Novel derivatives of flavones, xanthones and coumarins

The present invention relates to a novel class of compounds which have structures related to certain naturally occurring and synthetic flavonoids and to pharmaceutical uses thereof.

Thus according to one aspect of the present invention, there is provided a compound of Formula (I):

z-och₂-c=cch₂nrr¹ (I)

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A or a pharmaceutically acceptable salt or solvate thereof wherein:

R and R¹ are the same or different and each represents lower Ci.₆ alkyl, or a carbocyclic group containing from 5 to 10 ring atoms, said ring atoms forming one or two rings wherein the or each ring contains 5 or 6 ring atoms, or

R and R¹ taken together with the nitrogen atom to which they are attached, form a four- to eight-membered heterocyclic ring which may contain one or more additional heteroatoms selected from N, O or S, said heterocyclic ring being optionally substituted with a lower Cm alkyl group or a benzyl group;

Z represents:

(A)

<img file="AU7000000A_D0001.tif" />

wherein

R² and R³ are each independently selected from:

(i) hydrogen, (ii) a substituted or unsubstituted, preferably aromatic, carbocyclic or heterocyclic group containing from 5 to 10 ring atoms, said ring atoms forming one or two rings, wherein the or each ring contains 5 or 6 ring atoms, any heteroatoms being selected from N, O and S, any substituents being independently selected from the group consisting of:

(a) Cl, (b) Br, (c) F, (d) OH, (e) NO₂, (f) CF₃, (g) Cm lower alkyl (in particular CH₃), (h) SCH₃, (i)NHCOCH₃, G)N(R⁶)(R⁸) wherein R⁶ and R⁸, are the same or different and each represents H or lower C-m alkyl, (k) OR¹⁰ wherein R¹⁰ represents H or lower C-i-6 alkyl which may be saturated or unsaturated and being unsubstituted or substituted with the group NRR¹ wherein R and R¹ is as defined above, and (l)OCOR¹¹ wherein R¹¹represents H or lower Ci_4 alkyl,

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PCT/EP00/08365 (iii) Cl, (iv) Br, (v) F, (vi) OH, (vii) NO₂, (viii) a saturated or unsaturated lower C-i-6 straight or branched hydrocarbyl group which may be unsubstituted or substituted by 1, 2 or 3 substituents selected from Cl, Br, F, OMe, NO₂ and CF₃, (ix) NHCOCH₃, (x) N(R⁶)(R⁸), (xi) SR¹⁰, (xii) OR¹⁰, and (xiii) OCOR¹¹ wherein R⁶, R⁸, R¹⁰ and R¹¹ are as defined above;

or

R₂ and R₃ taken together with the carbon atoms to which they are attached form a carbocyclic or heterocyclic ring having 5 or 6 ring atoms, any heteroatom being selected from N, 0 or S, said carbocyclic or heterocyclic ring being saturated or unsaturated, and being unsubstituted or substituted with one or more substituents selected from Cl, Br, F, OH, NO₂, CF₃, C_w lower alkyl, SCH₃, NHCOCH3, N(R⁶)(R⁸), OR¹⁰ and OCOR¹¹ wherein R⁸, R⁸, R¹⁰ and R¹¹ are as defined above; and

R⁴ represents hydrogen, or OR¹⁰ wherein R¹⁰ is as defined above or (B)

<img file="AU7000000A_D0002.tif" />

wherein R⁵ represents hydrogen or a lower C1-6 straight or branched hydrocarbyl group which may be unsubstituted or substituted by 1, 2 or 3 substituents selected from Cl, Br, F, OMe, NO₂ and CF₃.

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MThus in one aspect the invention provides compounds having the structure (IA’):

O R⁴

OCH₂-C=CCH₂NRR¹ (IA’) wherein

R² and R³ are each independently selected from:

(i) hydrogen, (ii) a substituted or unsubstituted, preferably aromatic, carbocyclic or heterocyclic group containing from 5 to 10 ring atoms, said ring atoms forming one or two rings, wherein the or each ring contains 5 or 6 ring atoms, any heteroatoms being selected from N, O and S, any substituents being independently selected from the group consisting of:

Cl, Br, F, OH, NO₂, CF₃, Cm lower alkyl (in particular CH₃), SCH₃, NHCOCH₃, N(R⁶)(R⁸), OR¹⁰ and OCOR¹¹, wherein R⁶, R⁸, R¹⁰and R¹¹are the same or different and each represents H or lower C₁₄ alkyl, (iii) Cl, (iv) Br, (v) F, (vi) OH, (vii) NO₂, (viii) a saturated or unsaturated lower Ci-6 straight or branched hydrocarbyl group which may be unsubstituted or substituted by 1, 2 or 3 substituents selected from Cl, Br, F, OMe, N0₂ and CF₃, (ix) NHCOCHs, (x) N(R⁶)(R⁸), (xi) SR¹⁰, (xii) OR¹⁰, and (xiii)OCOR¹¹wherein R⁶, R⁸, R¹⁰ and R¹¹ are as defined above;

or

R₂ and R₃ taken together with the carbon atoms to which they are attached form a carbocyclic or heterocyclic ring having 5 or 6 ring atoms, any heteroatom being selected from N, O or S, said carbocyclic or heterocyclic ring being saturated or unsaturated, and being unsubstituted or substituted with one or more substituents selected from Cl, Br, F, OH, NO₂, CF₃, Cm lower alkyl, SCH_3i NHCOCH₃, N(R⁶)(R⁸), OR¹⁰ and OCOR¹¹, wherein R⁶, R⁸, R¹⁰ and R¹¹ are as defined above; and

R⁴ represents hydrogen, or OR¹⁰ wherein R¹⁰ is as defined above.

