Hz). 4.17-4.1 I (m, O.SH, H-Z(3, H-3(3), 4.03 {t; 0.75H, H
tea. J;.~ = 6 Hz). 3.80-3.67 (m. 1.25H, H-4a, H-5aa, H-Sa[3), 3.53 x_39 (m, I.75.H, H-Sbcx.
H-4(i, H-Sb(i), ?.0G (s, 3H. CI33-a and CH3-Vii). Anal. (Cz8H3aO5S ~ Q.75 H=O) C. H.
SUBS'CITUTE_SHEEC (RULE 26) WU 00/04866 ~ 02464681 2004-04-07 i~C?IUS~9/16630 I-(2,3,5-Tri-O-benzyl-4-thio-~x,~~n-arabinofuranosyl) cytosine ('7). To a suspension of 1-n-acetyl 2.3.5-tri-C7-benzyl-4-thio-n-arabinofuranose (478 mg, 1 mmol) and cytosine ( I 11.0 m~, 1 nnrial) in anhydrosis acetonitrile (25 rr~mol) were added consecutively hexamethyld'iszla2ane (HMDS.162rng,1 tnmoi) and chlorotrirnethylsilane a (TMSCI, 4;4 mg, 4 mmol), and the mixture was stirred at room temperature for Q.5 , hours. This solution was cooled to -78 ° C.
Trimethylsilyltrifluoromethane sulfonate (267 nag, I .2 mmol) was added and the resulting solution was stirred at -78 ° C for another 2.5 h, after which time the reaction was essentially complete. The mixture was warmed to room temperature, concentrated to a small volume (5 mL), diluted v~rith methylene chloride (50 mL) and then washed with water (20 rnL) followed by saturated sodium bicarbonate and water. The organic layer was dried over MgSO:, and evaporated to dryness. The residue was purified by chromatography over silica gel (SO
a. elution with CHCI3/MEOH 98:2) to afford 7 (4I2 mg 77.5%) as a coloriess syrup TLC (95:5 CHC13IME(.7lEi) R,t. 0.65; MS zle 536 (M+Li)t.
'H NIV1TZ (CDCI3) ~ $.22 (d, 1, H-dp, 3 ° 7.~6); (d; 1, H-6a; J5,6 =
7.5 I32); x.38-?.09 (m, 30, aromatic H's); 5.65 (d, 1, H-1'~, 3~.,~. = 5.7 I3.z); 6.36 (d, 1, H-I'a, Jt.,. _ 1_2 Hz);
5.44 (d. 1. H-SQ); 5.26 (d, 1, H-Sp); 4.97-4.33 (averlapgirtg multiplets, 12;
CaH3CH,), 4.26 (dd. I H-2'p, J,..s- = G.S Hz); 4.22 (m, 1, H-3'~, J3~.4. = 1 Hz); 4.1b (dd; 1, H-3'~, J;..,. = 6.4 I-iz); 4.13 (m, I. H-Z'a, J2._3. = 1.7 Hz); 3.91 (m, 1_ ~I-4'~);
3.78 (m, 1. H
5~.,); 3.73-3.63 (m. 2. H-S'e); 3.55 (tn, I, H-5'a); 3.46 (in, 1. H-4'~.
SUBS'TIfliT6 SH~61" (RLSt~ 26) WO 00104866 PC'TItJS99116b30 I-(4-'JChio-a, ~-u-arabiu~oturanosyl) cytosine (8). To a solution of boron irichloride ( 1 M solution) in dry dichloromethane (7mL, 7mmo1) cooled to -78 ~ C, -was added dropwise over a period of 30 min, a solution of compound 7 (26S mg, 0.5 mmol) in clxy diclUoromethane (10 mL). Stinting was continued overnight at -20 ~C. The solvent was removed in vacuo and the residue was coevagorated with dichloromethane (4 x ?0 mL). The residue was neutralized with saturated NaHCOs (25 tnL) and washed with chloraforn~ (IS mL). The aqueous iayer'was applied to a canon exchange (H') column and eluted with water to remove salts and tlxen eluted with 1N NHdOH to obtain the desired compound 8 (11U rng, 85°~°0) MS z!e 260(M+I~i)".
~H NMR (Me-,SO-ab) 8 7.9~ (d, .67, H-6-ø, J5.6 = 7.5 Hz); 7.9a (d. 4.33, H-6a, JS_6 --7.5 Hz); 7.17-7.03 (overlapping bs, 2, NH's); 6.33 (d, 4.67, H-i' ø, d, :,. =
4.6 Hz); 5.86 (d. 0.33, ~-I-1'oc, J,.,- = 7.3 Hz); 5.77 (d, 0.33, H-Stx): 5.70 (d, 0.67, H-5 j3): 5.61 arid 5.59 (overlapping doubiets, 1, 2'-OHø, J.,.~.,~ = 5.1 Hz, 2'-OHa. J,.,-_oH =
5.9 Hz);
5.47 (d, 0.33, 3'-OHa, J3. ~,~H = 5.1 Hz); 5.38 (d, 0.67. 3'-fJHø, J 3'.3'-(3H
= 4.2 Hz);
5.08 (t. 0.67. 5'-f)H~3; Jg~_S'.OH ~ 5.4 Hz); 4,90 (t, 0.33, S"-UHa, JS~,~,_oH
~ 5:2 Hz); 4.00-3.93 {m. 1.&7, H-2'Q, H-2's, H-3'~; 3.$6-3.76 (tn, l, IH S'« and H-5'p~; 3.67-3.55 (xn;
1. H-5' ~, + H-3' «); 3.49-3 .3 3 (m, 0.67; H ~' «, H-5' «); 3 . L7 (m. 0.67, H-4' ~).
SU86'i'ITUi"E SHEET (RULE 26~
WO OO/Od866 ~ 02464681 2004-04-07 pCTNS99/16630 :y 1-(S-D-dimethoxytrityl-4-than-Q-n-strabinofuranosyl) cytosine (9). To a solution of compound 8 (I00 mg, 0.38 mmoi) in dry pyridine (10 mL) was added 4,4'-~
Dimeth.oxytritylchloride (135 mg, 0.6 mtnol) and the solution was stirred for 2 It at room temperatuze_ The reaction mixture was evaporated to dryness and the crude visas dissolved in ethyl acetate (Z0 mL) and washed with water and evaporated to dryness to afford a solid which was purified on silica geI column (CHCI3/MeOH 98:2) to obtain pure compound 9 (96 mg, 90% based on 1:1 mixhire of oc, (3-mixture of 8). MS
~Je SG8.3 (M+Li)':
'H NMR (Me,SO-db) S 7.77 (d, 1, H-6, Jss ~ 7.5 Hz); 7.42-?.23 (m, 9, aromatic H's);
?.17 (bs, I, NH); 7,05 (bs, I; NH); 6.91-6.88 (rn, 4, aromatic H's); 6.36 (d, 1, H-1', .1,. ,. = 4.8 Ha); 5.65 (d, I, H-S); 5.76 (d, 1, 2'-OH, J2.,i..os, = 4.6 Hz);
5.43 (d, 1, 3'-OH, ~Ta-.:--cm ' 3.3 Hz); 3.98-3:9I (m, Z, H-2'; H-3'); 3.75 (s, G. OCIri;); 3.39-3.25 (m, B, H' 4'. H-~').
