This invention relates to improved anthracycline derivatives, to processes and to intermediates for preparing the derivatives, to pharmaceutical compositions containing them and to the use of these derivatives as antitumor agents in mammals. More specifically, the present invention relates to 8- heteroanthracycline derivatives.

BACKGROUND OF THE INVENTION

Anthracycline antibiotics including doxorubicin and daunorubicin are important chemotherapeutic agents in the treatment of a broad spectrum of neoplastic conditions. While daunorubicin (1) is clinically used mainly against acute childhood and adult leukemias, doxorubicin (2) , also known as adria ycin, has the widest spectrum of antitumor activity of all chemotherapeutic agents (Weiss, R.B., Sarosy, G., Clagett-Carr, K. , Russo, K. and Le land-Jones, B., Cancer Chemothβr. Pharmacol., 1£, 185-197, 1986; Arcamone, F., Doxorubicin, Academic Press, New York, 1980) .

SUBSTITUTE SHEET

The usefulness of known anthracycline antibiotics is compromised by dose limiting toxicities such as myelosuppression (Crσoke, S.K., Anthracyclines; Current Status and New Developments, Academic Press, N.Y. 1980) and cardiotoxicity (Olson, R.D. et al, Proc. Natl. Acad. Sci., USA £5 3585-3589, 1988 and references therein) as well as the resistance from treated tumors (Mi naugh, E.G. et al. Cancer Research, 42.. 8-15, 1989; McGrath, T. et al, Biochemical Pharmacology, 23.497-501, 1989) . In view of the proven effectiveness of known anthracyclines in the treatment of cancer, efforts have been undertaken to develop anthracycline analogs with either an improved therapeutic index or with reduced cross-resistance.

Several thousands of anthracycline derivatives have been obtained either from streptomyces biosynthesis or via the semisynthetic modification of known natural anthracycline antibiotics (Arcamone, F. , Doxorubicin, Academic Press, N.Y. 1980; Thomson, R.H., Naturally Occurring Quinones III: Recent Advances, Chapman and Hall, New York 1987; Anthracyclines: Current Status and New Developments, Academic Press, New York, 1980; Brown, J.R. and Iman, S.H., Recent Studies on Doxorubicin and its Analogues, Prog. Med. Chem. 21 170-236, 1984; Brown, J.R. Adriaaycin and Related Anthracycline Antibiotics, Prog. Med. Chem., 15. 125-164, 1978). The majority of known anthracyclines show two types of structural differences: (i) the substitution pattern of the aglycone tetracyclic ring system, and (ii) the structure and number of glycosides attached at C-7 or

SUBSTITUTE SHEET

c-10 (doxorubicin numbering) . Some examples of the structural diversity of anthracycline antibiotics are shown in Figure 1.

UBSTITUTE SHEET

In contrast to the great number of derivatives obtained from these two kinds of structural modifications, there has been little effort towards the synthesis and biological evaluation of ring-A heteroanthracycline derivatives. Some 9-oxa-hetero- anthracyclines (2-5) were prepared by Koch et al but antitumor activity was not significant (Heterocycles, 26(2), 341-5, 1987; Heterocycles_2£(4) , 879-82, 1987). Mitsher et al found that N-(trifluoroacetyl)-4- demethoxy-9-azadaunorubicin (£) had no antitumor activity (J. Med. Chem.. 29 (1) . 1277-81, 1986).

Pyranonaphthoquinones such as nanaomycin A (2) and kalafungin (£) occur naturally and show potent antibacterial as well as antifungal activity (Moore, H.W. and Czβrniak, R. , Medicinal Research Reviews, 1(3), 249-280, 1981 and references therein). Granaticin (2.) has been reported to show antitumor activity (Chang, C.J. , Floss, H.G., Soong, . and Chang, C.T., J. Antibiot., 2_lt 156, 1975).

SUBSTITUTE SHEET

The present invention provides, heteroanthracyclines which are structurally distinguished from the prior art compounds by the nature of ring A of the anthracyclinone moiety. More specifically, the compounds of the present invention are structurally distinguished from the prior art compounds by having an hetero atom at position 8 of the ring A of the anthracyclinone. This structurally distinct class of compounds exhibits therapeutic activity, in particular anticancer and antitumor activity, are active against some adriamycin-resistant tumor cells, and also may potentially display less myelosupreββion•

In one aspect of the invention, there is provided a compound of the formula (10):

EET

10

wherein

X _r and X ₂ are independently selected from the group consisting of

O,

S,

ON(R) , wherein R is selected from the group consisting of hydrogen,

C _l . ₁₆ alkyl,

C]_ ₁₆ acyl and,

C ₁ „ ₁₆ alkyla ine, X ₃ is selected from the group consisting of

O,

S, so, so ₂ ,

NO, and

NR, wherein R is selected from the group consisting of

^C l-1« ^acv1 '

Cι- ₁₆ aUcyl, ^c l-lβ "TT ¹ ' ^c ι-lβ haloacyl, and hydrogen;

SUBSTITUTE SHEET

R _χ , R ₂ , R ₃ , R ₄ , R ₃ and R ₈ are independently selected from the group consisting of hydrogen, hydroxyl,

C _λ _ ₁₆ alkyl,

C ₁ _ _lβ alkoxyl, ^c ₃ - ₈ cycloalkyl, tosyl, triflate, trifluoroacetate, halogen, nitro, cyano, ^C ι- ₁₆ arylacyl,

am inoa Iky laminoa lcoho 1 of formula NH(CH ₂ ) _N NH(CH ₂ ) _a OH where n and m independently range from 1 to 4, aminoalkylaminoalkylhalide of formula NH(CH ₂ ) _n NH(CH ₂ ) _m X where n and m independently range from 1 to 4 and X is a halogen, amino which may be unβubβtituted or mono- or di-substituted by c ₁ . ₈ alkyl, C ₃ _ _β cycloalkyl, acyl, tri luoroacy 1 , aralkyl or aryl, C^.g alkenyl, C _1-β alkynyl, halaoalkylnitrosureido of the formula NH(CO)N(NO) (CH ₂ ) _n CH ₂ X, wherein n is 0 to 4 and X is a halogen, thiol, and a group of the formula -0-C(R)-0 wherein R is selected from the group consisting of

SHEET

hydrogen,

Ci- ₁₆ alkyl, ^c 3~8 cycloalkyl, alkoxyalkyl, aralkyl, araloxyalkyl, aryloxyalkyl and aryl; 6 is selected from the group consisting of hydrogen, Cι_!6 alkyl, C3-8 cycloalkyl, acyl of the formula -C(R)=0, or its dioxolane or dioxane ketal wherein R is selected from the group consisting of hydrogen,

Cl-16 alkyl, ^c 3-8 cycloalkyl, hydroxyalkyl, alkoxyalkyl, araloxyalkyl, acyloxyalkyl, amino which may be unsubstituted or mono- or di-substituted by Cχ_g alkyl,

C3-8 cycloalkyl, acyl, trifluoroacyl, aralkyl or aryl, and a naturally occurring amino acid, for example alanine, arginine, cysteine, glycine, leucine, lysine, methionine and the like, or a synthetic amino acid; a group of the formula -C(R)(0Cι_5 alkyl)2 where R is as defined above a group of the formula -C(OR)=0, wherein R is selected from the group consisting of hydrogen,

BSTITUTE SHEET

^c ι-i6 alkyl,

C ₃ _ ₈ cycloalkyl, hydroxyalkyl, alkoxyalkyl, aryloxyalkyl, araloxyalkyl, aryl and aralkyl; a group of the formula -CH ₂ C(OR)«o, wherein R is selected from the group consisting of hydrogen, ^C ι- ₁₆ alkyl, C ₃ _ ₈ cycloalkyl, hydroxyalkyl, alkoxyalkyl, aryloxyalkyl, araloxyalkyl, aryl, aralkyl, and amino, which may be unsubstituted, mono- or di-substituted by C^g alkyl, C ₃ . ₈ cycloalkyl, acyl, trifluoroacyl, aralkyl or aryl;

R ₆ ^» is selected from the group consisting of hydrogen, C _l . ₁₆ alkyl, halogen, amino, hydroxy, ^c l-1 ₆ alkoxy, thiol, cyano,

TE SHEET

sulfide, acyl of the formula -C(R)-o, wherein R is selected from the group of hydrogen, C _x _ ₁₆ alkyl, ^c ₃ -β cycloalkyl, hydroxyalkyl, araloxyalkyl, alkoxyalkyl, acyloxyalkyl, amino which may be unsubstituted or mono- or di- substituted by alkyl, C ₃ . ₈ cycloalkyl, acyl, trifluoroacyl, aralkyl or aryl, and a naturally occuring amino acid, for example alaninβ, arginine, cysteine, glycine, leucine, lysine, methionine and the like, or a synthetic amino acid;

A group of the formula -C(OR)«o, wherein R is selected from the group consisting of hydrogen,

C _1-16 alky ,

C ₃ . _β cycloalkyl, hydroxyalkyl, alkoxyalkyl, aryloxyalkyl, araloxyalkyl, aryl and aralkyl;

^C ι-16 al* ^β nyl Y and S ₇ are independently selected from the group consisting of

TITUTE SHEET

11

hydrogen, halogen, hydroxyl, ^c l-1 ₆ alkoxyl, C _λ _ ₁₆ alkyl, ^C 2-16 acetylenyl, C ₃ _ ₈ cycloalkyl, C ₂ _ ₁₆ alkenyl, cyano, a group of the formula -0-C(R)«0, wherein R is selected from the group consisting of hydrogen,

C _1-16 alkyl, ^c ₃ -β cycloalkyl, and alkoxyalkyl, aryl; an acyl of the formula -C(R)«0, wherein R is selected from the group consisting of hydrogen, thiol,

^C ι-1 ₆ thioalkyl,

^C l-16 alkyl# C ₃ -e cycloalkyl, hydroxyalkyl, alkoxyalkyl, araloxyaIky1, acyloxyalkyl, amino which may be unsubstituted or mono- or di-substituted, and a naturally occurring amino acid as defined above or a synthetic amino acid; a group of the formula -C(0R)«0, wherein R is selected from the group consisting of

TUTE SHEET

hydrogen,

C ₁ . ₁₆ alkyl, and

C ₃ _ ₈ cycloalkyl; and mono or oligosaccharides commonly present in other anthracyclines, for example one or more sugars selected from rhodosamine, cinerulσse-B, L-cinerulose, O-cinerulose, cinerulose-A, amicetose, aculose, rednose, rhodinosβ, 2-deoxyfucose, and a saccharide of

wherein

R ₉ and R ₁₀ are independently selected from the group consisting of hydrogen, halogen, hydroxy, acetoxy,

C _1-16 alkoxy,

C _1-16 alkyl,

C ₃ _ ₈ cycloalkyl, and thiol;

R _χl is selected from the group consisting of amino, which may be unsubstituted or mono or di- subβtituted by C ₁ _ ₈ alkyl, C ₃ _ ₈ cycloalkyl, acyl, tri luoroacyl, aralkyl or aryl and a naturally occuring amino acid, for example alanine, arginine, cysteine, glycine, leucine, lysine,

SUBSTITUTE SHEET

methionine and the like, or a syntehtic amino acid; mono or dibenzylated amino, acylated amino, trifluoroacylated amino, πorpholino, cyano substituted morpholino, mono-, di-, tri- or tetra-methoxy substituted morpholino, mono-, di, tri-or tetra-acetoxy substituted morpholino, hydroxyl, hydrogen, halogen, acetoxy, ^C _l - ₁₆ alkoxyl, C ₃ _ ₈ cycloalkyl, thiol, sulfide, a group of the formula NH(CH ₂ ) _N CH(0R) ₂ , wherein R is independently selected from the group consisting of a C ₁ _ ₁₆ alkyl, c _1-16 acyl or C ₇ _ ₁₆ aroyl and wherein n is 0 to 5, chloroalkylnitrosoureido of the formula NH(CO)N(NO) (CH ₂ ) _n CH ₂ Cl, wherein n is 0 to 4; R ₁₂ is selected from the group consisting of hydrogen, hydroxyl or its tetrahydropranyl ether (-OTHP) , halogen, mono or oligosaccharides commonly present in other anthracyclines such as those defined above for R ₇ ,

EE

ammo, mono or dialkylated amino in which each alkyl contains 1 to 16 carbon atoms,

C _1-16 alkoxy, ^c ₃ - ₈ cycloalkyl, benzoate which may be unsubstituted or substituted with nitro, for example p-nitrobenzoate, acetoxy, and trifluoroacetoxy. chloroalkyl-nitrosoureido as defined above for

^R u

Preferred compounds of formula (10) are those wherein

Xj, and X ₂ are independently selected from the group consisting of

O, s, and

NH;

X ₃ is selected from the group consisting of

O,

S,

SO,

S0 ₂ ,

NH, and

NO; R _χ , R _a R ₃ , R , Rg and K ₈ are independently selected from the group consisting of hydrogen, hydroxyl,

C _1-4 alkoxy1, tosyl, tri late,

SHEET

fluorine, chlorine, aminoa Iky 1 aminoa lcohol of formula

NH(CH ₂ ) _n NH(CH ₂ ) _ffl OH where n and m independently range from 1 to 3, aminoalkyla inoalkylchloride of formula

NH(CH ₂ ) _n NH(CH ₂ ) _m Cl where n and m independently range from 1 to 3. amino, chloroalkylnitrosoureido of the formula

NH(C0)N(N0) (CH ₂ ) _n CH ₂ Cl, wherein n is 0 to 4, and a group of the formula -0-C(R)«0, wherein R is selected from the group consisting of hydrogen, C^g alkyl, and aryl;

R ₆ is selected from the group consisting of hydrogen, C _λ _ ₄ alkyl, acyl of the formula -C(R)-0, wherein R is selected from the group consisting of hydrogen,

C ₁ . _β alkyl, hydroxylalkyl, alkoxyalkyl, acyloxyalkyl and amino which may be unsubstituted or mono- or di-substituted with C ₁ . ₈ alkyl, C ₃ _ ₈ cycloalkyl, acyl, trifluoroacyl, aralkyl or aryl; a group of the formula -C(OR)-0, wherein R is

TE SHEET

selected from the group consisting of hydrogen, Ci. _Q alkyl, aryl, aralkyl; and a group of the formula -CH ₂ C(OR)«0, wherein R is selected from the group consisting of hydrogen, straight or branched C ₁ _ ₈ alkyl, and amino which may be unsubstituted or mono- or di-substituted with C ₂ _ ₈ alkyl, C _3-8 cycloalkyl, acyl, trifluoroacyl, aralkyl or aryl; R _β ' is selected from the group consisting of hydrogen, fluorine, amino,

C _1-4 alkoxy, sulfide, acyl of the formula -C(R)«0, where R is selected from the group of hydrogen,

C _1-8 alkyl, hydroxyalkyl, acyloxyalkyl, amino, cyano, a group of the formula -C(OR)«0, wherein R is selected from the group consisting of hydrogen,

C _j . ₈ alkyl, aryl,

C _j . _β alkenyl;

ITUTE SHEET

Y and R ₇ are independently selected from the group consisting of hydrogen, halogen, hydroxyl, C _1-8 alkoxy, ^c ₂ - ₈ acetylenyl, C ₂ _ ₈ alkenyl, cyano, a group of the formula -0-C(R)»0, wherein R is selected from the group consisting of hydrogen, and C _j .g alkyl; acyl of the formula -C(R)-0, wherein R is selected from the group consisting of hydrogen, thiol, C ₁ . ₈ alkyl, hydroxyalkyl, amino; a group of the formula -C(0R)-0, wherein R is selected from the group consisting of hydrogen, and Ci.galkyl, and a saccharide of formula

wherein

R ₉ and R ₁₀ are independently selected from the group

TUTE SHEET

consisting of hydrogen, fluorine, chlorine, and hydroxyl;

R _u is selected from the group consisting of amino which may be unsubstituted or mono- or di-substituted with C _j . _g alkyl, C ₃ _ _β cycloalkyl, acyl, trifluoroacyl, aralkyl or aryl; morpholino, cyano substituted morpholino, mono-, di-, tri-, or tetra-methoxy substituted morpholino, hydroxyl, mono or dialkylated amino with 1 to 16 carbons,

C _1-8 alkoxyl, a group of the formula NH(CH ₂ ) _N CH(OR) ₂ wherein R is independently selected from a group consisting of C^ _β alkyl, C _1-8 acyl or C ₇ _ ₁₂ aroyl and wherein n is 1 to 5; chloroalkylnitrosoureido of the formula

NH(CO)N(NO) (CH ₂ ) _n CH ₂ Cl wherein n is 0 to 4 , and fluorine; and

R ₁₂ is selected from the group consisting of hydroxyl or its tetrahydopyranyl ether, halogen, mono or oligosaccharide commonly present in other anthracyclines, for example one or more sugars selected from rhodosamine,

HEET

cinerulose-B, L-cinerulose, O-cinerulose, cinerulose A, amicetose, aculose> rednose, rhodinose, 2-deoxyfucose, daunosamine and trifluoroacetyldaunosamine, amino, mono or di ethylated amino,

C _i .g alkoxy, benzoate, p-nitrobenzoate, acetoxy and trifluoroacetoxy.

More preferred compounds of formula (10) are those wherein

X _1# and X ₂ , are independently selected from the group consisting of

0, and

NH; X ₃ is selected from the group consisting of

0,

S,

SO,

NH,

NO; ^R i' ^R ₂ ' ^R ₃ ' ^R «' ^R ₅ ^{and R} β ^are independently selected from the group consisting of hydrogen, hydroxy, methoxy, aminoethylaminoethanol aminoethylaminoethylchloride chloroalkylnitrosoureido of the formula

NH(C0)N(N0) (CH ₂ ) _n CH ₂ Cl, wherein n is 0 to 2 ,

E SHEET

amino, and fluorine;

R ₆ is selected from the group consisting of C _1-4 alkyl, acyl of the formula -C(R)-0, wherein R is selected from the group consisting of methyl, hydroxymethy1, acyloxymethyl and amino; a group of the formula -C(OR)»0, wherein R is selected from the group consisting of hydrogen, methyl and ethyl; a group of the formula -CH ₂ C(OR)«0, wherein R is selected from the group consisting of hydrogen, methyl and ethyl; Rg' is selected from the group consisting of hydrogen, fluorine, amino, methoxy, acyl of the formula -C(R)«0, wherein R is selected from the group of hydrogen, C _x _. _s alkyl, hydroxyalkyl, amino, cyano,

TUTE SHEET

a group of the formula -C(OR)=o, wherein R is selected from the group consisting of hydrogen,

C^g, alkyl, aryl,

C^, alkenyl;

Y and R ₇ are independently selected from the group consisting of hydrogen, halogen, hydroxy, methoxy, cyano, acetate, acetyl and a saccharide of formula

wherein _j and R ₁₀ are independently selected from the group consisting of hydrogen and fluorine;

R _u is selected from the group consisting of amino, di ethyla ino,

EET

trifluoroacetamido, morpholino, cyano substituted morpholino, mono-, di-, tri-, or tetra-methoxy substitiuted morpholino, a group of the formula NH(CH ₂ ) _N CH(0R) ₂ wherein R is selected from a group consisting of C ₁ _ ₄ alkyl ^c ι- ₄ acyl or C ₇ _ ₈ aroyl and wherein N is 2 to 5, chloroalkylnitrosoureido of the formula

NH(C0)N(N0) (CH ₂ ) _n CH ₂ Cl, wherein n is 0 to

4, and hydroxyl; and

R ₁₂ is selected from the group consisting of hydroxyl or its tetrahydopyranyl ether, benzoate, p-nitrobenzσate, amino, and fluorine.

Further preferred compounds of the formula (10) are those wherein X _χ and X ₂ are both oxygen; X ₃ is selected from the group consisting of

0,

S,

SO,

NH,

NO;

R ₁ R ₂ , R ₃ and R each are hydrogen;

R _s and R ₈ are independently selected from the group

STITUTE SHEET

consisting of hydrogen, hydroxyl, amino, aminoethylaminoethanol, Rg is selected from the group consisting of methyl, ethyl, acyl of the formula -C(R)=0, or its dioxolane or dioxane ketal wherein R is selected from the group consisting of methyl, hydroxymethyl, acetoxymethyl, and amino; a group of the formula C(OR)=0, wherein R is selected from the group consisting of hydrogen and methyl; and a group of the formula -CH2C(0R)=0, wherein R is selected from the group consisting of hydrogen and methyl; Rg' is selected from the group consisting of hydrogen, fluorine, amino, cyano, and a group of the formula -C(OR)=0, wherein R is selected from the group consisting of

Cι_4 alkyl, Y and R7 are independently selected from the group

SHEET

24

consisting of hydrogen, hydroxyl, bromine, chlorine, cyano, acetate, acetyl and a saccharide of

wherein R ₉ and R ₁₀ are independently selected from hydrogen and fluorine;

R _1X is selected from amino, dimethylamino, trifluoroacetamido, morpholino, cyano substituted morpholino, mono-, di-, tri- or tetra-methoxy substituted morpholino and a group of the formula NH(CH ₂ ) _N CH(OR) ₂ wherein R is selected from a group consisting of methyl, acyl or benzoyl and wherein N is 3 to 5 _r chloroalkylnitrosoureido of the formula NH(C0)N(N0)(CH ₂ ) _n CH ₂ Cl, wherein n is 0 to 4, and

ITUTE SHEET

R ₁₂ is selected from hydroxyl, benzoate and p- nitrobenzoate.

The term "alkyl" as employed herein includes both straight and branched chain radicals of up to 16 carbons, for example methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4, 4-dimethylpentyl, octyl, 2,2,4- trimethylpentyl, nonyl, decyl, undecyl, dodecyl, the various branched chain isomers thereof, as well as such groups including one or more halo substituent, such as F, Cl, Br, I or CF ₃ , an alkoxy substituent, an aryl substituent, an alkyl-aryl substituent, a haloaryl substituent, a cycloalkyl substituent or an alkylcycloalkyl substituent.

The term "cycloalkyl" as used herein means a cycloalkyl group having 3 to 8 carbons, for example cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl, cycloheptyl and cyclooctyl.

The term "aryl" as employed herein refers to monocyclic or bicyclic aromatic groups containing from 6 to 10 carbons in the ring portion, such as phenyl, naphthyl, substituted phenyl or substituted naphthyl, wherein the substituent on either the phenyl or naphthyl may be for example C _1-4 alkyl, halogen, C _1- alkoxy or nitro.

The term "halogen" as used herein means chlorine, bromine, fluorine or iodine.

The term "aralkyl" as used herein refers to alkyl groups as discussed above having an aryl substituent, such as benzyl, p-nitrobenzyl, phenethyl, diphenylmethyl, and triphenylmethyl.

SHEET

The term "aroyl" as used herein refers to a group of the formula -COAr wherein Ar denotes an "aryl" group as defined above.

The term "alkoxy" or "aralkoxy" as used herein includes any of the above alkyl or aralkyl groups linked to an oxygen atom.

The term "alkoxyalkyl" as used herein means any alkyl as discussed above linked to any alkoxy as discussed above, for example methoxymethyl.

The term "aryloxyalkyl" as used herein means any alkyl as discussed above linked to an aryl as discussed above by an oxygen atom, for example phenoxymethy1.

The term "araloxyalkyl" as used herein means an aralkyl as discussed above linked to an alkyl as discussed above by an oxygen atom, for example benzyloxymethyl.

The term "acyloxyalkyl" as used herein means an C _1-8 acyl group linked to an alkyl group as discussed above by an oxygen atom, for example acetσxymethyl.

The term "hydroxyalkyl" as used herein means an alkyl group as discussed above bonded to a hydroxyl group, for example hydroxymethyl.

It will be appreciated by those skilled in the art that when R ₅ »R ₈ » hydroxyl and X ₁ =X ₂ =0 that compounds of formula (2SL) exist in the form of the thermodynamically favored tautomers of formula (ϋ) . Therefore, compounds of formula (H) are included within the scope of the invention.

BSTITUTE SHEET

27

(X _j -X ₂ » 0 and R ₅ = R = OH )

This invention also includes all the possible isomers and mixtures thereof, including diastereoisomeric mixtures and racemic mixtures, resulting from the possible combination of R or S stereσchemical centers, when pertinent, at C-7, C-9 and C-10 as well as in all the chiral centers present in the sugar moiety.

This invention also comprises novel compounds which are prepared as intermediates or precursors of compounds of formulas (10) and (11) . Such intermediate compounds are described hereinafter in connection with processes of preparing compounds of formulas (10) and

(11).

The compounds of formula (10) can be prepared by the process illustrated in Reaction Scheme I.

12 13

TE SHEET

Heteroanthracyclines of general formula (10) in which Y is a saccharide are best prepared by using scheme I. With reference to Reaction Scheme I, an aglycone of formula (12) in which _x to R ₈ are as defined herein is reacted with a sugar derivative of formula (13) in which R _g to R ₁₂ are as defined herein and L is a displaceable atom or group. Suitable groups L include halogen, for example iodine, bromine or chlorine, an unsubstituted or substituted benzoyl group such as p-nitrobenzoyl, and -OR or -SR, where R is an unsubstituted or substituted alkyl group, for example a C _l . ₁₆ alkyl group such as methyl, ethyl or butyl, or R is an unsubstituted or substituted acyl group such as a C ₁ _ ₁₆ acyl group such as acetyl, or R is an unsubstituted or substituted aryl group. Such sugars are obtained by derivatizing known saccharides of the family of anthracycline antibiotics which are available from commercial or natural sources, (see for example, Manneret, C. , Martini, A., Pais, M., Carbohydrate Research, 166, 59- 70, 1987 and references therein; Acton, E.M., Tong, G.L., Mosher, C. . , and Wolge uth, R.L., J. Med. Chem. 27, 638- 645, 1984 and references therein; Arcamone F. , Cancer Research, 45, 5995-5999, 1985 and references therein).

The aglycone of formula (12) is typically reacted with the appropriate sugar derivative of formula (13) in a compatible solvent such as methylene chloride using a Lewis acid such as titanium tetrachloride, stannic chloride, of trimethylsilyltrifluoromethane-sulfonate. Alternatively, as it is known in the art of anthracycline chemistry, when the leaving group of the sugar moiety is a halogen, the Koenigs-Knorr glycosidation or its modification may be used.

A method of preparing the compounds of formula (12) in which X _lr X ₂ , X ₃ * 0 and R ₆ ' = hydrogen, is illustrated in Reaction Scheme II.

SHEET

Re ⁱ ction Scheme II

SUBSTITUTE SHEET

With reference to Reaction Scheme II, 2,5- dimethoxyben-zaldehyde dioxane acetal (15) can be prepared by treating at reflux 2,5-dimethoxybenzaldehyde (14) with 1,3-propanediol in benzene or any other suitable solvent and with an acid catalyst such as p-toluenesulfonic acid. The dioxane acetal (15) can then be treated with an alkyl lithium such as n-butyllithium and the lithio salt reacted with an appropriate alkyl halide of the formula R ₇ CH ₂ X, wherein X is halogen and R ₇ is as defined above, but compatible with the necessary reaction conditions. In the event that R ₇ is not compatible, functional group interconversion can be carried out at a latter step by using methods which are well known by one familiar with the art of organic synthesis.

Subsequent aqueous acidic treatment can lead to an appropriate 2,5-dimethoxy-6-alkylbenzaldehyde such as (16). Photochemical irradiation of an intermediate such as (16) in a solution of S0 ₂ in an aryl solvent such as benzene can give a dihydrothiophene-2,2-dioxide of formula (17) which can then be reduced with a borσhydride. Following acidic treatment, a £-sultine of formula (18) can be obtained. This intermediate can then be coupled via cyclocondensation with an appropriately functionalized aldehyde (19) and consequently yield dimethoxyisochroman intermediates such as (20) . The intermediates (20) can subsequently be chlorinated with an hypochlorite such as t- butylhypochlorite to give compounds of formula (21) _* Oxidative demethylation of (21) with an adequate oxidant such as eerie ammonium nitrate can give chloropyranoquinones of formula (22) . These quinones can then be coupled under basic conditions with adequately functionalized homophthalic anhydrides such as (23) to give pyranoquinone tetracyclic derivatives of formula (24). The

TITUTE SHEET

free phenol can then be protected to give tetracyclic compounds of the general formula (25) . Tetracyclic derivative (25) can then be brominated with a free radical brominating agent such as n-bromosuccinimide in the presence of an initiator such as UV light and in a chlorinated solvent such as carbon tetrachloride. The resulting unstable bromides can then be treated directly with an aqueous-ethereal solvent system to yield the pyrano-tetracyclic aglycones of formula (12) The aglycσnes of formula (12) can further be transformed to a variety of structures by using synthetic methodologies well understood in the art of anthracycline synthesis. Any functional group interconversion or removal of protecting groups is preferentially carried out under neutral or basic conditions at this stage or later in the synthesis, as convenient.

A variation for preparing the compounds of formula (12) is illustrated in Reaction Scheme III.

EET

Reaction Scheme Ifl

25

12

SUBSTITUTE SHEET

With reference to Reaction Scheme III, dimethoxy¬ isochroman intermediates such as (20) can be directly oxidatively demethylated with an oxidizing reagent such as eerie ammonium nitrate in a polar solvent system such as acetonitrile in water. The resulting pyranoquinones of formula (26) can then be coupled with appropriately functionalized homophthalic anhydrides (23) in an aprotic solvent with basic catalysis, preferentially lithium diisopropyla ide or sodium hydride. Tetracyclic derivative (27) can be separated from (24) . The free phenol can then be protected to give compounds of the general formula (25) . Bromination and solvolysis as described can give the aglycones of formula (12).

Preferred processes for the preparation of the compounds of formula (12) are illustrated in reaction Scheme IV. In route a of Scheme IV, the lithio salt, obtained after treatment of 2,5-dimethoxybenzaldehyde dioxane acetal (15) with an alkyl lithium, is reacted with an epoxide of general formula (31) optionally in the presence of a Lewis acid such as boron trifluoride etherate to give the adduct of formula (32) .

Route b _. represents an alternative approach for the preparation of adduct (32) . Consequently, the addition of an aldehyde of general formula (28) to the lithio salt of 2,5-dimethoxybenzaldehydedioxane acetal (15) can give an adduct of formula (29). In aldehyde (28), R' ₂ is a protecting group, which includes, but is not limited to, methoxyraethyl, methoxyethyl, methyl, benzyl, trityl, t- butyldimethylsilyl, t-butyldiphenylsilyl or other groups conveniently used for the protection of alcohols in the art of organic synthesis. The hydroxyl in formula (29) can be transformed to a variety of functionalities by using general methods obvious to those familiar with organic

34

synthesis. Therefore, compounds of formula (30) can be obtained which upon deprotection of the secondary protected alcohol, by using known methods, yield the desired substituted benzaldehydedioxane acetal adduct (32) .

The adduct of formula (32) is then cyclized in the presence of a mild aqueous acid to give 1-hydroxyisochroman of the formula (33)-

The tetracyclic aglycones (39) and (40) can be prepared according to route c. Pyranoquinσnes of formula (34) are prepared by oxidizing isochromans of formula (33) with an agent such as silver oxide or eerie ammonium nitrate. Quinones of formula (35) , in which the hydroxyl has been protected with a group R' ₂ , selected but not limited to, methyl, ethyl, methoxymethyl, methoxyethy1, trimethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, benzyl, p-nitrobenzyl and trityl, can then be added to an homophthalic anhydride of formula (23) in the presence of strong base, or to a benzo onoketene of formula (23') under U.V. irradiation (Krohn K., et al, Liebigs Ann. Chem. 943- 948 [1988]), to yield, after deprotection, the tetracyclic aglycones of general structures (39) and (40) .

The tetracyclic aglycones (39) and (40) can also be prepared according to route d. from isochroman (33) which after bis chlorination with a reagent such as t-butyl hypochlorite gives isochromans of formula (36) . Subsequent oxidation with an agent such as eerie ammonium nitrate yields bischloropyranoquinones of formula (37) . Addition of these quinones to o-quinodimethanes, generated thermolytically from benzosulfones such as (38) , can give tetracyclic aglycones of formula (39) and (40) . Compounds of the formula (12) are then accessible from these aglycones through functional group interconversion of the phenol group.

TE SHEET

Keaciion Scheine (V

SUBSTITUTE SHEET

36

A method of preparing compounds of formula (12) wherein R ₈ =R ₅ =H, is illustrated in Reaction Scheme V.

Reaction Scheme V •

— 4 (12,R _g -R _j -H)

With reference to Reaction Scheme V, the cycloaddition reaction between an o-quinσdimethane reactive intermediate (42) , which can be generated by heating a precursor such as ό"-sultine (41) (prepared by the method described for compound (18) in Reaction Scheme II), and a pyranoquinone such as (26) can yield pyranoquinone structures such as (43) , after consecutive treatment with silica gel. Bromination and- solvolysis as described for intermediate (23) can give pyranoquinone aglycones with no substituents on ring C such as (44) .

The process for the preparation of compounds of formula (10) in which R ₅ «R ₈ -OH, which tautomerizes into compounds of formula (il) , is illustrated in Reaction Scheme VI.

SUBSTITUTE SHEET

Reaction scheme VI

11. (R ₆ ^, -H.Y-ι_cch__rk_e,X ₃ -0)

SUBSTITUTE SHEET

38

ith reference to Reaction Scheme VI, the treatment of pyranoquinone derivatives such as (45) , prepared by the method described for the compound (24), with lead tetraacetate in glacial acetic acid can give the acetσxylated pyranoquinones with structures such as (46) . These can then be acetylated by treatment with acetic anhydride or acetyl chloride in the presence of a base such as pyridine. The resulting tetracyclic intermediates of formula (47) can be brominated and solvolyzed to give the diacetoxy pyranoquinone aglycone structures (48) . Glycosidation can then give the bisacetoxypyranoquinone glycosides of formula (49) . These, upon alkaline removal of the acetyl groups, yield glycosides (50) which would exist preferentially in the tautomeric form illustrated as structure (11) .

Preferred processes for the preparation of compounds of formula (11) and (44) are illustrated in Reaction Scheme VII. In Reaction Scheme VII pyranoquinones such as (34) can be added to o-quinodimethanes, obtained from the thermolysis of «S-sultine (41) , as described in reaction scheme V, to give directly pyranoanthraquinone aglycones of general formula (44). The cycloaddition reaction between a bisbenzoketene derivative such as (52) , conveniently generated from benzocyclobutanediones of formula (51) using a method as described by Krohn, [Liebig's Ann. Chem. 943- 948 (1988)], can give, in the presence of formula (34) and under ultraviolet irradiation, tetracyclic derivatives such as (53) , which will tautomerize to the more favorable structure (54). Glycosidation of aglycones of formula (54) as described in reaction Scheme I can give the glycosides of formula (11) .

SUBSTITUTE SHEET

Reaction Scheme VII

44

SUBSTITUTE SHEET

40

An alternative approach for the preparation of compounds of general formula (12) is shown in scheme VIII. With reference to scheme VIII, 2,5-dimethoxybenzoic acid is transformed to the benzamide of formula (55a) by first converting the acid, into an acid chloride, with oxalyl chloride in the presence of a base such as pyridine in a solvent such as dichloromethane, and then treating with diethyl amine in ether. The lithio salt of (55a) is then generated with a strong base such as sec-Butyllithium in a convenient solvent such as tetrahydrofuran in the presence of TMEDA and reacted with an electrophile, L-CH ₂ R ₇ , wherein R ₇ is as defined herein and L is a displaceable atom or group such as an halogen. The resulting benzo derivative of formula (55b) is then treated with a strong base such as lithium diisopropyl amide or n-Butyllithium in a solvent such as tetrahydrofuran and added to a carbonyl electrophile of formula (56) to give an adduct of formula (57) . The latter can then be cyclised to an isochromanone derivative of formula (58) by treating compound (57) with an acid. Reduction of (58) with a hydride such as DIBAL in a compatible solvent such as dichloromethane can give the 1-hydroxylated isochroman of formula (33) . Oxidative demethylation of (33) in a solvent system such as acetonitrile-water with, for example, eerie ammonium nitrate can give an isochromandione such as (34) which can then be transformed to a tetracyclic derivative such as (12) as explained in scheme IV. Functional group interconversion of the hydroxyl group of compound (12) into Y can be accomplished readily by employing methodology common to one familiar with the art of organic synthesis.

SUBSTITUTE SHEET

SUBSTITUTE SHEET

For example, glycosidation as- described herein in scheme I of tetracyclic derivative (12) can give structures of formula (10) in which Y is a saccharide; acetylation or benzoylation of compound (12) can give structures of formula (10) in which Y is O-COR and R is an alkyl or aryl; alkylation of the hydroxyl in derivatives of formula (12) can be accomplished with various known electrophiles, for example alkyl halides, orthoformates, or others, with or without catalysis, to give compounds of formula (10) in which Y is an alkoxy1; the hydroxyl group in formula (10) can be converted into a displaceable atom or group L in which L is selected among known leaving groups such as a halide, obtained for example by treating the alcohol with triphenylphosphine in the presence of carbon tetrachloride, carbon tetrabromide or iodine, or a sulfonate such as a mesylate, a tosylate or a triflate, obtained for example by treating the alcohol with esyl chloride, tosylchloride, triflie anhydride, or the like, in the presence of a base such as pyridine or triethylamine and in an adequate solvent for example benzene or dichlorometane, or L is any other appropriate leaving groups. Displacement of L with various nucleophiles would give compounds of formula (10) with different functional groups Y. For example, Y-CN can be obtained by displacement of L with a cyanide; Y-alkyl, alkenyl, or alkyne, can be obtained by displacement with a carbanion. The above examples are not intended to limit this invention in any way.

In the event that such methodology is not compatible with the other substituents at the R ₁ ,-R ₈ positions around the tetracyclic structure (12), then quinone (34) can be transformed into the isochromandione of formula (59) , with the desired Y group as defined herein, by applying readily available techniques in organic chemistry. Reaction of an

SUBSTITUTE SHEET

43

homophtalic anhydride of formula (23) with an isochroman such as (59) in the presence of a strong base such as lithium diisopropyla ide or sodium hydride in a solvent such as tetrahydrofuran can then give the tetracyclic intermediate of formula (60) . The phenol of this latter compound can then be transformed into various functional groups by using techniques available to the organic chemists, thus giving the tetracycle of formula (10).

