Protoflavonoid and flavonoid derivatives and their use as xanthine oxidase inhibitors

Abstract

The present invention relates to novel protoflavonoid and flavonoid derivatives as xanthine oxidase inhibitors of formula (I), to their preparation and therapeutic application.

Description

[0001] P 142069 TEP

[0002] Protoflavonoid and flavonoid derivatives and their use as xanthine oxidase inhibitors

[0003] SUMMARY OF THE INVENTION

[0004] The present invention relates to novel xanthine oxidase inhibitors of formula (I), to their preparation and therapeutic application.

[0005] BACKGROUND OF THE INVENTION

[0006] Xanthine oxidase (XO) is the enzyme responsible for the catabolism of purine in humans (Hille, R.; Hall, J.; Basu, P. The Mononuclear Molybdenum Enzymes. Chem. Rev. 2014, 114 (7), 3963-4038.). The catalytic process starts (Scheme 1) with the oxidation of hypoxanthine to xanthine, followed by the transformation of xanthine to uric acid, and as a result, molecular oxygen level decreases and reactive oxygen species (ROS) are formed such as the superoxide anion (O2~) and hydrogen peroxide (H2O2) (Lund, A. K. Oxidants and Endothelial Dysfunction. In Comprehensive Toxicology, Elsevier, 2010; pp 243-274.)

[0007] Hypoxanthine Xanthine Uric acid

[0008] xanthine oxidase

[0009]

[0010] H2O + O2H2O2

[0011] Scheme 1

[0012] The under-excretion or over-production of uric acid leads to elevated levels of serum uric acid, termed hyperuricemia, which has been established as the major etiologic factor in gout (Chen, C.; Lti, J.-M.; Yao, Q. Hyperuricemia-Related Diseases and Xanthine Oxidoreductase (XOR) Inhibitors: An Overview. Med Sci Monit 2016, 22, 2501-2512.; Day, R. O.; Kamel, B.; Kannangara, D. R. W.; Williams, K. M.; Graham, G. G. Xanthine Oxidoreductase and Its Inhibitors: Relevance for Gout. Clinical Science 2016, 130 (23), 2167-2180.). However, the higher expression of XO elevates the amount of reactive oxygen radicals, thus the enzyme is often associated with the development of other diseases including heart attack, stroke, hypoxic tissue damage, atherosclerosis, inflammation, metabolic diseases and hypertension (Dawson, J.; Walters, M. Uric Acid and Xanthine Oxidase: Future Therapeutic Targets in the Prevention of Cardiovascular Disease? Br J Clin Pharmacol 2006, 62 (6), 633-644.; Pacher, P; Nivorozhkin, A.; Szabo, C. Therapeutic Effects of Xanthine Oxidase Inhibitors: Renaissance Half a Century after the Discovery of Allopurinol. Pharmacol Rev 2006, 58 (1), 87-114. ). In humans, the inhibition of XO reduces the production of uric acid, and several medications that inhibit XO are indicated for the treatment of hyperuricemia and gout (Pacher, P; Nivorozhkin, A.; Szabo, C. Therapeutic Effects of Xanthine Oxidase Inhibitors: Renaissance Half a Century after the Discovery of Allopurinol. Pharmacol Rev 2006, 58 (1), 87-114.).

[0013] In general, XO-inhibitory agents can be classified into two different groups: purine analogues (e.g. allopurinol) and others (e.g. febuxostat) (Becker, M. A.; Schumacher, H. R.; Wortmann, R. L.; MacDonald, P. A.; Palo, W. A.; Eustace, D.; Vernillet, L.; Joseph-Ridge, N. Febuxostat, a Novel Nonpurine Selective Inhibitor of Xanthine Oxidase: A Twenty -Eight-Day, Multicenter, Phase II, Randomized, Double-Blind, Placebo-Controlled, Dose-Response Clinical Trial Examining Safety and Efficacy in Patients with Gout. Arthritis Rheum 2005, 52 (3), 916-923.). Regardless of their chemical origin, however, several adverse side-effects had been described in relation to the currently available XO-inhibitors over the years:

[0014] (1) For example, allopurinol’s most common documented side-effects include skin rash and gastrointestinal distress that can range in severity. A rare, but potentially fatal side-effect is allopurinol hypersensitivity syndrome (AHS) that consists of hepatitis, eosinophilia, and interstitial nephritis. Furthermore, other, less common side-effects include granulomatous hepatitis, liver necrosis, cholestatic jaundice, interstitial nephritis, and vasculitis (Day, R. O.; Graham, G. G.; Hicks, M.; McLachlan, A. J.; Stocker, S. L.; Williams, K. M. Clinical Pharmacokinetics and Pharmacodynamics of Allopurinol and Oxypurinol: Clinical Pharmacokinetics 2007, 46 (8), 623-644.).