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A preferred group of compounds are those wherein R, R¹ and R⁴ are as defined for

Formula (IA’) above, and

R² and R³ are each independently selected from:

(i) hydrogen, (ii) a substituted or unsubstituted, preferably aromatic, carbocyclic or heterocyclic group containing from 5 to 10 ring atoms, said ring atoms forming one or two rings, wherein the or each ring contains 5 or 6 ring atoms, any heteroatoms being selected from N, O and S, any substituents being independently selected from the group consisting of:

(a) Cl, (b) Br, (c) F, (d) OH, (e) NO₂, (f) CF₃, (g) C1-4 lower alkyl (in particular CH₃), (h) SCH₃, (i) NHCOCH3, (j) N(R⁶)(R⁸) wherein R⁶ and R⁸, are the same or different and each represents H or lower Cm alkyl, (k) OR¹⁰ wherein R¹⁰ represents H or lower C-i-₆ alkyl which may be saturated or unsaturated and being unsubstituted or substituted with the group NRR¹ wherein R and R¹ is as defined above, and (I) OCOR¹¹wherein R¹¹ represents H or lower C-m alkyl, (iii) Cl, (iv) Br, (v) F, (vi) OH, (vii) NO₂, (viii) a saturated or unsaturated lower Ci_6 straight or branched hydrocarbyl group which may be unsubstituted or substituted by 1, 2 or 3 substituents selected from Cl, Br, F, OMe, NO₂ and CF₃, (ix) NHCOCH₃, (x) N(R⁶)(R⁸), (xi) SR¹⁰, (xii) OR¹⁰, and (xiii)OCOR¹¹wherein R⁶, R⁸, R¹⁰ and R¹¹ are as defined for Formula (I).

Within this group, R² and R³ can both represent hydrogen. A further preferred group of compounds are those wherein one of R¹ or R² is hydrogen, and the other is selected from the group consisting of: (i) a substituted or unsubstituted, preferably aromatic, carbocyclic or heterocyclic group containing from 5 to 10 ring atoms, said ring atoms forming one or two rings, wherein the or each ring contains 5 or 6 ring atoms, any heteroatoms being selected from N, O and S, any substituents being independently selected from the group consisting of:

Cl, Br, F, OH, NO₂, CF₃, C1-4 lower alkyl (in particular CH₃), SCH₃, NHCOCH3, N(R⁶)(R⁸), OR¹⁰ and OCOR¹¹, wherein R⁶, R⁸, R¹⁰ and R¹¹ are the same or different and each represents H or lower C1-4 alkyl, (ii) Cl, (iii) Br, (iv) F, (v) OH, (vi) NO₂, (vii) a saturated or unsaturated lower Ci-₆straight or branched hydrocarbyl group which may be unsubstituted or substituted by

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PCT/EP00/08365 (o

1, 2 or 3 substituents selected from Cl, Br, F, OMe, NO2 and CF₃, (viii) NHCOCH3, (ix) N(R⁶)(R⁸), (x) SR¹⁰, (xi) OR¹⁰, and (xii) OCOR¹¹ wherein R⁶, R⁸, R¹⁰ and R¹¹ are as defined for Formula (I).

Within this preferred group of compounds, a further preferred group of compounds are those wherein R² hydrogen and R³ is selected from the group consisting of: (i) a substituted or unsubstituted, preferably aromatic, carbocyclic or heterocyclic group containing from 5 to 10 ring atoms, said ring atoms forming one or two rings, wherein the or each ring contains 5 or 6 ring atoms, any heteroatoms being selected from N, O and S, any substituents being independently selected from the group consisting of:

Cl, Br, F, OH, NO₂, CF₃, C1-4 lower alkyl (in particular CH₃), SCH₃, NHCOCH₃, N(R⁶)(R⁸), OR¹⁰ and OCOR¹¹, wherein R⁶, R⁸, R¹⁰ and R¹¹ are the same or different and each represents H or lower Cm alkyl, (ii) Cl, (iii) Br, (iv) F, (v) OH, (vi) NO₂, (vii) a saturated or unsaturated lower C-i-6 straight or branched hydrocarbyl group which may be unsubstituted or substituted by 1, 2 or 3 substituents selected from Cl, Br, F, OMe, NO₂ and CF₃, (viii) NHCOCH₃, (ix) N(R⁶)(R⁸), (x) SR¹⁰, (xi) OR¹⁰, and (xii) OCOR¹¹ wherein R⁶, R⁸, R¹⁰ and R¹¹ are as defined for Formula (I).

A further preferred group of compounds are those wherein R³ is hydrogen and R² is selected from the group consisting of: (i) a substituted or unsubstituted, preferably aromatic, carbocyclic or heterocyclic group containing from 5 to 10 ring atoms, said ring atoms forming one or two rings, wherein the or each ring contains 5 or 6 ring atoms, any heteroatoms being selected from N, O and S, any substituents being independently selected from the group consisting of:

Cl, Br, F, OH, NO₂, CF₃, Cm lower alkyl (in particular CH₃), SCH₃, NHCOCH₃, N(R⁶)(R⁸), OR¹⁰ and OCOR¹¹, wherein R⁶, R⁸, R¹⁰ and R¹¹ are the same or different and each represents H or lower Cm alkyl, (ii) Cl, (iii) Br, (iv) F, (v) OH, (vi) NO₂, (vii) a saturated or unsaturated lower Ci_₆straight or branched hydrocarbyl group which may be unsubstituted or substituted by 1, 2 or 3 substituents selected from Cl, Br, F, OMe, NO₂ and CF₃, (viii) NHCOCH₃, (ix) N(R⁶)(R⁸), (x) SR¹⁰, (xi) OR¹⁰, and (xii) OCOR¹¹ wherein R⁶, R⁸, R¹⁰ and R¹¹ are as defined for Formula (I).

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ΤΑ further preferred embodiment of the present invention are compounds wherein R²represents a substituted or unsubstituted, preferably aromatic, carbocyclic or heterocyclic group containing from 5 to 10 ring atoms, said ring atoms forming one or two rings, wherein the or each ring contains 5 or 6 ring atoms, any heteroatoms being selected from N, O and S, any substituents being independently selected from the group consisting of: Cl, Br, F, OH, NO2, CF3, C-m lower alkyl (in particular CH3), SCH3, NHCOCH3, N(R⁶)(R⁸), OR¹⁰ and OCOR¹¹, wherein R⁶, R⁸, R¹⁰ and R¹¹ are as defined as for Formula (I). For these compounds, R³ is preferably selected from the group consisting of H, Cl, Br, F, OH, NO2, a saturated or unsaturated lower C-m straight or branched hydrocarbyl group which may be unsubstituted or substituted by 1, 2 or 3 substituents selected from

Cl, Br, F, OMe, NO₂ and CF₃,

NHCOCH₃, N(R⁶)(R⁸), SR¹⁰, OR¹⁰, and OCOR¹¹ wherein R⁶, R⁸, R¹⁰ and R¹¹ are as defined for Formula (I).