SUBSTITUTE SHEET

Reaction Scheme IX

SUBSTITUTE SHEET

45

A more general approach for the preparation of compounds of general formula (10) is shown in scheme IX. With reference to scheme IX known compounds of formula (61) , in which L is a displaceable atom or group such as an halogen or a esylate, tosylate, or triflate, can be reacted with an intermediate of formula (62) in the presence of base to give an adduct of formula (63) . The required acyclic compounds of formula (62) are either known or easily obtainable. Cyclization of (63) to give (64) can be accomplished in an aprotic solvent such as tetrahydrofuran or ether and in the presence of a non nucleophilic base such as sodium hydride or lithium diisopropylamide. The ester group of (64) can then be transformed into various groups, as defined herein for R ₆ , by using known methodology. Intermediates of general formula (65) can then be used to prepare the desired tetracycie (10) by following route a. Thus oxidative demethylation of (65) with an oxidant such as eerie ammonium nitrate would give quinones of formula (66) , which can then be coupled with various homophtalic anhydrides of formula (23) to give tetracyclic heteronaptacenediones of formula (68) . Oxidation of (68) , for example via free radical bromination with n-bromosuccinamide or bromine in carbon tetrachloride or other compatible solvents, followed by treatment of the bromide with water can lead to aglycones of formula (12) . Depending on X ₃ , alternative oxidation procedures may be required. For example, when X ₃ «S it is desirable to oxidise the sulfur heteroatom to the sulfoxide (X ₃ -S0) , and then to carry out a Pummeror rearrangement with subsequent hydroxide treatment. Such approaches are common and well described in the literature. The conversion of compound (12) into (10) can be accomplished as described herein in other schemes.

SUBSTITUTE SHEET

Intermediate (65) can also be used in the direct preparation of the tautomeric form of (10), in the case when R ₅ =R ₈ =OH, by following route b. With reference to route b, electrochemical reduction of intermediate (65) in methanol in the presence of sodium methoxide can give the bisketal intermediate of formula (69) . Mono deprotection in the presence of a weak acid in aqueous media can give the quinone monoketal of formula (70) . This latter intermediate can then be coupled under strongly basic conditions achieved for example with NaH in aprotic media, with known benzofuranones of formula (71) in which Z is an electron withdrawing group such as cyano or phenylsulfone. The resulting heteronaphtacenediones of formula (72) can then be converted to the desired tautomer (11) by using the same methodology as described herein for compounds (25) or (68).

SUBSTITUTE SHEET

An alternate method for preparing key bicyclic intermediates of formula (65) is depicted in scheme X. With reference to scheme X, 2,5-dimethoxybenzaldehyde dioxane acetal is treated with a strong base such as n- butyllithium in an aprotic solvent such as diethyl ether, and the resulting lithio salt is alkylated with formaldehyde which can be conveniently generated from p- formaldehyde. The resulting hydroxyl in intermediate (73) can then be converted into a leaving group, for example such as a esylate, by mesylating with esyl chloride in the presence of a base such as pyridine in aprotic media. The leaving group of compound (74) can then be displaced with a nucleophile such as (62), as discussed in scheme IX herein, to give key intermediates of formula (75) . These latter compounds can then be deprotected in acidic aqueous media to give benzaldehydes of formula (76) . Cyclization of these intermediates can be accomplished with bases such as methoxide, carbonates, sodium hydride or lithium diisopropylamides in compatible solvents. Intermediates of formula (77) can then be further transformed to the desired key compounds of formula (65) by following either routes a. or h.

In route a, the hydroxyl group of benzoderivative (77) can be transformed into functional groups R ₇ to give (64) and subsequently (65), by using simple derivatizing techniques commonly used by the one familiar with organic synthesis. Route b can be employed when R ₆ ' is a hydrogen. In this case, dehydration of (77) to give (78) can be carried out under basic or acidic media. Transformation of the ester functionality of structure (78) into R ₆ , as defined herein, is possible by using known methods. The resulting derivatives of formula (79) can then be oxidized to give compounds of general formula (65) by using known oxidation techniques.

SUBSTITUTE SHEET

Reaction Scheme XI

SUBSTITUTE SHEET

50

A preferred mode for the preparation of compounds with general structure (10) is shown in scheme XI. With reference to scheme XI, compounds of formula (61) are coupled, under basic conditions in a suitable solvent such as benzene or tetrahydrofuran, with intermediates of formula (80) to give adducts such as (81) . Compounds of formula (80) , in which Pg is a protecting group such as benzoyl, and R ₆ ^» or R ₆ are preferably electron withdrawing groups, are accessible by derivatising known compounds. Deprotection of (81) with sodium hydroxide in protic solvent can give intermediates of formula (82) which can thereafter be cyclized under basic media in aprotic solvent to give the bicyclic intermediates of formula (65) .

Although key intermediates such as (65) are readily preparable as described herein, it is best to use compounds of formula (81) for the preparation of quinone derivatives such as (85) or (87) . Thus, compounds of formula (81) can be oxidized to give (83) by using known methods. For example, benzylic bromides can be oxidised with sodium bicarbonate in dimethylsulfoxide or with other known reagents to give aromatic aldehydes. Deprotection of (83), for example benzoate hydrolysis with sodium hydroxide, can directly give hydroxylated heterocyclic compounds of formula (84) . These latter derivatives are easily oxidatively demethylated with an agent such as eerie ammonium nitrate to give quinones of formula (85) . Compounds of formula (10) are readily prepared from (85) by using methodology described herein in other schemes.

Alternatively, functional group interconversion of the hydroxy into Y can be accomplished from quinone (85) as described herein in other schemes, or from benzo derivatives (84) to give (86) by employing known techniques. Compounds of formula (10) can then be obtained from (86) by following the same procedure as described for (84).

SUBSTITUTE SHEET

Reaction Scheme XII

ii

SUBSTITUTE SHEET

A shorter and more direct approach for the preparation of compounds of general formula (11), which is the tautomer of (10) when R5=R3=OH, is shown in scheme XII. With reference of scheme XII a known quinizarin derivative of formula (88) is converted to anthraquinone (90), in which OPg is a protected phenol and L is a displaceable atom or group, by protecting the hydroquinone in (88) as an alkoxyl, an acyl, a silyl or an ether by using known protecting methodology, and then treating for example the resulting protected quinizarin of formula (89) with n- bromosuccinamide or bromine, in a solvent such as carbon tetrachloride under free radical catalysis. The conversion of compound (90) into (11) can be achieved by using method¬ ology already described herein in other schemes. For exam¬ ples, compound (90) can be converted into (11), via (91), (96) and (95), by using the method as described for the conversion of (61) into (86) in scheme XI; compound (90) can be converted into (93), via (91), by using the method as described for the conversion of (61) into (65) in scheme XI; compound (94) can be obtained from (90), via (92) and (93), by following the sequence as described for converting (61) into (65) in scheme IX. The same methods for trans¬ forming (67) into (10) can be used for converting (94) into (11).

It will be appreciated that the anthracyclines of formulae (10) and (11) can be transformed into other struc¬ tures at the Xι and X2 positions using synthetic methods known in the art.

It will be further appreciated that compounds of formula (10) in which X3=S0 or SO2 can be obtained by oxidizing any of the intermediates in which X3=S, as convenienced by the synthesis. Convenient reagents for this oxidation are m-chloroperbenzoic acid, hydrogen

SUBSTITUTE SHEET

peroxide or any other known reagents. Compounds in which X ₃ =NO can be obtained from those where X ₃ =NH via oxidation. It will also be appreciated that the following reactions may require the use of, or conveniently may be applied to, starting materials having protected functional groups, and deprotection might thus be required as an intermediate or final step to yield the desired compound. Protection and deprotection of functional groups may be effected using conventional means. Thus, for example, amino groups may be protected by a group selected from aralkyl (e.g. benzyl), acyl or aryl (e.g. 2,4- dinitrophenyl) , subsequent removal of the protecting group being effected when desired by hydrolysis or hydrogenolysis as appropriate using standard conditions. Hydroxyl groups may be protected using any conventional hydroxyl protecting group, for example, as described in "Protective Groups in Organic Chemistry", Ed. J.F.W. McOmie (Plenum Press, 1973) or "Protective Groups in Organic Synthesis" by Theodora W. Greene (John Wiley and Sons, 1981) . Examples of suitable hydroxyl protecting groups include groups selected from alkyl (e.g. methyl, t-butyl or methoxymethyl) , aralkyl (e.g. benzyl, diphenylmethyl or triphenylmethyl) , heterocyclic groups such as tetrahydro-pyranyl, acyl, (e.g. acetyl or benzoyl) and silyl groups such as trialkylsilyl (e.g. t-butyldimethylsilyl) . The hydroxyl protecting groups may be removed by conventional techniques. Thus, for example, alkyl, silyl, acyl and heterocyclic groups may be removed by solvolysis, e.g. by hydrolysis under acidic or basic conditions. Aralkyl groups such as triphenylmethyl may be similarly removed by solvolysis, e.g. by hydrolysis under acidic conditions. Aralkyl groups such as benzyl may be cleaved, for example, by treatment with BF ₃ /etherate and acetic anhydride followed by removal

SUBSTITUTE SHEET

of acetate groups.

In the above processes, the compounds of formula (10) and (11) are generally obtained as a mixture of diastereoiso ers. These isomers may be separated by conventional chromatography or fractional crystallization techniques.

Where the compound of formula (10) or (11) is desired as a single isomer, it may be obtained either by resolution of the final product or by stereospecific synthesis from isomerically pure starting material or any convenient intermediate.

Resolution of the final product, or an intermediate or starting material therefor, may be effected by any suitable method known in the art: see for example, "Stereochemistry of Carbon Compounds", by E.L. Eliel (McGraw Hill, 1962) and "Tables of Resolving Agents", by S.H. Wilen.

The compounds of the formula (10) and (11) possess anti-cancer and anti-tumor activity. The compounds are also believed to possess antibacterial, antifungal and antiviral activity. While it is possible to administer one or more of the compounds of the invention as a raw chemical, it is preferred to administer the active ingredient(s) as a pharmaceutical composition.

In another aspect, the invention therefore provides pharmaceutical compositions primarily suitable for use as antitumor and anticancer agents, comprising an effective amount of at least one compound of the invention or a pharmaceutically acceptable derivative thereof in association with one or more pharmaceutically acceptable carriers and optionally other therapeutic and/or prophylactic ingredients. All the pharmaceutically acceptable salts for example the HCl and tartaric acid salts of the compounds useful as antitumor agents in

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mammals, including humans, are included in this invention.

It will be appreciated by those familiar with the art of clinical oncology that the compound(s) of this invention can be used in combination with other therapeutic agents, including chemotherapeutic agents (Cancer: Principles and Practices of Oncology, 3rd Edition, V.T. DeVito Jr., S. Hellman and S.A. Rosenberg; Antineoplastic Agents edited by W.A. Re ers, John Wiley and Sons, N.Y., 1984). Thus, it will be understood that the compounds or pharmaceutical compositions of the invention may be formulated with the therapeutic agent to form a composition and administered to the patient or the compounds or compositions and the therapeutic agent may be administered separately, as appropriate for the medical condition being treated.

Therefore, for therapeutic purposes, a compound . or composition of this invention can be used in association with one or more of the therapeutic agents belonging to any of the following groups:

l) Alk lating agents such as:

2-haloalkylamines (e.g. elphalan and chlorambucil) ; 2-haloalkylsulfides; N-alkyl-N-nitrosoureas (e.g. carmustine, lomustine or semustine) ; aryltriazines (e.g. decarbazine) ; mitomycins (e.g. mitomycin C) ; ethylhydrazines (e.g. procarbazine) ; bifunctional alkylating agents (e.g. mechlorethamine) ; carbinola ines (e.g. sibiromycin) ; streptσzotocins and chlorozotocins; phosphσramide mustards (e.g. cyclophosphamide) ; urethane and hydantoin mustards

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56

2) Antimetabolites such as: mercaptopurines (e.g. 6-thioguanine and 6-

(methylthio]purine) ; azapyrimidines and pyri idines; hydroxyureas; 5-fluorouracil; folic acid antagonists (e.g. amethopterin) ; cytarabines; prednisones; diglycoaldehydes

3) Intercalators such as: bleomycins and related glycoproteins; anthracylines (e.g. doxorubicin, daunorubicin, epirubicin, esorubicin, idarubicin, aclacinomycin A) ; acridines (e.g. m-AMSA) ; hycanthones; ellipticines (e.g. 9-hydroxyellipticine) ; actinomycins (e.g. actinocin) ; anthraquinones (e.g. l,4-bis[(arainoalkyl)- amino]- 9,10- anthracenediones) ; anthracene derivatives (e.g. pseudourea and bisanthrene); phleomycins; aureolic acids (e.g. mithramycin and olivomycin);

4) Mi otic inhibitors such as: dimeric catharanthus alkaloids (e.g. vincristine, vinblastine and vindesine); colchicine derivatives (e.g. trimethylcolchicinic acid)

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57

epipodophyllotoxins and podophylotoxins (e.g. etoposide and teniposide); maytansinoids (e.g. maytansine and colubrinol); terpenes (e.g. helenalin, tripdiolide and taxol) ; steroids (e.g. 4β-hyroxywithanolide E) ; quassiniods (e.g. bruceantin) ; pipobroman; methylglyoxals (e.g. methylglyoxalbis-

(thiosemicarbazone);

5) Hormones (e.g. estrogens, androgens, tamoxifen, nafoxidine, progesterone, glucocorticoids, itotane, prolactin) ;

6) Immunostimulants (e.g. human interferons, levamisole and tilorane) ;

7) Monoclonal and polyclonal antibodies;

8) Radiosensitising and radioprotecting compounds (e.g. metronidazole and misonidazole) ;

9) Other miscellaneous cytotoxio agents such as: camptothecins; quinolinequinones (e.g. streptonigrin and isopropylidene azastreptonigrin) ; cisplatin and related platinum complexes; tricothecanes (e.g. trichodermol or vermicarin A); cephalotoxines (e.g. harringtonine) ;

BSTITUTE SHEET

10) Cardioprotecting compounds, such as ( ±)-l,2-bis(3,5- dioxopiperazin-1-yl) propane, commonly known as ICRF- 187, and ICRF-198;

11) Drug-resistance reversal compounds such as P- glycoprotein inhibitors, for example Verapamil;

12) Cytotoxic cells such as lymphokine activated killer - cells or T-cells,

13) Immunostimulants such as interleukin factors or antigens.

14) Polynucleotides of sence or antisencing nature.

15) Polynucleotides capable of forming triple helices with DNA or RNA.

16) Polyethers

17) Distamycin and analogs.

The above list of possible therapeutic agents is not intended to limit this invention in any way.

The pharmaceutical compositions of the invention can be in forms suitable for oral, rectal, nasal, topical (including buccal and sublingual) , vaginal or parenteral (including intraarterial intraperitoneal, intramuscular, subcutaneous and intravenous administration) by inhalation or by insufflation. Where appropriate, the formulations may be conveniently presented in discrete dosage units and may be prepared by any method well known in the art of pharmacy. All methods include the step of bringing into

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association the active compound with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.

For injectable use, the pharmaceutical composition forms include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and roust be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, chremophor-el, twin 80, glycerol, dimethyl sulfoxide (DMSO) , propylene glycol, and liquid polyethylene glycol, and the like suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active ingredient or ingredients in the

SUBSTITUTE SHEET

required amount in the appropriate solvent with various of the other ingredients enumerated above, as required followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Pharmaceutical formulations suitable for oral administration may conveniently be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution; as a suspension; or as an emulsion. The active ingredient may also be presented as a bolus, electuary or paste. Tablets and capsules for oral administration may contain conventional excipients such as binding agents, fillers, lubricants, disintegrants, or wetting agents. The tablets may be coated according to methods well known in the art. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils) or preservatives.

As used herein, the expression "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal

SUBSTITUTE SHEET

agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except isofar as any conventional media or agent is incompatible with the active ingredient, its use in the present compositions is contemplated. Supplementary active ingredients can be incorporated into the inventive compositions.

It is especially advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used in the specification and claims herein refers to physically discrete units suited as unitary dosages for the animal subjects to be treated, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as disclosed in detail in this specification.

The dosage of the principal active ingredient for the treatment of the indicated conditions depends upon the age, weight and condition of the subject being treated; the particular condition and its severity; the particular form of the active ingredient, the potency of the active ingredient, and the route of administration. A daily dose of from about 0.001 to about 100 mg/kg of body weight given singly or in divided doses of up to 5 times a day or by

SUBSTITUTE SHEET

continuous infusion embraces the effective range for the treatment of most conditions for which the novel compounds are effective and substantially non-toxic. For a 75 kg subject, this translates into between about .075 and about 7500 mg/day. If the dosage is divided for example, into three individual dosages, these will range from about .25 to about 2500 mg. of the active ingredient. The preferred range is from about 0.1 to about 50 mg/kg of body weight/day with about 0.2 to about 30 mg/kg of body weight/day being more preferred.

The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active ingredient in amounts ranging from about 0.1 to about 1000 mg., with from about 1.0 to about 500 mg. being preferred. Expressed in proportions, the active ingredient is generally present in from about 0.1 to about 500 mg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

Antitumor treatment comprises the administration of any of the compounds of this invention in an acceptable pharmaceutical formulation at the effective therapeutic dosage. It is understood that chemotherapy can require the use of any of the compounds of this invention bound to an agent which facilitates targeting the compound to the tumor cells. The agent may be chosen from, for example, monoclonal or polyclonal antibodies, proteins and liposomes. The compounds of this invention could also be administered as monomeric, dimeric or oligomeric metal

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63

chelate complexes with, for example iron, magnesium or calcium.

The compounds of the invention exhibit antitumor or anticancer activity, most notably, antitumor or anti- cancer activity with human breast cancer, leukemia, colon cancer, lung cancer, renal cancer, ovarian cancer, CNS cancer and melanoma. This list of conditions is however not exclusive, and it is believed that the compounds of the invention will exhibit activity against other tumors and cancers, such as for example pancreatic cancer and bladder cancer.

Certain of the above described intermediates employed in the synthesis of compounds of the invention are also of interest from a pharmacological standpoint. Com¬ pounds believed to possess antibacterial, antifungal, anti¬ viral, antitumor and anticancer activity include the following compounds as outlined in the different reaction schemes: (10), (11), as well as the compounds (12), (17), (18), (20), (21), (22), (24), (25), (26), (27), (29), (30), (32), through (41), (43) through (50), (53) and (54), (58), (59), (60), (64) to (70), (72), (77) to (79), (83) to (87), (91) to (96). As with the compounds (10) and (11), the intermediates are preferably administered as a pharmaceuti¬ cal composition for the treatment of the conditions listed above, and may be administered in the dosages noted above for the compounds (10) and (11). Moreover, the interme¬ diates may be administered as pharmaceutically acceptable salts or as metal chelate complexes where appropriate, and may be administered as a mixture with other of the interme¬ diates compounds, and/or with compounds of the formula (10) or (11), and/or with one or more of the therapeutic agents or agents targeting cancer or tumor cells.

UBSTITUTE SHEET

EXAMPLES

The invention will now be further described with reference to the following Examples.

EXAMPLE 1 Step 1: 2,5-Di_nethoxybenzaldehydedioxane acetal

A solution containing 200g (1.2 mmol) of 2,5- di ethoxybenzal-dehyde, 150g (2.0 mmol) of 1,3-propanediol, and l.Og of p-toluene-sulfonic acid in 1.0 L of benzene was refluxed until no more water could be isolated in the Dean- Stark water separator (6 hours) . The reaction mixture was then cooled and washed with 400 ml of saturated aqueous sodium bicarbonate, 200 ml of water and 200 ml of saturated aqueous sodium chloride. The organic layer was then dried over MgS0 ₄ and the solvent was removed in vacuo. Distillation of the residue under reduced pressure (B.P. 167°C at 1 mmHg) gave 263.7g (98% yield) of a slightly yellow oil characterised as 2,5-dimethoxybenzaldehydedioxane acetal. H NMR (200 MHz,- CDC1 ₃ )5: 1.40 (d , IH, HCHa), 2.24 (m, IH, HCHe), 3.77 (s, 3H, OCH ₃ ) , 3.78 (s, 3H, 0CH ₃ ) , 4.00 (dt, 2H, O-HCHa), 4.24 (dd, 2H, O-HCHe ) , 5.84 (s, IH, 0-CH-O) , 6.82 (dd, 2H, ArH), 7.19 (d, IH, ArH) .

Step 2; 2,5-Di_aethoxy-6-_nethylbenza_ldehyde

To a cooled (-40°C) solution containing 84.Og (0.37 mmol) of 2,5-dimethoxybenzaldehydedioxane acetal in 2.0 L of dry diethyl-ether was added with stirring and under argon 240 ml of a 2.5M n-butyl lithium solution in hexanes. The mixture was stirred for four hours at -25°C and then 24 hours at -10 ^β C. Then to the cooled (-25 ^β C) stirred reaction mixture under argon was added 90.Og of methyl iodide and stirred overnight at room temperature. The solution was then washed twice with 300 ml of water, once with 300 ml of saturated sodium chloride and dried over MgS0 ₄ . The organic

SUBSTITUTE SHEET

solvent was evaporated and the residue was dissolved in 50Oml of ether and stirred for 1-1/2 hours with 500 ml of IN aqueous HCl. The organic layer was separated and washed twice with 200 ml water, once with 200 ml of brine and dried over MgS0 ₄ . Evaporation of the solvent gave a yellow oil which was flash chromatographed with 2.5% ethyl acetate in toluene. A 67% yield (45g) of 2,5-di_methoxy-6- methylbenzaldehyde was obtained (MP: 61-61.5°C). ~E NMR (200 MHz, CDClj) δ: 2.46 (s, 3H, CH ₃ ), 3.78 (s, 3H, 0CH ₃ ) , 3.83 (s, 3H, 0CH ₃ ), 6.90 (dd, 2H, ArH), 10.58 (s, IH, CHO).

Step 3: 5,8-dimethoxy-1-hydroxy-1,3-dihydrobenzo[c] thiophen-2,2-dioxide

Pττv_«»riιι-re 1; Under an argon atmosphere, a deoxygenated solution containing 1.60g (8.9 mmol) of 2,5-dimethoxy-6- methyl-benzaldehyde, 11.Og of S0 ₂ in 100 ml of thiophene free benzene was irradiated at 350 nm for 36 hours. The precipitated crystals (2.01g,.93% yield) were filtered and found sufficiently pure for further use.

Procedure 2: For larger scale, under argon, a deoxygenated solution containing 10.0 g (55.5 mmol) of 2,5- dimethoxy-6-methylbenzaldehyde, 50g of SO, in 600 ml of thiophene free benzene was irradiated with a medium pressure mercury immersion lamp with pyrex filtration for four days. The resulting sludge was extracted three times with 400 ml of IN NaOH and the combined aqueous layer was washed twice with 200 ml of methylene chloride. The aqueous layer was then neutralised with concentrated aqueous HCl and the resulting mixture was then extracted three times with 500 ml of methylene chloride. The combined organic layer was then washed once with 200 ml water, 200 ml of saturated aqueous sodium bicarbonate, 200 ml of water, 200 T"o ^' brine, and then dried over MgS0 ₄ . Following evaporation of solvent,

SUBSTITUTE SHEET

11.2g (83%) of pure 5,8-dimethoxy-l-hydroxy-l,3-dihydrobenzo [c]thiophene-2,2-dioxide was obtained (MP: 140°C decomposes) -E NMR (200 MHz, CDC1 ₃ ) δ:3.57 (s, 3H, 0CH ₃ ) , 3.60 (s, 3H, 0CH ₃ ), 4.44 (dd, 2H, CH ₂ ), 5.65 (s, IH, CH) , 6.85 (dd, 2H, ArH).

Step 4; 4,7-di_methoxy-3,8-dihydrobenzo[b]-1,2-oxathiin-2- oxide.

Following a slightly modified procedure (CharIton J.L., and Durst T., Tet. Lett., 25.(46), 5290 - 1984), to a stirred and cooled (0°C) solution of 7.30g (30 mmol) of the 1- hydroxysulfone, prepared in step 3, in 275 ml of methanol was added in portions over fifteen minutes 5.65 g of sodium borohydride. The mixture was stirred for one hour and then warmed at 50°C for five minutes. The reaction mixture was then evaporated to dryness and to the residue was added 200 ml of concentrated aqueous HCl. After warming at 50°C for five minutes, 300 ml of water was added and the aqueous mixture was extracted three times with 300 ml of CH ₂ C1 ₂ . The combined organic layer was washed twice with 200 ml of water, once with 200 ml of brine and dried over MgS0 ₄ . After evaporation of the solvent, the residue was found to be sufficiently pure to be used in the next step (MP: 90.0- 91.0°C). -E NMR (200 MHz, DMSO-d.) δ: 3.63 (d, IH, J=16 Hz, CHjSO), 3.80 (s, 3H, 0CH ₃ ), 3.81 (s, 3H, 0CH ₃ ), 4.13 (d, IH, J-16 Hz, CH ₂ SO), 5.14 (dd, 2H, CH ₂ 0-), 6.78 (s, 2H, aryl-H) .

Step 5; Ethyl(5,8-dJL___ethoxyisochroman-3-yl)formate

In a 250 ml triple necked round bottom flask, equipped with a Dean-Stark, was refluxed a solution containing 0.6g (0.3 mmol) of ethylglyoxalate (Kelly et al, Synthesis, 544, 1972) in 100 ml of benzene until no more water could be separated. A solution containing 4.39g (19.2 mmol) of the sultine from step 4 in 75 ml of benzene was then added

SUBSTITUTE SHEET

dropwise over 3 hours. During the addition argon was bubbled in the reaction mixture. Reflux was continued overnight and after cooling, the excess glyoxalate was extracted from the mixture with four portions of 200 ml water. The benzene layer was dried and evaporated to give a residue from which was separated 3.13g (61%) of dimethoxyisochroman after flash chromatography from ethyl acetate/toluene. (MP: 59.8°C). H NMR (200 MHz, CDC1 ₃ ) δ: 1.34 (s, 3H, J»7.1 Hz, CH ₃ ) , 2.78 (broad dd, IH, J-17.0, 10.8, 1.3 Hz, HCHaCHC»0), 3.10 (ddd, IH, J-16.9, 3.9, 1.44 Hz, HCHe, CHC«0), 3.77 (s, 3H, OCH ₃ ),

3.80 (s, 3H, 0CH ₃ ), 4.26 (dd, IH, J-10.8, 3.9 Hz, OCHC=0), 4.30 (q, 2H, J=7.1 Hz, -0CH ₂ ) , 4.68 (broad dt, IH, J=16.0, 1.4, 1.3, ArHCHaO-) 5.07 (broad d, J=16.0 Hz, ArHCHeO-) , 6.66 (dd, J-8.9 H, ArH) .

Step 6; Ethyl(7-chloro-5,8-dimethoxyisochro_πan-3- yl)formate

Under argon and at room temperature, was added dropwise 0.820g (7.6 mmol) of t-butylhypochlorite to a stirred solution containing 1.973g (7.4 mmol) of the isochroman from step 5 in 75 ml of anhydrous CH ₂ C1 ₂ . The reaction mixture was stirred for 3 hours and then washed successively with 25 ml portions of saturated aqueous sodium thiosulfate, water and brine. After drying over Na ₂ S0 ₄ , the organic layer was evaporated and the residue was flash chromatographed with 2.5% ethyl acetate in toluene as the eluting solvent mixture. The title compound was obtained in 46% yield (1.02g). (MP: 95.0°C) -E NMR (200 MHz, CDC1 ₃ ) δ: 1.35 (t, 3H, J=7.1 Hz, CH,), 2.93 (broad dd, J=16.8, 10.3 Hz, HCHaCHC=0), 3.16 (broad dd, J-16.9, 3.2 Hz, HCHeCHC-0), 3.78 (s, 3H, 0CH ₃ ) ,

3.81 (s, 3H, OCH ₃ ), 4.27 (dd, J-10.2, 3.3 Hz, -CH) , 4.31 (q, 2H, J«7.lHz, OCHj), 4.63 (broad d, IH, J=16.2 Hz, HCHaO), 5.01 (d, IH, J-16.2 Hz, HCHeO), 6.73 (s, IH, ArH).

SUBSTITUTE SHEET

Step 7: Ethyl(7-chloro-5,8-dioxo-5,β-dihydroiso-chroman- 3-yl)formate

To a stirred solution of l.Og (3.7 mmol) of the chloroiso-chroman from step 6 in 20 ml of acetonitrile was added dropwise a solution containing 6.25 g (11.4 mmol) of eerie ammonium nitrate in 20 ml of water. The mixture was stirred overnight and then diluted with 50 ml of CH ₂ C1 ₂ . The organic layer was separated and the aqueous phase was extracted twice with 25 ml CH ₂ C1 ₂ . The combined organic layer was washed once with 50 ml H ₂ 0, 50 ml brine and then dried over Na ₂ S0 ₄ . After evaporation of solvents, flash chromatography of the residue with a solvent gradient of 5 to 20% ethyl acetate in toluene yielded 495 mg (55%) of the titled compound. (MP: 83.5°C) *H NMR (200 MHz, CDC1 ₃ ) δ: 1.27 (t, 3H, J-7.1 Hz, CHj), 2.58 (ddt, IH, J-19.0, 8.8, 3.2 Hz, HCHaCHC»0), 2.83 (d multiplets, 1Ξ, HCHeCHC-0), 4.17 (dd, IH, J-8.7, 3.3 Hz, OCHC-O), 4.20 (q, 2H, J»7.0 Hz, OCH ₂ ), 4.44 (dt, IH, J-18.8, 3.4 Hz, HCI O), 4.78 (d multiplet, IH, J-18.8, 2.6, 1.7 Hz, HCHeO), 6.95 (s, IH, C=CH).

Step 8: Ethyl [ll-acetoxy-5,12-dioxo-3,4,5 _# 12-tetra___ydro- anthraceno [2,3-c] pyran-3-yl]formate

To a cooled (0*C) and stirred solution of 203 mg of dry diisopropyla ine in 7 ml of dry tetrahydrofuran under argon was added 0.74 ml of a 2.5 M solution of n-butyl lithium in hexanes. The mixture was cooled to -78°C and stirring was continued for 1/2 hour. A solution containing 325 mg (2.0 mmol) of homophthalic anhydride in 7 ml THF was slowly added over five minutes. Next was added in one portion a solution containing 500 mg (1.85 mmol) of the chloroquinone from step 9, in 9 ml THF. The reaction mixture was then stirred for 20 min at -78*C, allowed to warm up to room temperature and stirred for one hour. The reaction was then quenched with 10 ml of saturated ammonium chloride, and partitioned between 10

SUBSTITUTE SHEET

ml of 5% aqueous HCl and 100 ml CH ₂ C1 ₂ . The organic layer was then separated and washed with 25 ml of water, 25 ml of brine and dried over Na ₂ S0 ₄ . Evaporation of solvents yielded the crude pyranoanthracyclinone which was immediately acetylated in 90 ml of CH ₂ C1 ₂ at room temperature for 10 hours with acetic anhydride (1.25 ml) in the presence of 100 mg of dimethylaminopyridine and 1.5 ml of pyridine. To this reaction mixture was then added 50 g of ice, and the isolated organic layer was washed consecutively with 25 ml portions of 5% aqueous HCl, water and brine. The organic solution was then dried over Na ₂ S0 ₄ and evaporated. The residue was then subjected to flash chromatography (5% ethyl-acetate in toluene) and gave 351 mg (34% yield) of the desired titled compound. (MP: 171-173°C) ^l E NMR (300 MHz,CDCl ₃ ) δ: 1.32 (t, 3H, J-7.1 Hz, CH,), 2.64 (s, 3H, C0CH ₃ ) , 2.80 (ddt, IH, J-19.1, 9.0, 3.0 Hz, HCHaCHC-O), 3.07 (d M, IH, J-18.9 Hz, HCHeCHC-O), 4.29 (dd, IH, J-9.0, 4.8Hz, OCHC-O), 4.28 (q,l H, J-7.1 Hz, OCHj), 4.59 (dt, IH, J-18.9, 3.2 Hz, HCHaO), 4.97 (broad d, IH, J-18.9 Hz, HCHeO), 7.72 (m, 2H, ArH), 8.20 ( , 2H, ArH), 8.58 (s, IH, ArH). CMR (75.44 MHz, CDC1 ₃ ) δ: 13.9, CH ₃ , 20.9, CH ₃ , 24.6, CH ₂ ; 61.6, 0CH ₂ ; 71.1, OCH; 123.9, 127.4, 130.3, 130.5, 130.6, aryl CH; 117.9, 128.6, 130.4,

135.6, 140.9, 144.4, 148.2, aryl C; 166.7, 170.4, ester C-O;

181.7, 182.5, quinone C-O. IR (FT, CDC1 ₃ ) ^v max: 1774 acetate C-O, 1750, ester C- ; 1667, 1643, quinone C-O. BRMS calculated for C ₂₂ H _lβ 0 ₇ : (M+-) 394.1053 found 394.1067.

Step 9s (1S,3S) and (1R,3R)-Ethyl [11-acetoxy-l-hydroxy- 5,12-dioxo-3,4,5,12-tetrahydroanthraceno [2,3-c] pyran-3-yl] formate

A mixture containing 257 mg (0.65 mmol) of the pyranotetra-cycle obtained in step 8, 121 mg of n- bromosuccinimide, and 15 mg of AIBN in 25 ml of carbon tetrachloride was refluxed for 2 hours. After cooling, the solvent was removed in vacuo and the residue was treated with

SUBSTITUTE SHEET

40 ml of a 1:1 THF/water solvent mixture for one hour. Most of the THF was then removed in a rotaevaporator and the residual aqueous mixture was extracted three times with 30 ml CHjClj. The combined organic layer was then washed with 25 ml aliquots of water and brine. After drying over Na ₂ S0 ₄ , the solvent was removed and the residue was flash chromatographed to give 178 mg (67%) of the titled aglycone. (MP: 190- 192*C). ^l H NMR (300 MHz, CDC1 ₃ ) μ235: 1.25 (t, 3H, J-7.1 Hz, CH ₃ ), 2.54 (s, 3H, OCOCH ₃ ) , 2.59 (dd, IH, J-11.5, 19.1 Hz, HCH&), 2.89 (dd, IH, J-4.0, 19.1 Hz, HCHe , 4.21 (q, 2H, J-7.1 Hz, OCH ₂ ), 4.73 (dd, IH, J-4.1, 11.6 Hz, OCH), 5.89 (bs, IH, OCH-OH), 7.34 (bs, IH, exchangeable OH), 7.84 (m, 2H, ArH), 8.25 (m, IH, ArH), 8.34 (m, IB, ArH), 8.63 (s, IH, ArH). CMR (75.44 MHz, DMSO- d«) δ: 14.0, CH ₃ ; 20.9, CH ₂ ; 61.0, OCH ₃ ; 64.4, OCH; 85.9, O-CH-OH; 120.0, 126.8, 130.8, 130.9, 135.1, aryl CH; 118.0, 128.5, 130.0, 142.1, 142.7, 147.7, aryl C; 169.3,

170.5, ester C-O; 180.8, 183.2, quinone C-O. IR (FT, CDC1 ₃ ) ^v max: 3575, bs, OB; 1774, 1750, ester C-O; 1670, quinone C-O. HRMS calculated for C„H _lt O _t : [M+-] 410.1002 found 410.1010.

Step 10; (1'S,1R,3S) and (l'S,IS,3R)-Ethyl[11-acetoxy-l- (2',3',6'- trideoxy-3'-trifluoroacetamido-4'-O-p- nitrobenzoyl-L- lyxohexopyranose)-5,12-dioxo-3,4,5,12-tetrahydr- oanthraceno-(2,3-c) pyran-3-yl] formate

To a stirred and cooled (-40°C) solution of 222 mg (0.41 mmol) of 2,3,6-trideoxy-3-trifluoroacetamido-l,4-di-0-p- nitrobenzoyl-α (or β)-L-lyxohexopyranoββ in 20 ml of a 3:1 CH ₂ C1 ₂ -Et ₂ 0 solvent system was added 0.15 ml of trimethylsilyltrifluoromethane sulfonate under argon atmosphere and in the presence of 4A molecular sieves. The mixture was stirred one hour at -5*C and then cooled to - 15*C. A solution containing 121 mg (0.30 mmol) of the pyranoaglycone from step 9 above in 10 ml CH ₂ C1 ₂ was added

SUBSTITUTE SHEET

next and the mixture was stirred for 20 hours at -15°C. The reaction mixture was then poured in 50 ml of a 1:1 ethyl acetate saturated sodium bicarbonate solvent system, filtered and the separated organic layer was washed with 10 ml of water, 10 ml of brine and dried over Na ₂ S0 ₄ . After removal of solvents, the residue was flash chromatographed with 10% ethyl acetate in toluene. The desired titled pyranoanthracycline glycosides were obtained as a 1:1 diastereomeric mixture in 65% yield (239 mg) . (MP: 160-162°C decomposes). B NMR (200 MBz, CDC1 ₃ ) of the (l'S, IS, 3R) diastereomer, δ: 1.24 (d, 3H, J-6.5 Hz, B ₃ C-6'), 1.37 (t, 3H, J-7.1 Hz, CH ₃ ), 2.06 (m, 2B, H ₂ C-2'), 2.58 (s, 3H, acetyl, CH ₃ ) 2.74 (dd, IH, J-17.5, 12 Hz, HCHaCHC-O) , 3.17 (dd, IH, J-19.4, 3.8 Hz, HCHeCHC-O), 4.32 (q, 2H, J-7.0 Hz, OCE_,CR_ ) , 4.42 (broad q, IH, J-7.1 Hz, BC-5'), 4.66 (m, IB, BC-3'), 4.74 (dd, IB, J-11.9, 3.8 Bz, OCHC-O) , 5.47 (broad s, IH, BC- 4'), 5.75 (broad s, IB, BC-1'), 6.05 (s, IB, 0-CB-O), 6.31 (d, IB, J-6.6 Bz, NB), 7.76 (m, 2B, ArH), 8.14 (dm, 2B, ArH), 8.31 (dd, 4B, p-nitroben-zoyl-B) , 8.60 (s, IH, ArH). *B NMR (200 MBz, CDClj) of the (l'S, 1R, 3S) diastereomer, δ: 1.26 (d, 3B, J-6.5 Bz, B ₃ C-6'), 1.35 (t, 3B, J- 7.1 Bz, 0CB ₂ CH,) , 2.06 ( , 2B, B ₂ C-2'), 2.59 (s, 3B, acetyl- CB ₃ ), 2.76 (dd, IB, J-18.8, 12 Bz, ECI&CBC-O), 3.15 (dd, IH, J- 19.4, 4.1 Hz, HCHeCHC-O), 4.31 (q, 2B, J-7.1 Bz, OCBjCB ₃ ) , 4.66 (m, 2H, overlaped BC-3' and BC-5'), 4.76 (dd, IH, J-11.9, 4.0 Hz, OCHC-O), 5.44 (broad s, IB, BC-4'), 5.65 (broad s, IB, BC- 1'), 6.42 (d, IB, J-7.4 Bz, NB) , 7.76 (m, 2B, ArH), 8.14 (dm, 2B, ArB) , 8.32 (dd, 4H, p-nitrobenzoyl -H), 8.60 (s, IB, ArH). CMR of the mixture (75.44 MBz, CDC1 ₃ ) δ: 13.9 and 14.0, C0 ₂ CH_C_H ₃ ; 16.6, 16.8, 6'-CB ₃ ; 20.9, acyl CB ₃ ; 24.1, 24.6, 2'-CH ₃ ; 29.4, 29.8, 4-CH ₃ ; 45.4, 45.5, CBNB; 61.7, 61.8, ester OCH ₃ ; 65.6, 66.3, 5'-OCB; 66.5, 4'-OCB; 71.8, 72.4, 3- OCB; 87.9, 92.7, 1-O-CB-O; 92.7, 98.0, l'-O-CB-O; 115.6, quartet, J-289.2 Bz, CF ₃ ; 124.0, 127.57, 127.64, 130.5, 130.6, 130.7, 131.18, 131.22, 135.6, aryl CH; 118.0, 128.4, 134.6, 134.7, 141.0, 141.5, 142.2, 143.1, 148.3, 151.10.