~EUBSTtTU'tE SHEEP (RULE 2~
1~V0 ~0/04866 ~ 02464681 2004-04-07 1-(4-Thio-(3-u-arabinofuranospl) cytosine (I4). Compound 9 (90 mg, 0.16 mmol) was treated with trifluoroaGetic acid (22 mg) in chlorafaxm (5 mL) at roam temperature foz I O min. The reaction mixture was neutralized with aq. NaHC03 and the aqueaits layer was applied on a catiou exchange column which was eluted first with water to remove salt and finally with IN NHdOH to afford compound 10 (35 mg, 85°/a), rnp218-Z20 °C
( li t' 3? 1 _322 °C); MS zle 26Q(M+H)+.
'H NMR (Me,SO-d6) 6 7.94 (d, 1, H-6, 3~.6= 7.5 Hz); ?.I2 (bs, 1, NH); ?.U4 (6s, 1.;
NHl'. _6.33 (d, 1. H-1', J' :,' = 4.6 H~); 5.70 (d, 1, H-5); 5.61 (bd, 1, 2'-OH, .T,.,,.ou =
3.1 Hz); 5.38 (6d. 1, 3'-OH, J3.,3,.cH '- 3:5 Hz); S.Q8 (bt, 1, 5'-OH, JS.,s..oH ~ 4.9 Hz);
4.U0-3.93 (m, 2, H-2', H-3'); 3.78 (m, I, H-S'a); 3.6I (m, 1, H-5'b); 3.16 (m, 'l, H-4').
Example 2 Preparatioct of 1-(4-Thin-t3-D-Arabinofnranosyl)~.FluoroCytosine 1-(2,3,5-Tri-D-benzyl-4-thio-a,tl-~?-arabiuofnranosyl)~-flunrocytosine ('7a).
To a suspension of 1-C?-acetyl 2,3,5-tri-O-benzyl-4-thio-D-arabinofmranose (478 m.g,1 ~nmol) and cytosine ( 129.0 mg, 1 mmol) in anhydrosis acetouitrile (25 mmol) were added consecutively I~zexamethyldisilazane (HIVIDS,162 mg, l mmol) and chlorotrimethylsilane (TMSCI. 434 mg. 4 mmol), and the mixture was stirred at room temperature for 0.5 llour5. Tlois solution was cooled to -78°C.
Trirnethylsilyltrifluoromethane sulfonate (2b'l mg, 1.2 mmol) was added and the resulting solution was stirred at -7&°C for 2fl another 2.5 h, after which time the reaction was essentially. complete.
The mixture was warnaed to room temperature, concentrated to a small volume (5 mL), diluted with ZI
~UBS~IME SHEET (RUCHE 26) .
WO 00I0~866 PCTJUS99/16630 tnethyleue clzlor~ide (SO mL) and then washed with water (20 mL) followed by~
saturated sodium bicarbonate and water. The organic layer was dried over. MgSO., and evaporated to dryness, The residue was purified by chromatography over silica gel (SO
g. elution with CHCI~IMEOH 98:2) to afford 7 as 2:1 oe.Ii mixture (425.2 m~
80.0%) as a colorless syntp TLC (95:5 CHCI3fM,EOH R,. 0.65; MS 7le S53 (IvI+Li)'.
'I-i NMR {CDCI3} 8 8.40 (d, I, H-6a,J=7.6); 8.10 (d, 1, H-6a, Jt,b ~ 7.5 Hz};
7..35-7.09 -(n3, 30. aromatic H's); 6.55 (d, 1, H-1'a, J, :~- = 5.7 Hz); 6.25 (d, 1, I~-1'n, 31. ,. = 1.2 ' Hz), 4.90 (d, 1. II-S~); 4.~8 (d, l, H-Sa}; 4.30-4.55 (overlapping multiplets.
12, C,~H;CHI), 4.15 (dd, 1 H-2'a, Ja~,~~ = 6.8 Hz); 4.i0 (m, 1. H-3'Q, 33,.x. = 'l Hz): 3.90 (dd, l, H ~'~,. J3~.,,- = 6.4 Hz); 3.75 (m, 1, H-2'a, Jz.w = 1.7 Hz); 3.70 (m, 1. H-4'~; 3.65 (m, I. H-5'~a): 3.50-3.55 (m,2,H-5'a); 3.45 (m, I, H-5'a}; 3.4D (m. 1, H-4'~, Z-(4-Trio-a-D-arabinofuranosyl)5-ifuorvrytosine (loa). To a solution of boron trichioride ( 1 M solution} in dry dichlorometbane (7 mL. 7 anmol} cooled to -78°C. ~~as added dropwise over a period of 30 min, a solution of corapound 7 (2273 mg.
4.5 IS mmol) in dry dichloromethane (10 mL).~ Stirring was continued overnight at -20°C:
tl3e solvent was removed in vacuo and the residue was coevaporated with dichloramethane (4 x 20 mL). The residue was neutralized with saturated NaHC03 (25 nL) and washed with chlorafomm (15 mL). The aqueous layer was applied to a cation exchange (H') column and eluted with ,hater to remove salts and then eluted with 1N
NH~OH to obtain the desired compound Sa as ?:l a.B mixture which upon crystallization with water gave pure IOa (32.2 mg, 25%) MS zle 278(M+H)', 'H NMR (Mt~SO-db) 8 8:15 (d, 1, H-6, Js.e = 7.5 Hz); 7.75 (bs. I, NH); 7.50 (bs. 1, NH); G ~5 (d, I, SUBSTCtUfE SHEEt (RU4E 2.6~
wo ooioa866 PCTlU59911~630 H-1', 1~ :,v= 4.t~ Hz); 5.65 (d, 1, H S); 5.60 (bd, 1,2'-OH. J=.,~-~H = 3.1 Hz); 5.X0 (bd., 1.3'-OH: J~ ,3'-OM = 3.5 Hz); 5.30 (bt, 1, 5'-OH, 3S..S..o~ = 4.9 Hz); 4.00-3.90 (in. 2. H-?', ~ .