Alternatively compound R³ may represent a substituted or unsubstituted, preferably aromatic, carbocyclic or heterocyclic group containing from 5 to 10 ring atoms, said ring atoms forming one or two rings, wherein the or each ring contains 5 or 6 ring atoms, any heteroatoms being selected from N, O and S, any substituents being independently selected from the group consisting of:

Cl, Br, F, OH, NO₂, CF₃, C-m lower alkyl (in particular CH₃), SCH₃, NHCOCH₃, N(R⁶)(R⁸), OR¹⁰ and OCOR¹¹, wherein R⁶, R⁸, R¹⁰ and R¹¹ are as defined for Formula (I).

For these compounds, R² is preferably selected from the group consisting of H, Cl, Br, F, OH, NO₂, a saturated or unsaturated lower Cm straight or branched hydrocarbyl group which may be unsubstituted or substituted by 1, 2 or 3 substituents selected from

Cl, Br, F, OMe, NO₂ and CF₃,

NHCOCH₃, N(R⁶)(R⁸), SR¹⁰, OR¹⁰, and OCOR¹¹ wherein R⁶, R⁸, R¹⁰ and R¹¹ are as defined for Formula (I).

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Where R² and/or R³ represents a substituted carbocyclic or heterocyclic group, the substituents on the carbocyclic or heterocyclic group are preferably selected from OH or OR¹⁰ wherein R¹⁰ is as defined for Formula (I).

A particularly preferred carbocyclic group is phenyl or phenyl substituted with 1 to 3 OH or OR¹⁰ groups. For these compounds, R¹⁰ preferably represents methyl or

-CH₂

<img file="AU7000000A_D0003.tif" />

Also preferred are compounds wherein one of R² or R³ represents H or a lower C1-6 10 straight or branched hydrocarbyl group, with methyl being especially preferred.

The invention also provides a compound of Formula (I) having the structure (IA”):

<img file="AU7000000A_D0004.tif" />

wherein R, R¹ and R⁴ are as defined as for Formula (I), and R² and R³ taken together 15 represent Ring Q, said Ring Q being a carbocyclic or heterocyclic ring having 5 or 6 ring atoms, any heteroatom being selected from N, O or S, said carbocyclic or heterocyclic ring being saturated or unsaturated and being unsubstituted or substituted with one or more substituents selected from Cl, Br, F, OH, NO2, CF3, Cm lower alkyl, SCH₃, NHCOCH3, N(R⁶)(R⁸), OR¹⁰ and OCOR¹¹, wherein R⁶, R⁸, R¹⁰ and

R¹¹ are as defined as for Formula (I).

For these compounds Ring Q preferably represents a carbocyclic or heterocyclic aromatic ring, any heteroatom being selected from N, 0 or S, said ring being unsubstituted or substituted with one or more substituents selected from Cl, Br, F,

OH, NO₂, CF₃, Cm lower alkyl, SCH₃, NHCOCH3, N(R⁶)(R⁸), OR¹⁰ and OCOR¹¹,

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PCT/EP00/08365 wherein R⁶, R⁸, R¹⁰ and R¹¹ are as defined as in Formula (I). Particularly preferred are those compounds wherein Ring Q represents a benzene or pyridine ring.

The substituent Z may be attached to any position in the aromatic ring. Thus the 5 compounds of Formula (IA’) or (IA”) described above include compounds having the structures (IA)x, (IA)y and (IA)z:

<img file="AU7000000A_D0005.tif" />

<img file="AU7000000A_D0006.tif" />

<img file="AU7000000A_D0007.tif" />

(IA)x , (IA)y , and (IA)z wherein R, R¹, R², R³ and R⁴ are as defined above.

For the compounds of Formula (IA’) or (IA”) described above, R⁴ preferably represents H, OH or OCH3.

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The invention further provides compounds of Formula (I) having the structure (IB):

<img file="AU7000000A_D0008.tif" />

wherein R and R¹ are as defined for Formula (I) and R⁵ represents H or a lower Ci-6 straight or branched hydrocarbyl group which may be unsubstituted or substituted by 1,2 or 3 substituents selected from Cl, Br, F, OMe, NO₂ and CF₃. In a preferred embodiment, R⁵ represents H or methyl.

For the compounds of Formula (IB) described above, the substituent Z may be attached to any position in the aromatic ring. Thus the compounds of Formula (IB) described above include compounds having the structures (IB)w, (IB)x, (IB)y and (IB)z:

<img file="AU7000000A_D0009.tif" />

(IB)w,

<img file="AU7000000A_D0010.tif" />

(IB)x ,

R⁵

<img file="AU7000000A_D0011.tif" />

O O (IB)y , and

OCH₂“C=CCH₂NRR¹

M

<img file="AU7000000A_D0012.tif" />

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PCT/EPOO/08365 (IB)z och₂-cecch₂nrr¹ wherein R, R¹ and R⁵ are as defined for Formula (I)

For the compounds of Formulae (I), (IA’), (IA”) or (IB), the substituent R and R¹ are the same or different and preferably each represents a Cm alkyl group or a C5-8 cycloalkyl group. Within this group of compounds, R and R¹ are preferably independently selected from methyl, ethyl, propyl, cyclopropyl or a cyclohexyl group.

In a preferred group of compounds, the R and R¹ groups taken together with the nitrogen atom to which they are attached, form a four- to eight-membered heterocyclic ring. Of these, it is preferred that R and R¹ taken together with the nitrogen atom to which they are attached, form a pyrrolidine, piperidine, piperazine, N-methylpiperazine, N-benzylpiperazine or a morpholine group.

It will be appreciated that the compounds of Formula (I) contain a basic amino function and thus may be converted to acid addition salts, with pharmacologically acceptable acids, e.g. hydrochloric acid and phosphoric acid. Such salts are also included in the present invention.

The compounds of Formula (I) may be conveniently prepared by a process comprising the steps of:

(i) reacting a hydroxy derivative, Z-OH, with propargyl bromide to form an alkyne, Z-OCH₂C=H; and (ii) reacting the alkyne Z-OCH₂OH with an amine HNRR¹. Such a process forms a further aspect of the present invention.

The invention further provides a compound of Formula (I) as defined above for use as a modulator of multiple drug resistance in cancer chemotherapy or an

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PCT/EP00/08365 &amp;

antiproliferative medicament. In particular, the compounds of Formula (I) are especially useful for the modulation of multiple drug resistance mediated by Pglycoprotein.

The compounds of Formula (I) as defined above may also be useful for the manufacture of a medicament for the treatment or prevention of neoplasms, particularly those located in the uterus, ovary or breast. Further the compounds Formula (I) may be especially useful for the manufacture of a medicament for the treatment of paclitaxel- and docetaxel-resistant cancer cells.