[0015] (2) Another good example is febuxostat, the most recently approved (2009), non-purine type XO-inhibitor. Despite being developed as an effective alternative for patients who are intolerant / contraindicated to allopurinol, the use of febuxostat can have numerous common side effects that include liver function abnormalities, dizziness, arthralgia, nausea, and rash (Edwards, N. L. Febuxostat: A New Treatment for Hyperuricaemia in Gout. Rheumatology 2009, 48 (suppl 2), ii 15-ii 19 ).

[0016] On these bases, significant research efforts are ongoing to find new, more effective and safer XO-inhibitory agents and natural products can be promising sources for discovery (Orhan, I. E.; Deniz, F. S. S. Natural Products and Extracts as Xantine Oxidase Inhibitors - A Hope for Gout Disease? CPD 2021, 27 (2), 143-158.). Among natural products, flavonoids were found to possess a high potential for the inhibition of XO (Lin, S.; Zhang, G.; Liao, Y; Pan, J.; Gong, D. Dietary Flavonoids as Xanthine Oxidase Inhibitors: Structure-Affinity and Structure- Activity Relationships. J. Agric. Food Chem. 2015, 63 (35), 7784-7794), for example chrysin, having the following structure (Lin, S.; Zhang, G.; Liao, Y; Pan, J. Inhibition of Chrysin on Xanthine Oxidase Activity and Its Inhibition Mechanism. International Journal of Biological Macromolecules 2015, 81, 274-282):

[0017] .0.

[0018] 2

[0019] 3

[0020]

[0021] Another rare, widely unknown group of natural products, protoflavonoids has also been investigated. Common structural elements of their representatives include a non-aromatic B-ring and a hydroxyl group at C-l' and typically, this structural moiety appears as a symmetric / ?-quinone dienone that may be partially or fully saturated. As mentioned, protoflavonoids are present in nature, however, total- and semi -synthetic methods are available that can satisfy the quantitative needs of their in-depth biological study (Hunyadi, A.; Chuang, D.-W.; Danko, B.; Chiang, M. Y; Lee, C.-L.; Wang, H.-C.; Wu, C.-C.; Chang, F.-R.; Wu, Y.-C. Direct SemiSynthesis of the Anticancer Lead-Drug Protoapigenone from Apigenin, and Synthesis of Further New Cytotoxic Protoflavone Derivatives. PLoS ONE 2011, 6 (8), e23922).

[0022] Regarding their bioactivities, protoflavonoids are generally known for their potent anti cancer effect, which represents their most deeply investigated bioactivity.

[0023] Protoapigenone 1 '-O-propargyl ether:

[0024]

[0025] was the first non-planar flavonoid identified by our group with an XO-inhibitory activity comparable that of allopurinol. Despite the success, selectivity of this activity was unsatisfying due to the in vitro cytotoxic side-effect of the compound close to its active concentration. Thus, there is still a need to identify compounds which possess high XO inhibitory activity and at the same time show increased selectivity in order to provide active and safe XO inhibitors, which have decreased side effects.

[0026] BRIEF DESCRIPTION OF THE INVENTION

[0027] According to the present invention it has been found that the semi-synthetic modification of protoflavonoids and flavonoids by the introduction of specific nitrogen-containing functionalities on the B ring can simultaneously result in the decrease of the products’ cytotoxic side-effects and the enhancement of their other, non-tumor related XO inhibitory bioactivities. Thus, in one aspect the present invention relates to the compounds of formula (I)

[0028]

[0029] wherein

[0030] is a simple or double bond;

[0031] if between the carbons to which R2 and R3 are attached is a simple bond the value of n is 1,

[0032] if between the carbons to which R2 and R3 are attached is a double bond the value of n is 0;

[0033] X is O, S or NRi, wherein, Ri is H or Ci-Ce alkyl;

[0034] R2 is H, OH, Ci-Ce alkyl or Ci-Ce alkoxy;

[0035] Rj is H, OH, Ci-Ce alkyl, Ci-Ce alkoxy or -OC(O)Ci-Ce alkyl;

[0036] R4 is H, Ci-Ce alkyl or-C(O)Ci-Ce alkyl, wherein the alkyl group in Ci-Ce alkyl or -C(O)Ci-Ce alkyl optionally contains one or more double or triple bond(s);