H-3'); 3.75 (m, 1, H-S'n); 3.65 (m, l, H-S'~); 3.1S (m, I, H-4').
' Ototani, H.; Whistler, R.L. Preparation and Antitumo'~r Activity of 4'-Thio Analogs 0~' ?.?'-Anhydro-1-l3-17-arabinofuranosylcytosme. J. Med. Chem. 1974, 17, 535-537.
SUB~~ SHED' (R!!L~ ~6, WO 00!04866 ~ 02464681 2004-04-07 PCTlUS99/I6630 Example 3 The following in vitro tests were carried out.
Table 1 iCSfl Molar Values - ' ThioaraC 5-fluorothiaaraC~'ts~e arabinc~side LOX IMV 1 Melanoma 7 x 10'6 6 x 10'6 1 x 10-6 PC-3 Prostate $ x 10'6 1 x 10''~ S x IO'~
ZR-75-I Mammary 3 x 10'6 6 x 10'' 4 x 10' CAKI-I Renal 3 x 10'~ 2 x IO's 6 x 1U-' DLD-1 Colon 7 x 106 . 8 x 10's 5 x I0'~
ID ~ NC1-H23 Lung 5 x 10'G ' 3 x 10-6 3 x IO'' , SK-MEL 28 Melanoma 7 x 10'6 - 2 x IO'6 S1~TB-7 (CNS) 1 x IO'a ~t 2 x 10' The above tests were carried out with a 72 hour exposure to the compound using Natural Red assay for all cell lines except SK-MEL 28 which was done with SRB assay.
WU 00!04866 . PC'1'1US99~116630 Example 4 The fallowing in viva tests were carried out to illustrate the effectiveness of ' the compounds of the present invention.
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' WO OO/OA866 PCTJUS99l1663t3 As can be appreciated from the above tests, in most cell lines, A12A-C
exhibits l3igher cytoxicity as comgaaed to that of the present invention which wauild tend to discourage use of the compounds of the present invention as a cancer treaement. However, surprisingly, the J.-(4-thin-i3-D-arabinofuranosyl)cytosine is more potent than ARA,-C and more selective in killing cancel cells in vivo.
Compounds employed according to the present invention exhibit better anticancer activity along with lower toxicity. This may be due to the fact that 1-(4-thio-~-Ll-arabinofuranosyl)cytosine does not get deaminated by cytidine dearninase as quickly as AraC does, which is shown below in Table 5 by using radio labeled substances:
Good substrate activity of cytidine deaminase with AraC has been a major problem with this drug because it not only decreases half life of drug but also gets converted to Ai'3Uri~lile Wnich is not an active substance.
Table 5 Cytidine deaminase activity I5 determined with radioactive substrates Compound 1~", V""x '~,n:~/~", AraC I47 9 0.06 l.-(4-thio-13-D-arabinofuranosyl2571 17 0.007 cytosine The following tests ware carried out which illustrate the abiliey of the thioarabinofuranosyl compounds according to tkte present invention to inhibit DhTA
replication in tttarumalian cells. In the following tests, dCyd refers to 2'-deoxycytidine, TCyd refers to thiodeoxycytidine, T-araC refers to 1-(4-thio(3-D-axabinofuranosyl) cytosine and Ara-C refers to ''cytosine arabinoside" _ The terms °'d CTP", "T-d CTP", "ara CTP" and "T ara CTP°' refer to the corresponding .. 2S u-iphosphates. The team "dLIItD" refers to 2-deoxyuridine. The term "F-d.Urd"
refers to fluoxinated deoxyuridine and "F-dUMP" refers to fluorinated deoxyuridine monophosphate.
WO OOIp4866 , PCTlLTS94/16534 Reference is made to Figs. la and 1b which illustrate the metabolism of d Cyd, T-d Cyd, ara C and T-era C to their respective triphasphates. Ln particular, ..
CEM cells were incubated with 2040 nM of [5-3H)T-dCyd, 5 nM [5-3H]araC, or 2S
nm [5 3H]T-araC for the times indicated. The radioactivity in the ~'-triphosphate , peak was deterrnin:ed using S,A~ T-1PLC. Por eack~ compound. the amount of radioactivity in the 5'-triphosphate peak was more than 903 of the total -radioactivity eluting from the S~ HPLC colunm..
Test results related to the inhibition of CEM cell growth are shown below in Table 6.
CEM cells were incubated with various concentrations of eaclx compound and the effect on cell IIUIrib~rS Wa5 determined using a Goulter Counter. The IC~o was detexmined from a plot of the perceat of control growth versus drug concentration. The data presented are the mean and standard deviation from 3 separate experiments. The araC-resistant cells were obtained by incubating CEM
IS cells in she presence of 150 nM araC for approximately 1 month at which rime the CEM cells were growing at control rakes.
Table 6 Inhibition of CEM CeII Growth by dCyd Analogs Compound 'Wild-type ara.C-resistant Resistam/VPT
ICso ~~
T-dCyd ~ 2200 ~ I400 ?240 t 7600 3 T-araC 24 ~ 9 125 t 84 5 araC 6 ~ 3 800 ~ 400 145 . WU Oul04866 CA 02464681 2004-04-07 ~'CTIU$gg~I6630 Test results related to metaboiism in wild type and araC resistant CEM cells are shown below in Table 7.
CEM cells (wild-type or araC-resistant cells) were incubated with 100 nM of [5-3H)dCyd, [,5-3H~T-dCyd, [5 3I3]araC, or [5 3H]T-araC for the times indicated.
' S The radioactivity in the 5'-triphasphate peak and the incurporauon of radioactivity is the acid-insoluble fraction (DNA) was determined. Fox each compound, the amouni of radioactivity in the 5'-triphosphate peak was more than 90 ~ of the total radioactivity eluting fmm the SA~~ HPLC column.
Table 7 IO Metabolism of dCyd and Its Anaiags in Wild-Tppe and araC Resistant CEM Cells Compound IncubationCeil Type Tri- DNA Total Time phosphate Hours gmoles11 06cells (gercent of WT) dCyd ~ a.25 wT 2.27 I.I9 3.~.~
Resistant 0.021 0.0I1(0.89)0.032 (0.93) (0.91) T-dCyd 0.25 WT 0. S77 1.29 Z. I7 Resistant 0.036 (4. 0.051 (4. 0.487 (4.0) I ) 0) .