SUBSTITUTE SHEET

151.14, aryl C; 157.1, quartet, J- 37.7, COCF ₃ ; 164.8, 165.2, 169.2, 169.8, .170.2, ester C-O; 181.2, 183.2, quinone C- . IR (FT, CDCl ₃ ) ^v max: 1775, 1737, bs, ester C-O; 1670, quinone C=0.

Step 11: (2R,1R,3S) and (l'S,IS,3R)-Methyl[11-hydroxy-l- (2',3,'6'- trideoxy-3'-trifluoroacetamido-L- lyxohexopyranose) -5 , 12-dioxo-3 ,4,5,12- tetrahydroanthraceno (2,3-C) pyran-3-yl] formate)

Under argon, at room temperature a solution containing

115 mg (0.13 mmol) of the glycosides from step 10 above in 10 ml of dry methanol was treated with 0.16 ml of a 1.0M NaOCB, methanolic solution for two hours. The reaction mixture was then quenched with three drops of saturated aqueous NB ₄ C1 and the solvent was evaporated to dryness. The residue was stirred with pentane for five hours and then filtered. The pentane insoluble portion was then taken up in ether and filtered. The ether was evaporated and the residue was flash chromatographed with a solvent gradient ranging from 50% ethyl acetate in toluene to 20% methanol in ethyl acetate.

The titled heteroanthracycline glycosides were obtained in

74% yield (56 mg) as a 1:1 mixture (MP: 147-150°C). ^l E NMR

(300 MBz, CDC1 ₃ ) of the (1'S,1R,3S) diastereomer, δ: 1.30 (d,

3B, J-6.9 Bz, B ₃ C-6'), 1.88 (m, IB, Bac-2'), 2.04 (m, IB,

BeC-2'), 2.72 (dd, IB, J-19.0, 12.0 Bz, BCB&CBC-O) , 3.18

(dd, IH, J-19.0, 3.9 Hz, HCHeCHC-O) , 3.66 (broad s, IH, HC-

3'), 3.86 (s, 3B, OCBj) 4.33 (m, 2H, BC-4'and BC-5'), 4.73

(dd, IB, J-11.8, 3.9 Bz, 0-CBC-O), 5.50 (broad s, IH, ΞC-1'),

5.88 (singlet, IH, 0-CH-O), 6.74 (broad d, IB, NB), 7.74 (m,

2B, ArH), 7.98 (m, IB, ArH), 8.18 (s, IB, ArH), 8.50 (m, IB,

ArH).

-E NMR (300 MBz, CDC1 ₃ ) of the (1'S,1S,3R) diastereomer, δ: 1.34 (d, 3B, J-7.0 Bz, B ₃ C-6'), 1.88 (m, IB, BaC- 2'), 2.04 ( , IB, BeC-2'), 2.72 (dd, IB, J-19.0, 12.0 Bz, BCHaCHC- O), 3.18 (dd, IH, J-19.0, 3.9 Hz, HCHeCHC-O) , 3.66 (broad s, IB,

SUBSTITUTE SHEET

72A

Example 1: Preparation of BCH-242: (l'S. IR. 3S) and (l'S, IS. 3R)-Methyl [ll-hydroxy-l-α' 3' 6'- deoxy-3-tπflι ^t roaceamido-L-lyxohexopyranose)-5. 12-dioxo-3.4,5,12-tetrahydroant__π_- ceno [2,3-c] pyπuι-3-yl] formate

SUBSTITUTE SHEET

73

HC-3'), 3.87 (s, 3H, OCB ₃ ), 4.33 (m, IH, HC-4'), 4.59 (broad q, IH, J-7.1 Hz, HC-5'), 4.73 (dd, IB, J-11.8, 3.9 Bz, O-CBC-

O), 5.60 (broad s, IB, BC-1'), 6.04 (s, IB, O-CB-O), 6.74

(broad d, IB, NB) , 7.74 (m, 2B, ArH), 7.98 (m, IH, ArB) , 8.18

(s, IB, ArB), 8.50 (m, IB, ArB). ΞRMS calculated for

C _2i B ₂₄ F ₃ NO ₁₀ : [M+] 579.1353, found 579.1358.

EXAMPLE 2 Step 1: p-Nitrobenzyl(5,8-di_nethoxyisochroB_an-3- yl)formate

The same methodology as used in Example 1 step 5 was used but with 40g (.18 mmol) of p-nitrobenzylglyoxalate hydrate. A 58% (4.1g) of the titled compound was obtained after flash chromatography (MP: 140-141°C). -E NMR (200 MBz,CDCl ₃ ) δ: 2.84 (dd, IB, J-16.3, 10.3 Bz,BCB^CBC- ) , 3.09 (dd, IB, J-16.2, 4.0 Bz, BCBeCBC-O), 3.77 (s, 3B, OCB ₃ ), 3.80 (s, 3H, OCHj), 4.39 (dd, IB, J- 10.3, 4.0 Bz, OCBC-O), 4.70 (d, IB, J-16.0B ₃ , BCB&O), 5.07 (d, IB, J-15.9 Bz, HCHeO) . 5.36 (broad s, 2B, CB ₂ ), 6.67 (broad s, 2B, ArB), 7.54 (d, 2B, ArB), 8.24 (d, 2B, ArB).

Step 2: Methyl(5,8-di_aethoxyisochroman-3-yl)formate

To a cooled (0*C) solution containing 500 mg (1.34 mmol) of the p-nitrobβnzylatβ from step 1 in 10 ml THF was added under inert atmosphere 15 ml of a 0.1 M sodium methoxide solution in methanol. After stirring ten minutes, a few drops of saturated aqueous ammonium chloride were added and the solvent was removed. Flash chromatography of the residue gave 282 mg (84%) of the desired isochroman. (MP: 89-90'C). *B NMR (200 MBz, CDC1 ₃ ) δ: 2.80 (broad dd, IB, J-16.9, 10.8 Bz, BCI&CHC-O), 3.12 (ddd, IH, J-17.0 3.9, 1.4 Hz, HCBeCBC-0, 3.78(s, 3B, OCB ₃ , 3.81(s, 3B, OCB ₃ ), 3.84 (s, 3H, C00CH ₃ ) , 4.30 (dd, IB,

SUBSTITUTE SHEET

-74

J-10.8, 3.9 Bz, OCBC-O), 4.70 (dt, IH, J-16.2, 1.5 Hz, HCHaO), 5.08 (d, IH, J-16.2 Hz, HCHeO)/ ⁶ - ⁶⁷ (dd, 2H, ArH). Step 3: Methyl(5,8-dioxo-5,8-dihydroisochroman-3- yl)formate

To a solution containing 265 mg (1.0 mmol) of the isochroman from step 2 in 5 ml of acetonitrile was added dropwise a solution of 1.726g of eerie ammonium nitrate in 5 ml of water at room temperature. After stirring for ten minutes, the mixture was diluted with 50 ml of methylene chloride. The organic phase was separated and the aqueous layer was extracted twice with 25 ml of CH ₂ C1 ₃ . The combined organic extract were washed once with water, once with brine and then dried over MgS0 ₄ . Evaporation of solvents gave 228 mg (98%) of residue which was found to be above 95% pure pyranoquinone. (MP: 55-58°C) *H NMR (200 MBz, CDC1 ₃ ) δ: 2.68 (dddd, IB, J-19.0, 9.0, 3.7, 2.8 Bz, BClaCBC-O) , 2.93 (d septet, IB, J-19.0 Bz, BCB&CBC-0 ) , 3.84 (s, 3B, OCB ₃ ), 4.31 (dd, IB, J-9.0, 4.4 Bz, CB) , 4.52 (dt, IB, J-18.7, 3.3 Bz, BCBaO), 4.86 (ddd, IB, J-18.9, 2.8, 1.6 Bz, HCHeO), 6.78 (dd, 2B, BC-CB) .

Step 4: Methyl [6-and ll-hydroxy-5,12-dioxo-3,4,5,12- tetrahydroanthraceno [2,3-c] pyran-3-yl]formate

A solution of 2.5M n-butyl lithium (.20 mmol) is added under argon at 0°C to a stirred solution of 0.07 ml of dry diisopropy-lamine in 2 ml of TBF and then stirred for 0.5 hour at -78*C. To the LDA was added dropwise over several minutes a solution of 73 mg (0.45 mmol) of homophthalic anhydride in 2 ml of TBF and then 100 mg (0.45 mmol) of the pyranoquinone from step 3 dissolved in 3 ml of THF; The resulting mixture was stirred 20 minutes at -78*-C, warmed to room temperature, and stirred for one hour. After quenching with 5 ml of saturated aqueous ammonium chloride the mixture was partitioned between 5 ml of 5% BCl

SUBSTITUTE SHEET

75 and 50 ml CH ₃ C1 ₂ . The organic layer was separated, washed with 10 ml of brine and dried over Na ₂ S0 ₄ . Flash chromatography of the residue obtained after evaporation of solvents gave the pyranotetracycles•in 54% yield. The less polar regioisomer had (MP: 202-204'C). *B NMR (300 MBz, CDCl _j ) δ: 2.80 (dd t, IB, J«l£.l., 9 Λ _f 3. 1 Bz, BCBaCBC-O) , 3.11 (dm, IB, J-19.1 Bz, BCBeCBC-O) , 3.84 (s, 3B, OCB ₃ ), 4.35 (dd, J-9.1, 4.3 Bz, OCBC-O), 4.68 (dt, IB, J-19.0, 3.3 Bz, BCBa-O), 5.06 (broad d, IH, J-18.9 Bz, ECBe-O) , 7.71 (m, 2B, ArB), 7.95 (m, IB, ArB), 8.13 (s, IB, ArB), 8.46 (m, IB, ArB), 13.70 (s, IB, exchangeable OB). CMR (75.44 MHz, CDClj), δ: 25.7, CB ₂ ; 53.1, CB ₃ 0; 63.3 CB ₂ 0, 72.2 CBO; 122.7, 125.4, 129.8, 131.1 and 131.9, aryl CB; 127.6, 128.2, 136.4, 143.0 and 143.4 aryl C; 163.1 and 171.0, aryl COB and ester CO; 182.4, 187.1 quinone CO. BRMS calcultaed for CuHijO, 339.0869 found 339.0853. The more polar regioisomer had (MP: 225-235°C dec.) -E NMR (300 MBz, CDC1 ₃ ) δ 2.85 (ddt, IB, J-19.0, 9.2 Bz, BCBaCBC-O) , 3.13 (dm, IB, J-19.0 Bz, BCBeCBC-O), 3.86 (s, IB, OCB ₃ ), 4.37 (dd, J-9.1 et 4.3 Bz, OCgC-O) , 4.68 (dt, IB, J-19.0, 3.4 Bz, BCBa-O), 5.04 (broad d, IB, J-21.1 Bz, BCBe-O), 7.73 (m, 2B, ArB), 7.97 (m, IB, ArB), 8.12 (s, IB, ArB), 8.50 (m, IB, ArB), 13.85 (s, IB, exchangeable OB).

Step 5: Methyl [6-acβtoxy-5,12-dioxo-3,4,5,12-tetrahydro- anthraceno [2,3-c] pyran-3-yl]formate

A mixture containing 60 mg (0.18 mmol) of pyranotβtracyclβ from step 4, 0.25 ml acetic anhydride, 0.3 ml pyridinβ and 6 mg of dimethylaminopyridine in 20 ml of CHjClj was stirred overnight at room temperature under argon atmosphere. The mixture was then diluted with 25 ml CH ₂ C1 ₂ and washed consecutively twice with 15 ml of water, twice with 10 mT ^" of IN HCl, once with 15 ml of water and dried over NaS0 ₄ . Flash chromatography of the residue obtained after flash chromatography yielded 55 mg (81%) of

SUBSTITUTE SHEET

76 the titled acetylated pyranotetracycle. (MP: 196-198°C) ^l E NMR (200 MHz, CDC1 ₃ ) δ: 2.60 (s, 3H, COCB ₃ ) , 2.81 (ddt, IB, J-18.7, 9.1, 3.3 Bz, HCBaCBC-O) , 3.10 (dm, IB, J-19.0 Bz, HCBeCBC-O), 3.85 (s, 3B, OCB ₃ ) , 4.31 (dd, IB, J-9.3, 4.2 Bz, CB), 4.64 (dt, IB, J-19.0, 3.2 Bz, BCBa-O) _ 5.02 (broad d, IH, J-19.0 Hz, HCHe -0, 7.73 (m, 2H, ArH), 8.05 (m, IH, ArH), 8.13 (m, IH, ArB), 8.58 (s, IB, ArB).

Step 6: (IS, 3S) and (IR, 3R)-Methyl [6-acetoxy-l- hydroxy-5, 12- dioxo-3,4,5,12-tetrahydro- anthraceno [2,3-c]pyran-3-yl)formate

A mixture containing 47 mg (0.12 mmol) of the acetylated pyranotetracycle from step 5, 0.23 mg of N- bromosuccinimide, and 0.1 mg of AIBN in 5 ml of CC1 ₄ was refluxed for two hours. The solvent was then removed in vacuo and to the residue was added 10 ml of a 3:1 TBF-B ₃ 0 solvent mixture. After stirring for one hour at room temperature, the mixture was extracted with three 10 ml portions of CB ₃ C1 ₃ . The combined organic extracts were washed once with 10 ml of water and dried over Na ₂ S0 ₄ . Flash chromatography of the residue obtained after removal of solvents gave 35 mg (71%) of the titled pyranόtetracyclic aglycone. (MP: 190°C decomposes) ^l E NMR (200 MBz, DMS0-d«) δ: 2.60 (dd, IB, J-19.6, 11.8 Bz, HClaCHC-O), 3.15 (dd, IB, J-19.6, 4.3 Bz, HCHe.CHC-0) , 3.74 (s, 3B, OCB ₃ ), 4.86 (dd, IB, J-11.5, 4.5 Bz, OCHC-O), 6.21 (d, IB, J-6.2 Bz, C10B), 7.34 (d, IB, J-6.3 Bz, exchangeable OB), 7.75 (m, 2B, ArB), 8.1 (m, 2B, ArB), 8.61 (s, IH, ArH).

SUBSTITUTE SHEET

77

Step 7s (1'S,1R,3S) and (l'S,IS,3R)-Methyl [6-acetoxy-l-

(2',

3 ' , 6 '-trideoxy-3 '-trif luoro-acetamido- '-0-p- nitrobenzoy 1-L-lyxohexopyranose ) -5 , 12-dioxo- 3,4,5,12- tetrahydroanthraceno [2,3-c] pyran-3-yl] formate

Glycosidation of the pyranoanthraquinone from step 6 could be carried out by the procedure as described for example 1 step 10. The titled pyranoanthraquinone glycoside could be obtained in an overall yield of 77% (MP: 158-160*C of 1'S,1S,3R and 225-227 ^β C of 1'S,1R,3S). B NMR (200 MHz, CDC1 ₃ ) of the 1'S,1S,3R) diastereomers, δ: 1.25 (d, 3H, J-6.8 Bz,B ₃ C-6'), 2.06 (m, 2B, B ₂ C-2'), 2.62 (s, 3B, acetyl CB ₃ ), 2.77 (dd, IB, J-19.5, 11.3 Bz, BCBaCBC-O), 3.19 (dd, IB, J-19.3, 3.7 Bz, HCHeCHC-O) , 3.89 (s, 3B, OCHj), 4.42 (broad q, IB, J-6.9 Bz, BC-5'), 4.65 ( , IB, BC-3'), 4.78 (dd, IB, J- 11.4, 3.7 Bz, OCBC-O), 5.48 (broad s, IH, HC-4'), 5.76 (broad s, IH, HC-1'), 6.05 (s, IB, O- CH-O), 6.35 (d, IB, J-6.4 Bz, NB) , 7.76 (m, 2B, ArB) 8.12 (m, 2B, ArH), 8.31 (dd, 4H, p-nitrobenzoyl-B) , 8.65 (s, IB, ArH).

-E NMR (200 MHz, CDC1 ₃ ) of the (1'S,1R,3S) diastereomer, δ: 1.40 (d, 3H, J-6.6 Bz, B ₃ C-6'), 2.06 (m, 2B, B ₃ C-2'), 2.61 (s, 3B, acetyl CB ₃ ) , 2.81 (dd, IB, J-19.0, 11.7, HCS&CHC-O), 3.19 (dd, IB, J-19.4, 3.9 Bz, BClgCBC- O,) 3.89 (s, 3H, OCH,), 4.72 (broad , 3H, overlaped BC-5', BC-3', OCBC-O), 5.46 (broad s, IB, BC-4'), 5.67 (broad s, IB, BC- 1'), 6.26 (s, IB, O-CH-O), 6.41 (d, IB, J-8.0 Bz, NB) , 7.72 (m, 2H, ArB), 8.18 (dm, 2B, ArB), 8.35 (dd, 4B, p- nitrobenzoyl-B), 8.67 (s, IB, ArB) .

SUBSTITUTE SHEET

77A

Example 2: Preparation of (l'S, IR, 3S) and (l'S, IS, 3R)-Methyl [6-acetoxy-l-(2\ 3', 6'-πideo y-3 ^, - trifluo_oace_____«lø-*'-0-p-_ritrobe_ _oy^ 4, 5, 12-tecra- hydioantfaia eno [2,3-c] pyran-3-ylJ foπnate

SUBSTITUTE SHEET

78 EXAMPLE 3

Step 1: Ethyl(6 and 11 hydroxy-5,12-d±oxo-3,4,5,12- tetrahydro-anthraceno[2,3-c]pyran-3-yl)] formate

A solution of 2.5M n-butyl lithium (.20 mmol) was added under argon at 0°C to a stirred solution of 0.07 ml of dry diisopropyla ine in 2 ml of TBF and stirred for 0.5 hour at -78°C. To the LDA was added dropwise over several minutes a solution of 73 mg (0.45 mmol) of homophtalic anhydride in 2ml of TBF and then 100 mg (0.45 mmol) of the pyranoquinone, from the previous step, dissolved in 3 ml of TBF. The resulting mixture was stirred 20 minutes at - 78°C, warmed to room temperature, and stirred for one hour. After quenching with 5 ml of saturated aqueous ammonium chloride the mixture was partitioned between 5 ml of 5% BCl and 50 ml CB ₃ C1 ₃ . The organic layer was separated, washed with 10 ml of brine and dried over Na ₃ S0 ₄ . Flash chromatography of the residue (10% EtOAc in toluene) obtained after evaporation of solvents, gave a polar component which was tentatively assigned as the 6- hydroxytetracycle, in 15% yield. (MP: 150-152°C). -E NMR (200 MHz, CDC1 ₃ ) δ: 1.36 (t, 3H, J-7.1 Bz, CB ₃ ) , 2.84 (ddt, IB, J-19.1, 9.1, 3.2 Bz, BClaCBC-0), 3.13 (d m, IB, J-19.1 Bz, BCIsCHC-0), 4.32 (q, 2H, J-7.1 Hz, 0CH ₃ ) , 4.34 (dd, J-9.1, 4.3 Hz, OCHC-0), 4.67 (dt, IH, J-19.0, 3.3 Hz, HCHa- O), 5.06 (broad d, IB, J-19.0 Bz, BCBe-O), 7.71 (multiplet, 2B, ArH), 7.94 (multiplet, IH, ArB), 8.11 (s, IB, ArH), 8.47 (multiplet, IH, ArH), 13.7 (s, IB, exchangeable OB). CMR (75.44 MBz, CDC1 ₃ ) δ: 14.0, CB ₃ ; 24.4, CB ₃ ; 61.6, 63.2, 0CB ₃ ; 71.7, OCB; 121.9, 125.0, 129.4, 130.7, 131.5, aryl CH; 127.2, 127.6, 131.0, 136.0, 141.7, 144.2, 162.7, aryl C; 170.4, ester C-O; 183.5, 187.5, quinone C-O. IR (FT, CDClj) ^v max: 3405, bs, OH; 1748, ester C-O; 1660, 1644, quinone C-O, 1609, C*C. BRMS calculated for C _3g B ₁₍ 0 ₍ : [M+*]» 352.0947 found 352.0997.

SUBSTITUTE SHEET

79

The less polar component, tentatively assigned as ethyl [11- hydroxy-5,12-dioxo-3,4,5,12-tetrahydroanthraceno (2,3- C) pyran- 3-yl] formate, was obtained in 32% yield. (MP: 149-150°C). ^l E NMR (300 MHz, CDC1 ₃ ) δ: 1.35 (t, 3H, J-7.1 Hz, CH ₃ ), 2.78 (ddt, IB, J-3.4, 9.1, 19.0 Bz, BCBa), 3.07 (d m, IB, J-19.0 Bz, BCBe) , 4.31 (overlaped of with dd, 3B, OCBj and OCB), 4.65 (dt, IB, J-3.3, 18.8 Bz, BCBa) 5.04 (bd, IH, J-18.8 Hz, HCIa), 7.71 ( , 2H, ArH), 7.93 (dd, IH, J-1.3, 7.3 Hz, ArH), 8.07 (s, IH, ArB), 8.43 (dd, IB, J-1.2, 7.2 Bz, ArB). CMR (75.44 MBz, CDC1 ₃ ) δ: 13.9, CH ₃ ; 24.8, CH ₃ ; 61.6, 62.6, OCB ₃ ; 71.6, OCB; 122.1, 124.8, 129.3, 130.6, 131.4, aryl CB; 106.1, 127.0, 127.6, 135.7, 142.6, 143.0, 162.6, CB; 170.3, ester C-O; 182.0, 186.0, quinone C-O. IR (FT, CDC1 ₃ ) ^τ max: 3590, broad, OB; 1748, ester C-0; 1662, 1645, quinone C-O, 1607, C=C. BRMS calculated for C ₃₀ H„0«: [M+-J-352.0947 found 352.0946. In addition, ethyl (5,12-dihydroxy-6,ll-dioxo-3,4,6,ll- tetrahy-droanthraceno [2,3-c] pyran-3-yl)formate BCB-650, could be isolated in 10% yield. (MP: 153-154°C). -E NMR (300 MHz, CDC1 ₃ ) δϊ 1.36 (t, 3H, J-6.0 Bz, CB ₃ ), 2.96 (ddt, IB, J-2.0, 10.1, 18.2 Bz, BCBa), 3.26 (ddd, IB, J-1.8, 4.0, 18.3 Bz, HCBg), 4.33 (q, 2B, J-6.2 Bz, OCB ₃ ) , 4.37 (dd, IB, J-4.0, 10.0 Bz, OCB), 4.82 (dt, IH, J-2.0, 17.6 Hz, O- BCHa), 5.23 (d, IB, J-17.5 Bz, O-HCHe) , 7.85 ( , 2H, ArH), 8.35 (m, 2B, ArB), 13.17 (s, IB, ArOB) , 13.34 (s, IB, ArOB).

Step 2; Ethyl [6-acetoxy-5 , 12-dioxo-3 ,4 , 5 , 12- tetrabydroan- thraceno (2,3-c) pyran-3-yl] formate

A mixture containing 60 mg (0.18 mmol) of the more polar pyranotetracycle, from the previous step, 0.25 ml acetic anhydride, 0.3 ml pyridine and 6 mg of dimethylaminopyridine in 20 ml of CB ₃ C1 ₃ was stirred overnight at room temperature under argon atmosphere. The

SUBSTITUTE SHEET

80 mixture was then diluted with 25 ml CB ₃ C1 ₃ and washed consecutively twice with 15 ml of water, twice with 10 ml of IN HCl, once with 15 ml of water and dried over NaS0 ₄ . Flash chromatography of the residue obtained after flash chromatography yielded 55 mg (81%) of the titled acetylated pyranotetracycle. (MP: 196-198 ^β C). *B NMR (300 MBz, CDC1 ₃ ), δ: 1.34 (t, 3H, J-7.2 Hz, CB ₃ ) , 2.63 (s, 3B, COCB ₃ ), 2.78 (ddt, IB, J-3.3, 9.1, 18.7 Bz, HCga), 3.10 (dm, IH, J-19.0 Hz, HCBe), 4.29 (q, 2B, J-7.2 Bz, OCB ₃ ), 4.31 (dd, IB, J-4.2, 9.3 Bz, OCH), 4.61 (dt, IH, J-3.2, 19.0 Hz, BCBa-O), 5.02 (bd, IB, J-19.0 Bz, BCHe-O) , 7.73 (m, 2H, ArB), 8.05 (m, IB, ArB), 8.13 (m, IH, ArH), 8.58 (s, IH, ArH). IR (FT,CDC1 ₃ ) ^τ max: 1773, 1751, ester C-0; 1667, 1644, quinone C-0; 1618, C-C. BRMS calculated for C ₃₃ B ₁₍ 0 ₇ : [M+] 394.1053 found 394.1020.

Step 3: (1S,3S) and (1R,3R)-Ethyl-[6-acetoxy-1-hydrαxy- 5,12- dioxo-3,4,5,12-tetrahydroanthraceno (2,3-c) pyran-3-yl] formate

A mixture containing 47 mg (0.12 mmol) of the acetylated pyranotetracycle 0.23 mg of N-bromosuccinimide, and 0.1 mg of AIBN in 5 ml of CC1 ₄ was refluxed for two hours. The solvent was then removed ifl vacuo and to the residue was added 10 ml of a 3:1 TBF-B ₃ 0 solvent mixture. After stirring for one hour at room temperature, the mixture was extracted with three 10 ml portions of CB ₃ C1 ₃ . The combined organic extracts were washed once with 10 ml of water and dried over Na ₃ S0 ₄ . Flash chromatography of the residue obtained after removal of solvents gave 35 mg (71%) of pyranotetracyclic aglycone (MP: 215-220°C decomposes). ^l E NMR (300 MHz, CDC1 ₃ ) δ: 1.35 (t, 3H, J-7.1 Hz, CH ₃ ), 2.61 (s, 3B, OCOCH,) 2.71 (dd, IH, J-19.6, 11.8 Hz, HCSftCHC- O), 3.15 (dd, IH, J-19.6, 4.3 Hz, HCBeCBC-O) . 4.32 (q, 2B, J-7.1 Hz, OCH ₃ ), 4.86 (dd, IB, J-11.5, 4.5 Bz, OCHC-O), 6.22 (s, IH, CHOH), 7.74 (m, 2H, ArB), 8.12 (m.

SUBSTITUTE SHEET

81

2H, ArB), 8.61 (s, IH, ArH). CMR (75.44 MHz, DMSO-d.) δ: 13.9, CH ₃ ; 20.9, CH ₃ ; 24.7, CH ₃ ; 60.9, OCB ₃ ; 64.6, OCB; 85.9, O-CB-O; 124.1, 126.7, 130.8,. 130.9, 131.0, aryl CH; 118.1, 126.6, 129.7, 135.3, 141.1, 143.9, 147.6, aryl C; 169.3, 170.5, ester C-O; 181.3, 182.6, quinone C-O. IR (FT, CDC1 ₃ ) ^v max: 3365, bs, OH; 1774, 1748, ester C-O; 1668, quinone C-O. BRMS calculated for C ₃₃ B _ιa O _t : [M+-] 410.1002 found 410.1009.

Step 4: (1'S,1R,3S) and (1'S,1S,3R)-Ethyl [6-acetoxy-l- (2',3', 6'-trideoxy-3'-trifluoroacetamido-4'-O-p- nitrobenzoyl-L-lyxohexopyranose)-5,12-dioxo- 3,4,5,12-tetrahydroan-thraceno (2,3-c) pyran-3- yl] formate

Glycosidation of the pyranoanthraquinone from step 3 above, could be carried out by following the procedure as described in example 1, step 10. The titled pyranoanthraquinone glycoside could be obtained in an overall yield of 77% (MP: 158-160°C of 1'S,1S,3R and 225- 227°C of 1'S,1R,3S). ~E NMR (300 MBz, CDC1 ₃ ) of the (1'S,1S,3R) diastereomers, δ: 1.25 (d, 3B, J-6.8 Bz, B ₃ C- 6'), 1.36 (t, 3B, J-7.0 Bz, CB ₃ ), 2.06 (m, 2B, B ₃ C-2'), 2.62 (s, 3B, acetyl, CB ₃ ) , 2.77 (dd, IB, J-19.5, 11.3 Bz, HCBaCBC-O), 3.19 (dd, IB, J-19.3, 3.7 Bz, BCBeCBC- O) , 4.31 (q, 2B, J-7.0 Bz, OCB ₃ ), 4.42 (broad q, IB, J-6.9 Bz, BO¬ S'), 4.65 (m, IB, BC-3'), 4.78 (dd, IB, J-11.4, 3.7 Bz, OCBC-O), 5.48 (broad s, IB, BC-4'), 5.76 (broad s, IB, BC- 1'), 6.05 (s, IH, O-CH-OJ, 6.35 (d, IH, J-6.4 Hz, NB) , 7.76 (m, 2H, ArB) 8.12 (m, 2B, ArB), 8.31 (dd, 4B, p- nitrobenzoyl-B), 8.65 (s, IB, ArB). ^l E NMR (300 MBz, CDC1 ₃ ) of the (1'S,1R,3S) diastereomer, δ: 1.40 (d, 3H, J-6.6 Hz,B ₃ C-6'), ^' Ϊ.37 (t, 3B, J- 7.0 Bz, CB ₃ ), 2.06 (m, 2B, B ₃ C- 2'), 2.61 (s, 3H, acetyl, CH ₃ ) , 2.81 (dd, IH, J-19.0, 11.7, HcH&CHC-O), 3.19 (dd, IH, J-19.4, 3.9 Hz, HCBeCHC-O) , 4.31 (q, 2B, J-7.0 Hz, OCH ₃ ), 4.72 (broad m, 3B, overlaped HC-

SUBSTITUTE SHEET

82

5', HC-3', OCHC-O), 5.46 (broad s, IH, HC-4'), 5.67 (broad s, IH, HC-1'), 6.26 (s, IB, O-CB-O), 6.41 (d, IB, J-8.0 Bz, NB), 7.72 (m, 2H, ArH), 8.18 (dm, 2H, ArB), 8.35 (dd, 4B, p-nitrobenzoyl-B) , 8.67 (s, IH, ArH). CMR of the diastereomeric mixture (75.44 MHz, CDC1 ₃ ) δ: 14.3 and 14.5, C0 ₃ CH ₃ CH ₃ ; 17.0 and 17.2, 6'-CH ₃ ; 21.4, acyl CH ₃ ; 24.9 and 25.4, 2'-CH ₃ ; 29.9 and 30.0, 4-CH ₃ ; 45.9 and 46.0, CBNB; 62.1 and 62.2, ester OCB ₃ ; 66.2 and 66.8, 5'-OCH; 66.9 and 67.1, 4'-OCB; 72.2 and 72.8, 3-OCB; 88.0 and 92.7, 1-O-CB- O; 92.8 and 98.7, 1'-O-CB-O; 113.1, quartet, J-287.1 Bz, CF ₃ ; aromatic CB: 124.5, 127.8, 128.1, 131.0, 131.2, 131.6,

131.7, 135.0, aromatic quaternary C: 118.5, 128.9, 130.9, 131.0, 131.2, 134.97, 138.16, 138.23, 139.5, 140.0, 144.9,

145.8, 148.85, 148.94, 151.7, 157.5, quartet J-37.3 Bz, C0CF ₃ ; 165.4, 165.7, 169.6, 169.7, 170.1, 170.5, ester C-O; 182.0, 182.3, 182.7, quinone C-O. IR (FT, CDC1 ₃ ) ^v max: 1774, 1737, broad, ester C-O; 1669, quinone C-O, 1532, amide.

Step 5s E t h y l [ 6 - l l - hy d r o xy - 5 , 12 - tetrahydxσanthraceno(2,3-c) ρyran-3-yl]formate and Ethyl [ll-acβtoxy-6-hydroxy-5, 12-dioxo- 3,4,5,12-tetrahydroanthraceno(2,3-c)pyran-3- yl]formate

A mixture containing 387 mg (1.1 mmol) of the unacetylated pyranotetracycle obtained in step 1 above, 2.5g lead tetraacetate, 60 ml of acetic acid, and 30 ml of CBjClj was stirred for 48 hours under argon at room temperature. The mixture was then diluted with 100 ml of CHjClj, extracted twice with 50 ml of water and dried over Na ₃ S0 ₄ . After removal of solvents, the residue was found to contain the titled compounds. CMR (75.44 MHz, CDC1 ₃ ) δ: 13.94, 20.66, 20.80, 24.98, 25.68, 61.57, 61.70, 63.60, 63.67, 71.97, 72.07, 126.76, 126.82, 127.48, 127.53, 134.19, 135.04, 135.07, 157.10, 159.03, 170.46, 170.52, 181.37, 188.78, 188.82, FT (IR, CDC1 ₃ ), ^v max: 1759.8,

SUBSTITUTE SHEET

83 1754.5, ester C-0; 1671.7, 1634.7, quinone C-0.

Step 6: Ethyl [6,ll-diacetoxy-5,12-dioxo-3,4,5,12- tetrahydroan- thraceno (2,3-c) pyran-3-yl] formate

The residue from step 5 above was added to a solution containing 5 ml of acetic anhydride, 6 ml of pyridine and 60 mg of dimethylaminopyridine in 50 ml of CB ₂ C1 ₃ . The mixture was stirred at room temperature overnight under argon and then added to 50g of ice. The aqueous layer was separated and extracted twice with 50 ml of CH ₂ C1 ₃ . The combined organic extracts were then consecutively washed once with 25 ml of water, twice with 25 ml of IN HCl, 25 ml of water, 25 ml of brine and then dried over Na ₃ S0 ₄ . After evaporation of solvents, flash chromatography of the residue yielded 175 mg (35%) of the titled bisacetylated pyranoanthraquinone. (MP: 203-205°C). -E NMR (200 MBz, CDC1 ₃ ) δ: 1.36 (t, 3B, J-7.1 Bz, CB ₃ ), 2.50 (s, 3B, COCB ₃ ) , 2.54 (s, 3B, COCHj), 3.05 (broad m, 2B,CB ₂ CBC«0, 4.35 (masked dd, IB, OCBC-O), 4.32 (q, 2B, J-7.1 Bz, OCBjCBa), 4.75 (broad d, IB, J- 6.7 Bz, BCBa-O), 5.11 (broad d, IH, J-16.5 Hz, BCB_e-0), 7.75 (m, 2B, ArB), 8.16 ( , 2B, ArB). IR (FT, CDC1 ₃ ) ^v max: 1771, broad s, ester CO; 1677, quinone C-O, 1591, C-C. BRMS calculated for C^S^Ox,: 451.1029 found 451.1061.

Step 7s (1S,3S) and (1R,3R) Ethyl [ll-acetoxy-1,6- dihydroxy- 5, 12-dioxo-3 , 4 , 5 , 12 - tetrahydroanthraceno (2,3-c) pyran- 3-yl] formate.

A mixture containing 75 mg (0.17 mmol) of the pyranotetracy- cle from step 6 above, 32 mg (0.17 mmol) of n-bromosuccinimide, 1 mg AIBN in 15 ml of CC1 ₄ was refluxed under argon for 2.5 hours. After removal of solvent, 25 ml

SUBSTITUTE SHEET

84 of a 4:1 TBF-B ₂ 0 solvent mixture was added to the residue and stirred for 0.5 hour. The mixture was then extracted three times with 25 ml of CH ₃ C1 ₃ and the combined extracts were washed with 25 ml of water, 25 ml of brine and dried over Na ₃ S0 ₄ . After evaporation of solvent, flash chromatography of the residue gave 61 mg (77%) of the desired bis acetylated tetracyclic aglycone. (MP: 220-250°C decomposes). ^X B NMR (300 MBz, DMSO-d.) δ: 1.25 (t, 3B, J-7.1 Bz, CB ₃ ), 2.42 (s, 3B, C0CB ₃ ) , 2.35 (bm, IB, BCBa) _. 2.47 (bm, IB, BCBe) , 4.21 (q, 2B, J-7.1 Bz, OCB ₃ ), 4.42 (m, IB, OCB), 6.11 (d, IB, J- 5.8 Bz, 0-CB.-OB) , 7.42 (d, IB, J-5.8 Bz, exchangeable, OB), 7.95 (m, 2B, ArB), 8.12 (m, IH, ArH), 8.22 (m, IH, ArH), 13.3 (s, IH, exchangeable, ArOH). IR (FT, CDCl ₃ ) ^v max: 3690, OB; 3500, 3700, bs, OB; 1764, 1730, ester C-O; 1668, 1636, quinone C-O; 1601, C-C. BRMS calculated for C ₃₃ B _lt 0,: [M+-] 425.0951 found 425.0948.