The compounds of Formula (I) may also advantageously be used as an antiproliferative medicament in combination therapies involving the combined use of a compound of Formula (I) with one or more anti-neoplastic or cytostatic agents, such as paclitaxel or docetaxel. The combination therapy may involve simultaneous or successive administration of a compound of Formula (I) with one or more antineoplastic or cytostatic agents, including anthracyclines, epipodophyllotoxins, actinomycin D, vinca alkaloids, colchicines, paclitaxel or docetaxel. Such combination therapy forms a further aspect of the invention.

The compounds of the invention may also be useful in the manufacture of a medicament for the treatment or prevention of menopausal disorders and osteoporosis.

The invention further provides a pharmaceutical composition comprising one of more of the compounds of Formula (I) in combination with one or more pharmaceutically acceptable excipients. Such a composition may also comprise one or more antineoplastic or cytostatic agents, such as paclitaxel or docetaxel.

The invention will now be described by way of illustrative examples and with reference to the accompanying formulae drawings.

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EXAMPLES

Example 1. General conditions to obtain the propynyloxy derivatives

HNRR¹, HCOH _ .

z-och₂c=ch -—--► Z-OCH₂C=CCH₂NRR¹^z CuSO₄^{z z} (I)

A mixture of hydroxy derivative (0.01 mol), K2CO3 (0.02 mol), Kl (0.01 mol), propargyl bromide (0.015 mol) and acetone (100 mL) was refluxed 10 h and hot filtered. The solvent was evaporated and the residue was crystallized with a suitable solvent.

Example 2. Preparation of 7-propynyloxy-4'-methoxyisoflavone

A mixture of 7-hydroxy-4'-methoxyisoflavone (2.68 g, 0.01 mol), K2CO3 (2.8 g, 0.02 mol), Kl (0.166 g, 0.001 mol), propargyl bromide (1.78 g, 0.015 mol) and acetone (100 mL) was refluxed 10 h and hot filtered. The solvent was evaporated and the residue was crystallized by toluene. This yields 2.75 g of a product with the following characteristics: m.p.145-146°C; ¹H NMR (CDCI₃) 5: 2.6 (m, 1H), 3.83 (s, 3H), 4.8 (s, 2H), 6.93-8.27 (m, 8H).

Example 3. Preparation of 7-propynyloxyisoflavone

A mixture of 7-hydroxyisoflavone (2.38 g, 0.01 mol), K2CO3 (2.8 g, 0.02 mol), Kl (0.166 g, 0.001 mol), propargyl bromide (1.78 g, 0.015 mol) and acetone (10O mL was refluxed 10 h and hot filtered. The solvent was evaporated and the residue was crystallized by toluene. This yields 2.1 g of a product with the following characteristics: m.p. 130-131°C; ¹H NMR (CDCI3) δ: 2.6 (m, 1H), 4.8 (s, 2H), 6.998.28 (m, 7H).

Example 4. Preparation of 7-propynyloxy-2-methyl-4’-methoxyisoflavone

A mixture of 7-hydroxy-2-methyl-4'-methoxyisoflavone (2.82 g, 0.01 mol), K₂CO₃ (2.8 g, 0.02 mol), Kl (0.166 g, 0.001 mol), propargyl bromide (1.78 g, 0.015 mol) and acetone (100 mL) was refluxed 10 h and hot filtered. The solvent was evaporated and the residue was crystallized by toluene. This yields 2.24 g of a product with the

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PCT/EP00/08365 following characteristics: m.p. 139-140°C; ¹H NMR (CDCI3) δ: 2.29 (s, 3H), 2.6 (m, 1H), 3.85 (s, 3H), 4.75 (s, 2H), 6.93-8.17 (m, 7H).

Example 5. Preparation of 7-propynyloxy-5-hydroxy-4’-methoxyisoflavone

A mixture of 5,7-dihydroxy-4'-methoxyisoflavone (2.84 g, 0.01 mol), K2CO3 (2.8 g, 0.02 mol), Kl (0.166 g, 0.001 mol), propargyl bromide (1.78g, 0.015 mol) and acetone (100 mL) was refluxed 10 h and hot filtered. The solvent was evaporated and the residue was crystallized by toluene. This yields 2.25 g of a product with the following characteristics: m.p. 174-176°C; ¹H NMR (CDCI3) δ: 2.6 (m, 1H), 3.86 (s, 3H), 4.8 (s, 2H), 6.47-7.91 (m, 7H), 12.90 (s, 1H).

Example 6. Preparation of 7,4’-dipropynyloxyisoflavone

A mixture of 5,7-dihydroxy-4'-methoxyisoflavone (2.54 g, 0.01 mol), K2CO3 (2.8 g, 0.02 mol), Kl (0.166 g, 0.001 mol), propargyl bromide (1.72 g, 0.015 mol) and acetone (100 mL) was refluxed 10 h and hot filtered. The solvent was evaporated and the residue was crystallized by toluene. This yields 2.31 g of a product with the following characteristics: m.p. 162-163°C; ¹H NMR (CDCI3) δ: 2.44 (m, 1H, CH), 2.57 (m, 1H), 4.54 (s, 2H), 4.56 (s, 2H), 6.85-8.08 (m, 8H).

Example 7. Preparation of 1 -propynyloxyxanthen-9-one

A mixture of 3-hydroxyxanthen-9-one (2.12 g, 0.01 mol), K2CO3 (2.8 g, 0.02 mol), Kl (0.166 g, 0.001 mol), propargyl bromide (1.78 g, 0.015 mol) and acetone (100 mL) was refluxed 10 h and hot filtered. The solvent was evaporated and the residue was crystallized by toluene. This yields 2.0 g of a product with the following characteristics: m.p. 168-169°C; ¹H NMR (CDCI3) δ: 2.56 (m, 1H), 4.94 (s, 2H), 6.958.33 (m, 7H).

Example 8. Preparation of 2-propynyloxyxanthen-9-one

A mixture of 2-hydroxyxanthen-9-one (2.12 g, 0.01 mol), K2CO3 (2.8 g, 0.02 mol), Kl (0.166 g, 0.001 mol), propargyl bromide (1.78 g, 0.015 mol) and acetone (100 mL) was refluxed 10 h and hot filtered. The solvent was evaporated and the residue was crystallized by toluene. This yields 2.25 g of a product with the following

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PCT/EP00/08365 characteristics: m.p. 153-154°C; ¹H NMR (CDCI3) δ: 2.58 (m, 1H), 4.8 (s, 2H), 7.358.38 (m, 7H).