I~ araC 1 WT I4.7 0.28 14.98 Resistant 0.19 ( 0.0I 8 0.208 ( I .4) I .2) {b.3) T-araC I WT 0.202 0.0056 0.2076 Resistant 0:051 {25)O.OD25 0.053$ (26) (45) W~ Q~~~866 CA 02464681 2004-04-07 ~ p~~g99116fi30 Results illustrating phtnphorylation in cell-free CEM extracts are shown below in Table 8.
A crude cell extract was prepared from wild-typo and araC~resistant CEM cells and the ability to phosphorylate dCyd, araC; and T-araC was determined. The number in parentheses is the , number of experiments performed. Reactions were performed in soiutioas containing 50 mM Tris (pN 8.Q); 5 tnN! ATP. 9.5 tnM MgCI:, 20 taM NaF, extract> and 1 ~m IS-'H]dCyd>
(5-~H)araC or .
[5'H]T-araC. At various times after the i~itiadon of the experiment,.an aliquot of each reaction volume was removed and placed on DE~81 filters. The filters were washed with 1 mM ammonium formate attd ethanol, and the amount of radioactivity on each filter was deterrained. The phosphorylation of each eompouad increased in a linear manner with respect to time. This assag was verified by HPLC.
Tal'le 8 Phosphorylation of dCyd, araC, attd T-at~C
in CeII-Free CEM E~ttraets Compound Wild Type araC-resistaatt CE.M Percern of wild-type cells pmoleslmg pmtefrirrtinnte 7.5 dCyd 240 ~ 33 (3) 3_5 t 0.2 (3) 1.~
araC 94 (1) L2 (I) 1.3 T-trraC U,83 (2) O.OIi (1) L3 Tabte 9 below t'llustrates deoxycydidine deaminase activity, In particular, deo~cycytidinc deamittase acciviry was pariftcd from Mvtt-d cells as described (Shewach et al, Mol. Pharmacot. 42:
SI8-524-; 1992). Each assay was done in dupl3catz and the kinetic constants were deternuned from tinear double-reciprocal plots of livelot:iry versus Ilconcentration of the substrate. The best line was SUBSTnt1'tE SHEEP (RULE 26~
WO alllti4866 ~ 02464681 2004-04-07 p~.~SggJ16534 detetmineti by linear regression from a! least S dacuttt points, and the IC", and ~t""~ were determined from the x and y intereepta.
Table 9 Substrate Characteristics of dCyd, araC, and T-araC
with dCyd Kinase Activity lsoiated from Mott-4 Celts Cottipound K," (uM) Relative V,~", v,nu~m dCyd 1.~ 1 ~:8 ' araC 15 t3.1 O.Ot?6 W-thin-araC 93 o_a6 0.005 Tabte IO below also illustrates deoxyeycidine deamiaase activity.
Deoxycyddine deaminase activiey was p~srified froth human placenta and the Km and V~x of dCyd, T-dCyd, araC, and T-araC were determined. Reaeuans were carried oat is salucians containing 20 raM potassium phosphate (pFl ?.4), 100 ntM KCI,~ various concentrations of radiolabeted nucleoside, and enzyme: The reacxions were stopped with acid, the substrate was separated from the product by paper chromatography, and the radiaacdvity in each vreze detezrnined.
The kinetic constants were determined from linear doublereciproeal plots of IJvetocity versus l~concentration of the substrate. The best line was determined by linear regression from at least 5 datum points, and the K~, and V~ were detettuiited froth the x and y intercepts. The data presented are the mean and standard deviation from 3 separate experiments.
suss-rrru~ shot t~utE Zs~
WO 00!048b6 ~ 02464681 2004-04-07 . ' pCT/US99/16630 Table IO
Suliactrmte Characterist3rs of dCyd, T-dCyd, araC, and T~raC
vrith dCyd Deaatlnase Activity Isolated fotri I3uman Placenta Compound FCC, (u.M) V,~ (pmoles/mg/tttin)V,~IF~, dCyd z3 t 2.7 . 13 t i.5 O.SS
T-dCyd 111 d: 7? 3? t 12 0.33 araC 238 t 13 t 1~ -~- 7 0.056 T-araC 2944 f 1023 21 t 6 0.0092 Results of half life tests in CEM cells are reported below in Table 11 After incubation of CEM celia for 1 hear with 100 nM of jS-~H]dCyd, [5-'HST
dG~d; j5-'H]araC, or [5 3H]T-anC, the cells were collected, washed with fresh tnedinm.
and resuspended in fresh mcdinm that did not contain radiolabeled nudeosides. Samples were calleceed at variaus times after the oells were resuspended in fresh medium, and the amount of radioactivity itt the 5'-criphosphate peak was determined using SAX HPLC. The data presented are the mean and standard deviation from 3 separate experiments.
Tsble II
xnitisl Half Life of dCTP, T-dCTP, araCTP, and T ar~CTP its CEM Cells Nucleotide Hours (SI?) acTP 0.9a . 0.16 T-dCTP 1.10 0.35 araCTP i.31 ~ 0.31 T-araCTP 10.8 t 1.80 SUB6TITUTE SHEEt (RULE 26) WO 00!0486b PC?fUS99/166~.i0 Results of tests related to retention of araC'TP and T-araCTP in CEM chlls are shown in Fig. 2. The tests were carried out as follows:
' After incubation of CEM cells for 1 hour with either 5 nM ~5-3H]araC yr 200 nm [S 3H]T-araC; the cells were collected, washed with fresh medium, and resuspended in fresh medium that did not contain radialabeled nucleosides.
Samples were collected at various times after the cells were resuspended in fresh medium, and the amount of radioactivity in the 5'-triphosphate peak was determined using SAX HPLC. Tn this experiment there was 0.639 pmoles araCTPllOb cells and 0.246 pmoles of T-araC'TP after the 1 hour incubation with radiolabeIed compound.
Results related to metabolism in CEM cells are reported below in Table 12.
In particular, CEM cells were incubated with 100 nM of [5 3HjdCyd, [5-3H]T-dCyd, [5 3H]araC, or [5-3H]T-araC for the times indicated. The complete metabolism of each compound was determined: The medium was analyzed for the original compound, its deaxninated. form using reverse phase HPLC, and H~~C7;
the IS acid-soluble extract was analyzed by SAX HPLC fox phosphorylated metabolites;
and the incorporation of radioactivity into acid-precipitable material (1?NA) was determined. All of the original radioactivity was accounted for in these fractions.