Step 8s (1'S,1R,3S) and (1'S,1S,3R) Ethyl [ll-hydroxy-6- acetoxy-l- (2 ' , 3 ' , 6'-trideoxy-3 '- trifluoroacetamido- 4'-O—p-nitrobenzoyl-L-lyxohexopyranose) 5,12- dioxo-

3,4,5,12-tetrahydroanthraceno (2,3-C) pyran-3-yl] formate

These compounds were obtained in 61% yield by following the same procedure as described in example 1, step 10 and using the aglycone from step 7 above. (MP: 155- 158*C of 1'S,1S,3R and 182-184°C of 1'S,1R,3S). ^X H NMR (200 MBz, CDClj) of the less polar (1'S,1S,3R) diastereomer, δ: 1.26 (d, 3H, J-6.5 Hz, H ₃ C-6'), 1.39 (t, 3B, J-7.1 Bz, CH,), 2.21 (m, 2B, B ₃ C-2'), 2.54 (s, 3B, 0-C-CB ₃ ), 2.88 (m, IB, BCl&CHC-O), 3.13 ( , IB, ECSeCBC- O), 4.37 (q, 2B, J-7.1 Hz, OCH ₃ ), 4.49 (broad q, IB, J-6.5 Hz, HC-5'), 4.67 (m, IH, HC- 3'), 4.83 (dd, IH, J-11.6, 4.45 Bz, OCBC-O), 5.49 (broad s, IB, BC-4'), 5.77 (broad s, IB, h<6 Bz, BC-

SUBSTITUTE SHEET

85

1'), 6.21 (s, IH ,0-CH-O), 6.24 (d, IH, J-9.1 Hz, NH), 7.84 (m, 2H, ArH), 8.60 (m, 2H, ArH), 8.33 (dd, 4H, p-nitroaryl- B), 13.54 (s, IB, exchangeable, OB). *H NMR (200 MHz, CDC1 ₃ ) of the (1'S,1R,3S) diastereomer δ: 1.29 (d, 3B, J-6.5 Bz, B ₃ C-6'), 1.37 (t, 3B, J-7.1 Bz, CB ₃ ), 2.08 (m, 2H, H ₃ C-2'), 2.88 (broad m, IH, HCHaCHC-O) , 3.11 (broad m, IB, HCIsCHC-O), 4.25 (q, 2H, J- 7.0 Hz, OCH ₃ ), 4.67 (m, IH, HC-3'), 4.81 (m, IH, HC-5'), 4.85 (m, IH, OCBC-O), 5.45 (broad s, IB, BC-4'), 5.71 (broad s, IH, H-C-l'), 6.37 (broad d, IB, J-9 Bz, NB) , 6.39 (s, IB, O-CH-O) , 7.85 (m, 2H, ArB), 8.30 (m, 2B, ArB), 8.33 (dd, 4H, p-nitroaryl-B), 13.66 (s, IB, exchangeable, OB). IR (FT, CDC1 ₃ ) ^v max: 3431, OH; 1737, bs, ester C-O; 1674, quinone C-O; 1595, C-C.

Ste ^p s (1'S,1S,3R) and (1'S,1R,3S) Methyl[11-hydroxy-l- (2' , 3',6'-trideoxy-3'-trifluoroacetamido-4'- hydroxy-L-lyxohexopyranose)-5,12-dioxo-3,4,5,12- tetrahydro-anthraceno[2,3-c]pyran-3-yl]formate, BCH-692 and BCH-691 respectively.

Deprotection as described in example 1, step 11 (done at room temperature) gave the titled compounds in 65% yield. The less polar component was assigned to BCH-692. -E NMR (300 MHz, CDC1,) δ: 1.31 (d, 3B, J-6.6 Hz, CB,) , 1.87 (dt, IH, J-3.8, 13.5 Hz, 2'-HCH.), 2.04 (dd, IB, J-5.2, 13.4 Hz, 2'-HC5ft), 2.76 (dd, IH, J-11.8, 19.5 Bz, BCBa) , 3.18 (dd, IH, J-3.9, 19.5 Hz, HCHs), 3.64 (bs, IB, 4'-CB), 3.87 (s, 3B, OCH,), 4.23 (bq, IB, J-6.6 Bz, 5'-CB), 4.39 (m, IB, 3'-CH), 4.75 (dd, IB, J-3.9, 11.8 Bz, O-CH) , 5.59 (bd, IH, J-3.5 Hz, l'-CH), 6.02 (s, IB, O-CB-O), 6.71 (bd, IH, J-9.1 Hz, NH), 7.72 (m, 2H, ARH), 7.96 (M, IH, ArB), 8.15 (s, IB, ArH), 8.48 (m, IH, ArB), 13.75 (s, IB, ArOB).

The more polar component was assigned to BCH-691. -E NMR (300 MHz, CDC1,) δ: 1.40 (d, 3H, J-6.6 Hz, CH ₃ ) , 1.88

SUBSTITUTE SHEET

86

(m, 2H, 2'-CH ₃ ), 2.75 (dd, IH, J-11.7, 19.7 Hz, HCHa), 3.17 (dd, IH, J-4.1, 19.7 Hz, HCHe.) / 3.64 (bs, IH, 4'-CB), 3.85 (s, 3H, OCH ₃ ), 4.31 (m, IH, 3'-CH), 4.58 (bq, IH, J-6.7 Hz, 5'-CH), 4.71 (dd, IB, J-4.1, 11.6 Hz, O-CH) , 5.49 (bs, IH, l'-CH), 6.19 (s, IH, O-CH-O), 6.72 (bd, IB, J-7.9 Hz, NH) , 7.70 (m, 2H, ArB), 7.97 (bd, IB, J-7.4 Bz, ArB), 8.17 (s, IB, ArB), 8.47 (bd, IB, J-7.4 Bz, ArB), 13.8 (s, IB, ArOB). Small amounts of BCB-673 (5-10%) could also be obtained. The spectral data was similar as the one obtained with BCH- 691 except for the presence of the ethyl ester group.

Step 10 s (1'S,1S,3R) and 1'S,1R,3S) Methyl (ll-hydroxy-6- methoxy- 1 - [ 2 ' , 3 ' , 6 ' - 1 ideoxy- 3 - trifluoroacβtamido-4 '-hydroxy- L- lyxohβxopyranose ] -5 , 12-dioxo-3 ,4,5,12- tetrahydroanthra- ceno [2,3-c] pyran-3 -yl ) formate , BCB-674 and BCH-675 respectively.

Deprotection was done as described in step 9 above but at -15° C. BCB-674 is assigned to the compound giving the following data: ^X H NMR (300 MHz, CDC1 ₃ ) δ: 1.33 (d, 3H, J-6.6 Bz, CH ₃ ), 1.89 (m, 2H, 2'-CB ₃ ), 2.89 (dd, IB, J-12.2, 18.6 Bz, HCla), 3.31 (dd, IB, J-4.2, 18.6 Bz, BCBe) , 3.62 (bs, IB, 4'-CH), 3.85 (s, 3H, 0CB ₃ ), 3.92 (s, 3B, 0CB ₃ ) , 4.35 (m, IH, 3'-CH), 4.53 (bq, IH, J-6.6 Hz, 5'-CH), 5.49 (bs, IB, l'-CH), 6.25 (s, IH, O-CH-O), 6.76 (bd, IH, NH) , 7.82 (m, 2H, ArH), 8.24 (m, 2H, ArB), 12.54 (s, IB, ArOB).

BCB-675 was assigned to the compound which gave the following data: -E NMR (300 MBz, CDC1 ₃ ) δ: 1.28 (d, 3B, J-6.6 Bz, CH ₃ ), 1.87 (m, IB, 2'-BCBJι), 2.04 (m, IB, 2'- HCHe), 2.86 (dd, IH, J-12.1, 18.4 Hz, HCHa), 3.32 (dd, IB, J-3.9, 18.3 Bz, HCIs), 3.63 (bs, IB, 4'-CH), 3.86 (s, 3H, OCB,), 3.88 (s, 3B, OCH,), 4.20 (bq, IH, J-6.6 Hz, 5'-CH), 4.33 (m, IH, 3'-CH), 4.83 (dd, IH, J-3.9, 12.1 Hz, O-CH), 5.51 (bs, IH, l'-CH), 6.13 (s, IH, O-CH-O), 6.75 (bd, IH,

SUBSTITUTE SHEET

87

J-8 Hz, NH) , 7.27 (m, 2H, ArH) , 8.27 (m, 2H, ArH) , 13.6 ( s , IH, ArOH) .

Step:11 (1'S,1R,3S) and (l'S,lS,3R)-(6-hydroxy-5,12- dioxo-1-(3'-trifluoroacetamido-1-daunosaminyl)- 3,4,5,12-tetrahydro-anthraceno[2,3-C]pyran-3- yl)formate

Under argon, a (28°C) solution containing 196 mg (0.25 mmol) of glycoside from step 4 above in 50 ml of dry methanol was treated with a total of 5 equivalent (0.25 ml) of a 4.37 M, NaOCH ₃ methanolic solution which were added according to the progress of the reaction. The reaction mixture was then quenched with 10 ml of saturated aqueous NH ₄ C1 and extracted with dichloromethane (2x50 ml). The combined organic layers were washed with water (2x75 ml) and dried (Na ₃ S0 ₄ ) . Purification by BPLC gave 7.8 mg (5% yield) of the 1'S,1R,3S diastereomer, BCH-681, (MP: 200"C dec). *H NMR (300 MHz, CDC1 ₃ ) δ: 1.35 (t, 3H, J-7.1 Hz, CH _J CH _J ), 1.42 (d, 3H, J-6.6 Bz, B ₃ C-6'), 1.89 (2B, BaC-2' and BeC-2'), 2.76 (dd, IB, J-19.6, 11.4 Bz, HCBaCBC-O) , 3.19 (dd, IB, J-19.6, 4.1 Bz, BCBeCBC-O) , 3.64 (broad s, IB, HC-4'), 4.31 (q, 2H, J-7.2 Bz, OCB _a CB ₃ ), 4.32 (m, IH, HC-3'), 4.61 (bq, IH, J-6.5 Bz, BC-5'), 4.69 (dd, IH, J-11.8 and 4.2 Hz, O-CH), 5.48 (broad s, IH, HC-1'), 6.17 (s, IB, O-CB-O), 6.73 (broad d, IB, NB), 7.74 (m, 2B, ArB), 7.96 (m, IB, ArB), 8.13 (s, IB, ArB), 8.51 ( , IB, ArB), 13.83 (s, IB, Ar-OB).

The l'S, IS, 3R diastereomer, BCB-684, was also obtained with 2% yield (2.9 mg) . (MPs 175°C melt and dec). ^l E NMR (300 MHz, CDC1 ₃ ) δ: 1.32 (d, 3H, J-6.6 Bz, B ₃ C-6'), 1.38 (t, 3B, J-7.2 Bz, OCB ₃ CBj) , 1.85 (m, IB, BaC- 2'), 2.02 (m, IB, BeC-2'), 2.62 (dd, IB, J-19.5, 11.8 Bz, BCBaCBC-O), 3.21 (dd, IH, J-19.5, 4.0 Bz, HCHeCBC-O), 3.65 (broad s, IB, HC-4'), 4.25 (bq, IH, HC-5'), 4.35 (m, 3H,

SUBSTITUTE SHEET

88

OCHjCHj and BC-4'), 4.75 (dd, IH, J-12.0, 3.8 Hz, OCHC-O), 5.61 (broad s, IH, HC-1'), 6.02 (s, H, O-CH-O), 6.72 (broad d, IH, NH), 7.75 (m, 2H, ArB), 7.98 (m, IB, ArB), 8.12 (s, IB, ArB), 8.52 (m, IB, ArB), 13.86 (s, IH, Ar-OH) .

BCH-706 could be isolated by HPLC in 2% yield. Its NMR spectrum is similar to the one obtained from BCB-684 except for the presence of a proton signal for the methoxy ester group at 3.87 pp instead of signals for the ethyl ester.

BCH-683 was also obtained in 17% yield and was assigned to the compound giving the following data. (MP: 190-215° C dec.)

^■ H NMR (300 MHz, CDC1 ₃ ) δ: 1.41 (d, 3H, J-6.6 Bz, CB ₃ ) , 1.89 (m, 2B, 2'-CH ₃ ), 2.78 (dd, IH, J-11.8, 19.2 Bz, BCga) , 3.20 (dd, IH, J-4.0, 19.6 Hz, BCBe) , 3.66 (bs, IB, 4'-CB), 3.87 (s, 3B, OCB ₃ ), 4.34 (m, IH, 3'-CH), 4.63 (bq, IB, J-6.6, 5'-CH), 4.73 (dd, IH, J-4.0, 11.8 Bz, O-CB) , 5.49 (bs, IB, l'-CB), 6.18 (s, IB, O-CB-O), 6.77 (bd, IB, J-7.1 Bz, NB) , 7.74 (m, 2B, ArB), 7.98 (dd, IB, J-2.4, 7.1 Bz, ArB), 8.14 (s, IB, ArB), 8.52 (dd, IB, J-2.4, 7.1 Bz, ArB), 13.8 (s, IB, ArOB).

Step 12s (1'S,1R,3S) and (1'S,1S,3R) Ethyl (6-hydroxy-ll- methoxy-5 , 12-dioxo-l- ( 3 '-trif luoroacetamido-1- daαnosaminyl ) -3 , 4 , 5 , 12-tetrahydroanthraceno [2,3- c]pyran-3-yl) formate

The reaction from step 11 above, carried out at -15° C, yielded (20% yield) a mixture of 1'S,1R,3S and l'S, IS, 3R ethyl ( 6 -hydroxy- 11 -methoxy- 5, 12-dioxo-l- ( 3 '- trif luoroaceta-mido- 1-daunosaminyl ) -3 , 4 , 5 , 12 - tetrahydroanthraceno [2,3-c] pyran-3-yl) formate. (MPs 176-180*C). ^l E NMR (300 MHz, CDC1 ₃ ) of the (l'S, IR, 3S) diastereomer δ: 1.34 (d, 3H, J-6.6 Bz, B ₃ C-6'), 1.38 (t, 3B, J-7.1 Hz, OCH ₃ CHa), 1.90 (m, IH, BaC-2'), 2.04 (m, IB,

SUBSTITUTE SHEET

88A

Example 3: Preparation of various pyrano modified hcterointhracyclinones and heteroanthncyclines

SUBSTITUTE SHEET

88E

Example 3 : (Continued )

SUBSTITUTE SHEET

88C

Example 3 : (Continued )

+ Diastereomer

SUBSTITUTE SHEET

89

BeC-2'), 2.94 (dd, IB, J-18.7, 12.2 Hz, HCHaCHC-O) , 3.33 (dd, IH, J-18.4, 3.3 Hz, BCBeCBC-O) , 3.65 (broad S, IB, BC- 4'), 3.95 (s, 3B, ArOCB ₃ ), 4.35 (q, 2B, J-7.1 Hz, OCBjCB ₃ ) , 4.42 (m, IB, BC-3'), 4.68 (bq, IB, HC-5'), 4.78 (dd, IH, J-11.9, 4.2 Bz, O-CB), 5.53 (broad s, IB, BC-1'), 6.34 (s, IB, O-CB-O), 6.76 (broad d, IB, NB) , 7.83 (m, 2B, ArB), 8.31 (m, 2B, ArB), 13.70 (s, IB, ArOB). *B NMR (300 MBz, CDC1 ₃ ) of the l'S, IS, 3R diastereomer δ: 1.31 (d, 3H, J-6.6 Bz, B ₃ C-6'), 1.38 (t, 3B, J-7.1 Bz, OCB _J CB _J , 1.90 ( , IB, BaC-2'), 2.04 (m, IB, BeC-2'), 2.91 (dd, IB, J-18.2, 12.0 Bz, BClaCBC-O), 3.34 (dd, IB, J-18.3, 3.7 Hz, HCHeCHC-O), 3.65 (broad s, IH, HC-4'), 3.94 (s, 3H, ArOCB ₃ ), 4.28 (bq, IB, BC-5'), 4.35 (q, 2B, J-7.1 Bz, OCHjCHj), 4.42 (m, IH, HC-3'), 4.81 (dd, IH, J-12.4, 4.0 Hz, O-CB), 5.59 (broad s, IB, BC-1'), 6.14 (s, IB, O-CB-O), 6.74 (broad d, IB, NH) , 7.83 (m, 2B, ArB), 8.31 (m, 2H, ArB), 13.59 (s, IH, ArOH) .

EXAMPLE 4

Step 1; p-nitrobenzyl (5,8-dioxo-5,8-dihydroisochroma___-3- yi) formate

p-Nitrobenzyl 5,8-dimethoxy-isochroman-3-yl formate was oxidized as described in example 2, step 3. The titled compound was obtained in 92% yield. (MP:133*C decomposes) -E NMR (200 MHz CDC ,) δ: 2.70 (ddt, IB, J-18.7, 9.0, 3.0 Bz, HCgaCHOO), 2.95 (d multiplet, IH, J-19.0 Hz, HCIfiCHC-O), 4.38 (dd, IH, J-8.9, 4.3 Hz, OCHC _* 0), 4.56

(dt, IH, J-17.1, 3.0 Hz, HCfift-O), 4.88 (ddd, IH, J-17.3, 2.8, 1.7 Hz, HCIA-O), 5.36 (broad s, 2H, CB ₃ ArB), 6.79

(dd, 2H, ArH), 7.57 (d, 2H, ArH), 8.29 (d, 2H, ArH).

SUBSTITUTE SHEET

90

Steo 2; p-nitrobenzyl [5 , 12-dioxo-3 , 4 , 5 , 12- tetrahydroanthraceno [2,3-c] pyran-3-yl]formate

A solution containing 669 mg (1.9 mmol) of pyranoquinone from step 1, 485 mg (2.9 mmol) of 3,6- dihydrobenzo [b]-l,2-oxathiin-2-oxide (Charlton ϋ.L. and Durst T., Tet. Lett., 2£, 5287, 1984) in 50 ml of xylenes was refluxed overnight. The solvent was then removed in vacuo. The residue was flash chromatographed with ethyl acetate in toluene and gave 536 mg (62%) of the titled pyranoanthraquinone. (MP: 214-215°C decomposes) ¹ H NMR (200 MBz, CDClj) δ: 2.84 (ddt, IB, J-18.9, 9.0, 2.7 Bz, HClaCBC-O), 3.12 (broad d, IB, J-19.0 Bz, HCHgCHC-O) , 4.40 (dd, IB, J-9.1, 4.4 Hz,0CHC- 0), 4.67 (dt, IH, J-19.0, 3.0 Hz, BCBa-O), 5.04 (broad d, IB, J-18.9HZ, HCls-O), 5.33 (broad s, 2B, ArCB ₃ ), 7.53 (d, 2B, ArB), 7.67 (m, 2H, ArH), 8.04 ( , 2H, ArB) 8.21 (d, 2B, ArB), 8.58 (s, IB, ArB, 8.62 (s, IH, ArH).

Ste ^p 3: (IS, 3S) and (IR, 3R) p-nitrobenzyl [1-hydroxy- 5,12- dioxo-3, 4, 5, 12- tetrahydroanthraceno [2,3-c] pyran- 3-yl] formate

A mixture containing 164 mg (0.37 mmol) of the pyranoanthra-cyclinone from step 2, 65 mg (0.37 mmol) of N- bromosuccinimide and 10 mg of AIBN in 25 ml of CC1 ₄ was refluxed for 2 hours. The solvent was then removed and the residue was stirred with 35 ml of a 7:3 solution of THF in water for ten hours. The mixture was then extracted three times with 25 ml aliquots of CH ₃ C1 ₃ . The combined organic layer was washed once with 10 ml of water, 10 ml of brine, and then dried over NaS0 ₄ . After evaporation of solvents, flash chromatography of the residue gave 118 mg (69%) of the desired pyranoanthraquinone aglycone. (MP: 275°C decomposes) H NMR (200 MHz, DMSO-d,) δ: 2.68 (dd, IH,

ITUTE SHEET

91

J-19.1, 11.5 Hz, HCBaCBC=0), 3.00 (dd, IH, J-19.4, 4.4 Hz, BCHeCHC-O), 4.89 (dd, IH, J-11.4,. 4.1 Hz, OCHC-O), 5.39 (broad s, 2H, ArCB ₃ ), 5.98 (d, IB, J-6.3 Bz, CHOB) , 7.40 (d, IB, J-6.1, exchangeable OB), 7.7 (d, 2B, ArB), 7.8 ( , 2B, ArB), 8.27 (d, 2B, ArB), 8.29 (m, 2B, ArB), 8.66 (s, IB, ArB), 8.67 (s, IB, ArB).

Step 4: (l'S, IR, 3S) and (l'S, IS, 3R)-p-nitrobenzyl [1- (2',3',6'-trideoxyaceta_βido-4'-O-p-nitrobenzoyl- L- lyxohexopyranose)-5,12-dioxo-3,4,5,12- tetrahydroanthra-ceno [2,3-c]pyran-3-yl]formate

These glycosides were obtained by the same procedure as described in step 10 of example 1 and by using the aglycone from step 3 of this example. (MP: 192-195°C for 1'S,1S,3R and 173-174 ^β C for 1'S,1R,3S). -E NMR (200 MBz,CDC1 ₃ ) of the (1'S,1S,3R) diastereomer, δ: 1.12 (d, 3B, J-6.3 Bz, B ₃ C-6'), 2.09 (m, 2B, B ₃ C-2'), 2.83 (dd, IB, J-19.4, 11.7 Bz, BClaCBC-0), 3.26 (dd, IB, J-19.1, 3.7 Bz, BCBeCBC-O), 4.4 (broad q, IB, J-6.1 Bz, BC-5'), 4.65 (m, IH, HC-3'), 4.89 (dd, 1H,J- 11.8, 3.6 Hz, OCHC-O), 5.42 (broad s, 3H, HC-4' and aralCB ₃ ), 5.82 (broad s, IH, W _h <6Hz, BC-1'), 6.13 (s, IB, _h <0.7 Bz, OCB-O) , 6.40 (d, IH, J-7.3 Hz, NH), 7.28 (d, 2H, p-nitrobenzyl-B) , 7.73 (m, 2B, ArB), 8.09 (m, 2B, ArH), 8.29 (d, 2H, p-nitrobenzyl-H) , 8.31 (dd, 4H, benzoyl H) , 8.64 (s, IB, ArH), 8.69 (s, IH, ArH). ^l E NMR (200 MHz, CDC1 ₃ ) of the (1'S,1R,3S) diastereomer, δ: 1.42 (d, 3H, J-6.4 Bz, B ₃ C-6'), 2.09 ( , 2B, B ₃ C-2'), 2.85 (dd, IB, J-19.4, 11.3 Bz, BCIaCBC-O) 3.25 (dd, IB, J-19.4, 4.3 Bz, BCBjgCBC-O), 4.63 (m, IB, BC-3'), 4.84 (overlaped, m, 2H, HC-5' and OCHC-O), 5.41 (broad s, 2H, aralCH ₃ ), 5.47 (s, IH, W _h «5 Hz, HC-4'), 5.69 (s, IH., _& <0.7Hz, O-CH-O), 6.57 (d, IH, J-7.2 Bz, NB) 7.73 (d, 2B, p-nitrobenzyl-H), 7.75 (m, 2B, ArB), 8.11 (m, 2B, ArH), 8.33 (d, 2H, p-nitrobenzyl-H), 8.37 (dd, 4H, p- nitrobenzoyl-B), 8.69 (s, IB, ArB).

SUBSTITUTE SHEET

92

Step 5: (1'S,1S,3S) Methyl ( 1 - [ 2 ' , 3 ' , 6 ' - t r ide oxy ac e t amido _- 4 ' - hydroxy-L- lyxohexopyranose] -5 , 12-dioxo-3 , 4,5, 12-tetra-

_% v hydroantraceno[2, 3-c ]pyran-3-yl) formate - BCH-672

Deprotection of the less polar glycoside, from step 4 above, by using the method described in example 1, step 11, gave the titled compound in 20% yield. *H NMR (300 MHz, CDC1 ₃ ) δ: 1.29 (d, 3B, J-3.3 Bz, CH ₃ ), 1.85 (dt, IH, J-3.8, 13.3 Hz, 2'-HCIa), 2.00 (dd, IB, J-5.4, 13.4 Bz, 2'-BCHe), 2.76 (ddd, IH, J-1.0, 11.8, 19.5 Bz, BCBa), 3.18 (dd, IH, J-3.8, 19.5 Hz, HCIe), 3.63 (bs, IB, 4'-CB), 3.86 (s, 3H, OCH ₃ ), 4.23 (bq, IH, J-6.6 Hz, 5'-CH), 4.35 (m, IH, 3'-CB), 4.75 (dd, IB, J-3.9, 11.8 Bz, O-CB), 5.61 (bd, IH, J-3.4 Hz, l'-CH), 6.04 (s, IH, O-CHO) , 6.71 (bd, IH, J-9.0 Hz, NH), 7.70 ( , 2H, ArH), 8.06 (m, 2H, ArB), 8.60 (s, IB, ArH), 8.67 (s, IH, ArB).

A second less polar component, tentatively assigned as (l'S IS, 3R) methyl (l-[2',3',6'-trideoxyacetamido-4'- hydroxy-L-lyxo-hexopyranose]-5 , 12-dioxo-3 ,4,5,12- t e t rahydroanthraceno [2,3-C] pyran-3-yl) formate, BCB-670, was obtained in 60% yield.

^l E NMR (300 MHz, CDC1 ₃ ) δ: 1.25 (d, 3H, J-6.6 Bz, CB ₃ ), 1.86 (dt, IB, J-3.84, 13.2, 2'-BCIa), 2.09 (dd, IB, J-5.2, 13.4 Bz, 2'-BCIs), 2.51 (dd, IB, J-11.9, 15.2 Bz, HCHa , 2.88 (dd, IH, J-2.9, 15.2 Hz, BCIa) , 3.61 (bd, IB, J-4.2 Bz, 4'- CB), 3.82 (s, 3B, OCB ₃ ), 4.21 (bq, IB, J-6.8 Bz, 5'-CB), 4.44 (m, IB, 3'-CB), 4.57 (dd, IH, J-2.74, 11.87 Hz, O-CH), 5.53 (bd, IH, J-3.6 Hz, l'-CH), 6.19 (s, IH, O-CH-O), 7.67 (bd, IH, J-8.6 Hz, NB) , 7.71 (m, 2B, ArB), 8.06 ( , 2H, ArH), 8.57 (s, IH, ArH), 8.58 (s, IH, ArH).

TUTE SHEET

•93

Step 6: (1'S,1R,3S) Methyl ( 1 - [ 2 ' , 3 ' , 6 ' - t r ideoxy ac e t amido- 4 ' - hydroxy-L- lyxohexopyranose] -5 , 12-dioxo-3 ,4,5, 12-tetra- hydroanthraceno ( 2 , 3-c ) yran-3-yl ) formate , BCH-

671.

Deprotection of the more polar glycoside, from step 4 above by using the method described in example 1, step 11, gave BCB-671 in 70% yield. *B NMR (300 MHz, CDC1 ₃ ) δ: 1.41 (d, 3H, J-6.6 Bz, CB ₃ ), 1.93 (m, 2B, 2'-CB ₃ ), 2.51 (dd, IH, J-12.0, 15.1 Hz, HCIa)/ 2.85 (dd, IB, J-3.0, 15.1 Hz, BCBe), 3.65 (bs, IB, 4'-CB), 3.80 (s, 3B, OCB ₃ ) , 4.46 (overlapped m, 3H, 3',5'-CH and O-CH), 5.42 (bd, IH, J-3.2 Hz, l'-CH), 6.32 (β, IH, O-CH-O), 6.69 (bd, IH, J-8.3 Bz, NB), 7.70 (m, 2B, ArH), 8.05 (m, 2H, ArH), 8.57 (s, IB, ArB) 8.58 (s, IB, ArH).

SUBSTITUTE SHEET

Example 4 : Preparation of (l'S, IS, 3R) and (l'S, IR, 3S) methyl [l-(2\ 3 ^* . ό'-trideoxy-trifluoroacetamido-

4 ^, -hyd_oxy-L-lyxohexo-pyranose)-5,-12-dioxo-3,4,5,12-tet rahydroanthraceno [2,3-c] pyran-3-yl] formate

94 EXAMPLE 5

Step 1: 5,8-dimethoxy-l-methylbenzo[C]l,2-oxathiin-2- oxide

Following a slight modification of Durst and Charlton procedure (Tel. Lett., 25 (46), 5290, 1984), 800 ml of a 2.5 M solution of methyl lithium in hexanes was added, at room temperature and under argon, to a solution containing 14.38 g (59 mmol) of hydroxysulfone from "example 1 - step 3 ^H in 2.5 1 of anhydrous THF. After stirring for 16 hours, 400 ml of methanol was added to the mixture. Solvents were removed under vaccuo and the residue was treated with 500 ml of 12 M BCl at 50°C for 3 min. The solution was then diluted in 1 1 of water and extracted three times with 300 ml of dichloromethane. The combined organic layer was washed twice with water, once with brine and dried over MgS0 ₄ . After removal of solvents, flash chromatography of the residue gave the titled sultine as a yellow solid in 47% yield. (MP: 152°- 153 ^β C). ~E NMR (200 MHz, CDC1 ₃ ) δ:1.58 (d, 3B, J-6.2 Hz, CH ₃ ) , 3.49 (d, IH, J-14.8 Hz, CH) , 3.79 (s, 3H, 0CB ₃ ), 3.80 (s, 3H, 0CB ₃ ) , 4.29 (d, IH, J-14.8 Hz, CH), 5.63 (q, IH, J-6.2 Hz, CH ₃ CH), 6.82 (s, 2Ξ, ArH). CMR (75.44 MHz, CDC1 ₃ ) δ:15.3, CH ₃ ; 52.2, CB ₃ ; 55.7, OCH ₃ ; 59.9, CB; 110.1, 110.6, aryl CB; 123.6, 123.8, 149.8, 149.9, aryl C. MS (Cl, NH ₃ ,137 ^β C). m/e: 246 (100, M+NH .

Step 2s Cis and trans p-nitrobenzyl (5,8-dimethoxy-l- methyliso— chrαman-3-yl) formate

These 1-methylated isochromans were obtained by following the same procedure as described for the isochroman of "example 1, step 5". Thus, the reaction between 4g (16.5 mmol) of the 1-methylated sultine from step 1 above and 18.76g (83 mmol) of p-nitrobenzylr glyoxalate hydrate resulted in a black residue which after

SUBSTITUTE SHEET

^• 95 flash chromatography (10% ethyl acetate - 25% toluene - 65% cyclohexane) gave the two titled diastereomeric isochromans in a 3:1 ratio. The trans isomer (2.8g, 44% yield) had: (MP:110°-111°C). -E NMR (200 MHz, CDC1 ₃ ) δ:1.53 (d, 3B, J-6.8 Bz, CHj), 2.85 (dd, IH, J-9.8, 16.8 Hz, Hcha) , 3.07 (dd, IB, J- 4.4, 16.7 Bz, HCBe.), 3.78 (s, 3B, OCB ₃ ), 3.79 (s, 3B, OCB ₃ ), 4.69 (dd, IB, J-4.7, 9.9 Bz, OCB), 5.29 (q, IB, J-6.7 Bz, OCHCB ₃ ), 5.33 (bs, 2H, OCH ₃ ), 6.68 (s, 2B, B- C- C-B), 7.49 (d, 2B, J-8.9 Bz, ArB), 8.22 (d, 2B, J-8.9 Hz, ArB). CMR (75.44 MBz, CDC1 ₃ ) δ:19.6, CB ₃ ; 25.5, CB ₃ ; 55.3, 55.4, OCB ₃ ; 65.0, OCB ₃ ; 67.2, 68.6, OCB; 107.8, 123.6, 128.2, aryl CB; 121.0, 128.0, 142.8, 147.6, 149.2, 150.6, aryl C; 171.4, ester C-O.

IR (FT, CDClj) ^v max: 1756, ester C-O, 1216, C-O. MS (Cl, NB ₃ , 191 ^β C) m/e: 405 (52, M+NB , 270 (100, M+NB ₄ -C ₇ B _s N0 ₃ ) . The cis isomer (247) (0.9g, 14% yield, oil) had: ^X H NMR (200 MHz, CDC1 ₃ ) 5:1.62 (d, 3B, J-6.4 Hz, CH ₃ , 2.73 (ddd, IB, J-2.1, 11.4, 16.0 Bz, BCS&), 3.17 (dddd, IB, J-1.2, 2.4, 16.0 Bz, BCBe), 3.79 (s, 3B, OCB ₃ ), 3.80 (s, 3B, OCB ₃ ), 4.23 (dd, IB, J-2.4, 11.4, OCB), 5.09 (bq, IH, J-6.5 Hz, CHCB ₃ ), 5.36 (bs, 2B, OCB ₃ ), 6.71 (bs, 2H, ArB), 7.80 (d, 2B, J-8.9 Bz, ArH), 8.25 (d, 2H, J-8.9 Hz, ArH). CMR (75.44 MHz, CDC1 ₃ ) 5:21.5, CH ₃ ; 26.7, CH ₃ ; 55.2, 55.6, OCH ₃ ; 65.0, OCHj, 71.5, 72.1, OCB; 108.4, 123.7, 128.3, aryl CB ₃ ; 107.9, 122.9, 142.8 148.1, 149.9, 150.4, aryl C; 170.8, ester C-O. IR (FT, CDC1 ₃ ) ^v max: 1755, ester C-O, 1219, C-O. BRMS calculated for C ₃₀ B ₃₁ NO ₇ 387.1318 found 387.1299.

Step 3s Ciβ-p-nitrobenzyl(5,8-dioxo-l-mβthyl-3,4,5,8- tetrahy-drobenzo (2,3-c]pyran-3-yl) formate

To 400 mg (1.0 mmol) of the cis isochroman from step 2 above in 10 ml of acetonitrile was added, dropwise over 5 min and with stirring, a solution containing 1.96 g (3.6 mmol) of eerie ammonium nitrate in 10 ml of water. After

SUBSTITUTE SHEET

- 96 five minutes the mixture was diluted with 25 ml of water and then extracted three times with 50 ml of methylene chloride. The combined organic layer was washed once with 50 ml of water, once with 25 ml of brine, and then dried over MgS0 ₄ . Following evaporation of solvent, the oily yellow residue was found to be pure (95% yield) isochromandione. Flash chormatography, with 20% ethyl acetate in toluene, reduced the yield (65%) considerably without significantly increasing the purity. ^l E NMR (200 MBz, CDC1 ₃ ) 5:1.57 (d, 3B, J-6.7 Bz, CB ₃ ), 2.57 (ddd, IB, J- 4.3, 10.8, 18.4 Bz, HCH_a) , 2.98 (bdt, IB, J- 2.8, 18.4 Bz, HClfi), 4.19 (dd, IB, J-2.75, 10.8 Bz, O-CB), 4.81 ( , IB, OCHCHj), 5.36 (bs, 2H, OCB ₃ ), 6.76 (dd, 2B, J-10.1 Bz, ArB), 7.57 (d, 2B, J-9.0 Bz, ArB), 8.24 (d, 2B, J-9.0 Bz, ArB). CMR (75.44 MBz, CDC1 ₃ ) 5:20.2, CB ₃ ; 25.2, CB ₃ ; 65.3, OCB ₃ ; 70.2, 71.1, OCB; 123.6, 128.4, aryl CB; 135.7, 136.8, CB; 138.4, 142.2, 143.4, 147.6, quaternary C; 169.2, ester C-O; 185.2, 185.4, quinone C-O.

Step 4; Trans-p-nitrobenzyl (5,8-dioxo-l-methyl-5,8- dihydro- isochroman-3-yl) fozaate

Ox dative demethylation of 160 mg (0.4 mmol) of trans- p- nitrobenzyl (5,8-dimethoxy-l-methylisochroman-3-yl) formate with 780 mg (1.4 mmol) of eerie ammonium nitrate, as described in step 3 above, give 137 mg of the titled isochromandione as a yellow oil. *B NMR (200 MBz, CDC1 ₃ ) 5:1.5 (d, 3H, J-6.8 Bz, CB ₃ ), 2.67 (ddd, IB, J-2.2, 8.9, 19.0 Bz, BCBA), 2.89 (ddd, IB, J-1.3, 4.6, 19.0 Bz, BCBeJ, 4.59 (dd, IB, J-4.7, 8.9 Bz, OCB), 5.04 (bq, IB, J-6.8 Bz, OC1CB,), 5.33 (bs, 2B, OCB ₃ ), 6.75 (dd, 2B, J-10.1 Bz, B- C-C-B), 7.54 (d, 2B, J-8.9 Bz, ArH), 8.24 (d, 2H, J- 8.9 Bz, ArB). CMR (75.44 MBz,CDCl ₃ ) 5: 19.4, CH,; 24.4, CB ₃ ; 65.5, OCB ₃ ; 66.7, 67.1, OCB; 123.8, 128.6, 136.0, 136.5, aryl CB; 137.3, 142.2, 143.5, 146.7, aryl C; 185.0, 185.3,

SUBSTITUTE SHEET

97- quinone C-0.

Step 5: (1R,3R) and (1S,3S) Cis-p-nitrobenzyl(5,12-dioxo- l-methyl-3,4,5,12-tetrahydroanthraceno(2,3- c)pyran-3-yl)formate

This compound was obtained by following the same procedure as described in "example 4, step 2". Thus, reaction of 250 mg (1.5 mmol) of 3,6-dihydrobenzo-(b)-l,2- oxathin-2-oxide with 268 mg (0.7 mmol) of the cis isochromandione from step 3 above gave 157 mg (49% yield) of the titled tetracycle. (MP: 118°-120 ^β C). ^l E NMR (200 MHz, CDC1 ₃ 5:1.69 (d, 3H, J-6.5 Hz, CH ₃ ), 2.74 (ddd, IH, J-3.7, 10.8, 18.5 Hz, Bcla), 3.24 (dt, IB, J-2.8, 18.5 Bz, HCBa), 4.26 (dd, IB, J-2.9, 10.9 Bz, OCB), 5.04 (m, IB, CHCHj), 5.38 (bs, 2B, OCB ₃ ), 7.59 (d, 2B, J-8.9 Hz, ArH), 7.7 (m, 2H, ArB), 8.05 ( , 2B, ArB), 8.26 (d, 2B, J-8.9 Bz, ArB), 8.60 (s, IB, ArB), 8.61 (s, IB, ArB), CMR (75.44 MBz, CDC1 ₃ ) 5:20.6, CB ₃ ; 26.2, CH ₂ ; 65.5, OCH,; 71.0, 71.4, OCH; 123.9, 128.6, 129.9, 129.7, 130.2, aryl CH; 127.8, 127.9, 128.2, 134.7, 141.1, 142.2, 146.7, 147.2, aryl C; 169.7, ester C-O; 182.2, 182.5, quinone C-O.

Step 6: (1S,3R) and (1R,3S) Trans-p-nitrobenzyl(5,12- dioxo-l-mβthyl-3,4,5,12-tetrahydroanthraceno[2,3- c] yran-3-yl)formate

Following the procedure as described in "example 4, step 2", the reaction between 175 mg (1.1 mmol) of 3,6- dihydrobenzo-(b)-l,2-oxathiin-2-oxide with 187 mg (0.5 mmol) of the quinone from step 4 above gave 119 mg (52% yield) of the titled compound. (MP: 131°-132°C). -E NMR (200 MHz, CDClj) 5:1.62 (d, 3H, J-6.8 Hz, CH,), 2.89 (ddd, IB, J-2.0, 8.9, 19.0 Bz, BCla). 3.11 (ddd, IB, J-1.0, 4.6, 19.1 Bz, HCIs, 4.69 (dd, IH, J-4.7, 8.8 Hz, OCH), 5.30 (bq,

UBSTITUTE SHEET

•98

IB, J-6.8 Bz, CHCH ₃ ), 5.35 (bs, 2B, OCB ₃ ) , 7.55 (d, 2H, J-8.7 Hz, ArH), 7.7 (m, 2H, ArH), 8.05 (m, 2H, ArH), 8.20 (d, 2B, J-8.7 Hz, ArH), 8.58 (bs, 2H, ArH). CMR (75.44 MHz, CDC1 ₃ ) 5: 19.6, CH ₃ ; 25.3, CH ₃ ; 65.5, 0CB ₃ ; 67.0, OCH; 123.9, 128.7, 128.8, 129.6, 130.2, aryl CH; 127.9, 128.0, 128.2, 134.8, 141.1, 142.3, 146.7, 147.6, aryl C; 170.3, ester C-O; 182.8, 182.9, quinone C-O.