Example 9. Preparation of 3-propynyloxyxanthen-9-one

A mixture of 3-hydroxyxanthen-9-one (2.12 g, 0.01 mol), K2CO3 (2.8 g, 0.02 mol), Kl (0.166 g, 0.001 mol), propargyl bromide (1.78 g, 0.015 mol) and acetone (100 mL) was refluxed 10 h and hot filtered. The solvent was evaporated and the residue was crystallized by toluene. This yields 2.25 g of a product with the following characteristics: m.p. 142-144°C; ¹H NMR (CDCI₃) δ: 2.61 (m, 1H), 4.84 (s, 2H), 6.988.38 (m, 7H).

Example 10. Preparation of 7-propynyloxyflavone

A mixture of 7-hydroxyflavone (2.38 g, 0.01 mol), K2CO3 (2.8 g, 0.02 mol), Kl (0.166 g, 0.001 mol), propargyl bromide (1.78 g, 0.015 mol) and acetone (100 mL) was refluxed 10 h and hot filtered. The solvent was evaporated and the residue was crystallized by toluene. This yields 2.58 g of a product with the following characteristics: m.p. 199-200°C; ¹H NMR (CDC1₃) δ: 2.6 (m, 1H), 4.8 (s, 2H), 6.758.18 (m, 9H).

Example 11. Preparation of 7-propynyloxy-3-methylflavone

A mixture of 7-hydroxy-3-methylflavone (2.52 g, 0.01 mol), K2CO3 (2.8 g, 0.02 mol), Kl (0.166 g, 0.001 mol), propargyl bromide (1.78 g, 0.015 mol) and acetone (100 mL) was refluxed 10 h and hot filtered. The solvent was evaporated and the residue was crystallized by toluene. This yields 2.32 g of a product with the following characteristics: m.p. 179-180°C; ¹H NMR (CDCI₃) δ: 2.15 (s, 3H), 2.69 (m, 1H), 4.8 (s, 2H), 6.95-8.25 (m, 8H).

Example 12. Preparation of 7-propynyloxy-4-methylcoumarin

A mixture of 7-hydroxy-4-methylcoumarin (1.76 g, 0.01 mol), K2CO3 (2.8 g, 0.02 mol),

Kl (0.166 g, 0.001 mol), propargyl bromide (1.78 g, 0.015 mol) and acetone (100 mL) was refluxed 10 h and hot filtered. The solvent was evaporated and the residue was crystallized by toluene. This yields 1.93 g of a product with the following

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PCT/EP00/08365 characteristics: m.p.140-141°C; ¹H NMR (CDCI3) 5: 2.4 (s), 2.69 (m), 4.8 (s, 2H), 6.15-7.58 (m, 4H).

Example 13. General conditions to obtain the aminopropynyloxy derivatives _ HNRR¹, HCOH _ _d

-och₂-c=ch-’->-z-och₂-c=cch₂nrr¹

CuSO₄ (I)

A solution of formaldehyde (0.5 ml), selected amine (6 mmol) and CUSO4 (0.1 g) in EtOH/H₂O (20 mL) was added to a solution of propynyloxy derivative (4.6 mmol) in EtOH/H₂O (20 mL). H2SO4 was added until pH 8 and the mixture was refluxed 24 h. NH₃ (30 mL) was added and the mixture was extracted with ether. After evaporation of the solvent the residue was purified by flash-chromatography (eluent: toluene/acetone 4/1) and crystallized by suitable solvent.

Example 14. 7-(4-Piperidinobut-2-yn)-oxy-4-methoxyisoflavone (see accompanying formula drawing VIB 15)

A solution of formaldehyde (1 ml), piperidine (0.85 g, 0.01 mol) and CUSO4 (0.2 g) in EtOH/H₂O (40 mL) was added to a solution of propynyloxy derivative (3.08 g, 0.01 mol) in Et0H/H₂O (40mL). H₂SO4 was added until pH 8 and the mixture was refluxed 24 h. NH₃ (60 mL) was added and the mixture was extracted with ether. After evaporation of the solvent the residue was purified by flash-chromatography (eluent: toluene/acetone 4/1) and crystallized by ligroin. This yields 1.63 g of a product with the following characteristics: m.p. 95-97°C; ¹H NMR δ: 1.73-1.98 (m, 2H), 1.52-1.68, (q, 4H), 2.4-2.55 (t, 4H), 3.3 (s, 2H), 3.85 (s, 3H), 4.85 (s, 2H), 6.9-8.25 (m, 8H).

Example 15. 7-(4-Morpholinobut-2-yn)-oxy-4'-methoxyisoflavone (see accompanying formula drawing VIB 17)

A solution of formaldehyde (1 ml), morpholine (0.87 g, 0.01 mol) and CUSO4 (0.2 g) in EtOH/H₂O (40 mL) was added to a solution of propynyloxy derivative (3.08 g, 0.01 mol) in EtOH/H₂O (40 mL). H₂SO₄ was added until pH 8 and the mixture was refluxed 24 h. NH₃ (60 mL) was added and the mixture was extracted with ether.

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After evaporation of the solvent the residue was purified by flash-chromatography (eluent: toluene/acetone 4/1) and crystallized by ligroin. This yields 1.62 g of a product with the following characteristics: m.p. 98-100°C; ¹H NMR δ: 2.43-2.61 (m, 4H), 3.3 (s, 2H), 3.6-3.78 (m, 4H), 3.78 (s, 3H), 4.75 (s, 2H), 6.9-8.3 (m, 8H).

Example 16. 7-[4-(4-Benzyl-piperazin-1 -yl)-but-2-yn]-oxy-4'-methoxyisoflavone (see accompanying formula drawing VIB 16)

A solution of formaldehyde (1 ml), benzylpiperazine (1.76 g, 0.01 mol) and CuSO₄(0.2 g) in EtOH/H₂0 (40 ml) was added to a solution of propynyloxy derivative (3.08 g, 0.01 mol) in EtOH/H₂O (40 mL). H₂SO₄ was added until pH 8 and the mixture was refluxed 24 h. NH₃ (60 mL) was added and the mixture was extracted with ether. After evaporation of the solvent the residue was purified by flashchromatography (eluent: toluene/acetone 4/1) and crystallized by ligroin. This yields 1.1 g of a product with the following characteristics: m.p. 98-100°C; ¹H NMR δ: 2.452.65 (m, 8H), 3.35 (s, 2H), 3.52 (s, 2H), 3.85 (s, 3H), 4.85 (s, 2H), 6.95-8.27 (m, 13 H).