Far each compound, the amount of radioactivity in the 5'-trighosphate peak was more than 9C1 % ' of the total radioaccavity eluting from the SAX HPLC column.
WC? OOlOd866 ~ 02464681 2004-04-07 . ' p~~s9911663U
Table Ix RQetabolism of dCyd, T-dCyd, scraC, and T-araC in CEM Cells Nucleoside Time OriginalDeazxtirtaiedH=O nucleoside- DNA
of IncubationCompoundCompottttd, TP
(hours) ~ (percent of mtal) dCyd 1 38 0 55 4 3 T-dCyd 1 90 0 Q 2 8 araC 1 g'7 0 4 13 0.4 T-aaaC 24 96 0 0 4 0.3 Resuhs from tests related to the effect of F-dUrd on metabolism are shown below in Tahle 13.
CEM cells were incubated with I00 nM of [S-'HJdCyd, [3-'H]T-dCyd, [~',li]araC, dr (~-'H]T-araC for the times indicated in the presence ox absence of 10 ~.m F-dUrd, which is metabolized to F-iIUMP (a potent iahibitor of thynudylate syuthetase). Tho complete meuaholism of each compound was deterntlned as described in the legend to Table 12, and the percent of deami>lated metabolites for each compound was determined_ 1a the absence of F..dUrd, the destitinated product 1~ for dCyd was [3H]13,0 due to the removal of ['H] at the 5 position of F-dUMP by chymidylate synchetase. Isi the presence of F-dUrd, the deaminsted product of dCyd, T-dCyd, and araC was in the medium.as dUrd. T-dUrd, or araU, respectively.
_ . CA 02464681 2004-04-07 W O Ii0/04866 PC?11J599116630 Table 13 Effect of F-dUrd on the M~abolism of dCyd, T dCyd, araC, or T-araC
Compound F-DUrd (~M) Intrubation Percent Deaminacion Time , ~~) S acya o ~ 32 ac~ya la 1 zz r-acya o s T-dCyd ~ 10 g ~
araC 0 g Q
exec to s to T-araC U 24 p T-araC IO 24 Q
Table I4 below reports results of tests concerning effects of DNA, 1t1~1A and protein syndtesis.
I5 CEM cells vrreze incubated with 60 EsM T-dCyd, 15Q nM azaC, or 7S0 uM T-araC.
Radiolabeled precursors of DNA [SHadThd, RNA (yi]Urd), or protein (['HJlenoine) were added to each treatment 3Q~ minutes after, the addidan of each compound. Samples were taken I , 2, 3 and 4 hours after the additlan of radialabeted precursors, and the incorpo~acion of radioactivity into RNA, DNA, or protein were determined as desezibed (Parker et al, Biocltent.
pharmncol. S5: I6~3-168I, t998). Ench number represents the average of two experiments.
SUBSTfNTE SHEET (MULE 26) WO Ot1/0486b CA 02464681 2004-04-07 ~ pCTlUS99I16G30 Table 14 Effect of T~dCyd, araC, and T-araC on DNA, RNA, and Protein Syntheses Compound DNA RNA ~ protein Percent of control 60 ~cM T-dCyd 3B 95 78 150 nM araC g 9b 96 750 nRri T ataC 16 8? 72 As can be appreciated ftoTn the above, thioarobitzofuranosyl compounds according to the present invention can be used to inhibit DNA replication in mammalian cells.
These results tend to suggest their ability as itttmunomodtiiatars, which would render them suitable for seating . .
autolmmune diseases. This is further supported by the fact that several guanosine analogs have bees shown tv stimulate the immune system (VW'eigic, W.O., CRD Crit.Rev.lmmunal., 1987, 7, 285; Lin ec al, .l.Med.Chetrl., 1985, 28, 1194-1198: Reitzet al, J.Med.Chem., 1994, 3?, 3561-35?8; Michael et al., l.Med.Chent., 1993, 3G. 3431-3436). Certain 3-B-ribofuranosylthiazalo(4.,5-d]pyrimidines hnve also been shown to have significant immunoactivity, including marine spleen cell proliferation and in vivo activity against Semliki Forest virus (Nagahara et aL, J
Med.Chem., 1990, 33, 40?-4iS).
The foregoing description of the invention illustrates and describes the present invention.
Additionally. the disclosure shows attd describes only the preferred embodiments of the invenuott but, as mantioned above, it is to be understood that errs irlvencion is capable of use in various other 24 combinations, modifications, and environments and is capable of chaztges or modifications within the scope of the inventive concept as expressed herein, comtttensurate with the above teachiFtgs andlor the skill or knowledge of the relevant axt. The embodiments described hezcinabove are further intended to explain best modes known of .SUBSTlf UTE SHEET ~RULF 2~
CA 02464681 2004-04-07 F(~T~f~,~9~'~~~3~
practicing the invention and to enable others skilled in the art to utilise the invernion in such, or ocher, embodiments and with the various modifications required by the gartieular applications or uses of the invention. Accordingly, the description as not intended to limit the invention to the form disclosed herein. Also, it is intended that the appended claims be construed to include alternative embodiments.
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' WO OO/OA866 PCTJUS99l1663t3 As can be appreciated from the above tests, in most cell lines, A12A-C
exhibits l3igher cytoxicity as comgaaed to that of the present invention which wauild tend to discourage use of the compounds of the present invention as a cancer treaement. However, surprisingly, the J.-(4-thin-i3-D-arabinofuranosyl)cytosine is more potent than ARA,-C and more selective in killing cancel cells in vivo.
Compounds employed according to the present invention exhibit better anticancer activity along with lower toxicity. This may be due to the fact that 1-(4-thio-~-Ll-arabinofuranosyl)cytosine does not get deaminated by cytidine dearninase as quickly as AraC does, which is shown below in Table 5 by using radio labeled substances:
Good substrate activity of cytidine deaminase with AraC has been a major problem with this drug because it not only decreases half life of drug but also gets converted to Ai'3Uri~lile Wnich is not an active substance.