[0037] ring B is selected from:

[0038]

[0039] wherein in (a)

[0040] are independently from each other a double or simple bond, R5 is H, OH, Ci-Ce alkyl, Ci-Ce alkoxy, or -C(O)Ci-Ce alkyl, wherein the alkyl group in Ci-Ce alkyl or Ci-Ce alkoxyl or -C(O)Ci-Ce alkyl optionally contains one or more double or triple bond(s), or Rs is phenyl, phenoxyl, benzyl, benzyloxy, or a halogen; Re, R?, R9 and Rio are independently selected from H, OH, halogen, Ci-Ce alkyl, Ci-Ce alkoxy and -OC(O)Ci-Ce alkyl, wherein the alkyl in Ci-Ce alkyl, Ci-Ce alkoxy or -OC(O)Ci-Ce alkyl optionally contains one or more double or triple bond(s);

[0041] Rs is selected from the following:

[0042] • =N-O-Ri6, wherein Rie is Ci-Ce alkyl optionally containing one double or triple bond, optionally substituted benzyl or phenyl,

[0043] • -NH-O-R17, wherein Rn is Ci-Ce alkyl optionally containing one double or triple bond, optionally substituted benzyl or phenyl,

[0044] • =N-NH-CO-NR27R28, wherein R27 and R28are independently selected from H, Ci-Ce alkyl optionally containing one double or triple bond, optionally substituted benzyl or phenyl,

[0045] • =N-NH-CS-NR29R3O, wherein R29 and R30 are independently selected from H, Ci-Ce alkyl optionally containing one double or triple bond, optionally substituted benzyl or phenyl,

[0046] • -NH2, and

[0047] • -NR18R19, wherein Ris and R19 are independently selected from Ci-Ce alkyl, optionally containing one or more double or triple bond, optionally substituted benzyl or phenyl;

[0048] wherein in (b)

[0049] R11, R12, Ri4 and Ris are independently selected from H, OH, halogen, Ci-Ce alkyl, Ci-Ce alkoxy and -OC(O) Ci-Ce alkyl, wherein the alkyl group in Ci-Ce alkyl, Ci-Ce alkoxy or -OC(O) Ci-Ce alkyl optionally contains one or more double or triple bond(s);

[0050] R13 is selected from the following:

[0051] • -NH-O-R20, wherein R20is C1-C6alkyl optionally containing one double or triple bond, optionally substituted benzyl or phenyl,

[0052] • -N=N-CO-NR23R24, wherein R23 and R24 are independently selected from H, Ci-Ce alkyl optionally containing one or more double or triple bond, optionally substituted benzyl or phenyl • -NH-NH-CS-NR25R26, wherein R25 and R26 are independently selected from H, Ci-Ce alkyl optionally containing one or more double or triple bond, optionally substituted benzyl or phenyl,

[0053] • -NH2, and

[0054] • -NR21R22, wherein R21 and R22 are independently selected from Ci-Ce alkyl, optionally containing one or more double or triple bond, optionally substituted benzyl or phenyl;

[0055] provided that the compound of formula (I) is not one of the following compounds:

[0056] OH O

[0057] OH O

[0058] OH 0

[0059]

[0060] NH2

[0061] HO

[0062]

[0063] OH O

[0064] or the salt of the compounds of formula (I).

[0065] The compounds of formula (I) may contain one or more asymmetric carbon atoms. They may therefore exist in the form of enantiomers or diastereomers. These enantiomers or diastereomers, and also the mixtures thereof, including the racemic mixtures form part of the invention.

[0066] The compounds of the formula (I) may exist in the form of bases or their addition salts with acids. Such salts also form part of the invention. These salts may be prepared with pharmaceutically acceptable acids, specifically with strong pharmaceutically acceptable acids. Nevertheless, the salts of other acids useful, for example for purifying or isolating the compounds also form part of the invention.

[0067] In the context of the present invention certain terms have the following definition:

[0068] Ci-Ce alkyl means an aliphatic hydrocarbon group which may be straight or branched having about 1 to about 6 carbon atoms in the chain, Particular alkyl groups have from 1 to about 4 carbon atoms. Examples of alkyl groups include methyl, ethyl, / / -propyl, z-propyl, / / -butyl, s-butyl, / -butyl, / / -pentyl, 3-pentyl, hexyl, a particular example of alkyl group is methyl.

[0069] Ci-Ce alkoxy means -O- Ci-Ce alkyl group wherein the Ci-Ce alkyl is as defined above.