Example 17. 7-(4-Pyrrolidinobut-2-yn)-oxy-4'-methoxyisoflavone (see accompanying formula drawing VIB 91)

A solution of formaldehyde (1 ml), pyrrolidine (0.71 g, 0.01 mol) and CUSO4 (0.2 g) in EtOH/H₂0 (40 mL) was added to a solution of propynyloxy derivative (3.08 g, O.O1 mol) in EtOH/H₂O (40 mL). H₂SO₄ was added until pH 8 and the mixture was refluxed 24 h. NH₃ (60 mL) was added and the mixture was extracted with ether. After evaporation of the solvent the residue was purified by flash-chromatography (eluent: toluene/acetone 4/1) and crystallized by ligroin. This yields 0.8 g of a product with the following characteristics: m.p. 111-112°C; ¹H NMR δ: 1.68-1.83 (m, 4H), 2.62.65 (m, 4H), 3.5 (m, 2H), 3.85 (s, 3H), 4.83 (m, 2H), 6.96-8.26 (m, 8H).

Example 18. 7-(4-Diethylaminobut-2-yn)-oxy-4*-methoxyisoflavone (see accompanying formula drawing VIB 90)

A solution of formaldehyde (1 ml), diethylamine (0.73 g, 0.01 mol) and CuSO₄ (0.2 g) in EtOH/H₂O (40 mL) was added to a solution of propynyloxy derivative (3.08 g, 0.01 mol) in EtOH/H₂0 (40 mL). H₂SO₄ was added until pH 8 and the mixture was refluxed

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h. NH3 (60 mL) was added and the mixture was extracted with ether. After evaporation of the solvent the residue was purified by flash-chromatography (eluent: toluene/acetone 4/1) and crystallized by ligroin. This yields 1.2 g of a product with the following characteristics: m.p. 73-75°C; ¹H NMR δ: 1 (t, 6H), 2.5 (q, 4H), 3.49 (s, 2H), 3.85 (s, 3H), 4.85 (s, 2H), 6.95-8.28 (m, 8H)

Example 19. 7-(4-Diethylaminobut-2-yn)-oxyisoflavone (see accompanying formula drawing VIB 92)

A solution of formaldehyde (1 ml), diethylamine (0.73 g, 0.01 mol) and CUSO4 (0.2 g) in EtOH/H₂O (40 mL) was added to a solution of propynyloxy derivative (2.94 g, 0.01 mol) in EtOH/H₂0 (40mL). H2SO4 was added until pH 8 and the mixture was refluxed 24 h. NH₃ (60 mL) was added and the mixture was extracted with ether. After evaporation of the solvent the residue was purified by flash-chromatography (eluent: toluene/acetone 4/1) and crystallized by ligroin. This yields 0.62 g of a product with the following characteristics: m.p. 79-80°C; ¹H NMR δ: 1.03 (t, 6H), 2.5 (q, 4H), 3.49 (s, 2H), 4.84 (s, 2H), 7.0-8.26 (m, 9H).

Example 20. 7-(4-Morpholinobut-2-yn)-oxyisofiavone (see accompanying formula drawing VIB 93)

A solution of formaldehyde (1 ml), morpholine (0.87 g, 0.01 mol) and CUSO4 (0.2 g) in EtOH/H₂O (40 mL) was added to a solution of propynyloxy derivative (2.94 g, 0.01 mol) in EtOH/H₂0 (40mL). H₂SO4 was added until pH 8 and the mixture was refluxed 24 h. NH₃ (60 mL) was added and the mixture was extracted with ether. After evaporation of the solvent the residue was purified by flash-chromatography (eluent: toluene/acetone 4/1) and crystallized by ligroin. This yields 1.5 g of a product with the following characteristics: m.p. 104-105°C; ¹H NMR δ: 2.5-2.6 (m, 4H), 3.35 (s, 2H), 3.75 (m, 4H), 4.85 (m, 2H), 6.95-8.22 (m, 9H).

Example 21. 7-(4-Morpholinobut-2-yn)-oxy-2-methyl-4'-methoxyisofIavone (see accompanying formula drawing VIB 105)

A solution of formaldehyde (1 mi), morpholine (0.87 g, 0.01 mol) and CuSC>4(0.2 g) in EtOH/H₂O (40 mL) was added to a solution of propynyloxy derivative (3.2 g, 0.01 mol) in EtOH/H₂O (40 mL). H₂SO₄ was added until pH 8 and the mixture was

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Example 22. 7-(4-Morpholinobut-2-yn)-oxy-5-hydroxy-4*-methoxyisoflavone (see accompanying formula drawing VIB 102)

A solution of formaldehyde (1 ml), morpholine (0.87 g, 0.01 mol) and CuSC>4 (0.2 g) in EtOH/H₂O (40 mL) was added to a solution of propynyloxy derivative (3.2 g, 0.01 mol) in EtOH/H₂O (40 mL). H2SO4 was added until pH 8 and the mixture was refluxed 24 h. NH₃ (60 mL) was added and the mixture was extracted with ether. After evaporation of the solvent the residue was purified by flash-chromatography (eluent: toluene/acetone 4/1) and crystallized by ligroin. This yields 0.84 g of a product with the following characteristics: oil, hydrochloric salt m.p. 120-123°C (methanol-ether); ¹H NMR δ: 2.3 (m, 4H), 3.3 (s, 2H), 3.7 (m, 4H), 3.85 (s, 3H), 4.85 (m, 2H), 6.48-7.90 (m, 7H), 12.85 (s, 1H).

Example 23. 7-(4-Bis-4-Morpholinobut-2-yn)-oxyisoflavone (see accompanying formula drawing VIB 97)

A solution of formaldehyde (1 ml), morpholine (0.87 g, 0.01 mol) and CUSO4 (0.2 g) in EtOH/H₂O (40 mL) was added to a solution of propynyloxy derivative (3.2 g, O.O1 mol) in EtOH/H₂O (40 mL). H2SO4 was added until pH 8 and the mixture was refluxed 24 h. NH₃ (60 mL) was added and the mixture was extracted with ether. After evaporation of the solvent the residue was purified by flash-chromatography (eluent: toluene/acetone 4/1) and crystallized by ligroin. This yields 1.06 g of a product with the following characteristics: m.p. 158-159°C; ¹H NMR δ: 2.55 (m, 8H),

3.34 (s, 4H), 3.74 (m, 8H), 4.7 (s, 2H), 4.85 (s, 2H), 6.98-8.26(m, 8H).