Table 5 Cytidine deaminase activity I5 determined with radioactive substrates Compound 1~", V""x '~,n:~/~", AraC I47 9 0.06 l.-(4-thio-13-D-arabinofuranosyl2571 17 0.007 cytosine The following tests ware carried out which illustrate the abiliey of the thioarabinofuranosyl compounds according to tkte present invention to inhibit DhTA
replication in tttarumalian cells. In the following tests, dCyd refers to 2'-deoxycytidine, TCyd refers to thiodeoxycytidine, T-araC refers to 1-(4-thio(3-D-axabinofuranosyl) cytosine and Ara-C refers to ''cytosine arabinoside" _ The terms °'d CTP", "T-d CTP", "ara CTP" and "T ara CTP°' refer to the corresponding .. 2S u-iphosphates. The team "dLIItD" refers to 2-deoxyuridine. The term "F-d.Urd"
refers to fluoxinated deoxyuridine and "F-dUMP" refers to fluorinated deoxyuridine monophosphate.
WO OOIp4866 , PCTlLTS94/16534 Reference is made to Figs. la and 1b which illustrate the metabolism of d Cyd, T-d Cyd, ara C and T-era C to their respective triphasphates. Ln particular, ..
CEM cells were incubated with 2040 nM of [5-3H)T-dCyd, 5 nM [5-3H]araC, or 2S
nm [5 3H]T-araC for the times indicated. The radioactivity in the ~'-triphosphate , peak was deterrnin:ed using S,A~ T-1PLC. Por eack~ compound. the amount of radioactivity in the 5'-triphosphate peak was more than 903 of the total -radioactivity eluting from the S~ HPLC colunm..
Test results related to the inhibition of CEM cell growth are shown below in Table 6.
CEM cells were incubated with various concentrations of eaclx compound and the effect on cell IIUIrib~rS Wa5 determined using a Goulter Counter. The IC~o was detexmined from a plot of the perceat of control growth versus drug concentration. The data presented are the mean and standard deviation from 3 separate experiments. The araC-resistant cells were obtained by incubating CEM
IS cells in she presence of 150 nM araC for approximately 1 month at which rime the CEM cells were growing at control rakes.
Table 6 Inhibition of CEM CeII Growth by dCyd Analogs Compound 'Wild-type ara.C-resistant Resistam/VPT
ICso ~~
T-dCyd ~ 2200 ~ I400 ?240 t 7600 3 T-araC 24 ~ 9 125 t 84 5 araC 6 ~ 3 800 ~ 400 145 . WU Oul04866 CA 02464681 2004-04-07 ~'CTIU$gg~I6630 Test results related to metaboiism in wild type and araC resistant CEM cells are shown below in Table 7.
CEM cells (wild-type or araC-resistant cells) were incubated with 100 nM of [5-3H)dCyd, [,5-3H~T-dCyd, [5 3I3]araC, or [5 3H]T-araC for the times indicated.
' S The radioactivity in the 5'-triphasphate peak and the incurporauon of radioactivity is the acid-insoluble fraction (DNA) was determined. Fox each compound, the amouni of radioactivity in the 5'-triphosphate peak was more than 90 ~ of the total radioactivity eluting fmm the SA~~ HPLC column.
Table 7 IO Metabolism of dCyd and Its Anaiags in Wild-Tppe and araC Resistant CEM Cells Compound IncubationCeil Type Tri- DNA Total Time phosphate Hours gmoles11 06cells (gercent of WT) dCyd ~ a.25 wT 2.27 I.I9 3.~.~
Resistant 0.021 0.0I1(0.89)0.032 (0.93) (0.91) T-dCyd 0.25 WT 0. S77 1.29 Z. I7 Resistant 0.036 (4. 0.051 (4. 0.487 (4.0) I ) 0) .
I~ araC 1 WT I4.7 0.28 14.98 Resistant 0.19 ( 0.0I 8 0.208 ( I .4) I .2) {b.3) T-araC I WT 0.202 0.0056 0.2076 Resistant 0:051 {25)O.OD25 0.053$ (26) (45) W~ Q~~~866 CA 02464681 2004-04-07 ~ p~~g99116fi30 Results illustrating phtnphorylation in cell-free CEM extracts are shown below in Table 8.
A crude cell extract was prepared from wild-typo and araC~resistant CEM cells and the ability to phosphorylate dCyd, araC; and T-araC was determined. The number in parentheses is the , number of experiments performed. Reactions were performed in soiutioas containing 50 mM Tris (pN 8.Q); 5 tnN! ATP. 9.5 tnM MgCI:, 20 taM NaF, extract> and 1 ~m IS-'H]dCyd>
(5-~H)araC or .
[5'H]T-araC. At various times after the i~itiadon of the experiment,.an aliquot of each reaction volume was removed and placed on DE~81 filters. The filters were washed with 1 mM ammonium formate attd ethanol, and the amount of radioactivity on each filter was deterrained. The phosphorylation of each eompouad increased in a linear manner with respect to time. This assag was verified by HPLC.
Tal'le 8 Phosphorylation of dCyd, araC, attd T-at~C
in CeII-Free CEM E~ttraets Compound Wild Type araC-resistaatt CE.M Percern of wild-type cells pmoleslmg pmtefrirrtinnte 7.5 dCyd 240 ~ 33 (3) 3_5 t 0.2 (3) 1.~
araC 94 (1) L2 (I) 1.3 T-trraC U,83 (2) O.OIi (1) L3 Tabte 9 below t'llustrates deoxycydidine deaminase activity, In particular, deo~cycytidinc deamittase acciviry was pariftcd from Mvtt-d cells as described (Shewach et al, Mol. Pharmacot. 42:
SI8-524-; 1992). Each assay was done in dupl3catz and the kinetic constants were deternuned from tinear double-reciprocal plots of livelot:iry versus Ilconcentration of the substrate. The best line was SUBSTnt1'tE SHEEP (RULE 26~
WO alllti4866 ~ 02464681 2004-04-07 p~.~SggJ16534 detetmineti by linear regression from a! least S dacuttt points, and the IC", and ~t""~ were determined from the x and y intereepta.
Table 9 Substrate Characteristics of dCyd, araC, and T-araC
with dCyd Kinase Activity lsoiated from Mott-4 Celts Cottipound K," (uM) Relative V,~", v,nu~m dCyd 1.~ 1 ~:8 ' araC 15 t3.1 O.Ot?6 W-thin-araC 93 o_a6 0.005 Tabte IO below also illustrates deoxyeycidine deamiaase activity.
Deoxycyddine deaminase activiey was p~srified froth human placenta and the Km and V~x of dCyd, T-dCyd, araC, and T-araC were determined. Reaeuans were carried oat is salucians containing 20 raM potassium phosphate (pFl ?.4), 100 ntM KCI,~ various concentrations of radiolabeted nucleoside, and enzyme: The reacxions were stopped with acid, the substrate was separated from the product by paper chromatography, and the radiaacdvity in each vreze detezrnined.