Step 7s (1R,3R) and (1S,3S) Cis-p-nitrobenzyl(5,12-dioxo- 7, 10-dimethoxy-l-methyl- ' 3,4,5,12- tetrahydroanthraceno [2,3-c] pyran-3-yl ) ormate

Following the procedure as described in "example 4, step 2", the reaction between 95 mg (0.4 mmol) of the sultine from example 1, step 4, with 71 mg (0.2 mmol) of the quinone from step 3 above gave 42 mg (41% yield) of the titled tetracycle. (MP: 154 ^β -156 ^β C). ~E NMR (200 MHz, CDC1 ₃ ) δ:1.68 (d, 3B, J-7.6 Bz, CB ₃ ), 2.75 (ddd, IB, J-3.6, 10.7, 18.6 Bz, CHfia), 3.27 (bdt, IH, J-2.8, 18.6 Hz, HCHe) , 4.00 (s, 3H, OCH,), 4.01 (s, 3B, OCH,), 4.28 (dd, IB, J-2.8, 10.7 Bz, OCB), 5.06 (m, IB, OCHCH,), 5.38 (bs, 2B, OCB ₃ ), 6.90 (s, 2B, ArB), 7.59 (d, 2B, J-8.9 Bz, ArB), 8.26 (d, 2B, J-8.9 Bz, ArB), 8.95 (s, IB, ArB), 8.98 (s, IB, ArB). IR (FT, CDC1 ₃ ) *max: 1757, este C-O; 1664, quinone C-O. MS (DCI, 240, NH,) m/e: 517 (100, M+) , 382 (68, M+- C ₇ H,N0 ₂ ) .

Step 8s (1R,3R) and (1R,3S) Tra___s-p-nitrobenzyl(5,12- dioxo-7 , 10-dimethoxy-l-methyl-3 , 4,5,12- tetxahydroanthraceno [ 2 , 3-c ] pyran-3-yl ) formate

Following the same procedure as described in "example 4, step 2", the reaction between 70 mg (0.3 mmol) of the sultine from "example 1, step 4", and 53 mg (0.15 mmol) of quinone from step 4 above gave 30 mg (44% yield) of the titled tetracycle. (MP: 180*-182*C). -E NMR (200 MBz,

UBSTITUTE SHEET

98A

Example 5: Preparation of 1-methylated tetrahydroanthraceno [2 -c] pyιaa-3-yl derivatives

SUBSTITUTE SHEET

99

CDC1 ₃ ) 5: 1.57 (d, 3H, J-6.8 Hz, CH ₃ ), 2.84 (ddd, IH, J-2.1, 9.1, 19.0 Hz, HCHa), 3.06 (ddd, IH, J-1.2, 4.6, 18.9 Hz, HCBe), 3.95 (s, 3H, OCH ₃ ), 3.96 (s, 3H, OCH ₃ ), 4.63 (dd, IH, J-4.69, 8.9 Hz, OCH), 5.29 (m, IH, OCHCH ₃ ), 5.30 (bs, 2H, OCBj), 6.86 (s, 2H, ArH), 7.50 (d, 2H, J-8.8 Hz, ArH), 8.16 (d, IH, J-8.8 Hz, ArH), 8.91 (s, IH, ArH), 8.92 (s, IH, ArH). CMR (75.44 MHz, CDC1 ₃ ) δ: 19.7, CH ₃ ; 25.4, CH ₂ ; 55.9, 2XOCB ₃ ; 65.5, OCB ₃ ; 67.1, 67.8, OCH; 107.6, 123.4, 123.9, 128.7, aryl CH; 127.4, 127.5, 128.6, 140.9, 142.3, 146.8, 150.9, aryl C; 170.4, ester C-O; 182.8, 182.9, quinone C-O. IR (FT, CDC1,) 'max: 1757, ester C-O; 1664, quinone C-O.

EXAMPLE 6

Step 1: 5,8-dimethoxyisochroαan-3-yl carboxylic acid

A solution containing 133 mg (0.5 mmol) of the ethylbenzyl-isochroman formate from "example 1, step 5", in

10 ml of THF was added 10 ml of a 1M aqueous sodium hydroxide solution. After stirring at room temperature for

0.5 hour, the mixture was evaporated down to 5 ml and then diluted with 25 ml of water. The aqueous layer was extracted three times with 20 ml aliquots of CH ₂ C1 ₂ , then acidified with concentrated aqueous HCl and reextracted four times with 50 ml of ethyl acetate. Only the combined ethyl acetate layers were kept, and after washing twice with water, evaporation of solvent gave 125 mg (99% yield) of the titled isochromanyl acid. (MP: 217-218'C). -E NMR

(200 MHz, DMSO-d δ: 2.88 (broad ddt, IH, J-9.9, 17 Hz,

HCB&CB), 3.15 (ddd, IH, J-1.4, 4.6, 17 Hz, BCBeCB), 3.91

(β, 3B, OCH,), 3.93 (a, 3B, OCH,), 4.46 (dd, IB, J-4.5,

9.9, OCBC-O), 4.75 (broad dt, IB, J-16.4 Bz, ArB, CH^O),

5.05 (broad dd, IH, J-16.5 Hz, ArHClsP), 6.92 (dd, 2B,

J-11.2 Bz, ArB). CMR (75.44 MHz, CDC1,) 5:26.7, CH ₃ ; 49.9,

OCH ₃ ; 55.9, 56.1, ArOCH ₃ ; 65.3, OCB; 108.7, 109.1, aryl CH;

123.1, 124.5, 150.6, 152.3, aryl CH; 210.2, C0 ₃ H.

SUBSTITUTE SHEET

100

Step 2: Methyl (5,8-djjoethoxyisochrα__an-3-yl) ketone

_. t

To a solution containing 150 mg (6.3 mmol) of isochromanyl acid (261) in 15 ml of anhydrous TBF was added dropwise, over 5 minutes at -78*C and under argon, 0.94 ml of a 1.4M methyl lithium in ether solution. The solution was stirred for 10 minutes at -78*C then warmed to room temperature and stirred for two more hours. Methanol (1 ml) was then added, followed with 25 ml of water, and the mixture was extracted three times with 50 ml of dichloromethane. The combined organic layer was washed with 25 ml water, 25 ml of saturated aqueous NaCl, and dried over MgS0«. After evaporation of solvent, flash chromatography of the residue (10% ethylacetate in toluene) gave 110 mg (74%) of the titled isochroman ketone. (MP: 84*-85*C). *B NMR (200 MHz, CHC1,) 5:2.32 (s, 3B, COCH,), 2.59 (bdd, IB, J-11.3, 17.00Hz, HCfla), 3.04 (ddd, IB, J-1.5, 3.8, 17.1, HCHe , 3.78 (s, 3H, OCH,), 3.79 (s, 3H, OCH,), 4.08 (dd, IH, J-3.8, 11.4 Hz, O-CH), 4.66 (bd, IH, J-15.9 Hz, HcSa-O), 5.04 (bd, IB, J-15.9 Hz, BCflfi-O) , 6.66 (dd, 2H, J-9.0 Bz, ArB). CMR (75.44 MHz, CDC1 ₃ ) 5:24.7, CH ₃ ; 25.9, CH ₃ ; 55.4, 55.6, ArOCH,; 64.7, OCH,; 79.1, OCB; 107.2, 107.7, aryl CB; 122.2, 123.9, 149.2, 151.0, aryl C; 208.4, C-O. IR (CDCl,) 'max: 1717, C-O.

Step 3s Methyl (5,8-dioxo-5,8-d_Lhydroisochroβan-3-yl) ketone

To a stirred solution containing 700 mg (3 mmol) of the isochroman from step 2 above in 20 ml of acetonitrile was added dropwise over 5 minutes, at room temperature, a solution containing 2.0g (3.6 mmol) of eerie ammonium nitrate in 20 ml of water. Stirring was continued for five minutes and then 100 ml of dichloromethane was added to the mixture. Successive washings of the organic layer were done with 50 ml of water and 50 ml of brine. After drying

101

over MgS0 _< , evaporation of solvent gave 560 mg (92% yield) of the yellow isochromandione, as a dark yellow oil. *H NMR (300 MBz, CDC1 ₃ ) 5:2.34 (s, 3B, C0CB ₃ ), 2.48 (dddd, IH, J-2.8, 4.0, 10.1, 19.2 Hz, BCfla). 2.87 (dm, IB, J-19.2 Bz, HCHβ), 4.06 (dd, IB, J-4.0, 10.1 Bz, OCB), 4.52 (dt, IH, J-3.3, 18.6 Hz, HC£a-0), 4.83 (ddd, IH, J-1.0, 2.7, 18.6 Bz, HCHe-O), 6.80 (dd, 2H, J-10.2 Bz, HC-CH) . CMR (75.44 MBz, CDCl,) 5: 23.1, CH,; 25.8, CH,; 62.7, OCH,; 77.7, OCB; 136.0, 136.4, CB; 183.3, 139.6, quaternary C; 185.17, 185.24, quinone C-O, 206.4, COCH,. IR (CDCl,) ^v max: 1722, COCB ₃ ; 1659, quinone C-0.

Step 4s Methyl(11-hydroxy-5 ,12-dioxo-3,4,5,12- tetrahydroanthra- ceno[2,3-c]pyran-3-yl) etone and Methyl (5-hydroxy-5, 12-dioxo-3,4,5,12- tetrahydroanthraceno[2,3-c] yran-3- yl) etone, BCH-687

These compounds were obtained by following the same procedure as described in step 1 of example 3 and using the methyl 5,8-dimethoxyiaochro_nan-3-yl ketone in this example. Flash chromatography of the residue gave a mixture of both regioiso ers (64%) which were not necessarily separated for the next step. The two regioisomera could however, be separated by preparative BPLC. The lass polar regioisomer had *B NMR (300 MHz, CDCl,) δ: (a, 3H, CB,), 2.62 (m, IB, BC&CBC-O), 3.08 (m, IB, BCBaCBC-O), 4.10 (dd, IB, J-10.0, 3.7 Bz, BCHa-O), 5.02 (broad d, IH, HCflβ-O), 7.73 (m, 2H, ArH), 7.97 (m, IB, ArB), 8.12 (a, IB, ArB), 8.50 ( , IH, ArH), 13.86 (a, IH, ArOB). The more polar regioisomer had ^l E NMR (300 MBz, CDCl,) 5: 2.35 (a, 3B, CB,), 2.59 (m, IH, HCIeCHCO), 3.05 (broad dt, J-19.0, 2.8 Bz, HCHaCBCO), 4.08 (dd, IB, J-10.0, 3.9 Bz, CH), 4.44 (dt, IH, J-22.5, 6.7, 373 Hz, BCfla-O), 5.03 (broad d, IB, J-18.7 Bz, BCHe-O), 7.71 ( , 2B, ArB), 7.95 (m, IB, ArB), 8.13 (a, IB, ArB), 8.46 ( , IB, ArB), 13.70 (a, IB, ArOH).

SUBSTITUTE SHEET

•102

The autoxidized product, methyl (5,12-dihydroxy-6,ll dioxo-3,4,6,11-tetrahydroanthraceno [2,3-c] pyran-3-yl) ketone BCB-688, was also obtained in 8% yield. (MP. 210* decomposes). ^l E NMR (300 MBz, CDCl,, CDCl,) 5: 2.40 (s, 3H, COCH,), 2.81 (m, IH, BCfla) , 3.22 (ddd, B, J-2.0, 4.0, 18.1 Bz, BCIfi), 4.15 (dd, IH, J-3.9, 10.6 Bz, OCB), 4.79 (dt, B, J-2.2, 17.4 Bz, OHCHa t 5.20 (dd, IH, J-l.l, 17.4 Bz, HCflfl), 7.84 ( , 2B, ArB), 8.36 (m, 2B, ArB), 13.18 (a, IB, ArOH), 13.31 (s, IH, ArOB).

Step 5s Methyl(6,1l-diacetoxy-5,12-dioxo-3,4,5,12- tetrahydroan-thraceno[2,3-c]pyran-3-yl)ketone

The titled compound waa obtained in 45% yield by following the same procedure as described in example 3 by sequentially carrying out step 7 and 8. ^l E NMR (300 MHz, CDCl,) δ: 2.35 (a, 3H, COCH,), 2.51 (a, 3H, OCOCH,), 2.54 (a, 3H, OCOCH,), 2.72 (m, IH, HCH.), 3.05 (m, IB, BCBs), 4.08 (m, IH, OCHC-O), 4.73 (bd, IH, OHCfla), 5.08 (bd, IB, OHCflft), 7.65 (m, 2H, ArH), 8.15 (m, 2H, ArH).

The mixture of monoacetoxylated compounda BCH-721 methyl (6-hydroxy-ll-acβtoxy and 6-acβtoxy-ll-hydroxy-5,12-dioxo- 3,4,5,12-tetrahydroanthraceno [2,3-c] pyran-3-yl) ketone, could be recovered in 10%.

Step 6s Methyl (11-acβtoxy-l ,6-dihydroxy-5,12-dioxo- 3,4,5,12-tetrahydroanthraceno[2,3-c]pyran-3- yl)ketone.

The titled compound waa obtained in 24% yield by following the same procedure as described in example 3, atep 9. *B NMR (300 MHz, CDCl,) δ: 2.38 (a, 3H, COCH,), 2.52 (a, 3B, OCOCH,), 3.04 (m, 2H, CB,), 4.55 '-

STITUTE SHEET

103

OCBC-O), 6.36 (ba, IB, OCgOH), 7.82 (m, 2H, ArB), 8.23 ( , 2H, ArB).

Step 7: (1'S,1R,3S) and (1'S,1S,3R) Methyl (l-[2',3',6'- trideoxy-3 ' -trif luoroacetamido-L-4 '-O-p- nitrobenzoyl-L-lyxohβxo-pyranoββ)-6-hydroxy-ll- ac e t o xy - 5 , 12 -di oxo - 3 , 4 , 5 , 12 - tetrahydroanthraceno ( 2 , 3-c ) pyran-3-yl ) ketone

Following the regular glycosylation procedure (step 10, example 1) the desired titled pyranoanthracycline gycoside 2 were obtained in 37% yield. ~E NMR (300 MHz, CDCl,) δ: 1.21 (overlapped d, 3H, 6'-CH ₃ ), 2.11 (m, 2H, 2'- CB ₃ ), 2.38 (s, 3B, -COCH,), 2.53 (a, 3B, -OCOCH,), 2.68 (m, IB, HCfla), 3.21 (m, IH, HCfift) , 4.39 ( , IB, 5'-CH), 4.63 (m, 2H, 3' and 4' CH) , 5.16 (dd, IB, J-4.1, 12.6 Bz, O-CB), 5.48 and 5.78 (ba, IB, l'-CH), 6.21 (a, IH, O-CH-O), 7.81 (m, 2B, ArB), 8.25 (overlapped m, 6H, ArB), 13.69 (s, IB, ArOB).

Step 8 s (l'S') Methyl [ 1- ( N-trlf luoroacyldaunosamlne ) -6- hydroxy-ll-acetoxy-5 , 12-dioxo-3 , 4 , 5 , 12- tetrahydroanthraceno (2,3-C) yran-3-yl ] ketone .

A cooled (-5*C) solution containing 48.4 mg (0.06 mmol) of the glycoside from atep (before) in 5 ml of dichloromethane waa treated with 5 ml of a 0.2 M NaOB methanolie solution for 15 minutes. The reaction mixture waa then quenched by acidification with 0.1N BCl to pB 6, extracted with CH,C1,

(2x15 ml) and the combined organic phaaea were waahad with water (2x30 ml) and dried (Na,S0 ₄ ). Purification by HPLC

(sphβrβx CN5U; hexane; ethylacetate ; 80%-20%) gave 5.3 mg

(14% yield) of the deaired compound. (MP. 235-236*C). ^l E

NMR (300 MHz, CDCl,) 5: 1.30 (d, 3H, J-6.7 Bz, CH,) , 1.87

(m, IB, 2'- BCflft), 2.12 (ra, IH, 2'-HCH_£), 2.37 (s, 3H, -

SUBSTITUTE SHEET

104

'a , 3 '.; , ' ^0C0CB•, ' ^{2,H ,B} ' ^la - "* ^» '< ³ -" ⁽ -.

5 -CH), 4.39 («, IB, 3--CH), 4.60 (dd, IH, J-3.8, 11.4, „- CB), S.S7 (b., IH, l'-CH), 6.13 ,., 1 _B , o-CH-O) , 6.67 (bd, IH, J-8.4 Hz, HH), 7.79 , _B , 28, ArB), 8.21 _(n , 1 _H , _M , , 8.29 (m, IH ArH), 13.49 (., IH, ArOH).

SUBSTITUTE SHEET

104A

SUBSTITUTE SHEET

1Q5-

EXAMPLE 7

Step 1; 3-formyl-5,8-dimethoxyisochroman

Ethyl-(5,8-dimethoxyisochroman-3-yl) formate (697 mg, 2.62 mmol) was dissolved in toluene (20 ml) and cooled to - 78°C. DIBAL (2.97 ml, 1.5M, 4.45 mmol) was cooled to -78°C and added slowly to the reaction mixture over a period of 15-20 minutes. A TLC taken right away after the addition revealed that the reaction waa over. Cold MeOB (4 ml) was added slowly (H ₂ evolutionl) and the mixture was extracted with ethyl acetate (3x50 ml). The organic phases were combined, washed with brine and dried over MgS0 ₄ . Flash chromatography of the crude residue gave the title products (483 mg, 83%). ^l E NMR (300 MBz, CDCl,) 6:2.63 (dd, IB, J-16.8, 11.3 Bz, HClfiCHC-O), 2.99 (dd, IB, J-17.0, 3.8 Bz, HCSfiCHC-O), 3.76 (s, 3B, OCH,), 3.77 (s, 3H, OCB ₃ ), 4.09 (dd, IB, J-10.7, 4.4 Bz, Bβ-O), 4.87 (dd, 2B, J-103.8, 15.9 Bz, BCBa.e-O . 6.66 (m, 2B, ArB).

Step 2: 3-(l-hydroxy-2-mβthoxymβthoxy)ethyl-5,8- dimethoxyiso- chrc

A solution of nBu ₃ SnCB ₃ OMOM (l,066g, 2.92 mmol) in 12 ml of TBF was cooled to -78*C under argon. The solution was stirred while n-BuLi (1,1 ml, 2.5M, 2.75 mmol) was added. After 30 minutes, 3-formyl-5,8-dimethoxyisochroman (483 mg, 2.18 mmol) was added. After 45 minutes, the cold reaction mixture was partitioned between water and ether. The organic phase was dried over MgS0 ₄ , filtered, and concentrated. Flash chromatography of the crude residue (hβxanβ/ethyl acetate; 1:1) gave the title compound in 66% yield (431 mg). The following spectral data were obtained from the mixture of two diaβtβrβoiβomβrβ. ^l E NMR (300 MBz, CDClj, ppm) δ: From 2.49 to 3.10 (m, 3B, BCHj_ _t S"CH-C-0 and OB), 3.35 (s, 3B, CB,0Cfl,), ∑rom 3.55 to 3.92 (xα, 6B, CB,-

SUBSTITUTE SHEET

- 106

OMOM, CBaeCB»Q and fifl-OB), 3.69 (s, 3B, OCB ₃ ) , 3.71 (s, 3B, OCB ₃ ), 4.55 ( , IH, HCHa-O) , 4.64 (s, 2H, OCgj-OCH,) , 4.90 (m, IH, HCBe-O), 6.57 (m, 2B, ArB). IR: (neat), 3464 (OB), 2940, 2830, 1480, cm' ¹ .

Step 3: 5,8-di___βthoxy-3-(methoxymethoxy)acetoisochroman

To a stirred solution of oxalyl chloride (3.37 ml, 38.67 mmol) in 80 ml of CB ₂ C1 ₃ was added dropwise a solution of DMSO (2.99 ml, 38.67 mmol) in 10 ml of CB ₃ C1 ₂ at -78*C under argon over a period of 5-10 minutes. The stirring waa continued for 15 minutea before adding a aolution of 3-(l-hydroxy-2-arthoxy-αethoxy)ethyl-5,8- dimβthoxyiβochroman (3,842g, 12.89 mmol) in 20 ml of CB ₃ C1 ₃ over a period of 5 minutes. After the mixture was stirred for 45 minutes at -78*C, triβthylaminβ (19.6 ml) was introduced. The reaction mixture was waxaed to room temperature over 1 hour, quenched with 100 ml of water. The aqueous layer was extracted with CB ₃ C1 ₃ (3x100 ml) and the combined organic phases were washed with water and brine, dried over MgS0 ₄ , filtered, and concentrated to a yellow residue that was purified by flash chromatography (hexanβ/ethyl acetate; 7:3) to give the titled compounds (3.295g, 86%). ^X H NMR (300 MHz, CDC1 ₃ ) 5:2.63 (dd, IH, J-13.9, 5.7 Hz, BCB_*CHC-0), 3.07 (dm, IB, BCHftCBCO) , 3.39 (s, 3H, CHjOCfi,), 3.75 (a, 3H, OCH,), 3.76 (a, 3H, OCB ₃ ) , 4.19 (dd, IH, J-11.4, 3.8 Hz, CH-O), 4.60 (a, 2H, CBjOCB ₃ ) , 4.79 (dd, 2H, J-111.0, 15.9 Bz), 6.63 (m, 2B, ArB). "C NMR (75.44 MHz, CDCl,) δ: 25.37, 56.01, 56.18, 65.31, 70.21, 97.06, 107.84, 108.37, 122.59, 124.33, 149.81, 151.62, 206.78. IR (neat) 2960, 2910, 1735 CO, 1620, 1580, 1540, 1480 car ¹ .

S*_?g 41 3-(■el ¹ In11jmethoxy)aceto-5,8-dioxoiaochroman

5,8-dimβthoxy-3-(methoxymethoxy)-acetoiaochroman

SUBSTITUTE SHEET

-107-

(3.155g,

10.59 mmol) was dissolved in acetonitrile (35 ml) and cooled to 0°C. A solution of eerie ammonium nitrate (CAN) (17.53g in 35 ml of B ₂ 0) was added dropwise with stirring. The ice bath was then removed and stirred for an extra 15 minutes. Water (30 ml) was added and the mixture was extracted with CH ₂ C1 ₂ . The organic phases were washed with water, dried over MgS0 ₄ , filtered and concentrated in vacuo to give a reddish oil which was found to be pure title compound without flash chromatography. (2.38g, 85% yield). ^l E NMR (300 MBz, CDC1 ₃ , ppm) δ: 2.52 (m, IB, BCIfi-CBC-0), 2.90 ( , IB, BCleCBC«0), 3.39 (s, 3B, OCBjOCJL,), 4.19 (dd, IB, J-10.3, 3.8 Bz, BC-O), 4.44 (td, IB, J-18.7, 3.7 Bz, BC1&-0), 4.54 (s, 2H, OCBj-OCH,), 4.70 (s, 3B, COCBj-OMOM) , 4.77 (dd, IB, J-18.6, 2.5 Bz, BCSfi-O), 6.75 (m, 2B, ArB).

Step 5s 3-(2-methoxymethoxy)acβto-6-hydroxy-l,2,3,4- tetrahydro-(2-oxygen)napthacene-5,12-dione and 3-(2-methoxymethoxy)acβto-11-hydroxy-1,2,3,4- tβtrahydro-(2-oxygen)-napthacene-5,12-dione

To a solution of LDA (9.42 mmol) in 30 ml of TBF was added dropwise a solution of homophthalic anhydride (1.53g, 9.43 mmol) in 30 ml of TBF at -78*C under argon. After 5 minutes, a solution of 3-(2-methoxymethoxy)aceto-5,8- dioxoiaochroman (2.3589, 8.14 mmol) in 35 ml of TBF was introduced. The stirring was continued at -78°C for 20 minutes and then at room temperature for 30 minutes. After the mixture was kept in the fridge overnight, it was quenched with sat. NB ₄ C1 solution (40 ml) and then carefully partitioned between IN BCl (15 ml) and CH ₂ C1 ₃ (150 ml). The organic layer was washed with brine, dried over NajS0 ₄ , and then concentrated to a crude residue which was purified by flash chromatography to give a mixture of the two regioisomers (46%). The following spectral data were obtained from the mixture of two regioisomers. ^: H NMR-

SUBSTITUTE SHEET

108

(300 MHz, CDCl;,), δ:2.58 (m, 2H, HCSftCBC-O) , 3.04 (dd, 2H, J-19.2, 3.26 Bz, BCIft-CHCO), 3.40 (s, 6B, OCH ₃ ), 4.22 ( , 2B, CB-O), 4.57 (s, 4B, OCfijOCHj), 457 (m, 2H, HCHA-O), 4.58 (a, 4H, COCBj-OMOM), 4.87 (m, 2H, BClft-O, 7.67 (m, 4B, ArB), 7.69 (m, 2B, ArB), 7.86 (m, 2B, ArB), 8.38 ( , 2B, ArB). IR: (neat) 2925, 2843, 1740 (CO), 1654, 1639, 1608, 1568 cm- ^χ .

Step 6s 6 and 11-acetoxy-3-(2-methoxymethoxy)aceto- 1,2,3,4- tetrahydro-(2-oxygen)naphtacene-5,12-dione

The rβgioiaomβric mixture from atep 5 above (l,962g, 5.13 mmol) was dissolved in CB ₂ C1 ₂ (130 ml). Acetic anhydride (26.7 ml), pyridine (26.7 ml) and DMA? (0,518g) were successively added and stirred at room temperature for 30 minutes. The reaction mixture was then poured onto ice water, and extracted with CB ₂ C1 ₂ . The organic layers were combined, washed with HCl (4%), water, dried over sodium sulfate, and concentrated to give a crude residue which was flash chromatographed using hexane/BtOAc (1:1) to give a mixture of the desired titled compounds in greater than 80% yield. The following apectral data were obtained from the two rβgioiaomβric mixtures. *B NMR (300 MHz, CDClj), δ:2.54 (m, 2H, HCfiaCBC-O), 3.10 (m, 2H, BCflftCBC-O) , 3.40 (s, 3H, CBjOCJB)), 3.41 (s, 3H, CHjOCHj), 4.23 (dd, 2H, J-10.48, 3.84 Hz, OCHC-O), 4.58 (s, 2H, CO-Cg,OMOM), 4.58 (s, 2H,

COC&OMOM), 4.58 (m, 2H, HCH_ft-0), 4.72 (s _r 4H, COCg,OCB ₃ ), 4.95 (m, 2B, HCIfO) _$ 7.72 (m, 4B, ArH), 8.19 (m, 4H, ArB), 8.54 (s, IB, ArH), 8.58 (s, IH, ArH).

SUBSTITUTE SHEET

109

Step 7: 3-(2-hydroxy-l-propyleneketal)aceto-6-acetoxy- 1,2,3,4-tetrahydro-(2-oxygen)naphthacene-5,12- dione and

3-(2-hydroxy-l-propyleneketal)-aceto-ll-acetoxy- 1,2,3,4-tetrahydro-(2-oxygen)naphthacene-5, 12- dione

The mixture of tetracycles from step 6 above (70 mg, 0.16 mmol) was dissolved in toluene (30 ml) followed by the addition of 1,3-propanediol (1 ml) and PPTS (2 mg) . The reaction mixture was refluxed overnight using a Dean Stark trap to remove the water formed during the process. The reaction mixture was extracted with CH2CI2. the organic phases were combined, washed with H2O, dried over sodium sulfate, and concentrated jLn vacuo. The crude residue obtained was flash chromatographed using EtOAc/CH2Cl2 (1:1) to give the desired title products (66 mg) in 94% yield. The following spectral data were recorded on the two regio- isomeric mixtures. !H NMR (300 MHz, CDCI3) δ:1.55 (m, 2H, 0-CH ₂ CH-H-CH ₂ -0), 1.85 (m, 2H, 0-CH ₂ -CH-H-CH ₂ θ) , 2.05 (m, 2H, OH), 2.25 (m, 2H, HCHaCHC<) , 2.58 (s, 3H, OCOCH3), 2.61 (s, 3H, OCOCH3), 2.80 (m, 2H, HCHeCHC<), from 3.55 to 4.15 ( , 14H, 0-CH ₂ -CH ₂ -0, CH ₂ 0H and HC-O), 4.52 (m, 2H, HCHa- O), 4.94 (m, 2H, HCHe-O), 7.69 (m, 4H, ArH), 8.07 (m, 4H, ArH), 8.52 (s, IH, ArH), 8.57 (s, IH, ArH). IR: (neat) 3454.6, 2929.2, 2878.6, 1766.4 (CO), 1658.0, 1612.4 cm ^-1 .

Step 8: 3-(2-acetoxy-l-propyleneketal)aceto-6-acetoxy- 1, 2,3,4-tetrahydro-(2-oxygen)naphthacene-5, 12- dione and

3-(2-acetoxy-l-propyleneketal) ceto-11-acetoxy-l, 2,3,4-tetrahydro-(2-oxygen)naphthacene-5,12- dione

A mixture of tetracyclic compounds of step 7 above (367 mg, 0.84 mmol) was dissolved in CH2CI2 (20 ml) followed by the successive addition of acetic anhydride

SUBSTITUTE SHEET

110

(1,0 ml), pyridine (1,0 ml) and DMAP (60.9 mg) . Within half hour, the reaction was complete. It was extracted with CH2Cl2« The organic phases combined, washed with H2O, dried over MgSθ4, and concentrated iri vacuo. The crude residue obtained was flash chromatographed using hexane/EtOAc (7:3) to give the desired products as a mixture (278 mg, 0.6 mmol, 69%). The following spectral data were recorded on the two regioisomeric mixtures. ^H NMR (300 MHz, CDCI3, ppm) δ:1.85 (m, 4H, OCH ₂ CH ₂ -CH2θ) , 2.11 (s, 6H, CH ₂ OCOCH ₃ ), 2.60 (s, 3H, OCOCH3), 2.63 (s, 3H, OCOCH3), 2.86 ( , 4H, HCHa.eCHC<) 3.98 (m, 10H, OCH2CH2- CH ₂ -0 and 0CHC<), 4.29 (d, 2H, J=11.87, CH ₂ - OCOCH3), 4,5 ( , 2H, HCHa-0) , 4.94 (dd, 2H, HCHeO), 5.05 (d, 2H, CH2OCOCH3), 7.70 (m, 4H, ArH), 8.10 (m, 4H, ArH), 8.56 (s, IH, ArH), 8.61 (s, IH, ArH), IR: (neat) 2922.3, 1774.8, 1745.6 (C=0), 1669.9, 1617.5 (C=0 quinone), 1431.1 cm" ¹ .

Step 9: 3-(2-acetoxy-l-propyleneketal)aceto-6-acetoxy-l- hydroxy-1,2,3,4-tetrahydro-(2-oxygen)naphthacene- 5,12-dione and

3-(2-acetoxy-l-propyleneketal)-aceto-ll-acetoxy- 1-hydroxy-l,2,3,4-tetrahydro-(2- oxygen)naphthacene-5,12-dione

A mixture of tetracyclic compounds of step 8 above (34 mg, 0.09 mmol) was dissolved in CCI4 (13 ml) followed by addition of NBS (32 mg, 0.18 mmol) and benzoyl peroxide (2 mg) . The reaction mixture was refluxed and irradiated by sun-lamp under argon for about one hour. It was then concentrated jji vacuo and treated with stirred in THF/H2O (6 ml, 1:1) before it was extracted with C^C^. The organic phases were combined, washed with H2O, dried over sodium sulfate and concentrated ^ri vacuo. The crude residue obtained was flash chromatographed using

SUBSTITUTE SHEET

111

EtOAc/CHjCl _j (1:1) to give the desired products as a mixture (26.5 mg, 0.05 mmol, 59%). The following spectral data were recorded on the two regioisomeric mixtures. -E NMR (300 MBz, CDC1 ₃ , ppm) δ: 1.85 (m, 4B, OCB ₂ -CI _J -CB ₂ -0), 2.12 (s, 6H, CBjOCOCI,), 2.60 (s, 3B, OCOCHj), 2.62 (s, 3H, OCOC1,), 2.86 (m, 4B, HCg^_£CHC<), 4.08 (m, 8B, OCB,-CB ₃ - CIi-O), 4.38 (d, 2B, CBj-OCOCB,), 4.46 (dd, 2B, OCBC<, 4.89 (broad d, 2B, J-12.03, CB ₂ -OCOCB ₃ ), 6.14 (broad d, 2H, J-12.64, CaOB 7.71 (m, 4B, ArB), 8.05 (m, 4B, ArB), 8.56 (a, IB, ArB), 8.59 (a, IB, ArB). IR: (neat) 3402.0 (OB), 2925.3, 1772.2; 1735.0, 1666.8, 1617.3, 1443.7 cm" ¹ .

Step 10s 3-(2-hy roxy)acβto-6-hydroxy-l,2,3,4-tβtrahydro- (2-oxygen)naphthacene-5,12-dione and

3-(2-hydroxy)acβto-ll-hydroxy-1,2,3,4- tβtrahydro-(2-oxygen)naphthacene-5,12-dione

A reaction mixture containing both tetracyclic regioisomers from step 5 above (500 mg, 30%, 0.39 mmol) was diaaolved in 20 ml of CB ₃ OB followed by addition of 10 ml of 2.5 BCl βolution. The mixture waa stirred at room temperature for 0.5h before it was extracted with CB ₂ C1 ₂ (150 ml). The organic layer was washed with H ₃ 0, dried over MgS0 ₄ , filtered and then concentrated to a residue that was purified by flash to give an inseparable mixture of the titled compounds. (20 mg, 15%). The following spectra were recorded on the two regioisomeric mixtures. *B NMR (300 MBz, CDCl,) 0:2.80 (m, IH, BCIaCHC-O), 3.1 (m, IB, HCIfiCHC-O), 3.67 (a, 2H, CB,OB), 3.84 (a, 3B, OCH,), 4.33 (m, IH, CH-O), 4.68 (m, IB, BClftO), 5.03 (m, IB, BCIfi- 0), 7.71 (m, 2H, ArB), 7.94 (m, IH, ArB), 8.11 (m, IH, ArH), 8.48 (m, IB, ArB), 13.70 (s, IB, exchangeable OB), 13.83 (a, IB, exchangeable OB).

SUBSTITUTE SHEET

112

Step 11: 6,ll-diacβtoxy-3-(2-methoxymethoxy)aceto- 1,2,3, -tetrahydro-(2-oxygen)naphthacene-5,12- dione

A mixture of tetracyclic compounds from step 5 above (273 mg, 0.71 mmol) was treated with pb(0Ac) ₄ (370 mg, 3.1 mmol) in dark in the presence of 10 ml CB ₂ C1 ₃ and 30 ml AcOB at room temperature for 2 days. The mixture was concentrated in vacuo. The residue was partioned between BjO and CHC1 ₃ . The organic layer was washed with brine, dried over MgS0 ₄ and concentrated to a residue which was then treated with Ac ₂ 0 (4 ml), pyridine (4 ml) and DMAP (68 mg) for 2 houra. 20 ml of B ₂ 0 and 50 ml of CB ₂ C1 ₃ were added. The organic layer waa separated, washed with NaCl solution and dried over MgS0 ₄ . Flash chromatography of the residue gave the titled compound (47 mg, 17%). The following spectral data were recorded on the two regiosomeric mixtures. ^X H NMR (300 MBz, CDC1 ₃ ) δ: 2.50 (s, 3B, OCOC1,), 2.51 (s, 3B, 0C0C&,), 2.25 (m, IB, BCBaCB- C-O), 3.10 (m, IB, BCfifiCHC-O), 3.40 ( a, 3H, CB ₂ 0CBj) , 4.27 (m, IB, CBO), 4.61 (s, 2B, CH _J OCH _J ) , 4.72 (s, 2B, COC&OMOM), 4.70 (m, IB, BCflfi-O), 5.05 (m, IB, HCHe-O), 7.73 (m, 2B, ArB), 8.16 (m, 2B, ArB). IR: (neat) 2934, 1775, 1742 (CO), 1683, 1598, cm" ¹ .

Step 12: 3-(2-hydroxy)acβto-6-acβtoxy-l-hydroxy-l,2,3,4- tetrahydro-(2-oxygen)naphtacene-5,12-dione and

3-(2-hydroxy)acβto-ll-acβtoxy-l-hydroxy-1,2,3,4- tetrahydro-(2-oxygen)naphtacene-5,12-dione

A mixture of tetracyclic compounds from step 11 above (30 g, 0.06 mmol) was dissolved in acetone and B ₂ 0 (12 ml, 1:1, 58.42) followed by the dropwise addition of BCl cone. (-4 ml). The mixture was stirred at room temperature for

SUBSTITUTE SHEET

113

several hours until the ■ ar ing material was consumed. NaBCOj solution (sat.) was then added until a pB of. 8 waβ obtained. The mixture waa then extracted with CH,Cl,, dried over sodium sulfate and concentrated in vacuo. The crude residue obtained was flash chromatographied using ethyl acetate:CH,Cl _j (1:1) to give the title compounds as a mixture (9.3 mg, 0.02 mmol, 39%). The following spectral data were obrainβd from the regioiaomeriα mixture*. *B NMR (300 MBz, CDCl,) δ.2.62 (s, 6H, OCOCH,), 3.15 ( , 20, HCflaCHC-O) , 3.62 (m, 2H, HCfifiCHC- ) , 4.62 (m, 4H, Cfl _j OH) , 4.88 ( , 2B, CH-O) , 6.16 (d, 2B, J-13.95 Hz, CBOH) , 7.74 (m, 4B, ArH), 8.11 (m, 4B, ArH), 8.62 ( , 2B, ArB). IR« (neat) 3367.6 (broad, OB), 2932.2, 2855.7, 1771.7, .1738.8 (C- ). 1664.9, 1617.9 (CO, quinone) cm' ¹ .