Example 24. 7-(4-Morpholinobut-2-yn)-oxyflavone (see accompanying formula drawing VIB 103)

A solution of formaldehyde (1 ml), morpholine (0.87 g, 0.01 mol) and CuSC>4 (0.2 g) in EtOH/H₂O (40 mL) was added to a solution of propynyloxy derivative (2.94 g, 0.01

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PCT/EP00/08365 mol) in EtOH/hfeO (40 mL). H2SO4 was added until pH 8 and the mixture was refluxed 24 h. NH3 (60 mL) was added and the mixture was extracted with ether. After evaporation of the solvent the residue was purified by flash-chromatography (eluent: toluene/acetone 4/1) and crystallized by ligroin. This yields 0.75 g of a product with the following characteristics: m.p. 126-127°C; ¹H NMR δ: 2.56 (m, 4H),

3.35 (s, 2H), 3.7 (m, 4H), 4.86 (m, 2H), 6.79-8.2 (m, 9H). Mass: m/z 374 (M+, 14.38), 238 (100), 137 (82.79).

Example 25. 7-(4-Morpholinobut-2-yn)-oxy-3-methylflavone (see accompanying formula drawing VIB 104)

A solution of formaldehyde (1 ml), morpholine (0.87 g, 0.01 mol) and CUSO4 (0.2 g) in EtOH/HsO (40 mL) was added to a solution of propynyloxy derivative (3.09 g, 0.01 mol) in EtOH/H₂O (40 mL). H2SO4 was added until pH 8 and the mixture was refluxed 24 h. NH₃ (60 mL) was added and the mixture was extracted with ether. After evaporation of the solvent the residue was purified by flash-chromatography (eluent: toluene/acetone 4/1) and crystallized by ligroin. This yields 0.78 g of a product with the following characteristics: m.p. 139-140°C; ¹H NMR δ: 2.13 (s, 3H), 2.6 (m, 4H), 3.35 (m, 2H), 3.8 (m, 4H), 4.03 (s, 2H), 6.85-8.10(m, 8H).

Example 26. 7-(4-Morpholinobut-2-yn)-oxy-4-methylcoumarin (see accompanying formula drawing VIB 95)

A solution of formaldehyde (1 ml), morpholine (0.87 g, 0.01 mol) and CUSO4 (0.2 g) in EtOH/H₂O (40 mL) was added to a solution of propynyloxy derivative (2.14 g, O.O1 mol) in EtOH/hhO (40 mL). H2SO4 was added until pH 8 and the mixture was refluxed 24 h. NH3 (60 mL) was added and the mixture was extracted with ether. After evaporation of the solvent the residue was purified by flash-chromatography (eluent: toluene/acetone 4/1) and crystallized by ligroin. This yields 1.9 g of a product with the following characteristics: m.p.125-126°C; ¹H NMR δ: 2.4 (s, 3H), 2.52 (m, 4H), 3.3 (m, 2H), 3.7 (m, 4H), 4.78 (m, 2H), 6.16-7.54(m, 4H).

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Example 27. 7-(4-Diethylaminobut-2-yn)-oxy-4-methylcoumarin (see accompanying formula drawing VIB 94)

A solution of formaldehyde (1 ml), morpholine (0.73 g, 0.01 mol) and CUSO4 (0.2 g) in EtOH/H₂O (40 mL) was added to a solution of propynyloxy derivative (2.14 g, 0.01 mol) in EtOH/H₂O (40 mL). H₂SO₄ was added until pH 8 and the mixture was refluxed 24 h. NH3 (60 mL) was added and the mixture was extracted with ether. After evaporation of the solvent the residue was purified by flash-chromatography (eluent: toluene/acetone 4/1) and crystallized by ligroin. This yields 1.9 g of a product with the following characteristics: m.p. 108-110°C; ¹H NMR δ: 1.04 (t, 6H), 2.42 (s, 2H), 2.5 (q, 4H), 3.7 (m, 2H), 4.8 (m, 2H), 6.18-7.57(m, 4H).

Example 28. 1 -(4-Morpholinobut-2-yn)-oxyxanthone (see accompanying formula drawing VIB 99)

A solution of formaldehyde (1 ml), morpholine (0.87 g, 0.01 mol) and CuS0₄ (0.2 g) in EtOH/H₂O (40 mL) was added to a solution of propynyloxy derivative (2.5 g, 0.01 mol) in EtOH/H₂O (40 mL). H₂SO₄ was added until pH 8 and the mixture was refluxed 24 h. NH₃ (60 mL) was added and the mixture was extracted with ether. After evaporation of the solvent the residue was purified by flash-chromatography (eluent: toluene/acetone 4/1) and crystallized by ligroin. This yields 1.8 g of a product with the following characteristics: m.p. 98-101°C; ¹H NMR δ: 2.53 (m, 4H), 3.34 (m, 2H), 3.73 (m, 4H), 4.98 (m, 2H), 6.98-8.33 (m, 7H).

Example 29. 1 -(4-Diethylaminobut-2-yn)-oxyxanthone (see accompanying formula drawing VIB 98)

A solution of formaldehyde (1 ml), diethylamine (0.73 g, 0.01 mol) and CuSO₄ (0.2 g) in EtOH/H₂O (40 mL) was added to a solution of propynyloxy derivative (2.5 g, 0.01 mol) in EtOH/H₂O (40 mL). H₂SO₄ was added until pH 8 and the mixture was refluxed 24 h. NH₃ (60 mL) was added and the mixture was extracted with ether. After evaporation of the solvent the residue was purified by flash-chromatography (eluent: toluene/acetone 4/1) and crystallized by ligroin. This yields 0.64 g of a product with the following characteristics: m.p. 70-72°C; ¹H NMR 6:1.02 (t, 6H), 2.5 (q, 4H), 3.45 (m, 2H), 4.96 (m, 2H), 6.98-8.33 (m, 7H).