The kinetic constants were determined from linear doublereciproeal plots of IJvetocity versus l~concentration of the substrate. The best line was determined by linear regression from at least 5 datum points, and the K~, and V~ were detettuiited froth the x and y intercepts. The data presented are the mean and standard deviation from 3 separate experiments.
suss-rrru~ shot t~utE Zs~
WO 00!048b6 ~ 02464681 2004-04-07 . ' pCT/US99/16630 Table IO
Suliactrmte Characterist3rs of dCyd, T-dCyd, araC, and T~raC
vrith dCyd Deaatlnase Activity Isolated fotri I3uman Placenta Compound FCC, (u.M) V,~ (pmoles/mg/tttin)V,~IF~, dCyd z3 t 2.7 . 13 t i.5 O.SS
T-dCyd 111 d: 7? 3? t 12 0.33 araC 238 t 13 t 1~ -~- 7 0.056 T-araC 2944 f 1023 21 t 6 0.0092 Results of half life tests in CEM cells are reported below in Table 11 After incubation of CEM celia for 1 hear with 100 nM of jS-~H]dCyd, [5-'HST
dG~d; j5-'H]araC, or [5 3H]T-anC, the cells were collected, washed with fresh tnedinm.
and resuspended in fresh mcdinm that did not contain radiolabeled nudeosides. Samples were calleceed at variaus times after the oells were resuspended in fresh medium, and the amount of radioactivity itt the 5'-criphosphate peak was determined using SAX HPLC. The data presented are the mean and standard deviation from 3 separate experiments.
Tsble II
xnitisl Half Life of dCTP, T-dCTP, araCTP, and T ar~CTP its CEM Cells Nucleotide Hours (SI?) acTP 0.9a . 0.16 T-dCTP 1.10 0.35 araCTP i.31 ~ 0.31 T-araCTP 10.8 t 1.80 SUB6TITUTE SHEEt (RULE 26) WO 00!0486b PC?fUS99/166~.i0 Results of tests related to retention of araC'TP and T-araCTP in CEM chlls are shown in Fig. 2. The tests were carried out as follows:
' After incubation of CEM cells for 1 hour with either 5 nM ~5-3H]araC yr 200 nm [S 3H]T-araC; the cells were collected, washed with fresh medium, and resuspended in fresh medium that did not contain radialabeled nucleosides.
Samples were collected at various times after the cells were resuspended in fresh medium, and the amount of radioactivity in the 5'-triphosphate peak was determined using SAX HPLC. Tn this experiment there was 0.639 pmoles araCTPllOb cells and 0.246 pmoles of T-araC'TP after the 1 hour incubation with radiolabeIed compound.
Results related to metabolism in CEM cells are reported below in Table 12.
In particular, CEM cells were incubated with 100 nM of [5 3HjdCyd, [5-3H]T-dCyd, [5 3H]araC, or [5-3H]T-araC for the times indicated. The complete metabolism of each compound was determined: The medium was analyzed for the original compound, its deaxninated. form using reverse phase HPLC, and H~~C7;
the IS acid-soluble extract was analyzed by SAX HPLC fox phosphorylated metabolites;
and the incorporation of radioactivity into acid-precipitable material (1?NA) was determined. All of the original radioactivity was accounted for in these fractions.
Far each compound, the amount of radioactivity in the 5'-trighosphate peak was more than 9C1 % ' of the total radioaccavity eluting from the SAX HPLC column.
WC? OOlOd866 ~ 02464681 2004-04-07 . ' p~~s9911663U
Table Ix RQetabolism of dCyd, T-dCyd, scraC, and T-araC in CEM Cells Nucleoside Time OriginalDeazxtirtaiedH=O nucleoside- DNA
of IncubationCompoundCompottttd, TP
(hours) ~ (percent of mtal) dCyd 1 38 0 55 4 3 T-dCyd 1 90 0 Q 2 8 araC 1 g'7 0 4 13 0.4 T-aaaC 24 96 0 0 4 0.3 Resuhs from tests related to the effect of F-dUrd on metabolism are shown below in Tahle 13.
CEM cells were incubated with I00 nM of [S-'HJdCyd, [3-'H]T-dCyd, [~',li]araC, dr (~-'H]T-araC for the times indicated in the presence ox absence of 10 ~.m F-dUrd, which is metabolized to F-iIUMP (a potent iahibitor of thynudylate syuthetase). Tho complete meuaholism of each compound was deterntlned as described in the legend to Table 12, and the percent of deami>lated metabolites for each compound was determined_ 1a the absence of F..dUrd, the destitinated product 1~ for dCyd was [3H]13,0 due to the removal of ['H] at the 5 position of F-dUMP by chymidylate synchetase. Isi the presence of F-dUrd, the deaminsted product of dCyd, T-dCyd, and araC was in the medium.as dUrd. T-dUrd, or araU, respectively.
_ . CA 02464681 2004-04-07 W O Ii0/04866 PC?11J599116630 Table 13 Effect of F-dUrd on the M~abolism of dCyd, T dCyd, araC, or T-araC
Compound F-DUrd (~M) Intrubation Percent Deaminacion Time , ~~) S acya o ~ 32 ac~ya la 1 zz r-acya o s T-dCyd ~ 10 g ~
araC 0 g Q
exec to s to T-araC U 24 p T-araC IO 24 Q
Table I4 below reports results of tests concerning effects of DNA, 1t1~1A and protein syndtesis.
I5 CEM cells vrreze incubated with 60 EsM T-dCyd, 15Q nM azaC, or 7S0 uM T-araC.
Radiolabeled precursors of DNA [SHadThd, RNA (yi]Urd), or protein (['HJlenoine) were added to each treatment 3Q~ minutes after, the addidan of each compound. Samples were taken I , 2, 3 and 4 hours after the additlan of radialabeted precursors, and the incorpo~acion of radioactivity into RNA, DNA, or protein were determined as desezibed (Parker et al, Biocltent.
pharmncol. S5: I6~3-168I, t998). Ench number represents the average of two experiments.
SUBSTfNTE SHEET (MULE 26) WO Ot1/0486b CA 02464681 2004-04-07 ~ pCTlUS99I16G30 Table 14 Effect of T~dCyd, araC, and T-araC on DNA, RNA, and Protein Syntheses Compound DNA RNA ~ protein Percent of control 60 ~cM T-dCyd 3B 95 78 150 nM araC g 9b 96 750 nRri T ataC 16 8? 72 As can be appreciated ftoTn the above, thioarobitzofuranosyl compounds according to the present invention can be used to inhibit DNA replication in mammalian cells.