^■ 113A-

E»mp«.7. ^^-<*^_ _ytbouata ^

^WΪ Λ » fay rexy eihyl bane subnuen

SUBSTITUTE SHEET

114'

EXAMPLE 8

Ste ^p 1; 3-carboιιetJ_oxy-5,8-d___nethoxyiβot oc]_xoman

l,4-dimβthoxy-2,3-dibromomβthylbβnzβnβ (lO.Og, 30.88 mmol) waβ diββolved in CB ₂ C1, and MβOB (750 ml, 6:4) followed by addition of ethyl 2-mercaptoacetate (4.02 ml, 37.06 mmol) with stirring under argon. The mixture was then cooled to 0*C followed by dropwiae addition of sodium methoxide (4.37M, 8.5 ml, 37.06 mmol) over a period of 2h uaing an automatic syringe pump. After 5 minutes the solvent waβ evaporated and the crude waa rediaβolved in TBF (400 ml) and cooled to 0*C again. NaOEt (2.10g, 30.88 mmol) waβ then added. The ice bath waβ removed and the reaction waβ stirred for 2 more hours. - The reaction mixture waβ then quenched with NB ₄ C1 (sat.) and extracted with ether. The organic phaseβ were combined, dried over MgS0 ₄ , filtered and concentrated. The crude residue was then treated with NaOMB in MeOB and TBF at 0*C for 2 hours. The reaction mixture waβ extracted with ether (100 mlx2) . The combined organic phases were washed with B,0, dried over MgSO _« , filtered and concentrated to a reaidue that was purified by flash chromatography to give title compound in 45% yield. ^l E NMR (300 MBz, CDCl,, ppm) o:2.97 (dd, IB, J-16.51, 8.79 Bz, BCBaCBC-O), 3.36 (dd, IB, J-16.7, 5.1 Bz, HCSeCBC-O), 3.73 (m, 2B, BCB& _t ft-O), 3.75 (s, 3B, OCB,), 3.78 (i, 6B, 2xOCH ₃ ), 3.85 ( , IB, BC-S), 6.69 (m, 2B, ArH).

Step 2t 3-carbcaethoxy-5,8-dioxoiβothiochr<

To a stirred βolution of iaothiochroman from step 1 above (253 mg, 0.94 mmol) in 3 ml of CB,CN waβ added

SUBSTITUTE SHEET

•115

dropwise a solution of eerie ammonium nitrate (1.550g, 283 mmol) in 3 ml of H ₃ 0. The reaction mixture waβ stirred at 0*C for 10 minutes and then at room temperature for 10 minutes, followed by extraction with CB,C1 ₂ (10 mlx3). The combined organic layers were washed with brine and water, dried over MgS0 ₄ , and then concentrated to a yellow residue (213 ag, 0.89 mmol) in 95% yield, which was found to be pure by BNMR. B NMR (300 MHz, CDCl,) 5:2.90 ( , 2B, BCHJ _I ΛCBC-O), 3.56 (m, 2B, BCIftft-S), 3.73 (s, 3B, OCB,), 3.74 (β, 3H, OCB,), 3.75 (m, IH, HC-S), 6.75 ( , 2H, ArB). "C NMR (75.44 MHz, CDCl,) ft: 22.2, 26.0, 37.4, 52.8, 136.2, 137.0, 140.0, 140.5, 172.0 (CO ester), 185.8, 187.0 (CO quinones). IR: (neat) 2956, 1736, 1658, 1607, 1442, 1409 car

Step 3t 3-rarbne__.tho.qp 6-hydroxy-l,2,3,4-tetrahydro-(2- aulfur)naphthacene-5,12-dione and 3-carbomβthoxy-ll-hydroxy-l,2,3,4-tβtrahydro(2- βttlfa )naphthacene-5,12-dione

To * βtirred βolution of LDA (0.98 mnol in 4 ml of TBF) waβ added dropwiββ a βolution of homophthalic anhydride (160.2 ag, 0.99 mmol) in 4 al of dry THF at -78*C under argon. After βtirring for 10 inutββ a βolution of quinone froa βtep 3 above (213.0 ag, 0.89 maol) in 4 al of THF waβ introduced. The βtirring waβ continued at -78°C for 20 minutββ and then at rooa temperature for lh. The mixture waβ quenched with eat _* NB ₄ C1 βolution (10 al) and then portioned between 0.5 N HCl (10 al) and CB,C1, (50 al). The organic layer waβ waβhed with brine, dried over Na,S0 ₄ and concβntratβd_ia £&&£&• The reβidue waβ purified by flaβh chromatography with hβxanβ and ethyl acetate (7:3) to give a mixture of titled compounda (125 ag, 40%). The following spectral data were obtained froa the two rβgioiβoaeric mixturββ. -E NMR (300 MHz, CDCl,), ft:3.09

EET

116

(dm, IB, HCHaCHC-O), 3.21 (dm, IB, BCBeCBC-O) , 3.73 (m, IB, CBS), 3.76 (s, 3B, OCB,), 3.77 (s, 3B, OCB,), 3.82 (m, 2B, BCBa.e-O) . 7.69 (m, 2B, ArB), 7.71 (m, IB, ArB), 8.09 (s, IB, ArB), 8.44 (m, IB, ArB), 13.80 (s, IB, exchangeable OB), 13.89 (s, IB, exchangeable OB) .

Step 4: 6-acetoxy-3-carbomethoxy-1,2,3,4-tetrahydro-(2- sulfur) napthacene-5,12-dione and ll-acetoxy-3-carbomethoxy-l,2,3,4-tetrahydro-(2- sulfur) napthacene-5,12-dione

A mixture of tetracycles from step 3 above (95 mg, 0.268 mmol) waβ treated with Ac,0 (2 ml), pyridine (2 ml) and catalytic amount of DMAP (34 mg) in 10 ml of CB ₃ C1 ₂ at room temperature. After stirring for 0.5h, the mixture was poured into 25 ml of ice water and then diluted with 25 ml of CBjClj. The organic layer waβ washed twice with 0.5 N BCl solution (2x20 ml) and then dried over Na ₂ S0 ₄ . Filtration followed by concentration j i vacuo provided a yellow reβidue that was purified by flash chromatography to give the titled products (56 mg, 53%). The following spectral data were obtained from the two isomeric mixtures. *B NMR (300 MHz, CDCl,, ppm) 5:2.60 (s, 3B, OCOCH,), 3.10 ( , 2H', BCBa.aCBCOi. 3.7 (m, IB, CB-S), 3.74 (s, 3B, OCB,), 3.75 (s, 3B, OCB,), 3.81 (m, 2B, BC1&*£-S), 7.73 (m, 2B, ArH), 8.06 ( , 2B, ArB), 8.59 (s, B, ArB). IR: (neat) 2960, 2922, 1770, 1738 (CO), 1662, 1634, 1623 (CO, quinone) cm" ^x .

116A

Examplt 8: Preptn ^ή on of teoifay rotnthπceno [*3-c] thiopynn-3-yl d _β iv _i tive*

out

BCHβM

SUBSTITUTE SHEET

117

EXAMPLE 9

Step 1: t-Butyl acetoacetate 2-benzoate

To a stirred solution of t-butyl acetoacetate (10 ml, 60 mmol) in benzene (120 al) and THF (60 al) waβ added NaB (1.5g, 61 mmol) in portionβ after 15 minutββ, benzoyl peroxide (2.91g, 12 mmol) diaaolved in benzene (30 ml) waa added. After 1 hour at room temperature, the reaction mixture waβ waβhed with 50 ml of βaturated aqueouβ ammonium chloride, 2x50 ml of aaturated aqueouβ aodium bicarbonate, and 50 ml of saturated aqueous sodium chloride. The organic layer waβ then dried over MgS0 ₄ and the aolvent waβ removed ia ∑asufi. The exceββ of t-butyl acetoacetate waβ removed by dietillation under reduced preββure. The reβidue (2.4g, 72%) iβ obtained aβ a βlightly yellow oil characterized aβ t-butyl acetoacetate 2-benzoate. ^l B NMR (300 MHz, CDCl,) ft:1.52 (β, 9H,-C(CB,),), 2.42 (s, 3B, - CB,), 5.63 (β, IH, -CB-O), 7.47 (dt, J-0.7 and 7.4 Bz, 2B, ArB), 7.61 (dt, J-0.7 and 7.4 Bz, IB, ArB), 8.13 (dd, J-1.4 and 7.1 Bz, 2B, ArH).

Step 2t 3,6-rt1aethoxy-2-broaomethylene-1-(3-t-Butyl- cβxboxylatβ), 3-benzoate, 2-butanone 4-yl) benzene

To a βtirred βolution containing 500 ag, (1.55 mmol) of 3,6-diaethoxy, 1,2-dibroaoaethylene benzene and 684 mg (2.46 mmol) of t-Butyl acetoacetate-2-benzoate in 11 ml of acetonitrile and 6.5 ml of THF waβ added lg of cesium carbonate. After 45 minutes, the reaction mixture was filtered over silica gel. The solvent waβ removed in vacuo. 948 mg of crude 3,6-dimethoxy,2-bromome-thylene 1-

SUBSTITUTE SHEET

118'

(3-t-Butyl carboxylate, 3-benzoate, 2-butanone 4-yl) benzene waβ obtained. *B NMR (300 MBz, CDCl,) 8:1.45 (s,

9B,

-C(CB,),), 2.41 (β, 3B, -COCB,), 3.53 (β, 3B, -OCB,), 3.82

(s, 3B, -OCH,), 3.83 (2xd, J-14.8 Bz, 2B, ArBCB _T -_ . 4.79

(8, 2H,

-CH ₃ -Br), 6.72 (2xd, J-9.0 Bz, 2B, ArH), 7.38 (a, 2H,

ArBCO-), 7.54 (a, IB, ArBCO-), 7.81 (a, 2B, ArBCO-).

Step 3> 3,6-diaβthoxy, 2-hros_αa_ethylene,l-(3-benzoate, 2- bαtanoαe,4-yl)-benzene

To a atirred aolution containing 948 mg of crude B- Ketoeβter in 12 al of acetone waβ βlowly added 6 ml of BBr 48%. The reaction mixture waβ heated to 50*C for 105 minutββ, then poured in 50 al of water and 50 al of ether, 100 al of a βaturatβd aqueous βodiua bicarbonate waβ βlowly added. The aqueouβ layer waβ extracted with 50 ml of ether. The organic layβrβ were waβhed with 50 ml of a saturated aqueouβ βodium chloride, then dried over MgS0 ₄ . Evaporation of the aolvent gave 828 mg of a βlightly yellow oil characterised aβ 3,6-dimethoxy 2-broaαae-thylene, l-(3- benzoate, 2-butanone, 4-yl) benzene. ^l E NMR (300 MBz, CDCl,) 5:2.29 (a, 3H, -COCH,), 3.24 (dd, J-9.0 and 14.3 Bz, IH, -CBj-CH-O-), 3.45 (dd, J-4.6 and 14.3 Bz, IB, -CH^-CH- 0-), 3.81 (β, 3H, -OCH,), 3.83 (β, 3H, -OCB,), 4.80 (2xd, J-9.7BZ, 2H, -CBj-Br), 5.51 (dd, J-4.6 and 9.0 Bz, IB, -CB- 0-), 6.77 (2xd, J-9.0 Hz, 2H, ArH), 7.43 (a, 2B, ArBCO-), 7.55 (a, IB, ArHCO-), 7.95 (a, 2H, ArBCO-).

SUBSTITUTE SHEET

^■ 119

Step 4: 3,6-dimethoxy 2-(3-benzoate, 2-butanone, 4-yl) benzaldehyde

To a stirred solution containing 828 mg of the crude benzylic bromide in 10 ml of dimethylsulfoxide was added 390 mg (4.65 mmol) of sodium bicarbonate. The reaction mixture was heated to 90°C for 25 minutes, then poured in 150 ml of water and extracted with 3x50 ml of ethyl ether. The organic layers were washed with 50 ml of a saturated aqueous sodium chloride, dried over MgS0 ₄ and the solvent waβ evaporated. The reβidue waβ flash chromatographed with hexanes-ethyl acetate 3-1 as the eluting solvent mixture. The titled compound was obtained in 60% yield (319 mg) for 3 steps. -E NMR (300 MBz, CDCl,) 5:2.34 (s, 3H, -COCB,), 3.65 (m, 2B, ArB-CB ₂ -), 3.81 (s, 3B, -OCB,), 3.84 (s, 3B, -OCB,), 5.42 (dd, J-5.1 and 9.1 Hz, IB, -CB-0-), 6.85 (d, J-9.2BZ, IB, ArB), 7.04 (d, J-9.2BZ, IB, ArB), 7.41 (m, 2B, ArBC0 ₂ -), 7.54 (a, IB, ArBCO,-), 8.00 (m, 2B, ArBCO-), 10.62 (s, IB, -CBO).

Step 5s 3,6-djjaethoxy 2-(3-faydroxy,2-butanone, 4-yl) benzaldehyde dimethyl acetal

To a stirred solution containing 319 mg (0.93 mmol) of keto-aldehyde in 10 ml of methanol and 2 ml of methyl orthoformate waβ added 30 mg of p-tolueneaulfonic acid. After 3.5 houra, 600 mg of potaββium hydroxide and 3 ml of water were added and the reaction mixture waβ heated to reflux for 4 hours. The solution waβ then cooled and the methanol evaporated, 25 ml of water and 25 ml of ethyl ether were added. The aqueouβ layer waβ extracted with 2x25 ml of ethyl ether. The organic layera were washed with 25 ml of a saturated aqueouβ sodium chloride and dried over MgS0 ₄ . Evaporation of the solvent gave 280 mg as an

UBSTITUTE SHEET

120

oil of crude 3.6-dimethoxy 2-(3-hydroxy, 2-butanone, 4-yl) benzaldehyde dimethyl acetal. -E NMR (300 MBz, CDCl,} 5:1.33 (s, IB, -CB,), 3.03 (dd, J-2.9 and 13.9 Bz, IB, CBj-CB-O-), 3.26 (s, 3B, -OCB,), 3.27 (m, 1B,-CH _J -CB-0), 3.30 (s, 3B, -OCB,), 3.31 (s, 3B, -OCB,), 3.53 (s, 3B, OCB,), 3.75 (s, 3B, -OCB,), 3.76 (s, 3B, -OCB,), 4.07 (m, IB, -CB-0-), 4.17 (d, J-6.6 Bz, IB,-OB), 5.86 (s, IB, ArBCI-O), 6.75 (2xd, J-9.0 Bz, ArB).

Step 6s Methyl l,5,8-triaethoxyisochroaan-3-yl-keton dimethyl acetal

To a stirred solution containing 280 mg of the crud product from step 5 in 4 ml of methanol was added 30 mg o pyridinium p-tolueneaulfonate. After 30 minutes, ^' 100 μl o triethylamine was added and methanol waβ evaporated. Th reaidue waβ dissolved in methylene chloride and filtere over silica gel. The solvent waβ evaporated and th reaidue waa purified (not neceββary, the crude is clean b NMR) by flash chromatography with hβnaneβ-ethyl acetate 2: aβ the eluting βolvent mixture. The isochroman analog wa obtained in greater then 80% yield (225 ag) for 2 steps -E NMR (300 MBz, CDCl,) 5: 1.40 (β, 3B, -CB,), 2.47 (dd J-11.8 and 17.6 Bz, IB, -CfiA-CB-), 2.79 (dd, J-3.8 and 17. Bz, IB, -Clt-CH-), 3.29 (s, 3H, -OCB,), 3.32 (s, 3B, OCB,), 3.62 (s, 3B,-OCB,), 3.78 (s, 3B, -OCB,), 3.81 (s, 3B -OCB,), 4.35 (dd, J-3.8 and 11.8 Bz, IB, -CB ₂ -Cfl-0), 5.6 (s, IH, ArB-Cfl-O), 6.73 (2xd, J-8.9 Bz, 2B, ArB).

BSTITUTE SHEET

121

Ste ^p 7: Methyl (methoxy-1 , 5 , 8-dioxo-5 , 8 - dihydroisochroman-3-yl) ketone

To a stirred solution of I (7 mg, 0.023 mmol) in acetonitrile (0.5 ml) at O'C was slowly added Ceric ammonium nitrate (CAN) (44 mg, 0.069 mmol) in water (0.5 ml). After 30 minutes, water (10 ml) waa added and the mixture waβ extracted with CB,C1 ₂ . The organic phases were washed with brine and dried over MgS0 ₄ , filtered and concentrated in vacuo to give a yellow oil (II) which did not need any purification. ^l E NMR (300 MBz, CDCl,) 5: 2.34 (s, 3B, -CO-CH,), 2.39 (dd, J-11.3 and 19.8 Bz, IB, -CHa- CH), 2.85 (dd, J-4.1 and 19.8 Bz, IB, -Cfie-CH) , 3.61 (s, 3B, -OMe), 4.48 (dd, J-4.1 and 11.3 Bz, IB, CB ₂ -Cl-0) , 5.52 (s, IB, ArB-Cfi-O), 6.78 (2xd, J-10.2 Bz, 2B, ArB).

Step 8 Methyl(l-aβthoxy-6 and ll-hydroxy-5,12-dioxo- 3,4,5,12-tetrahydroanthraceno (2,3-c). pyran-3- yl)ketone

To a stirred mixture of NaB (8.7 mg, 0.36 mmol) in TBF (2 al) at 0*C waβ added homophtalic anhydride (57 mg, 0.35 mmol). After 10 minuteβ, I (79 mg, 0.35 mmol) in TBF (2 ml) waβ added and the reaction mixture waa warmed up to room temperature. After 1 hour BCl IN (10 ml) was added and the mixture waβ extracted with CB ₂ C1 ₂ . The organic phaseβ were waβhed with brine, dried over MgS0 ₄ , filtered and concentrated in vacuo. The reβidue waβ purified by flaβh ehroaatography uβing toluene-acetone 95:5 gave 13 mg (10% yield) of II. *H NMR (300 MBz, CDCl,) 5: 2.36 (s, 3B, -CO-CH,), 2.55 (dd, J-11.8 and 19.7 Hz, IB, -CB_a-CB-0) , 3.07 (dd, J-4.1 and 19.7 Bz, IB, -Cla-CB-O), 3.68 (s, 3B, - OCB,), 4.56 (dd, J-4.1 and 11.8 Bz, IB, -CB-CO) , 5.71 (s.

SUBSTITUTE SHEET

122

IB, -O-CB-O) , 7.7 (m, 2B, ArB) , 7.95 (dd, J-1.6 and 7. 1 Bz , IH, ArH) , 8.09 ( s, IH, ArH) , 8.46 (dd, J-1.6 and 8.7 Bz, IB, ArB) .

Step 9: Methyl 5,8-di___βthoxy-l-hydroxyiβochroaan-3-yl ketone

To a βtirred aolution of crude product from atep 5 (1.89g; 5.49 maol) in a mixture of acetone (80 ml) and water (25 al) waβ added p-toluane βulfonic acid aonohydrate (70 ag; .37 maol). After βtirring at room temperature for 3 hours, triethylaaine (2 al; 1.43 maol) was added and stirred for 5 minutββ. The mixture waβ evaporated until volume went down to 10 al. A mixture of acetone (80 al) and water (15 al) waβ added followed by addition of p- toluene sulfonic acid aonohydrate (70 ag; .37 mmol). After 3.5 hourβ, triethylaaine (3 al; 2.15 mmol) waβ added, evaporated to a email voluaβ, extracted with ether (3x100 al) . The extract waβ waβhed with brine (50 al), dried over MgS0 ₄ and evaporated yielding quite pure 1- hydroxyiaochroβan (1.3g; 94%), (MF. 136-138*C). NMR (CDCl,; 300 MBS) 5: 2.32 (3B, β, CH,0), 2.51 (IB, dd, J-12.4, 17.5 Bz, H-4), 2.97 (IB, d, J-3.5 Hz; -OH), 3.06 (IB, dd, J-4.1, 17.5 Hz, H-4), 3.77, 3.82 (3H, a each, ArOCB,), 4.70 (IB, dd, J-4.1, 12.4 Bz, B-3), 6.22 (IB, d, J-3.4 Bz, IB), 6.71, 6.76, (IH, d each, J-9.0 Hz, ArH).

SUBSTITUTE SHEET

123

Step 10: Methyl ( hydroxy-1 , 5 , 8-dioxo-S , 8 - dihydroiaochro8_an-3-yl) ketone

To a stirred solution of I (123 mg, 0.488 mmol) in acetonitrile (10 ml) at 0*C waβ βlowly added Ceric ammonium nitrate (CAN) (802 mg, 1.46 mmol) in water ( 6 ml). After 30 minutes, water (40 ml) was added and the mixture was extracted with CB ₂ C1 ₂ . The organic phases were washed with brine and dried over MgS0 ₄ , filtered and concentrated in vacuo to give a yellow oil (II) which did not need any purification (106 mg, 98%). -E NMR (300 MBz, CDCl,) 5: 2.31 (β, 3B, -CO-CB,), 2.39 (dd, J-11.5 and 19.6 Bz, IB, - CBa-CB), 2.88 (dd, J-4.2 and 19.6 Bz, IB, -Cflβ-CB) , 3.90 (broad a, IH, -OH), 4.65 (dd, J-4.2 and 11.5 Hz, IB, CH ₂ - Cfi-O), 6.04 (β, IB, ArB-Cl-O), 6.80 (2d, J-10.2 Bz, 2B, ArB).

Step 11s Methyl(l-hydroxy-6 and ll-hydroxy-5,12-dioxo-3,4- 5,12- tetrahydroanthraceno(2,3-c) yran-3- yl) etone

To a stirred βolution of homophtalic anhydride (87 mg, 0.536 mmol) in THF (6 ml) at 0*C waβ added a solution of sodium biβ (trimβthylβilyl) amide (0.536 mmol, 536 μl) 1M in THF. After 5 minutes the reaction mixture was cooled down to -78*C and a βolution of I (obtained fro hydroxy- quinonβ (119 ag, 0.536 m ol), mβthoxypropene (300μl) and catalytic amount of pyridiniua p-toluβneβulfonatβ) in CB ₂ C1, (2 al). After 1 hour at -78*C the reaction mixture

-» waβ waraed up to room teaperature for 2.5 hourβ then BCl IN (5 al) waβ added and after 5 minutes the mixture was extracted with CB,C1 ₂ . The organic phases were washed with brine, dried over MgS0 ₄ , filtered and concentrated in

SUBSTITUTE SHEET

124

vacuo. The residue was purified by flash chromatography using toluene-acetone 90:10 gave 12 mg (7% yield) of II. *B NMR (300 MBz, DMSO) 5: 2.26 (s, 3B, -CO-CB,), 2.49 (dd, J-11.5 and 19.4 Bz, IB, -Clft-CB-O), 2.85 (dd, J-4.2 and 19.4 Bz, IB, -Clfi-CB-O), 4.64 (dd, J-4.2 and 11.5 Bz, IB, - CB-CO), 5.95 (d, J-6.2 Bz, IB, -O-CB-O), 6.26 (d, J-6.2 Bz, IB, -OB), 7.81 (m, 2H, ArB), 8.13 (s, IB, ArB), 8.22 (d, J-7.7 Bz, IB, ArB), 8.36 (d, J-8.0 Bz, IB, ArB), 13.71 (s, IB, ArOB).

SUBSTITUTE SHEET

124A

E»mplt9 ^. Shoπιy _n *e«o ιnt_nceno[2_3<)pyπιι.3. _y |^ycooe. with a methyl ketone side chain

÷

SUBSTITUTE SHEET

125

EXAMPLE 10

Step 1: N,N-diethyl(2,5-d___eethoxy)benzaaide

To a stirred solution of 2,5-dimethoxybenzoic acid (6.61g, 36.3 mmol) in 50 ml of CB ₂ C1 ₂ was added a solution of (C0C1) ₂ in CHjClj (2.0M, 20 ml, 40.0 mmol) at 0*C under argon. After addition of catalytic amount of pyridine (0.2 ml) an evolution of gas was observed. The reaction was warmed up to room temperature and stirred for 3 hours. The volatile were removed and the reβidue waa dissolved in 300 ml of diethyl ether. Diethylaaine (5.26 ml, 50.8 mmol) was introduced dropwise at 0*C under argon. The resulting mixture was stirred at rooa temperature for another 3 hours, and then quenched with saturated K ₂ CO, solution. The organic layer was washed with 2N NaOB and water, dried over MgS0 ₄ , filtered and then evaporated. Flash chromatography of the reβidue with CB ₂ C1, and CB,C0 ₂ Et (1:1) provided pure title compound (8.34g, 35.1 mmol) in 97% yield aa white solid. ^X B NMR (CDCl,) 5: 1.05 (3H, t, J-7.1 Bz), 1.24 (3B, t, J-7.1 Bz), 3.16 (2B, q, J-7.1 Bz), 3.56 (2H, ), 3.77 (3H, a), 3.78 (3H, s), 6.77 (IB, m) , 6.84 (2B, ).

Ste ^p 2s H,H-diethyl(2,5-di_Bβthαxy-6-aethyl)benzaaide

To a stirred solution of TMEDA (2.23 ml, 14.8 mmol) in 25 ml of TBF waβ added β-BuLi (1.3M, 11.35 ml, 14.8 mmol) at -78*C under argon. After 20 minutββ, a solution of N,N- diβthyl(2,5-di_aβthoxy)bβnzamidβ (1.4g, 5.9 mmol) in 5 ml of TBF waβ introduced dropwiaβ at -78*C. The stirring was continued at

-78*C for 1 hour before Mel (1.40 ml, 22.5 mmol) was added. The resulting mixture waβ stirred at -78*C for 1 hour, followed by addition of 20 ml saturated NB ₄ C1 solution.

TUTE SHEET

126

The reaction mixture was extracted with Et ₂ 0. The combined organic layers were dried over MgS0 ₄ , filtered and concentrated. Flash chromatography of the residue with CH ₂ C1 ₂ and ethyl acetate (1:1) gave pure title compound (1.42g, 5.66 mmol) in 96% yield. -E NMR (CDCl,) δ: 1.02 (3B, t, J-7.2 Bz), 1.26 (3B, t, J-7.2 Hz), 2.11 (3H, s), 3.12 (2H, q, J-7.2 Hz), 3.45 (2H, ), 3.75 (3H, s) , 3.79 (3B, s), 6.68 (IB, d, J«8.9 Hz), 6.76 (IH, d, J=8.9 Hz).

Step 3: 3,4-dihydro-5,8-dimethoxy-3-vinylisocoumarin

To a stirred solution of the benzamide from step 2 above (3.51g, 14.0 mmol) in 140 ml of THF at -78°C under argon was added n-butyllithium (2.5M, 9.0 ml, 22.5 mmol) dropwise, generating a red solution. After stirred for 30 minutes, acrolein (2.25 ml, 33.6 mmol) was added. The stirring was continued at -78°C for 1 hour before it was quenched with 100 ml of saturated NH ₄ C1 solution. The mixture was extracted with CB ₂ C1 ₂ , and the combined organic layers were washed with H ₂ 0, dried over MgS0 ₄ , filtered and concentrated to a residue. Without further purification,, the residue was dissolved in 180 ml of benzene and then heated to reflux in the presence of camphorsulfonic acid (2.93g, 12.6 mmol) for 5 days. After cooled to room temperature, 200 ml of NaHC0 ₃ (5%) and 200 ml of CH ₂ C1 ₂ were added. The organic layer was washed with H ₂ 0, dried over MgS0 ₄ , filtered and then concentrated. Flash chromatography of the residue with CH ₂ C1 ₂ , hexane, and ethyl acetate (2:4:1) provided the title isocoumarin (1.28g, 5.47 mmol) in 39% overall yield. -E NMR (CDC1 ₃ ) 5:2.76 (IH, dd, J-16.9, 10.9 Bz), 3.22 (IB, dd, J=»16.9, 3.3 Bz), 3.83 (3B, s), 3.91 (3B, s), 4.85 (IB, m) , 5.29 (IB, dd, J-10.6, 1.1 Bz), 5.44 (IH, dd, J-17.3, 1.1 Hz), 6.0 (IH, m), 6.88 (IB, d, J=9.1 Hz), 7.06 (IH, d, J=9.1 Hz).

SUBSTITUTE SHEET

127

Step 4: 3,4-dihydro-l,5,8-trimethόxy-3-vinylisocoumarin

To a stirred solution of the isocau arin from step 3 above (9.4 mg, 0.4 mmol) in 8 ml of toluene was added dropwise a solution of DIBAL-H (1M, 0.50 ml, 0.50 mmol) at -78°C under argon. The resulting mixture was stirred at - 78°C for 1.5 hour before Na ₂ S0 10 H ₂ 0 (100 mg, 0.31 mmol). After the reaction was warmed to room temperature, it was filtered to remove the solids, and washed with acetone. The filtrate was evaporated to give an oily residue which contained the corresponding lactol. Without further purification, the residue was treated with 5 ml of CH ₃ 0H, 1 ml of CB(OMe) ₃ , and PTSA (30 mg, 0.16 mmol) at room temperature for 10 minutes. The solvents were removed by rotavapor and the residue was purified by flash chromatography with hexane and ethyl acetate (7:3) to give pure title isocoumarin (65 mg, 0.26 mmol) in 65% overall yield. -E NMR (CDCl,) 5: 2.47 (IB, dd, J-17.2, 12.0 Bz), 2.85 (IB, dd, J-17.2, 3.6 Bz), 3.56 (3B, s), 3.78 (3B, s), 3.82 (3B, s), 4.63 (IB, m), 5.24 (IB, dt, J-10.6, 1.4 Bz), 5.43 (IB, dt, J-17.3, 1.4 Bz) , 5.63 (IB, s), 6.05 (IB, m), 6.70 (IB, d, J-8.9 Bz), 6.76 (IB, d, J-8.9 Bz).

Step 5s l-methoxy-3-vinylisochroman-5,8-dione

To a stirred solution of the isocoumarin from step 4 above (30 mg, 0.12 mmol) in 2 ml of CB ₃ CN was added dropwise a solution of (NB ₄ ) ₂ Ce(N0 ₃ } ₍ in 1 ml of H ₂ 0 at 0°C. After 5 minutes, the reaction was warmed to room temperature and stirred for another 30 minutes. The reaction mixture was diluted with 10 ml of CH ₂ C1 ₂ and 10 ml

SUBSTITUTE SHEET

128

of B ₂ 0. The organic layer was washed with B ₂ 0, dried over MgS0 ₄ , filtered and then concentrated to a residue that was purified by flash chromatography (hexane/ethyl acetate; _* « 7:3) to provide pure product (15 mg, 0.07 mmol) in 57% yield along with a by-product (10 mg, 0.05 mmol, 40%). The by-product could be converted to desired product in 92% yield by treating it with CB(OMe) ₃ in MeOB with FTSA at room temperature. B NMR (CDCL ₃ ) 5: 2.29 (IB, dd, J-19.4, 11.4 Bz), 2.65 (IB, dd, J-19.4, 3.6 Bz), 3.55 (3B, s), 4.52 (IB, m), 5.27 (IB, dd, J-10.5, 1.4 Bz), 5.41 (IB, dd, J-17.3, 1.4 Bz), 5.43 (IB, s), 5.98 (IB, m), 6.74 (IB, d, J-10.2 Bz), 6.78 (IB, d, J-10.2 Hz).

Step 6: 6-hydroxy-l-methoxy-l,2,3,4-tetrahydro-3-vinyl- (2-oxygen) naphthacene-5,12-dione

To a stirred solution of i-Pr ₂ NH (0.254 ml, 1.8 mmol) in 6 ml of THF was added a solution of n-BuLi (2.5M, 0.725 ml, 1.8 mmol) at 0°C under argon. After stirring for 20 minutes, the newly made LDA solution was cooled to -78°C and stirred for additional 5 minutes. A solution of homophthalic anhydride (294 mg, 1.8 ml) in 4 ml of TBF was added dropwise, causing the color change from yellow, green and then to yellowish. Upon stirring for 5 minutes after completion of the addition, a solution of the quinone from step 5 above (332 mg, 1.5 mmol) in 4 ml of TBF was quickly injected. The resulting mixture was stirred at -78°C for 20 minutes at room temperature for 2 hours and then placed in the fridge overnight. The mixture was quenched with 10 ml saturated NH ₄ C1 solution and partitioned between IN HCl and CHjClj. The organic layer was washed with B ₂ 0, dried over MgS0 ₄ , filtered and then concentrated to a residue

SUBSTITUTE SHEET

- 129

that was purified by flash chromatography with hexane, CB ₂ C1 ₂ , ethyl acetate (2:2:1) to give title compound (276 mg, 0.82 mmol) in 55% yield as a single regioisomer. MF: 150°C (decomposes). -E NMR (CDC1 ₃ ) 5:2.46 (IH, dd, J-19.2, 11.4 Hz), 2.89 (IB, dd, J-19.4, 3.7 Hz), 3.61 (3B, s), 4.60 (IB, m), 5.30 (IB, dd, J-10.6, 1.3 Hz), 5.45 (IH, dd, J-17.3, 1.4 Hz), 5.63 (IB, s), 6.04 (IH, m), 7.70 (2H, m), 7.95 (IB, d, J-7.4 Bz), 8.11 (IB, s), 8.48 (IB, d, J-7.3 Bz), 13.83 (IB, s); "C NMR (CDC1 ₃ ) 5: 27.80, 56.85, 66.90, 94.11, 117.31, 122.41, 125.43. 128.64, 129.68, 130.42, 131.11, 131.87, 136.63, 137.49, 142.12, 144.99, 163.21, 181.95, 188.66.

Step 7: 6-acetoxy-l-methoxy-l,2,3,4-tetrahydro-3-vinyl- (2- oxygen)naphthacene-5,12-dione

To a stirred solution of the tetracyclic quinone from step 6 above (230 mg, 0.68 mmol) in 35 ml of CB ₂ C1 ₂ were added 7 ml of pyridine, 7 ml of Ac ₂ 0, and 123 mg of DMA? (1.00 mmol) at room temperature. The mixture was then stirred for 2 hours before it was poured into a mixture of ice water (20 ml) and CB ₂ C1 ₂ (20 ml). The organic layer was washed with B _a O, 0.1N BCl, dried over MgS0 ₄ , filtered and then concentrated to a crude residue that was purified by flash chromatography to give the title compound (178 mg, 0.47 mmol) in 69% yield as yellow solid. MF 228-229*C, IR (neat) 1208, 1611, 1643, 1667, 1763, 2912 CM" ¹ , ~E NMR (CDClj) 5: 2.41 (IB, dd, J-19.0, 11.0 Bz), 2.61 (s, 3B), 2.82 (IB, dd, J-19.0, 3.5 Bz), 3.61 (3B, s), 4.58 (IB, m), 5.29 (IB, dd, J-11.0, 1.5 Bz), 5.44 (IB, dd, J-17.0, 1.5 Bz), 5.64 (IB, s), 6.01 (IB, m), 7.72 (2B, m), 8.06 (IH,

SUBSTITUTE SHEET

130

m), 8.14 (IH, ), 8.59 (IH, s), ¹³ C NMR (CDC1 ₃ ) 5:21.14,

27.78, 56.22, 93.43, 116.62, 123.77, 127.00, 128.61,

130.01, 130.16, 130.24, 130.31, 135.52, 136.93, 139.80, 145.38, 168.90, 181.70, 182.50.

Step 8: 6-acetoxy-l-hydroxy-l,2,3,4-tetrahydro-3-vinyl- (2-oxygen)naphthacene-5,12-dione

To a stirred solution of the tetracycle form step 7 above (16 mg, 42 mmol) in 3 ml of .CH ₂ C.l ₂ was added a solution of B-bromo-9-BBN in CB ₂ C1 ₂ (1M, 0.105 ml, 105 mmol) at 0°C under argon. After stirred at 0°C for lh, 3 ml of ice water was added. The reaction mixture was extracted with CB ₂ C1 ₂ , washed with water, dried over MgS0 ₄ , filtered, and then concentrated to a crude residue that was purified by flash chromatography (hexane, CB ₂ C1 ₂ , ethyl acetate, 2:2:1) to give title compound (4 mg, 11 mmol) in 26% yield. IR (neat) 1191, 1610, 1640, 1770, 2919, 3440 CM ^_1 ₃ , -E NMR (CDClj) 5: 2.43 (IB, dd, J-19.0, 11.0 Hz), 2.60 (3B, s), 2.86 (IB, dd, J-19.0, 3.5 Bz), 4.61 (IB, M) , 5.28 (IB, dd, J-11.0, 1.5 Bz), 5.42 (IB, dd, J-17.0, 1.5 Bz), 5.62 (IB, br, s), 5.81 (IB, s), 6.00 (IB, m), 7.73 (2B, m), 8.67 (IB, m), 8.14 (IB, m), 8.63 (IB, s).

SUBSTITUTE SHEET

13 OA

Example 10: Prepara ^t ion of tetrahydroanthraceno [2,3-c] pyran-3-yl derive _t ive wi _t h a vinyl side chain

SUBSTITUTE SHEET

^• 131

EXAMPLE 11

Step 1: Methyl 2-methoxyisopropyloxy-acetate

To a stirred solution of methyl glycolate (5 ml, 64.8 mmol) and 2-methoxypropene (12.5 ml, 130.5 mmol) in dry dichloromethane (15 ml) at 0°C was added catalytic amount of pyridinium p-toluene-sulfonate (100 mg). The mixture was stirred at 0°C for 2 hours. Saturated sodium bicarbonate solution was added and methylene chloride layer was separated, dried over anhydrous magnesium sulfate. Solvent was evaporated and the product was distilled over anhydrous potassium carbonate under reduced pressure (20 mmBg) at 62°-64°C (yield - 9.9g, 94%), -E NMR (300 MHz, D ₄ - acetone) 5:1.28 (6H, s), 3.15 (3B, s), 3.67 (3B, s), 4.01 (2B, s).

Step 2: Methyl 3-(2 bromomethyl-3'-6'-d_L___ethoxy)phenyl-2- methoxyisopropyloxy propionate

To a solution of cyclohexylisoprσpylamine (1.6 ml, 9.8 mmol) in dry TBF (30 ml) at 0°C was added 2.5M BuLi in hexanes (3.95 ml, 9.8 mmol). After stirring at 0°C for 10 minutes, the mixture was cooled to -78*C. The protected methyl glycolate from step 1 (1.59g, 9.7 mmol) in TBF (6 ml) was added. After stirring for 15 minutes at -78°C, l,2-dibromomethyl-3,6-dimethoxybenzene (1.5g, 5.1 mmol) in dry TBF (16 ml) was added followed by addition of BMPA (2 ml), in 10 minutes. The mixture was stirred at -78°C for 1 hour. Temperature was then raised to -40°C slowly during the next 50 minutes. Saturated NB ₄ C1 solution (20 ml) was added, TBF was removed at 25°C and the residue was extracted with ether. Ether extract was washed with sat.

SUBSTITUTE SHEET

132

NaBC0 ₃ , dried over MgS0 ₄ , evaporated and the residue was chromatographed over silica gel washed with 5% triethylamine (eluent: hexanes/EtOAc - 4:1), yielding 1.37g (66%) of the title compound. H NMR (300 MHz, d _s -acetone) 5:1.05 (3H, s, CH ₃ ), 1.15 (3H, s, CH ₃ ), 2.78 (3H, s, OMe) , 3.01 (IH, dd, J-8.9, 13.5 Hz, H-3), 3.66 (3H, s, C0 ₂ Me) , 3.83 (6B, s, ArOCH ₃ ), 4.43 (IH, dd, J-5.0, 8.8 Hz, H-2), 4.81 (2H, s, ArCHjBr), 3.21 (IH, dd, J-5.1, 13.5 Hz, H-3), 6.88, 6.95 (IH, d, each, J-9 Hz, ArH).