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Example 23. 2-(4-Morpholinobut-2-yn)-oxyxanthone (see accompanying formula drawing VIB 101)

A solution of formaldehyde (1 ml), morpholine (0.87 g, 0.01 mol) and CUSO4 (0.2 g) in EtOH/H₂O (40 mL) was added to a solution of propynyloxy derivative (2.5 g, 0.01 mol) in EtOH/H₂O (40mL). H₂SO₄ was added until pH 8 and the mixture was refluxed 24 h. NH3 (60 mL) was added and the mixture was extracted with ether. After evaporation of the solvent the residue was purified by flash-chromatography (eluent: toluene/acetone 4/1) and crystallized by ligroin. This yields 1.8 g of a product with the following characteristics: m.p. 105-106°C; ¹H NMR δ: 2.53 (m, 4H), 3.33 (m, 2H), 3.7 (m, 4H), 4.84 (m, 2H), 7.39-7.83 (m, 7H).

Example 31. 2-(4-Diethylaminobut-2-yn)-oxyxanthone (see accompanying formula drawing VIB 100)

A solution of formaldehyde (1 ml), diethylamine (0.73 g, 0.01 mol) and CUSO4 (0.2 g) in Et0H/H₂O (40 mL) was added to a solution of propynyloxy derivative (2.5 g, 0.01 mol) in EtOH/H₂O (40 mL). H₂S0₄was added until pH 8 and the mixture was refluxed 24 h. NH₃ (60 mL) was added and the mixture was extracted with ether. After evaporation of the solvent the residue was purified by flash-chromatography (eluent: toluene/acetone 4/1) and crystallized by ligroin. This yields 0.64 g of a product with the following characteristics: m.p. 66-68°C; ¹H NMR 5: 1.08 (t, 6H), 2.54 (q, 4H), 3.5 (m, 2H), 4.86 (m, 2H), 7.35-8.38 (m, 7H).

Example 32. 2-(4-Morpholinobut-2-yn)-oxyxanthone (see accompanying formula drawing VIB 96)

A solution of formaldehyde (1 ml), morpholine (0.87 g, 0.01 mol) and CUSO4 (0.2 g) in EtOH/H₂O (40 mL) was added to a solution of propynyloxy derivative (2.5 g, 0.01 mol) in EtOH/H₂O (40 mL). H₂SO₄ was added until pH 8 and the mixture was refluxed 24 h. NH₃ (60 mL) was added and the mixture was extracted with ether. After evaporation of the solvent the residue was purified by flash-chromatography (eluent: toluene/acetone 4/1) and crystallized by ligroin. This yields 1.5 g of a product with the following characteristics: m.p. 126-128°C; ¹H NMR δ: 2.56 (m, 4H), 3.4 (m, 2H), 3.7 (m, 4H), 4.86 (m, 2H), 6.97-8.37 (m, 7H).

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BIOLOGICAL EVALUATION

Compounds VIB 16, VIB 94, VIB 99 and VIB 100 were tested for their cytotoxicity against drug-resistant cancer cells, both alone, and in combination with paclitaxel.

The results of these studies are shown below.

When tested alone these compounds were found to possess relatively low cytotoxicity (IC50 > 30 μΜ) against drug-resistant cancer cells.

The compounds were then evaluated in combination with paclitaxel for their cytostatic activity against the drug-resistant breast cancer cells MDA-435/LCC6-MDR. In the experiments, the compounds were used in combination with paclitaxel, the paclitaxel being at a concentration of 1 μΜ. The IC50 of paclitaxel decreases by 2-4 fold when used in combination with each of compounds, i.e. from 426 nM to 210-110 nM compared with paclitaxel alone. Consequently, in the presence of these compounds, paclitaxel can recover its excellent inhibitory activity against the drug-resistant cancer cells.

<td> Compound</td><td> ICso/nM</td><td> % Reduction in ICsoOf paclitaxel</td>

<td> Paclitaxel</td><td> 426</td><td> -</td>

<td> VIB 16 + Paclitaxel</td><td> 136</td><td> 67</td>

<td> VIB 94 + Paclitaxel</td><td> 210</td><td> 50</td>

<td> VIB 99 + Paclitaxel</td><td> 200</td><td> 53</td>

<td> VIB 100 + Paclitaxel</td><td> 110</td><td> 70</td>

Table 1

Experimental

The treatment consisted of concurrent exposure of MDA-435/LCC-MDR cells to paclitaxel in the presence or absence of the compounds reversing agent (1 μΜ) for

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Aif72 h in vitro. Assessment of cytotoxicity, i.e. cell growth inhibition, was determined according to the methods of Skehan, et al. as discussed in J. Nat. Cancer Inst., 82,

1107, 1990.

Briefly, cells were plated between 400 and 1200 cells/well in 96 well plates and incubated at 37°C for 15-18 h prior to drug addiction to allow attachment of cells. Compounds were solubilized in 100% DMSO and further diluted in RPMI-1640 containing 10 mM HEPES. After a 72 h incubation, 100 ml of ice-cold 50% TCA was added to each well and incubated for 1 h at 4°C. Plates were then washed 5 times with tap water to remove TCA, low-molecular weight metabolites and serum proteins. Sulforhodamine B (SRB) (0.4%, 50 ml) was added to each well. Following a five minute incubation at room temperature, plates were rinsed 5 times with 0.1% acetic acid and air dried. Bound dye was solubilized with 10 mM Tris Base (pH 10.5) for 5 min on a gyratory shaker. Optical density was measured at 570 nm.

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<img file="AU7000000A_D0013.tif" />

VIB 15

<img file="AU7000000A_D0014.tif" />

<img file="AU7000000A_D0015.tif" />

VIB 16

<img file="AU7000000A_D0016.tif" />

VIB 91

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Zl*

<img file="AU7000000A_D0017.tif" />

VIB 90

<img file="AU7000000A_D0018.tif" />

VIB 92

<img file="AU7000000A_D0019.tif" />

VIB 93

<img file="AU7000000A_D0020.tif" />

VIB 105

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<img file="AU7000000A_D0021.tif" />

VIB 102

<img file="AU7000000A_D0022.tif" />

VIB 97 or °

<img file="AU7000000A_D0023.tif" />

VIB 103

<img file="AU7000000A_D0024.tif" />

VIB 104

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<img file="AU7000000A_D0025.tif" />

VIB 95

<img file="AU7000000A_D0026.tif" />

VIB 94

<img file="AU7000000A_D0027.tif" />

<img file="AU7000000A_D0028.tif" />

VIB 98

Pd

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<img file="AU7000000A_D0029.tif" />

VIB 100

<img file="AU7000000A_D0030.tif" />

VIB 96

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