These results tend to suggest their ability as itttmunomodtiiatars, which would render them suitable for seating . .
autolmmune diseases. This is further supported by the fact that several guanosine analogs have bees shown tv stimulate the immune system (VW'eigic, W.O., CRD Crit.Rev.lmmunal., 1987, 7, 285; Lin ec al, .l.Med.Chetrl., 1985, 28, 1194-1198: Reitzet al, J.Med.Chem., 1994, 3?, 3561-35?8; Michael et al., l.Med.Chent., 1993, 3G. 3431-3436). Certain 3-B-ribofuranosylthiazalo(4.,5-d]pyrimidines hnve also been shown to have significant immunoactivity, including marine spleen cell proliferation and in vivo activity against Semliki Forest virus (Nagahara et aL, J
Med.Chem., 1990, 33, 40?-4iS).
The foregoing description of the invention illustrates and describes the present invention.
Additionally. the disclosure shows attd describes only the preferred embodiments of the invenuott but, as mantioned above, it is to be understood that errs irlvencion is capable of use in various other 24 combinations, modifications, and environments and is capable of chaztges or modifications within the scope of the inventive concept as expressed herein, comtttensurate with the above teachiFtgs andlor the skill or knowledge of the relevant axt. The embodiments described hezcinabove are further intended to explain best modes known of .SUBSTlf UTE SHEET ~RULF 2~
CA 02464681 2004-04-07 F(~T~f~,~9~'~~~3~
practicing the invention and to enable others skilled in the art to utilise the invernion in such, or ocher, embodiments and with the various modifications required by the gartieular applications or uses of the invention. Accordingly, the description as not intended to limit the invention to the form disclosed herein. Also, it is intended that the appended claims be construed to include alternative embodiments.
Claims (19)
1. A method far inhibiting DNA replication in a mammalian cell which comprises:
wherein each R individually is H, an aliphatic aryl group or an aromatic acyl group;
A is selected from the group consisting of wherein X is selected form the group consisting of hydrogen, fluorine, alkoxy, alkyl, haloalkyl, alkenyl, haloalkenyl, alkynyl, amino, monoalkylamino, dialkylamino, cyano and nitro.
wherein each R individually is H, an aliphatic aryl group or an aromatic acyl group;
A is selected from the group consisting of wherein X is selected form the group consisting of hydrogen, fluorine, alkoxy, alkyl, haloalkyl, alkenyl, haloalkenyl, alkynyl, amino, monoalkylamino, dialkylamino, cyano and nitro.
2. The method of claim 8 Wherein each R is H.
3. The method of claim 8 wherein A is
4. The method of claim 10 wherein X is H.
5. The method of claim 8 wherein X is H.
6. The method of claim 8 wherein said compound is 1-(4-thio-.beta.-D-arabinofuranosyl) cytosine:
7. A method for producing a compound of the formula 1 which comprises:
A) reacting a 2,3,5-tri-O-aryl or alkyl-4-xylose diaryl or dialkyl dithioacetal in the presence of a leaving group at the 4 hydroxyl position to produce the corresponding 1, 4-dithio-D-arabinofuranoside;
B) reacting said arabinofuranoside from step A) to acidolysis to form the corresponding O-acetyl-4-thio-D-arabinofuranose;
C) reacting said O-acetyl-4-thio-D-arabinofuranose from step B) with a compound selected from the group consisting of a cytosine; a 5-aza compound, a aza compound and blocked derivatives thereof to form the corresponding 4-thio-.alpha.,.beta.-D-arabinofuranosyl compound;
D) converting said arabinofuranosyl compound from step C) by hydrolysis to the corresponding thio sugar derivative; and E) separating out the a form of the anomeric mixture of step D) to thereby obtain the compound of formula 1.
A) reacting a 2,3,5-tri-O-aryl or alkyl-4-xylose diaryl or dialkyl dithioacetal in the presence of a leaving group at the 4 hydroxyl position to produce the corresponding 1, 4-dithio-D-arabinofuranoside;
B) reacting said arabinofuranoside from step A) to acidolysis to form the corresponding O-acetyl-4-thio-D-arabinofuranose;
C) reacting said O-acetyl-4-thio-D-arabinofuranose from step B) with a compound selected from the group consisting of a cytosine; a 5-aza compound, a aza compound and blocked derivatives thereof to form the corresponding 4-thio-.alpha.,.beta.-D-arabinofuranosyl compound;
D) converting said arabinofuranosyl compound from step C) by hydrolysis to the corresponding thio sugar derivative; and E) separating out the a form of the anomeric mixture of step D) to thereby obtain the compound of formula 1.
8. The method of claim 14 wherein said compound is step C) is cytosine.
9. The method of claim 14 wherein said hydrolysis in step D) comprises reacting said arabinofuranosyl compound from step C) with boron trichlorides.
10. The method of claim 14 wherein step E) comprises reacting the racemic mixture from step D) with 4,4'-dimethoxytritylcholoride to thereby produce 5'-O-dimethoxytrityl-4'-thio-.alpha.-.beta.-D-arabinofuranosylcytosine, separating 5'-O-dimethoxytrityl-4'-thio-.beta.-D arabinofuranosylcytosine; and then reacting S'-O-dimethoxytrityl-4'-thio-.beta.-D arabinofuranosylcytosine with trifluoroacetic acid to thereby obtain the compound of formula 1.
11. The method of claim 14 wherein said dithioacetal compound in step A) comprises 2,3,5-trio-O-benzyl-xylose dibenzyl dithioacetal.
12. The method of claim 14 wherein said dithioacetal is obtained by reacting L-xylose with methanol to produce methyl L-xylofuranoside and reacting said methyl L-xylofuranoside with benzyl bromide.
13. The method of claim 1 wherein A is:
14. The method of claim 20 wherein X is H.
15. The method of claim 7 wherein each R is H.
16. The method of claim 7 wherein A is:
17. The method of claim 23 wherein X is H.
18. The method of claim 7 wherein X is H.
19. The method of claim 7 wherein said compound is 1-(4-thin-.beta.-D-arabinofuranosyl) cytosine.
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| US9386998P | 1998-07-23 | 1998-07-23 | |
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| CA002338415A CA2338415C (en) | 1998-07-23 | 1999-07-23 | Preparation of thioarabinofuranosyl compounds and use thereof |
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