Step 3: Methyl 3-(2 -bromomethyl-3'-6'-di__ιethoxy)phenyl- 2-hydroxy propionate

To a solution of methyl 3-(2'-bromomethyl-3'-6'- dimethoxy)-phenyl-2-methoxy-isopropyloxy propionate (29g, 71 mmol) in diethyl ether (40 ml) was added IN HCl (20 ml). After stirring at ambient temperature for three hours, ether layer was separated, washed with brine, dried over MgS0 ₄ and evaporated to give the title product as an oil in quantitative yield. *B NMR (300 MHz, CDC1 ₃ ) 5:3.10 (IH, dd, J-8.7, 13.9 Hz, H-3), 3.29 (IH, dd, J-13.8, 4.3 Hz, H-3) 3.77 (3B, s, C0 ₂ Me), 3.79, 3.83 (3B, s, each, ArOMe), 4.47 (IB, dd, J-8.7, 4.5 Hz, H-2), 4.72 (2H, s, ArCH _j ) , 6.74, 6.80 (IH, d, each, J-9.0 Hz, ArH).

Step 4 S 5 _f B-dinwt-hfvr -i Λe_r*hrrsm»n-~i~γ1 --F iT "- _. ■" acid

To a solution of methyl-3(2 -bromomethyl-3 ^/ -6 ^/ - dimethoxy) pheny1-2-hydroxy propionate (30 mg, .09 mmol) in dry THF (3 ml) was added sodium hydride (60% dispersion in oil, 25 mg, .6 mmol). The mixture was stirred at 50°C for 2.5 hours. Small amount of ice was carefully added, extracted with dichloromethane. Aqueous portion was acidified with dil. BCl, extracted with EtOAc, washed with brine, dried and evaporated (yield -85%). ^X H NMR (300 MHz,

SUBSTITUTE SHEET

133

CDC1 ₃ ) 5: 2.74 (IH, dd, J=11.2, 17.1, H-2, H-4), 3.19 (IH, ddd, J-1.3, 3.8, 17.1 Hz, H-4), 3..74, 3.77 (3H, s, each, OMe), 4.26 (IH, dd, J=3.9, 11.2, H-3) 4.7 (IH, d, J=15.9, H-l), 5.06 (IH, d, J=15.9, H-l), 6.63 and 6.67 (IH, d, each, J-12.2, aromatic protons).

Step 5: Methyl 3-(2'-formyl-3',6'-dimethoxy)pheny1-2- methoxyisopropyloxypropionate

Methyl 3-(2'-bromomethyl-3',6'-dimethoxy)phenyl-2- methoxyisopropyloxy propionate (1.2g, 2.9 mmol) was dissolved in dry DMSO (25 ml) and sodium bicarbonate (622 mg, 7.4 mmol) was added. The mixture was stirred at 95°C for 45 minutes. It was cooled, quenched with water (125 ml), extracted with ether (3x200 ml). Extracts were washed with brine (25 ml) dried over MgS0 ₄ . Solvent was evaporated, yielding 9g of the title compound (90%). -E NMR data (300MHz, d _β -acetone) 5:1.16 (3H, s, CH ₃ ), 1.18 (3H, s, CBj), 2.99 (3B, s, 0CB ₃ ) , 3.53 (3B, s, C0 ₂ Me), 3.82 (3B, s, ArOB), 3.88 (3B, s, ArOMe), 7.04 (IB, d, J-10.0 Bz, ArB), 7.21 (IB, d, J-10.0 Bz, ArB), 10.50 (IH, s, CBO) .

Step 6s Methyl-5,8-dimethoxy-l-hydroxyisochroman-3-yl- foπnate

To a solution of methyl-S-^'-formyl-S'-e'- dimethoxy)pheny1-2-methoxyisopropyloxy-propionate (4.2g, 12.4 mmol) in TBF (75 ml) containing water (4 ml) was added pyridinium p-toluenesulfonate (230 mg) . After stirring at room temperature for 1.75 hours, triethylamine (3 ml) and

SUBSTITUTE SHEET

134

saturated NH ₄ C1 solution (60 ml) were added. THF was removed at 25°C and the mixture was extracted with ether (3x150 ml). Ether extract was washed with brine, dried and evaporated yielding 3.9g of 1-hydroxyisochroman (95%) (quite pure from NMR spectrum). (MP. 127-129°C) 'E NMR (300 MHz, CDC1 ₃ ) 5: 2.69 (IH, dd, J-12.3, 17.6 Hz, H-4), 3.07 (IH, broad peak, OH), 3.13 (IH, dd, J-17.5, 3.8 Hz, H- 4), 3.78 (3Ξ, s, C0 ₂ Me), 3.82, 3.83 (3B, s, each, ArOMe), 4.91 (IB, dd, J-12.3, 3.8 Hz, H-3), 6.24 (IH, far, s, H-l), 6.71 and 6.77 (IH, d, each, J-8.9 Hz, ArH).

Step 7: l-Hydroxy-3-carbmethoxy-5,8-dioxoisochroman

To a stirred solution of the isochroman from step 6 above (232 mg, 0.865 mmol) in 10 ml of CB ₃ CN was added a solution of cerium ammonium nitrate (1.422g, 2.59 mmol) in 7 ml of B ₂ 0 at 0°C. The reaction mixture was slowly warmed to room temperature and stirred for 15 minutes. After it was quenched with B ₂ 0 (20 ml), the mixture was extracted with CH ₂ C1 ₂ (2 x 50 ml). The combined organic layers were dried over MgS0 ₄ , filtered, concentrated to a residue that was found to be pure title compound and used for next reaction without further purification (201 mg, 0.844 mmol, 98%). -E NMR (CDClj) 5: 2.55 (IB, m), 2.95 (IB, dd, J-19.5, 4.0 Bz), 3.15 (IB, br s), 3.84 (3B, s), 4.86 (IB, m), 6.04 (IB, β), 6.79 (2H, m) .

SUBSTITUTE SHEET

135

Step 8: 3-Carbmethoxy-1,11-dihydroxy-1,2,3,4-1etrahydro- (2-oxygen) naphthacene-5,12-dione

3-Carbmethoxy-1,6-dihydroxy-l,2,3,4-tetrahydro- (2- oxygen)naphthacene-5,12-dione

To a freshly made LDA solution (1.7 mmol, 5 ml of THF) at -78°C was added dropwise a solution of homophathalic anhydride I (189.7 mg, 1.17 mmol) in 4 ml of THF under argon over 15 minutes. After stirring at -78°C for 5 minutes, a solution of quinone II (1.15 mmol) in 4 ml of THF was injected. Stirring was continued at -78°C for 1 hour and then at room temperature for 2 hours. After kept in fridge for overnight, the whole mixture was poured into 25 ml of IN HCl solution. The reaction mixture was extracted with CB ₂ C1 ₂ (20 ml x 3). The combined orgaic phases were washed with water, dried over MgS0 ₄ , filtered and then concentrated to a crude residue that was purified by flash chromatographied to give a mixutre of III and IV (1:1, 130.0 mg, 32%). The following spectral data were obtained from the two regioisomeric mixtures. I.R. (neat) 146g, 1501, 1612, 1658, 1742, 2952, 3428 cm" ¹ . -E NMR (CDC1 ₃ ) 5: 2.71 (2B, m) , 3.18 (2B, m) , 3.18 (2B, m), 3.87 (3B, s), 3.88 (3B, s), 4.92 (2B, m), 6.23 (IB, s), 6.27 (IB, s), 7.71 (4B, m), 7.95 (2B, d, J-7.7 Bz), 8.10 (IB, s), 8.12 (IB, s), 8.46 (2B, d, J-7.6 Bz), 13.75 (IE, s), 13.77 (IB, s).

Step 9: Methyl (1,5,8-tr__methoxyisochroπ_an-3-yl)formate

The compound from step 6 above was treated at room temperature with excess trimethyl orthoformate in methanol for twelve hours. A quantitative yield of the titled compound was obtained. -E NMR (300 MBz, CDC1 ₃ ) 5: 2.71 (dd, IB, J-12.4, 17.3 Bz, BCBa), 3.10 (dd, IB, J-4.0, 17.3 Bz, HCBe.), 3.59 (s, 3B, C0 ₂ CB ₃ ), 3.79 (s, 3B, 0CH ₃ ), 3.81

SUBSTITUTE SHEET

136

(s, 3H, 0CH ₃ ), 3.85 (s, 3H, 0CH ₃ ) , 4.80 (dd, IH, J-4.0, 12.4 Hz, O-CH), 5.71 (s, IH, O-CH-O) , 6.74 (2d, 2H, HC-H) .

Step 10: Methyl ( 1-methoxy-5 , 8-dioxo-5 , 8- dihydioisochroman-3-yl)formate

This compound was obtained in 90% yield by using the procedure in step 7, example 9. ^X H NMR (300 MHz, CDC1 ₃ ) 5: 2.54 (ddd, IH, J-1.2, 11.7, 19.5 Hz, HCHa), 2.88 (dd, IH, J-4.1, 19.5 Hz, HCBe), 3.53 (s, 3H, C0 ₂ CH ₃ ), 3.57 (s, 3H, OCH ₃ ), 4.10 (dd, IH, J-4.1, 11.7 Hz, OCHO), 5.49 (s, IH, O- CH-O), 6.75 (2d, 2H, HC-CH) .

SUBSTITUTE SHEET

136A

Example 11: Shor ^t synthesis of anthraceno [2,3-c] pyran-3-yI derivatives w wiith an ester side chain

SUBSTITUTE SHEET

137

EXAMPLE 12

Step 1: 2,5-dimethoxy-6-hydroxymethyl benzaldehyde dioxane acetal

To a solution of 2 , 5-di_methoxy benzaldehyde dioxane acetal (10.5g, 46.9 mmol) in dry ether (200 ml) at -10°C was added 2.5M BuLi (30 ml, 75 mmol). The mixture was stirred et -7°C for 5.5 hours. Dry paraformaldehyde (2.6g) was quickly added and the mixture was stirred at 0°C for 2 hours. It was stirred at room temperature overnight, poured into saturated NH ₄ C1 solution and diluted with ether. Ether-layer was separated, washed with brine (50 ml), dried over MgS0 ₄ and evaporated. The crude product was chromatographed over silica gel with CH ₂ C1 ₂ and EtOAc as eluent (7:1) yielding pure product (5.5g, 46%) as an oil. -E NMR (300 MHz, CDC1 ₃ ) 5:2.30 (2H, m, CB ₂ of dioxane acetal), 3.23 (IB, t, J-6.8 Bz, CH ₂ OH) , 3.78, 3.80 (3H, s, each, -OMe), 3.98 (2B, m, CH ₂ of dioxane acetal), 4.26 (2Ξ, dd, J-4.6, 11.3 Hz, CH ₂ of dioxane acetal), 5.03 (2B, d, J-6.8 Bz, CHjOH) , 6.21 (IH, s, methine proton of dioxane acetal), 6.77, 6.86 (IH, d each, J-10 Bz, ArB).

Step 2: 2 , 5-diκethoxy-6-mesyloxymethylbenzaldehyde dioxane acetal

To a solution of 2,5-dimethoxy-6-hydroxymethyl benzaldehyde dioxane acetal (19.8g, 78 mmol) in dry CB ₂ C1 ₂ (225 ml) at 0°C was added triethylamine (22 ml, 156 mmol) and methanesulfonyl chloride (9 ml). The mixture was stirred at 0°C for 2 hours. Saturated NB ₄ C1 solution (50 ml) was slowly added. CH ₂ C1 ₂ layer was separated, washed

SUBSTITUTE SHEET

- 138

with water (25 ml) and brine (25 ml) dried over MgS0 ₄ , evaporated (25g, 96%). MP: 87-?0°C, ~E NMR (300 MHz, CDC1 ₃ ) 5:3.22 (3H, S, -OS0 ₂ Me), 3.77, 3.80 (3H, s, each, OMe), 3.95 (2H, m, CHj of dioxane acetal), 4.24 (2H, dd, J-4.6, 11.3 Hz, CH ₂ of dioxane acetal), 5.77 (2H, s, CHj- OMS), 6.18 (IH, s, Methine proton of dioxane acetal), 6.88 (2H, tightly bored, aromatic ), 2.35 (2B, m, CH ₂ of dioxane acetal) .

Step 3: Methyl-2-(2 'formyl-3 ' , 6 '-dimethoxy benzyloxy)acetate

A mixture of methyl glycolate (3.2 ml, 41 mmol) and sodium methoxide in methanol (4.37M solution, 7.3 ml, 32 mmol) was held at 50°C for 4.5 hours under nitrogen. Methanol was removed, and dry DMSO (5 ml) was added. The mixture was cooled to 10°C. The mesylate (4.9g, 14.9 mmol) from step 2 in DMSO (50 ml) was added slowly, and the mixture was stirred at room temperature overnight. Ice (-50g) was added and the mixture was extracted with ether (200 ml x 3), washed with water (30 ml), dried and evaporated. The residue waβ dissolved in ether (100 ml) and 50 ml IN BCl was added. The mixture was stirred at room temperature for 1.5 hour. Ether layer was separated, washed with brine (20 ml), dried and evaporated. Crude product was chromatographed over silica gel (hexanes/ethyl acetate: 2:1 as eluent), yielding 1.89 of pure product (45%). (MP: 177-179 ^β C). IB NMR data (300 MBz, CDC1 ₃ ) 53.73, 3.81, 3.84 (3H, s, each, OMe), 4.16 (2H, s, CB,- CO _J CB _J ), 4.97 (2B, s, benzylic CB,), 6.94, 7.08 (IB, d, each, J-9.2 Bz, aromatic protons), 10.58 (IB, s, -CBO) .

SUBSTITUTE SHEET

139

Step 4: 3,4-dihydro-5,8-di___ethoxy-isochroman-3-yl-formic acid

A mixture of sodium (280 g, 12.2 mmol) and dry toluene (15 ml) was heated to reflux under nitrogen. Methyl 2-(2 -formyl-3 ^/ , 6'-dimethoxy beπzyloxy) acetate (1.26g, 4.7 mmol) in toluene (15 ml) was added dropwise. The mixture was held under reflux for 5 hours, cooled, and then transferred slowly into methanol (10 ml). Methanol was removed. THF (25 r.l) and 2N NaOH (25 ml) were added, the mixture was stirred for 1.5h at room temperature. THF was removed, extracted with CH ₂ C1 ₂ (50 ml). Aqueous portion was acidified with dil. HCl, extracted with ethyl acetate (2x150 ml) washed with brine (25 ml) dried and evaporated. Crude product in CH ₂ C1 ₂ (25 ml) was treated with p-toluenesulfonyl chloride (500 mg) pyridine (1 ml) overnight at 5°C. Product obtained after usual work-up was heated with pyridine (5 ml) at 120°C for 3 hours. The mixture was cooled diluted with water, neutralised with 3N BCl, extracted with ethyl acetate (3x50 ml). Solvent was removed and the product was dissolved in CH ₂ C1 ₂ (50 ml). It was extracted with 10% NaHC0 ₃ (2x25 ml). The aqueous extract was acidified with dil. HCl, extracted with EtOAc (2x75 ml) washed with brine, dried and evaporated yielding fairly pure solid product (300 mg, 27%). NMR (300 MHz, CDC1 ₃ ) 5:3.77, 3.79 (3H, s, each, OMe), 5.26 (2H, s, benzylic CBj), 6.71, 6.77 (IH, d, each, J-9.1 Hz, aromatic proton), 7.32 (IH, s, olefinic proton).

Step 5: 5,8-d_Lmethoxy-isochroman-3-yl-formic acid

To a solution of isochroman (5 mg, .02 mmol) in dry methanol (5 ml) magnesium turnings (10 small pieces) were

SUBSTITUTE SHEET

140

added gradually (one at a time) over 4 hours. The mixture was stirred overnight, acidified with dil. HCl. Methanol was removed and extracted with ether. Ether extract was washed with brine, dried and evaporated. The residue (1 mg) showed identical NMR spectrum as that of 5,8-dimethoxy- isochroman-3-yl-formic acid. (MP: i64-166°C).

Step 6: Ethyl(1-formyl-trimethyleneacetal-3.6-dimethoxy) benzylthioacetate

To a stirred solution of 2-bromomethyl-3,6- dimethoxyben- zaldehyde trimethylene acetal (650 mg, 2.05 mmol) were added a solution of 2-mercaptoacetate (0.25 ml, 2.28 mmol) in 2 ml of CH ₃ 0B and a solution of NaOMe (4.37 M in CBjOB, 0.47 ml, 2.05 mmol) at 0°C under argon. The resulting mixture was slowly warmed to room temperature and stirred for 6 hours. 20 ml of B ₂ 0 and 100 ml of Et ₂ 0 were added. The aqueous layer was extracted with ether (3x20 ml) . The combined organic layers were washed with H ₂ 0, dried over MgS0 ₄ , filtered and then concentrated to a residue which was found to be almost pure title produce and used for next reaction without further purification.

Step 7: Methyl(1-formyl-3,6-dimethoxy)benzylthioacetate

The reaction residue from step 6 above was dissolved in 20 ml of ether and 15 ml of IN HCl solution. After stirred at room temperature for 2 hours, the organic layer was separated, washed with 5% NaBC0 ₃ solution, dried.

SUBSTITUTE SHEET

141

filtered and then concentrated. Flash chromatography of the residue with hexane and ethyl acetate (7:3) provided title product (474 mg, 1.67 mmol) in 81% overall yield for two steps. -E NMR (CDC1 ₃ ) 5: 3.32 (s, 2H) , 3.72 (s, 3H) , 3.82 (s, 3H), 3.87 (s, 3H), 4.29 (s, 2H), 6.89 (IH, d, J-9.0 Hz), 7.08 (IH, d, J-9.0 Hz), 10.60 (IH, s); ¹³ C NMR (CDC1 ₃ ) 5: 26.45, 34.47, 52.57, 56.54, 56.70, 111.46, 117.70, 123.71, 130.34, 152.11, 157.96, 171.74, 193.12.

Step 8: 3 , 4-dihydro-3-carbmethoxy-5 , 8 - dimethoxyisothiochroman

To a stirred solution of the aldehyde from step 7 above (400 mg, 1.4 mmol) in 30 ml of benzene was added NaOMe powder (152 mg, 2.8 mmol). The resulting mixture was heated to reflux for 3 hours. The solid was removed by filtration, washed with benzene. The filtrate was evaporated to a residue that was purified by flash chromatography to give the title compound (166.1 mg, 0.63 mmol) in 45% yield along with recovered starting material (83 mg, 20%). -E NMR (CDC1 ₃ ) 5: 3.82 (s, 3B), 3.83 (s, 3B), 3.87 (s, 3B), 3.94 (s, 2B), 6.73 (IB, d, J-9.0 Hz), 6.91 (IH, d, J-9.0 Bz), 8.14 (IB, s); "C NMR (CDC1 ₃ ) 5: 23.81, 52.74, 56.30, 56.47, 109.96, 114.13, 119.38, 123.36, 128.30, 128.97, 149.68, 151.45, 166.17.

Step 9: 3-carbaethoxy-5,8-d_____ethoxyisothiochrom__n

To a stirred solution of the isothiochroman from step 8 above (129 mg, 0.48 mmol) in 20 ml of MeOB was added about 50 mg of magnesium chips. The reaction mixture started bubbling after 20 minutes induction period at room

SUBSTITUTE SHEET

142

temperature in water bath. Magnesium chips were added until all the starting material consumed. 10 ml of Nat. NB ₄ C1 solution, 10 ml of H ₂ 0, and 50 ml of ether were added. The organic layer was separated, washed with H ₂ 0, dried over MgS0 ₄ , filtered and concentrated to a crude residue that was purified by flash chromatography to give the title compound (105 mg, 0.39 mmol ₎ in 82% yield. The spectral data of title compound were found to be identical with the compound obtained in step 1, Example 8.

SUBSTITUTE SHEET

142A

Example 12 : Alternative approaches to isochroman and isothiochroman derivatives

" ^' I

' . 'J

'I '1

SUBSTITUTE SHEET

143

EXAMPLE 13 :

Step 1: 2,5-dimethoxy-6-hydroxyethylbenzaldehydedioxane acetal

To a cooled (-40°C) solution containing 1.68g (7.4 mmol) of 2,5-dimethoxybenzaldehydedioxane acetal in 50 ml of anhydrous diethylether was added, with stirring and under argon, 4.8 ml of a 2.5M n-butyl lithium in hexanes solution. The mixture was stirred for five hours at -5°C, 537 mg (6.0 mmol) of CuCN was then added, and stirring was continued for one more hour. To the mixture was then added l.Og of ethylene oxide and stirred overnight at 4°C. The mixture was then washed with 20 ml of water, 20 ml of saturated aqueous sodium chloride and dried over MgS0 ₄ . Flash chromatography of the residue obtained after removal of solvent gave 547mg (27% yield) of 2,5-dimethoxy-6- hydroxyethylbenzaldehyde dioxane acetal as a white solid. MP: 135-136 ^β C. -E NMR (200 MBz, CDC1 ₃ ) 5:1.48 (bd, IH, J-13.6 Hz, HCBe), 2.32 (m, IB, BCBa), 3.48 (dt, 2B, J-4.76, 12.0 Bz, ArCBj), 3.79 (s, 6B, 2XOCB ₃ ) , 3.86-4.08 (overlapped m, 4B, 2XOBCBe and CB ₂ 0B), 4.29 (m, 2B, 2XOBCS&), 6.28 (s, IB, O-CB-O), 6.79 (dd,. 2B, J-9.0 Bz, ArB). CMR (75.44 MBz, CDC1 ₃ ) 5: 25.9, 29.5, CB ₂ ; 55.7, 56.3, OCB ₃ ; 62.3, CB ₂ 0B; 67.9, 2XOCB ₂ ; 97.6, O-CB-O; 109.5, 111.8, aryl CH; 126.3, 129.2, 150.8, 152.8, aryl C. IR (FT, CDClj) *Max: 3350, 3550, bs, OB; 1257, 1089, C-O. BRMS calculated for C ₁₄ B ₂₀ 0 _S : [M ⁺ -] 268.1311 found 268.1316.

SUBSTITUTE SHEET

^• 144 -

Step 2: 5,8-Dimethoxy-l-hydroxyisochroman

A solution containing 150 mg (0.56mmol) of 2,5- dimethoxy-6- hydroxyethylbenzaldehydedioxane acetal in 5 ml of THF was stirred for one hour at room temperature with 5 ml of 0.2M aqueous HCl. The mixture was thendiluted with 25 ml of dichloromethane, washed successively with 25 ml aliquots of aqueous sodium bicaxbonate, water, brine and then dried over MgS0 ₄ . Flash chromatography of the residue obtained after removal of solvent gave 97 mg (82% yield) of the titled 1-hydroxyisochroman. (MP: 217°-219°C). -E NMR (200 MHz, CDC1 ₃ ) 5: 2.73 (m, 2B, CB ₂ ), 3.09 (bs, IB, exchangeable, OB), 3.84 (s, 3B, OCB ₃ ) , 3.88 (s, 3H, OCH ₃ ), 4.03 (m, IH, OBCB), 4.27 (m, IB, OBCB), 6.12 (bs, IB, OCH), 6.77 (dd, 2H, J-7.2 Bz, ArB). CMR (75.44 MBz, CDC1 ₃ ) 5: 22.2, CB ₃ ; 55.5, 55.7, OCB ₃ ; 56.8, OCB ₂ ; 87.4, OCH; 107.9, 109.4, aryl CH; 124.2, 124.8, 150.4, 150.6, aryl C. IR (FT, CDC1 ₃ ) ^v Max: 3650, bs, OB; 1260, C-0. BRMS calculated for CnB^O, [M*-] 210.0892 found 210.0871.

Step 3: l-Bydroxy-5,8-dioxo-5,8-dihydroiaochroaan

To a stirred solution containing 75 mg (0.36 mmol) of 5,8- dimβthoxy-1-hydroxyisochroman from step 2 above in 2 ml of TBF was added dropwise over four minutes, a solution containing 546 mg (1.0 mmol) of eerie ammonium nitrate in 2 ml of water. The mixture was stirred for five minutes and then diluted with 10 ml of dichlorome-thane. The separated organic phase was washed with 5 ml of water, 5 ml of brine and dried over MgS0 ₄ . Following evaporation of solvent, the yellow oil (62 mg, 96% yield) was found to be pure isochromandione. IB NMR (200 MBz, CDC1 ₃ ), 5: 2.54 (m, 2B, CH ₂ ), 3.51 (d, IB, J-4.4 Bz, exchangeable, OB), 4.09 (m.

SUBSTITUTE SHEET

- 145 -

2H, OCH ₂ ), 5.88 (d, IH, J-4.4 Hz, O-CH-O), 6.79 (dd, 2H, J-10.1 Hz, H-C-C-H). CMR (75.44 Mhz, CDC1 ₃ ) 5: 21.5, CH ₂ ; 56.4, OCH ₂ ; 85.3, OCH; 136.3, 136.4, aryl CH; 138.4, 141.3, CH; 185.3, 186.4, quinone C=0.

Step 4s 2 , 5 - D i m e t h o x y - 6 - ( 2 - hydroxypropyl)benzaldehydedioxane acetal

Application of the procedure described in step 1 of this example gr.ve 238mg (32% yield) of the desired benzaldehydedioxane acetal from 739mg (3.3 mmol) of 2,5- dimethoxybenzaldehydedioxane acetal and excess propylene oxide, after flash chromatography from 5% ethyl acetate in toluene. (MP: 160 ^β -161°C). ^l E NMR (300 MHz, CDC1 ₃ ) 5: 1.33 (d, 3H, J-6.1 Bz, CB ₃ ) , 1.49 (bd, IB, J-13.7 Bz, BCBe), 2.30 (m, IB, HCHa), 3.10 (dd, IH, J-2.4, 14.1 Hz, HCl), 3.47 (dd, IH, J-9.5, 13.9 Hz, HCB), 3.78 (s, 6H, OCH ₃ ), 4.02 (overlapped m, 3H, BO-CH and 2XHCBe-0), 4.30 (m, 2B, 2XCB ₂ -0), 6.27 (s, IB, O-CB-O), 6.78 (dd, 2B, J-9.0 Bz, ArB). CMR (75.44 MBz, CDC1 ₃ ) 5: 25.1, CB ₃ ; 25.8, 35.7, CB ₂ ; 55.6, 56.3, OCB ₃ ; 67.5, CBOB; 67.6, 67.7, OCB ₂ ; 97.4, O-CB-O; 109.4, 111.7, aryl CB; 125.9, 129.3, 150.7, 152.7, aryl C. BRMS calculated for C _ls B ₂₂ O _s : [M*-] 282.1467 found 282.1449. IR (FT, CDC1 ₃ ) 'Max: 3300-3550, bs, OB; 1257, 1094, C-O.

Step 5: 5,β-Diβethoxy-l-hydroxy-3-methylisochroman

Application of the procedure described in step 2 of this example gave 215 mg (86% yield) of the 3-methyl substituted isochroman from 250 mg of 2,5-dimethoxy-6-(2-

SUBSTITUTE SHEET

146

hydroxypropyl) benzaldehydedioxane acetal after flash chromatography with 20% ethyl acetate in hexanes. (MP: 240°-241°C). ^X B NMR (200 MHz, CDC1 ₃ ) 5: 1.41 (d, 3H, J-6.2 Hz, CH ₃ ), 2.34 (dd, IH, J-11.4, 17.4 Hz, HCHa), 2.81 (dd, IH, J-3.25, 17.3Hz, HCHe), 3.02 (d, 1Ξ, exchangeable, J-3.4 Bz, OB), 3.81 (s, 3H, 0CH ₃ ), 3.85 (s, 3H, OCB ₃ ), 4.37 (m, IB, OCB), 6.12 (d, IH, J-3.3 Hz, O-CH-O), 6.74 (dd, 2H, J-8.9 Bz, ArB). CMR (75.44 MBz, CDC1 ₃ ) : 21.3, CB ₃ ; 29.8, CH ₂ ; 55.7, 55.8, OCH ₃ ; 62.6, O-CB; 88.6, O-CB-O; 107.9, 109.5, aryl CB; 124.6, 150.47, 150.53, aryl C. IR (FT, CDC1 ₃ ) "Max: 3630, bs, OH; 1260, C-O. HRMS calculated for C ₁₂ H„0 ₄ : [M*-] 224.1049 found 224.1036.

Step 6: l-Hydroxy-5, 8-dioxo-3-methyl-5 , 8- dihydroisochroman

Oxidative demethyllation of 336 mg (1.5 mmol) of 5,8- dimethoxy-1- hydroxy-3-methylisochroman with 2.94 g (5 mmol) of ceric ammonium nitrate as described in step 3 of this example gave 262 mg (90% yield) of isochromandione as a yellow oil. ~E NMR (200 MBz, CDC1 ₃ ) 5: 1.38 (d, 3B, J-6.3 Bz, CB ₃ ), 2.15 (ddd, IB, J-1.2, 11.0, 19.5 Bz, HCHa) , 2.62 (dd, IH, J-3.3, 19.5 Hz, HCIe), 3.16 (d, IH, J-4.3 Bz, exchangeable, OB), 4.31 (m, IB, 0-CBCB ₃ ), 5.91 (d, IB, J-4.3 Bz, O-CB-O), 6.78 (dd, 2H, J-10.2 Hz, ArH).

Step 7: l,2-oxiranebutane-3-one dioxane ketal

In a three neck, round bottom flask (29.776g, 0.35 mmol) l,2-oxiranebutane-3-one, which had been previously

SUBSTITUTE SHEET

147

obtained following House's procedure (Organic synthesis Vol. Ill, p. 552), was mixed with 1,3-propanediol (118 ml, 1.59 mmol) and benzene (350 ml). To this mixture pyridinium tosylate was added (8.49g, 0.033 mmol). The mixture was refluxed, with water separation by a Dean-Stark trap, until the starting ketone has been completely used. The solution was washed twice with saturated sodium hydrogen carbonate solution, once with saturated sodium chloride solution, the organic phase was dried over sodium sulfate, and the bulk of the solvent was evaporated. The remaining mixture was distilled under reduced pressure to give the pure protected epoxy ketone in 22% yield. (BP: 20 mmflg, 75°C). ^X E NMR (300 MHz, CDC1 ₃ ) ppm: 1.28 (s, 3H, CHj), 1.60 (multiplet, 2H, dioxane ring), 2.61 (dd, IH, HCH-O-CB), 2.66 (dd, IB, HCH-O-CB), 3.00 (t, IB, BCB-O-CB , 3.91 (multiplet, 4B, dioxane ring). "C NMR (75.44 MBz, CDC1 ₃ ) ppm: 19.80 (CB ₃ , R ₃ C-CB ₃ ), 25.42 (CB ₂ , -O-CBj-CHj-CBj- 0-), 43.27 (CH ₂ , CBj-O-CB-), 56.46 (CB ₂ -0-CB) , 60.84 (CB ₂ ,- O-CHj-CHj-CBj-O-), 61.15 (CH ₂ , -Q-CB,-CB.-CB,-Q-_ , 96.65 (quaternary C, CB ₃ C(0(CB ₂ ) ₃ 0) . IR (FT, CDC1 ₃ ) cm" ¹ : 1494 (multiple bands, CB ₂ -0, oxirane ring. BRMS Clcd for C ₇ B ₁₂ 0 ₃ : [M*-]= 144.0786 found 144.0746.

Step 8: 1,4-dihydroxy-3-(ethane-1-one-dioxaneketal)-5,8- dimethoxy isochroman

A solution of 2.5M n-butyllithium (4.01 mmol) is added under argon at -15 ^β C, to a stirred solution of 0.5g of 2,5- dimethoxy benzaldehyde dioxane acetal (2.23 mmol) in 20 ml of anhydrous ether and stirred for 5 hours at -7°C. At this point, 1.48 ml of BF ₃ etherate (12.04 mmol) are added to 0.868g of l,2-oxiranebu-tane-3-one dioxane ketal (6.02 mmol) at -78°C, with stirring. To this new mixture is added the lithio salt just formed. The reaction is left

SUBSTITUTE SHEET

148

stirring at -78°C overnight. Then the mixture is quenched with 20 ml of HH ₄ C1 sat. Ether is added and the phases are separated. The organic layer is washed twice with 75 ml of water. Once with NaCl sat. and dried over MgS0 ₄ . Flash chromatography of the residue obtained gave the desired isochroman (13% yield). ^l E NMR (300 MHz, CDC1 ₃ ) ppm: 1.54 (s, 3H, CB ₃ ), 1.58 (multiplet, IH, -0-CH ₂ -HaCH-CH ₂ -0-) , 1.88 (multiplet, IB, -0-CB ₂ -BCBe-CB ₂ -0-), 2.71 (dd, IB, HCH- CB(OB)), 2.78 (dd, IB, BCB-CB-O), 3.76 (broad s, 6B, 2X 0 ₃ CB ₃ ), 3.95 (multiplet, 4B, -0-CH,-CB,-CH ₇ -0- ) . 5.75 (s, IH, dioxane ring), 6.70 (dd, 2H, ArB). "C NMR (75.44 MBz, CDC1 ₃ ) ppm: 21.53 (-C-CB ₃ ) ppm: 21.53 (-C-CH ₃ ), 25.33 (0- CB ₂ -CB ₂ -CB ₂ -0-), 55.66, 56.10 (0-CB ₂ -CB ₂ -CB ₂ -0-), 59.99 (CB ₂ - CH-O), 63.43, 68.21 (0-CH ₃ ) , 76.56 (CH ₂ -CH-0), 94.16 (CH- OB), 99.11 (C-quaternary, dioxane ring), 108.60, 109.86 (CB, aromatic). IR: (FT, CDC1 ₃ ) cm" ¹ : 3670 (OB).

Step 9: l-hydroxy-3-(ethane-1-one dioxane ketal)-5,8- dioxo-5,8-dihydroisochroman

A solution of 63.59g ceric ammonium nitrate (1.16x10" *mmol) in 1 ml of B ₂ 0 is added dropwise to a stirred solution of 12 mg of l-hydroxy-3-(ethane-1-one dioxane ketal)-5,8-dimethoxy isochroman (3.87 x lO' nmol) in 1 ml of acetonitrile, at room temperature. After 8 minutes, the mixture is diluted with 10 ml of CH ₂ C1 ₂ . The layers are separated. The aqueous layer is extracted twice with CHjClj and the organic layers are washed twice with water, once with NaCl sat., and dried over MgS0 ₄ . The residue obtained after evaporation of solvent gave a mixture of compounds including the desired quinone. IR: (FT, CDC1 ₃ ) cm ^-1 : 3600-3700 (broad OB), 1664.1 (C-O, stong band, quinone).

SUBSTITUTE SHEET

149

Step 10: 2 , 5-dimethoxy-6- ( l-hydroxy-2 , 3 - isopropylidenepropane) benzaldehyde dioxane acetal

A solution of 2.5M n-butyllithium (4.014 mmol) is added under argon at -15°C, to a stirred solution of 0.5g of 2,5-dimethoxy benzaldehyde dioxane acetal (2.23 mmol) in 20 ml of anhydrous ether and stirred for 5 hours at -7°C. To this mixture is added 0.78g of isopropylidene-D- glyceraldehyde (6.02 mmol) at -78°C. Isopropyli-dene was previously prepared using David Y. Jackson's procedure (Synthetic Communications, .18.(4), 337, 1988). The new mixture is warmed up to room temperature and left reacting overnight. Ether is added and the solution is washed twice with B ₂ 0, once with NaCl sat., and dried over MgS0 ₄ . Flash chromatography of the residue obtained after evaporation of the solvent gave the desired product. ¹ B NMR (300 MHz, CDC1 ₃ ) ppm: 1.35 (multiplet, IH, -0-CH ₂ -HaCB-CB ₂ -0-), 1.42 (broad s, 3B, CB ₃ ), 1.50 (broad s, 3H, CB ₃ ) , 2.25 (multiplet, IB, -0-CB ₂ -BCHe-CB ₂ -0-) , 3.76, 3.79 (2 s, 6H, 0-CB ₃ ), 3.90 (multiplet, 4B, -O-CBj-CHj-CBj-O-) , 4.24 (2 dd, 2B, CB ₂ group on isopropylidene), 4.85 (q, IB, CB group on isopropylidene), 5.76 (t, IB, -CB(OB)) , 6.21 (s, IB, dioxane ring) 6.81 (dd, 2H, aromatic). IR: (FT, neat) cm" -z 3400 (OB, broad band). BRMS Clcd for C _lt B ₃₍ 0 ₇ : [m*-]-354.1678 found 354,1670.

Step 11: 1,4-dihydroxy-3-hydroxymethyl-5,8-dimethoxy¬ isochroman

Treatment of 2,5-dimethoxy-6-(l-hydroxy-2,3- isopropylidene-propane) benzaldehyde dioxaneacetal with

SUBSTITUTE SHEET

150 silica gel gave the desired isochroman (23% yield). *E NMR (300 MBz, CDC1 ₃ ) ppm: 3.48 (dd, IH, CH(OH)-CH-HC-OH), 3.81, 3.86 (2 s, 6H, 0-CH ₃ ), 4.03 (t, IH, -CH(OH)-CH-HCH-OH) , 4.51 (broad d, IH, -CH-(OH)-CH-CH _. OH) , 4.89 (dd, IH, (OH)CH-CH-CBjOB), 6.50 (s, IH, -CH-0-CH-CH ₂ OH) , 6.81 (dd, 2H, aromatic). "C NMR (75.44 MHz, CDC1 ₃ ) ppm: 56.11 (- CH ₂ OH), 56.39 (OH)CB(O)-CH-CH ₂ OH) , 63.64, 66.07 (2x-0-CB ₃ ), 76.92 ((OB)CB-CH-CB ₂ OB), 95.13 ((OH)CH-0-CH-CH ₂ OB), 111.40, 11.75 (CH, aromatic), 122.02, 125.36 (C, aromatic; attached to pyranyl ring), 149.80, 152.36 (C, aromatic; bearing a methoxy group). IR: (FT, neat) cm" ¹ : 3520 (OB, not too intensed due to hydrogen bonding.) ΞRMS Clcd for C ₁₂ H ₁₄ 0 _s : (M*--H ₂ 0) - 238.0841 found 238.0871.

Step 12: 1,4-dihydroxy-3-hydroxymethyl-5,8-dioxo-3,4,5,8- tetrahydrobenzo (2,3c) pyran

A solution of ceric ammonium nitrate (1.40xl0~ ⁴ mmol) in 1 ml of H ₂ 0 is added dropwise to a stirred solution of 12. mg of l,3-dihydroxy-3-hydroxymethyl-5,8-dimethoxy isochroman in 2 ml of acetonitrile, at room temperature. The reaction is followed by TLC; the product migrates at same height as the starting material, but is much more U.V. active than the starting material and therefore can be differentiated. After about 10 minutes of stirring, the reaction is over. CB ₂ C1 ₂ (10 ml) is added, and the layers are separated. The aqueous layer is extracted twice with B ₂ 0, once with NaCl sat., and dried over MgS0 ₄ . The residue obtained after evaporation of the solvents gave the desired isochroman (628 yield) (MP: 138°-140°C). ^l H NMR (300 MHz, CDCl,) ppm: 3.44 (dd, IH, J-8.43, 2.08 Hz,

SUBSTITUTE SHEET

151

CB(OB)-CB-CB(OB)), 4.04 (dd, IH, J-8.38, 6.70 Hz, CH(OBO-0- CH-HCH(OB)), 4.32 (broad s, IB,

-CH(OB)-CB-CH ₂ (OH)), 4.87 (ddd, IH, J-6.55, 2.16, 1.21 Hz, (OH) CH-CH-CH ₂ (OH)), 6.20 (broad s, IH, (OH)CH-O-CB- CB ₂ (OB)), 6.80 (dd, 2B, aromatic). IR: (FT, CDC1 ₃ ) cm" ¹ : 3600-3650 (OB, medium band) , 1670.7 (C-O, quinone).

SUBSTITUTE SHEET

151A

Example 13: Preparation of other key isochromandiones useful in the synthesis oi various heteroanthracyciinones

SUBSTITUTE SHEET

^■ 152

EXAMPLE 14

Step 1: 5, 12-dimethoxy-3-carbornethoxy-1,2,3,4- tetrahydro-(2-sulfur)-naphtacene-6,11-dione

1,4-Dimethoxy-2,3-dibromomethyl-anthraquinone (3.5g, 7.76 mmol) was dissolved in CB ₂ Cl ₂ /MeOB (300 ml, 6:4) followed by the addition of ethyl 2-mercaptoacetate (1,02 ml, 9.31 mmol) with stirring under argon. The mixture was then cooled to 0°C followed by dropwise addition of sodium methoxide (4.37M, 2.13 ml, 9.31 mmol). The reaction mixture was stirred for an additional lh, and then concentrated in vacuo. The crude residue was redissolved in THF and cooled to 0°C, followed by the addition of NaOEt (0.63g, 9.31 mmol). The ice bath was then removed and the reaction was stirred until it had warmed to room temperature. The reaction mixture was then quenched with NB ₄ C1, and extracted with CB ₂ C1 ₂ . The organic layers were combined, washed with B ₂ 0, dried over MgS0 ₄ and concentrated in vacuo to give a residue which was flash chromatographed using hexane, EtOAc, and CB ₂ C1 ₃ (5:1:6) giving the desired products (679 mg) in 22% yield. ^l E NMR (300 MHz, CDCI ₃ ) 5: 1.28 (t, 3B, J-7.0 Bz), 3.17 (dd, IB, J-16.62, 7.83, BSla-C-O), 3.44 (dd, IB, J-16.7, 5.5 Bz, BCBeCBC-O) . 3.76, 3.77 (m, 2B, OCBC-O or HCHa-S), 3.78 (s, 6B, OCB,), 3.99 (m, IB, BCBe-Sϊ. 4.20 (q, 2B, J-7.0 Bz), 7.74 (m, 2H, ArB). 8.17 (m, 2B, ArB).

SUBSTITUTE SHEET

153

Step 2: 3-carbethoxy-5,12-dihydroxy-l,2,3,4-tetrahydro- (2-sulfur)-naphthacene-6,11-dione

To a stirred solution of I (20 mg, 0.05 mmol) in 5 ml of CH _j Clj at -78°C was added dropwise BBr ₃ (2.04 ml) under argon. The reaction mixture was stirred at -78°C for 1.5 hour before it was quenched with 5 ml of B ₂ 0 and 10 ml of CB ₂ C1 ₂ . The organic layer was separated, washed with Ξ ₂ 0, dried over MgS0 ₄ , filtered and concentrated to residue which was found to be pure II (19 mg, 0.05 mmol, 100%). ~R NMR (CDC1 ₃ ) 5: 1.29 (3H, t, J-7.0 Bz), 3.28 (2B, m), 3.90 (3B, m), 4.22 (2B, q, J-7.1 Hz), 7.80 (2H, m) , 8.31 (2B, m), 13.49 (IB, s), 13.50 (IB, s), ^: H NMR (C ₍ D _C ) 5:0.90 (3H, t, J-7.1 Bz), 3.08 (IB, m), 3.33 (IH, m), 3.42 (IH, ), 3.88 (4H, m), 7.85 (2B, ) , 8.14 (2B, m) , 13.78 (IB, s), 13.86 (IB, s), ¹³ C NMR (CDC1 ₃ ) 5: 14.13, 22.74, 26.11, 39.32, 61.74, 126.93, 133.48, 134.36, 155.10, 156.30, 168.01, 186.64.

Step 3: 1,4-diacetyl-2,3-dimethylanthraquinone

To a stirred suspension of 2,3-dimethylquinizarin (5.00 g, 18.637 mmol) in dry CB ₂ C1 ₂ (250 ml) was added acetic anhydride (40 ml) pyridine (40 ml) and dimethylaminopyridine (DMAP, 1.7g) at room temperature under argon. After two hours, the reaction mixture was poured into a mixture of ethyl acetate and ice (1:1, 800 ml), extracted with CB ₂ C1 ₂ , and the combined organic layers were dryed over MgS0 ₄ . Flash chromatography with toluene and ethylacetate; (95.5%) gave 2.517g of pure compound (38% yield). (MP: 222-224 ^β C). -E NMR (CDC1 ₃ ) 5: 2.27 (s, 6B, C j, 2.53 (s, 6B, COCBj), 7.72 (m, 2B, ArB), 8.15 (m, 2B, ArB).

SUBSTITUTE SHEET

154

Step 4: 1,4-diacetyl-2,3-bis-(bromomethyl)-anthraquinone

A mixture of diacetylated quinizarin from step 1 above, (2.221g, 8.801 mmol), NBS (3.956g, 22.002 mmol) and benzoyl peroxide (178 mg) in CC1 ₄ (350 ml) was refluxed and irradiated under argon with a 100W lamp for 5 hours. Additional NBS (0.5 mmol) and benzoyl pero::ide (0.2 mmol) were added. Reflux was continued for 3 hours and the solution was then cooled. Evaporation of the solvent in vacuo followed by flash chromatography with toluene and ethylacetate (97.5%: 2.5%) gave 3.095g of the titled compound (88% yield). (MP: 225-227°C). -E NMR (CDC1 ₃ ) 5: 2.56 (s, 6B, OCOCB ₃ ), 4.59 (br.s, 4B, CB_Brl . 7.75 (m, 2B, ArB), 8.14 (m, 2B, ArB).

SUBSTITUTE SHEET

154A

Example 14: Preparation of pyrano and thiopyrano modified anthracyclinones from 2,3-dunetnyl quinizaiiπe

SUBSTITUTE SHEET

155

Example 15

Antitumor Activity

BCB-242 was subjected to an in vitro cytotoxicity evaluation against 57 tumor cell lines to assess its potential as an anticancer drug. These assays were carried out at the Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute (Bethesda, Maryland, U.S.A.). The method consists of incubating varying concentrations of the drug with an inoculum of cells for two days. At the end of the assay, the number of viable cells is estimated with a dye, sulforhodamine B. The protocol is described in M.R. Boyd, "Status of the NCI Preclinical Antitumor Drug Discovery Screen", Principles and Practices of Oncology, 3, pp. 1-12, 1989. The 57 cell lines included two mouse leukemia lines (P388 parental and adriamycin resistant) and 56 human cell lines.

There are three parameters calculated: GI ₅₀ , the log molar cone, of drug required to inhibit cell growth by 50%; TGI, the log molar cone, of drug required to completely block cell growth; LC _S0 , the log molar concentration of drug required to reduce the original cell number by 50%. It should be noted that the first two parameters (GI ₃₀ and TGI) relate to antiproliferation effects while the third one (LC ₃₀ ) indicates true cytotoxicity. A value of -4 or greater is considered to denote resistance (inactivity) . The results are presented in the Table below.

A number of interesting observations can be made regarding the activity profile of BCB-242. This analog is able to inhibit cell growth of both mouse leukemia cell lines (P388 parental and adriamycin resistant) to an equal

SUBSTITUTE SHEET

156 extent (-5.78 vs -5.63). Likewise, BCH-242 shows a similarcytotoxic effect against MCF-7 parental and adriamycin resistant lines (-5.27 vs -5.15). The ability of BCH-242 to show activity against cell lines having known drug resistance suggests that the compound may be acting by an advantageous mechanism and that the compound may be clinically useful.

In addition, the various human and mouse leukemia lines, although sensitive to the antiproliferative effect of BCB-242, display some resistance to the cytotoxic effect of the drug. However, most of the cell lines of solid tumors ultimately show cytotoxicity to the drug. Since the potency of drugs against experimental leukemia usually predicts clinical bone marrow toxicity, it suggests that BCB-242 might be active clinically against certain solid tumors while exhibiting a minimal myelotoxic effect.

SUBSTITUTE SHEET

156A

ANTIPROLIFERATIVE AND CYTOTOXID EFFECTS OF BCH-242 IN TUMOR CELL LINES

^L °5lo TGI Log ₁₀ LC50

-5.35 > -4.00 -5.30 > -4.00 -5.35 > -4.00 -5.49 -5.09 -5.35 -4.47

-5.00 -4.42 -5.47 -5.20 -5.31 -4.78 -4.75 -4.37 -5.43 -5.17 -5.18 -4.59 -5.49 -5.23 -4.53 -4.16 -5.40 -5.09 -5.68 -5.34

-5.52 -5.24 -5.35 -5.01

-5.63 -5.31 -5.49 -5.17 -5.24 -4.72 -5.45 -5.12 -5.45 -5.09 -4.97 -4.49 -5.27 -4.75 -5.54 -5.19

-4.59 -4.40 -5.15 -4.59 -5.36 -5.03 -4.88 -4.44 -5.06 -4.52 > -4.00 > -4.00 -5.14 -4.59 -5.50 -5.22

-5.49 -5.24 -5.49 -5.24 -5.51 -5.21 -5.51 -5.24 -5.37 -5.11 -5.55 -5.26 -5.44 -5.18 -5.50 -5.25

-5.42 -5.10 -5.52 -5.23 -5.48 -5.22 -5.46 -5.23 -5.49 -5.19 -4.77 -4.38

156B

Log _1Q TGI LOG _1Q LC50

Mean -5.65

SUBSTITUTE SHEET

157

Exam l e I fi

In Vitro Clonogenic Assays of Heteroanthracyclines

A series of heteroanthracycline analogs were evaluated by the NCI at Bethesda, Maryland, USA. The same protocol as described in example 15 for BCB-242 was used. Tables 1 and 2 summarise the GI _S0 and LC ₅₀ average values obtained from a group of cell lines, per type of cancer. The original NCI data for the various derivatives, is provided herein in tables 3 to 11.

With reference to table 1, the heteroanthracycline derivatives show a weaker antiproliferative activity than daunomycin (DNM) or adriamycin (ADR) in the different type of cancers. Bowever, the ability of the heteroanthracycline agents to kill the solid tumor cells is clearly denoted in Table 2. Cytotoxycity ranges from a mean potency of -4.09 in BCB-674 to as high as -5.32 in BCB-242 in solid tumors. Most notably is the fact that average cytotoxicity in leukemia is generally much lower than with DNM or ADR. This is a desirable feature and may indicate a lower bone marrow toxicity than with the clinical agents, DNM and ADR. Since myelotoxicity limits the single dosage that can be administered to a patient, these results suggest that higher and more therapeutically useful single dosages would be permissible with the heteroanthracyclines of this invention in the chemotherapy against solid tumors.

SUBSTITUTE SHEET

TABLE 1

Average LOG Gl Hu iple cell lines

10 50

m

TABLE 2

Average LOG LC Multiple cell lines

10 50

_♦ ♦ _♦ ......... ♦-.. + + .. ₊ .- _♦ -. «.

(BCH no. I Leukemia I NSC lung I SC lung ( Colon | CNS | Melanoma I Ovarian I Renal I Mean Potency j ( j j j I I I I I (in sol id tumors]

I ONH |-4.30t0.36 (-5.07*0.37 (-5.58*0.09 (-4.83*0.37 (-5.08*0.42 |-5.64i0.17 (-4.78*0.20 |-4. 8*0.46 |-5.11i0.34 I

I A0R (-4.76*0.10 (-5.01*0.26 (-5.39*0.18 (-4.86*0.27 (-5.03*0.35 (-5.56*0.29 |-4.74*0.05 (-4.87*0.38 |-5.07t0.2β ( m ( 242 (>-4.00 1-5.21*0.38 (-5.58*0.27 (-5.59*0.36 (-4.86*0.24 (-5.41*0.07 1 -5.35*0.19 (-5.22*0.21 (-5.32t0.20 I

I 670 |>-4.00 (-4.62*0.27<1R)*(-4.41(1R) J-4.60*0.29(2R)(-4.58*0.15 (-4.69*0.29( 1R)(-4.40*0.13 (-4.53*0.16 (-4.57*0.09 I

( 671 (>-4.00 (-4.52*0.41 (-4.26*0.20 |-4.80*0.50(1R)|-4.37*.24 (-4.67*0.57 (-4.47*0.35 (-4.63*0.24 |-4.53*0.17 | I 672 (-4.37*0.15 (-4.56*0.08 (-4.40*0.20 (-4.55*0.18 (-4.50*0.16 | -4.67*0.18 (-4.59*0.13 (-4.62*0.08 |-4.56*0.0β f I 673 |>-4 ^* 00 |-4.66*0.43 (-4.44*0.04 (-4.46*0.45 (-4.57*0.22 (-4.87*0.50 (-4.66*0.50 (-4.96*0.32 (-4.66*0.18 j

( 674 (> ^* 4 ^* Oθ (-4.16*0.10 (-3.92*0.14 (-4.01*0.27 (-4.20*0.06 (-4.05*0.17 (-4.08*0.17 (4.24*0.14 1-4.09*0.11 j

I 675 |>-4.00 (-4.25*0.23 (-4.21*0.04 (-4.07*0.22 (-4.21*0.11 (-4.10*0.23 (-4.20i0.16 1 -4.25*0.10 (-4.18*0.07 |

I 681 N-4.00 (-4.64*0.41 (-5.18 (-5.14*0.27 (-4.65(1)10.24 (-5. 0*0.12 (-4.78*0.18 |-4.66(1)*0.40 (-4.91*0.27 (

I 683 (<-4.00 (-4.55*0.42 (-5.08 J-4.98(1)*0.38 |4.50(1)*0.21 (5.15*0.22 (-4.80*0.33 (-4.58*0.36 |-4.81ι0.25 ( O ( 689 |<-4.00 |-4.3β(*0.06)3R J-4.37(*0.05) (-4.41(10.03) J-4.39(*O.0S)2R|-4.3β(*O.O4) (-4.371*0.0 ) |-4.43(ι0.01)2R|-4.39(*0.02) | tΛ 'Represents the number of cell lines uhich are considered to be refractory towards the compound's cytotoxicity (LOG LC <-4.00).

___■_■ 1° ⁵⁰

rn H

C 09 m

H

H

C H

m

TABLE 3 CONT'D

m C

H

C H O I

m

C w m H

H

H m

Co

TABLE 4 CONT'D

m

09 en H

-H

C

H rn

rn

CO

C w CO

H

CO m

TABLE 5 CONT'D

CO

C w CO

H

CO

CO

C w CO

H

C

H m

Co X m

TABLE 6 CONT'D

CO oo

CO

H

H

C H

C

X rn

National Cancer Institute Developmental Therapeutics Program NSC: 636529-N/ 1 Units: Molar SSPL: Exp. ID: 901QSC22 TABLE 7 Mean Graphs BGH-674 Report Date: November 26, 1990 Test Date: October 22, 1990

Panel/Cell Line LogιnG15Q LogmTGl LogιnLC5Q

Leukemia

CCRF-CEM -4.97 -4.44 > -4.00

HL-60(TB)

K-562 -4.89 -4.34 > -4.00

MOLT-4 -5.18 -4.55 > -4.00

RPMI-8226 -5.21 -4.57 -4.00

SR-WJU Non-Small Cell Lung Cancer

CO A549/ATCC -4.84 -4.49 -4.15

C EKVX -4.74 -4.42 -4.10 w HOP-18 -4.77 -4.50 -4.22

HOP-62 m HOP-92 H NCI-H226

NCI-H23

NCI-H460

NCI-H522

C LXFL-529L Small Cell Lung Cancer H DMS 114

DMS 273 Colon Cancer

COLO 205

Co DLD-1 X HCC-2998

HCT-116

HCT-15

HT29 M12

KM20L2

SW-620 CNS Cancer

SF-268

SF-295

SF-539

TABLE 7 CONT'D

CO

C

00 CO

H

-H

C H

CO

X

CO

C

00 CO

H

H

C m

C

X m

TABLE 8 CONT'D

CO

00 CO

H

H

C

H m

CO

X m m

TABLE 9 CONT'D

CO

C oo

CO

H

H

C

H m

CO

X m rn

CO

00 CO

H

H C H

CO

X

TABLE 10 CONT'D

CO

C

00 CO

H

H

C

H m co

X

m

C oo co H

H C m

Co

X rn H

TABLE 11 CONT'D

CO

C

00 CO

H

H

C

H m

Co

X m m

m

C oo co

H

C

H m co X

TABLE 12 CONT'D

SF-539

SNB-19 -5.22 -4.91 -4.61

SNB-75 -5.20 -4.89 -5.06 -4.59

SNB-78 -4.79

U251 -5.60 -4.52 -4.99

XF 498 -5.11 -4.44 -4.84 -5.55 -4.57 Melanoma -5.05 -4.67

LOX IMVI

MALME-3M -5.73 -5.06

M14 -5.96 -4.31 -5.48 -5.85 -4.86

M19-MEL -5.29 -4.75

CO SK-MEL-2 -5.95 -5.54 -5.63 -4.90

SK-MEL-28 -5.02 -4.64

SK-MEL-5 -5.53 -5.02 -5.87 -4.61

UACC-257 -5.38 -5.91 -4.75

UACC-62 -5.36 -5.86 -4.71 Ovarian Cancer -5.24 -4.47

IGROV1

OVCAR-3 -5.74 -5.08 -5.78 -4.37

OVCAR-4 -5.26 -5.44 -4.78

OVCAR-5 -5.01 -5.05 -4.65

OVCAR-8 -4.80

SK-OV-3 -5.94 -4.56 -5.46 Renal Cancer -5.05 -4.64 -4.79

786-0 -4.53

ACHN -5.39 -4.98 -4.62

CO CAKI-1 -5.39 -4.99 -4.64

X RXF-393 -5.12 -4.84

RXF-631 -5.69 -4.55 -5.11

SN12C -5.39 -4.70 -4.95 -5.86 -4.56 m TK-10 -5.31 Uθ-31 -5.02 -4.77 -4.77 -5.20 -4.52 -4.91

MG_MID -4.61

Delta -5.58 -5.10

Range 0.45 -4.57 0.51 1.02 0.33 0.86 0.59

169

Example 17

In Vitro Clonoαenic Assays of Heteroanthracyciinones.

Tables 13 and 14 summarize the GI _S0 and LC ₅₀ average values obtained from a group of cell lines, per type of cancer, for various heteroanthracyciinones. The original results obtained from the NCI, Bethesda, Maryland, for these compounds are shown in tables 15 to 22. The same protocol as used for BCH-242 in example 15 was employed in this example.

It is most interesting to note that some of the heteroanthrocyclinones (BCH-651, BCH-657, BCH-660) listed in tables 13 and 14 show strong antiproliferative (GI _S0 ) and potent cytotoxic (LC _S0 ) activities. In the case of BCH-657 and BCH-660 the mean LC _S0 potency in solid tumors is similar to the one observed for DNM and ADR. The same trend observed with the heteroanthracyclines of example 16, where antileukexαic activity is depressed, recurs with the heteroanthracyciinones of this example. This aspect may be beneficial to the clinical use of heteroanthracyciinones in the chemotherapy of solid tumors.

BCH-687, a compound in which there are no benzylic substituents, has significant antiproliferative (GI _sα ) and cytotoxic ( C _aα ) activities in solid tumors. This is unprecedented because known anthracyclinones which lack benzylic substituents are normally devoid of anticancer activity.

SUBSTITUTE SHEET

TAILE 13

Average LOG Gl Hu iple cell lines

10 50

t

I rn

TABLE 14

Average LOG LC HuUiple celt lines

10 50

» ,

m C

CD to H

H m t m rn

TABLE 15 CONT'D

to w to

H

H

C

to

to

CD to

H

H

to

TABLE 16 CONT'D

SF-539 -5.02 -4.63 -4.26

SNB-19 -4.93 -4.51 -4.09

SNB- 5 -4.74 -4.38 -4.02

SNB-78 -5.06 >-4.00 > -4.00

U251 -5.57 -5.15 -4.23

XF 498 -4.84 -4.56 -4.27 Melanoma

LOX IMVI -5.59 -4.92 -4.41

MALME-3M -5.69 -5.20 -4.58

M14 -5.61 -5.14 -4.56 to M19-MEL -5.52 -5.05 -4.28

C SK-MEL-28 -5.24 -4.60 -4.04

SK-MEL-5 -5.67 -5.31 -4.88 w UACC-25 -5.60 -5.16 -4.53 to UACC-62 -5.52 -5.07 -4.38 Ovarian Cancer

H IGROV1 -4.95 -4.39 > -4.00

OVCAR-3 -5.63 -5.24 -4.37

H OVCAR-4 -5.28 -4.74 -4.35

OVCAR-5 -4.79 -4.53 -4.26 C OVCAR-8 -5.51 -4.96 -4.33 Renal Cancer H 786-0 -5.08 -4.61 -4.18

A498 -4.73 -4.46 -4.20

ACHN -5.13 -4.69 -4.31 to CAKI-1 -4.84 -4.49 -4.13

RXF-393 -5.56 -5.04 -4.47 X RXF-631 -4.91 -4.11 > -4.00

SN12C -5.19 -4.69 -4.29

TK-10 -4.60 -4.40 -4.19

Uθ-31 -4.93 -4.61 -4.30

MG MID -5.26 -4.67 -4.23 Delta 0.47 0.71 0.80

Range 1.13 1.38 1.04

to C

CD to

H

C

H m m

I rn m

TABLE 1 CONT'D

m C

CD to

H

H C

to

TABLE 18 CONT'D

m C m

H

H

C H to

X

National Cancer Institute Developmental Therapeutics Program NSC: 636532-Q/ 1 Units: Molar SSPL: Exp. ID: 901QSC22 TABLE 19 Mean Graphs BCH-658 Report Date: November 26, 1990 Test Date: October 22, 1990

Panel/Cell Line LogmG150 Log _ln TGl Log _ln LC5Q

Leukemia

CCRF-CEM >-4.00 > -4.00 >-4.00

HL-60(TB)

K-562 >-4.00 > -4.00 > -4.00

MOLT-4 > -4.00 > -4.00 > -4.00

RPMI-8226 > -4.00 > -4.00 > -4.00 m SR-WJU Non-Small Cell Lung Cancer

A549/ATCC >-4.00 > -4.00 > -4.00

EKVX > -4.00 >-4.00 > -4.00

CD HOP-18 > -4.00 > -4.00 > -4.00 to HOP-62

HOP-92 -4.70 -4. 16 > -4.00

NCI-H226 > -4.00 >-4.00 >-4.00

NCI-H23 > -4.00 > -4.00 > -4.00

NCI-H460 > -4.00 > -4.00 > -4.00

NCI-H522 -4.52 > -4.00 > -4.00

LXFL-529L > -4.00 > -4.00 >-4.00 Small Cell Lung Cancer

DMS 114 -4.53 > •4.00 >-4.00

DMS 273 > -4.00 > ^■ 4.00 > -4.00 to Colon Cancer

COLO 205 >-4.00 >-4.00 X DLD-1 >-4.00 >-4.00

HCC-2998

HCT-116 > -4.00 >-4.00

HCT-15 > -4.00 >-4.00

HT29 > -4.00 > -4.00

KM12 > -4.00 > -4.00

KM20L2 > -4 00 > -4.00

SW-620 > -4 oo > -4.00 CNS Cancer

SF-268 -4.23 >-4.00 >-4.00

SF-295 -4.09 > -4.00 > -4.00

TABLE 19 CONT'D

> - . - . > -4.00

> -4.00 > -4.00 > -4.00

> -4.00 > -4.00 > -4.00

> -4.00 > -4.00 > -4.00 -4.67 > -4.00 > -4.00

> -4.00 > -4.00 > -4.00

> -4.00 > -4.00 >-4.00 -4.08 > -4.00 >-4.00

> -4.00 >-4.00 > -4.00

-4.42 > -4.00 > -4.00

> -4.00 > -4.00 >-4.00

> -4.00 > -4.00 > -4.00 -4.54 -4.02 > -4.00 -4.24 > -4.00 > -4.00

> -4.00 > -4.00 > -4.00 -4.10 > -4.00 >-4.00

> -4.00 > -4.00 >-4.00

-4.15 -4.01 -4.00 0.54 0.33 o.oo 0.70 0.34 o.oo

m w to

H H

to

X rn

TABLE 20 CONT'D

m C

CXI to

H

H

C

to

X rn

m C to

H m to

X rn rn

177H

in O O O o MSH io OM π o o σv vo rt O O ^- o en o t- O O o r~ r~ rt O o o o CN CM CM en O en cn rt O O C rt O O O en o O O CM O O rt en

m C oσ to

H

H

C H to X

TABLE 22 CONT'D

to C to

H

H

H m to

X m

178 -

Example 18

In Vitro Activity of Heteroanthrocvclinones in Cell Lines Displaying Multi Druσ Resistance .MDR.

The anticancer activity of heteroanthracyciinones and heteroanthracyclines from this invention was confirmed from a second independent assay. This biological study was carried out at the Oncology department of the faculty of medicine at McGill University, in Montreal, Canada. It involved the use of adriamycin sensitive cells lines as well as multidrug resistant ones. The following describes the methodology used for anticancer testing. Biological results follow the protocol.

SCREENING TEST

For the IN VITRO test, we chose a tumor clonogenic assay which measures the ability of tumoral cells to form colonies (multicellular growth units) as a result of cell division in semisolid agar support (2,3). A more elaborate version of this fundamental assay, the soft agar colony formation disk diffusion assay was used. This assay defines the relative activity of one particular drug versus a tumoral cell line type. The effect of the test drug against tumoral cells could be measured by its inhibitory capacity against colony formation after the delivery of the compound, in different concentrations on an filter disk(4).

COLONY FORMATION DISK DIFFUSION ASSAY

SUBSTITUTE SHEET

179

A modified technique of this assay was used in order to investigate the antiproliferative activity of candidate • compounds. Tumoral cells from solid tumors and ascitic lines were plated separately in a two layer agar matrix. Cell lines to be tested were suspended in an upper layer of 0.4% Noble agar (Difco#00142-01) in RPMI-1066 (Gibco#3201875) at a yield varying between 10 ⁴ to 2 X 10 ^s cells per 60 mm Petri dish. Plating efficiency was determined for each line in order to achieve an optimal number of colonies per dish. Plates were examined before drug administration to confirm a uniform dispersion of single cells. The bottom layer consisted of 0.8% Noble agar, 0.8% Tryptic Soy Broth (difco#0037001) in CMRL- 1066/Fischer's. The bottom layers were used within 2 and 9 days of preparation. Plated dishes (bottom layer of agar, upper layer of cells and administered drug were incubated in humidified atmosphere at 37°C, 5% C0 _a for 10-20 days depending upon the time necessary to observe adequate colonies (defined as a cell aggregate of more than 50 cells) both control and drug inhibited. EJrugs were administered on a paper filter disk (Whatman No. 1) of 6.5 mm diameter placed on the upper layer at 1/3 distance from the edge of the dish. The anti-clonogenic activity of the compounds was measured as the zone of inhibition from the disk edge to the most proximal colonies. This zone of inhibition was checked on an inverted microscope (4OX) and was measured with a micrometer mounted in the eyepiece. The ocular micrometer was calibrated with a stage micrometer set for a particular combination of ocular and objective lenses.

Our calibration values were converted to those already used in other laboratories in order to have comparable values. 200 units of activity are equivalent to 6.5mm distance from the disk edge to the first colonies (5). Each concentration was run in triplicate and dispersion of

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180 the values was surprisingly small for the type of biological tests. We made a modification of the initial technique by not plating a solid tumor and a leukemia simultaneously in the same Petri dish. Anti-clonogenic tests performed on leukemia cell line have helped in the discovery of the majority of current clinically used cytostatic drugs. Our tests on solid tumor lines (animal or human) may help define active compounds with solid tumor specificity. This specific activity would be indicated by a higher level of cloning inhibition in solid tumors versus leukemia.; (6) .

MATERIALS AND METHODS

CELL LINES: 5 murine tumor lines and 2 rat tumor lines and 5 human tumor lines were used (Table 1).

ANIMAL TUMORS: 2 transplantable solid tumors, one reticulum cell sarcoma and one leukemia with and ADR resistant form were passaged IN VIVO. P388 and P388/ADR (7) routinely passaged in DBA/2, M5076 (8), B16 melanoma (9), Lewis lung carcinoma (9) routinely passaged in C57B1/6, were supplied by NCΪ, DCT Tumor Repository, NC -FCRF, Frederick, MD. Rat breast carcinoma MATB WT (18) and MATB/ADR (18) obtained from the Oncology Dept. of the Montreal General Hospital were passaged IN VITRO. Murine tumors were maintained IN VIVO in order to diminish the selection of subpopulations of cells normally present in the heterogenous IN VIVO tumor. The actual drug sensitivity assay was done IN VITRO. For these experiments cells from freshly excised tumors or from ascitic fluids were maintained in RPMI-1066.

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MICE: Inbred C57B1/6 females and DBA/2 females (20±2g) were obtained from Charles River Laboratory, Que. and used as tumor recipient.

CELL PREPARATION. The protocol described by Corbett et al (6) was used. Tumors were excised aseptically from the host and cut in 200-300 mg fragments in ΞBSS (Gibco# 3104020). The fragmented tumor was forced through a 60 mesh sieve and the sieve rinsed twice with cold HBSS. The cell suspension was gently centrifuged (100-150) g/5 min), resuspended in HBSS and once again centrifuged. The cell pellet was finally suspended in an adequate volume of medium. Difficult to disrupt tumors were digested with 0.25% Trypsin EDTA (Gibco#6105305) . The viability was over 90% for all cell lines prior starting experiments except for the LL line and P399/ADR which showed a varying degree of viability between 40 and 60%.

HUMAN TUMORS. MCF7 (10) and MCF7/ADR(11) HT29(12) were obtained from NCI, DCT Tumor Repository, NCI-FCRF, Frederick, MD. LS180 (13) and BE-1(14) were obtained from the Oncology Dept. of the Montreal General Hospital. Cell lines were initially maintained in RPMI-1640 supplemented with 10% fetal bovine serum and 100 units of Penicillin- Streptomycin (Gibco#6005140) . Cell lines were routinely passaged twice a week by trypsinization and maintained in 25, or 75-sq. cm Falcon Plastic flasks at 37°C, 5% C0 ₂ in humidified atmosphere.

DRUGS: Doxorubicin hydrochloride available from a commercial source was used as an internal control. For some line the DOX control was run simultaneously for both new synthesised agents. Our values for DOX as an internal

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182 control were consistently in accord with references(15,16) .

Both compounds were dissolved in DMSO (1 mg/lOOul) and brought to final volume (250ul) with Millipore deionized sterile water. Further dilutions were made with Milliwater. A volume of 25 ul of freshly prepared solution was placed on the filter disk and allowed to dry. A second control with vehicle (DMSO /Milliwater) was run simultaneously. Control disks with vehicle were found to produce no zone of inhibition. For the determination of drug action the compounds were tested in serial ten-fold dilutions. The results were expressed as units of inhibition of colony formation (Zone Units) produced at the same mass concentrations as DOX. Dilutions of the compounds as well as of DOX were stored in sterile Cryovials (Gibco#366656) at -20°C for no longer than 3 days.

REFERENCES

1. Hamburger, A.W., Salmon, S.E. Primary bioassay of human tumor stem cells. Science, 197:461-463, 1977.

2. Von Hoff, D.D., Clark, G.M. Stogdill, B.J. Sarodosy, M.F., O'Brien, M.T., Casper, J.T., Mattox, D.E., Page, C.P., Crux, A.B., Standbach, J.F. Prospective clinical trial of a human tumor cloning system. Cancer Res., 43:1926-1931,1983.

3. Corbett, T.H., Valeriote, F.A. Rodent Models in experimental chemotherapy. In "Rodent Tumor models in experimental cancer therapy", Ed. R.F. Kallman, Pergamon Press, 1987.

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183 -

4. LoRusso, P., Wozniak, A.J., Polin, L., Capp, D., Leopold, W.R., Werbel, L.M., Biernat, L., Dan, M.E. Antitumor efficacy of PD115934 (NSC 366140) against solid tumors of mice. Cancer Res., 50, 4900-4905, 1990.

5. Corbett, T.H., Valeriote, F.A. Baker, L.Ξ. Is the P388 murine tumor no longer adequate as a drug discovery model ? In "Investigational New Drugs", 55:3-20, Martinus Nijhoff Publishers, Boston, 1987.

6. Schabel, F.M., Jr., Skipper, H.E., Trader, M.W., Laster, W.R., Jr., Griswold, D.P., Jr. and Corbett, T.Ξ. Establishment of cross-resistance profiles for new agents. Cancer Treat. Rep. 67:905-922, 1983.

7. Hart, I.R., Talmadje, J.E., Fidler, I.J. Metastatic behaviour of a murine reticulum cell sarcoma exhibiting organ specific growth. Cancer Res., 41:1281-1287. 1981.

8. Cour enay, V.D. A soft agar colony assay for Lewis lung tumour and B16 melanoma taken directly form the mouse. Br. J. Cancer, 34:39-45, 1976.

9. Soule, H.D., Vazquez, J., Long, A., Albert, S., and Brennan, M. A human cell line from a pleural effusion derived from a breast carcinoma. J. Natl. Cancer Inst., 51:1409-1416, 1973.

10. Cohen, J.S., Lyon, R.C., Chen, C, Faustino, P.J., Batist, G., Shoemaker, M., Rubalcaba, , E., Cowan, K.H. Differences in phosphate metabolite levels in

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^• 184

drug-sensitive and -resistant human breast cancer cell lines determined by ₃₁ P. magnetic resonance spectroscopy. Cancer Res., 46:4087-4090, 1986.

11. Fogh, J., Trempe, G. "New human tumor cell lines". Chap.5 In: Fogh J. (Ed.) Human tumor cells in vitro, pp. 115-159. New York,. Plenum Publishing Corp., 1975.

12. Tom, B.H., Rutzky, L.P., Jakstys, R., Oyasu, C, Kahan, B.D. Human color.ic adenocarcinoma cells. 1. Establishment and description of a new line, IN VITRO, 12:180-191, 1976.

13. Goldin, A., Schepartz, S.A., Venditti, J.M., DeVita, V.T., Jr. Historical development and current strategy of the National Cancer Institute Drug Development Program, Chap. 5, In: "Methods in cancer research". Vol. XVI, Academic Press, 1979.

14. Alberts, D.S., Chen, H-S.G. Tabular summary of pharmacokinetic parameters relevant to IN VITRO drug assay. In: "Cloning of human tumor stem cells ", pp. 351-359, Ed. S.E. Salmon, Progress in Clinical and Biologcal Research, Vol. 48, 1980.

15. Goodman, G.E. Modulation of IN VITRO activity of Adriamycin* and Vinblastine by Verapamil, In: " Human tumor cloning", Ed. S.E. Salmon and Trent, J.M., Proceedings of the Fourth Conference on Human Tumor Cloning, pp.389-394, 1984.

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^• 18S" BIOLOGICAL RESULTS

The results obtained with the tumor clonogenic assay, in which a semisolid agar support was used, confirm the antitumor activity of the heteroanthracyciinones and heteroanthracyclines described herein (tables 23 and 24). In the human breast cancer lines, MCF7 and MCF7/ADR, a similar level of activity occurred for BCH's:687, 692, 699, 700, 701 and 706 between both cell lines, indicating a lack of cross resistance with adriamycin (Table 23). Most notably is the fact that BCH-684 and 712 were more active in the adriamycin resistant cell line, MCF7/ADR, than in the sensitive one. Similar results were obtained with the rat breast tumor cell lines MATB.WT and MATB/ADR. In this case BCH's: 677, 681, 684, 700, 705 and 712 displayed no cross resistance with adriamycin in MATB; BCH's: 704, 710 and 711 displayed a greater cytotoxicity in the resistant breast tumor cell line, MATB/ADR, than in the sensitive line. The results obtained with the breast tumor lines indicate that the heteroanthracyciinones and heteroanthracyclines of the present invention operate via an advantageous cytotoxic mechanism. A range of activity was observed from the compounds described herein in this example. The most active were BCH's: 684, 687, 691, 692, 700, 704, 706 and 710. BCH 710 shows specificity for the MATB/ADR rat breast ADR resistant adenocarcinoma and mouse ovarian reticulum cell sarcoma. Specificity for breast cancer was demonstrated by BCH's 700 and 704. Good broad spectrum activity was observed from BCH's: 684, 691, and 692, except in the leukemic cell lines P388 and P388/ADR, confirming the specificity of these compounds for solid tumors. Human colon adenocarcinoma HT-29, LS180 and BEl responded well with BCH-687, an analog in which no benzylic

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- 186 substituents are present. This is noteworthy because analogous compounds in the field, of anthracyclines are normally not significantly cytotxic towards tumor cells.

The data presented in this example supports the results presented in examples 15, 16 and 17. Heteroanthracyciinones and heteroanthracyclines, with an heteroatom in ring A as described herein, display a range of antineoplastic activity with specificity towards solid tumors. Cross resistance with doxorubicin or dannorubicin in some cases is not observed. The fact that cytotoxicity in hematologic malignancies is low in comparison with solid tumors suggest that myeclotoxicity should be decreased. This recurrent feature suggests that the therapeutic index of the compounds of the present invention has been increased. Therefore compounds of the present invention should be clinically useful because of reduced bone marrow toxicity and because of their demonstrated activity towards some resistant tumor cell lines.

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TABLE 23

CONCENTRATION: IOJIG/DISK

b) Compounds were not tatted In cat lines with a alath Indicated above.

TABLE 24

CONCENTRATION: lOOj-G/DISK