[0070] An alkyl optionally containing one or more double or triple bond(s) is for example allyl (-CH2-CH=CH2) or propargyl (-CH2-C≡CH).

[0071] Phenoxyl means -O-phenyl group, benzyloxy means -O-CEE-phenyl.

[0072] Halogen means chlorine, fluorine, bromine or iodine, specifically chlorine.

[0073] Optionally substituted benzyl means a -CH2-phenyl group wherein the phenyl is optionally substituted by one or more substituents, such as OH, halogen, C1-C5 alkyl, C1-C5 alkoxy, -NH2, or -N(C1-C5alkyl)2. Optionally substituted phenyl means a phenyl group optionally substituted by by one or more substituents such as as OH, halogen, C1-C5 alkyl, C1-C5 alkoxy, -NH2, or -N(Ci-Cs alkyl)2. Among the compounds of formula (I) according to the invention a first group of compounds is composed of the compounds of formula (la)

[0074]

[0075] wherein

[0076] are independently from each other a simple or double bond;

[0077] if between the carbons to which R2 and R3 are attached is a simple bond the value of n is 1,

[0078] if between the carbons to which R2 and R3 are attached is a double bond the value of n is 0;

[0079] X is O, S or NRi, wherein, Ri is H or Ci-Ce alkyl;

[0080] R2 is H, OH, Ci-Ce alkyl, Ci-Ce alkoxy;

[0081] Rj is H, OH, Ci-Ce alkyl, Ci-Ce alkoxy or -OC(O)Ci-Ce alkyl;

[0082] R4 is H, Ci-Ce alkyl or-C(O)Ci-Ce alkyl, wherein the alkyl group in Ci-Ce alkyl or -C(O)Ci-Ce alkyl optionally contains one or more double or triple bond(s);

[0083] Rs is H, OH, Ci-Ce alkyl, Ci-Ce alkoxy or -C(O)Ci-Ce alkyl, wherein the alkyl group in Ci-Ce alkyl or Ci-Ce alkoxy or -C(O)Ci-Ce alkyl optionally contains one or more double or triple bond(s), or Rs is phenyl, phenoxyl, benzyl, benzyloxy, or halogen;

[0084] Re, R7, R9 and Rio are independently selected from H, OH, halogen, Ci-Ce alkyl, Ci-Ce alkoxy and -OC(O) Ci-Ce alkyl, wherein the alkyl in Ci-Ce alkyl, Ci-Ce alkoxy or -OC(O) Ci-Ce alkyl optionally contains one or more double or triple bond(s);

[0085] Rs is selected from the following: • =N-0-R16, wherein R16is C1-C6alkyl optionally containing one double or triple bond, optionally substituted benzyl or phenyl,

[0086] • -NH-O-R17, wherein R17 is Ci-Ce alkyl optionally containing one double or triple bond, optionally substituted benzyl or phenyl,

[0087] • =N-NH-CO-NR27R28, wherein R27 and R28are independently selected from H, Ci-Ce alkyl optionally containing one double or triple bond, optionally substituted benzyl or phenyl,

[0088] • =N-NH-CS-NR29R3O, wherein R29 and R30 are independently selected from H, Ci-Ce alkyl optionally containing one double or triple bond, optionally substituted benzyl or phenyl,

[0089] • -NH2, and

[0090] • -NR18R19, wherein Ris and R19 are independently selected from Ci-Ce alkyl, optionally containing one or more double or triple bond, optionally substituted benzyl or phenyl.

[0091] Among the compounds of formula (I) according to the invention a second group of compounds is composed of the compounds of formula (lb)

[0092]

[0093] wherein

[0094] X is O, S or NRi, wherein, Ri is H or Ci-Ce alkyl;

[0095] Rj is H, OH, Ci-Ce alkyl, Ci-Ce alkoxy or -OC(O)Ci-Ce alkyl;

[0096] R4 is H, Ci-Ce alkyl or-C(O)Ci-Ce alkyl, wherein the alkyl group in Ci-Ce alkyl or -C(O)Ci-Ce alkyl optionally contains one or more double or triple bond(s);

[0097] R11, R12, Ri4 and Ris are independently selected from H, OH, halogen, Ci-Ce alkyl, Ci-Ce alkoxy and -OC(O) Ci-Ce alkyl, wherein the alkyl group in Ci-Ce alkyl, Ci-Ce alkoxy or -OC(O) Ci-Ce alkyl optionally contains one or more double or triple bond(s); R13 is selected from the following:

[0098] • -NH-O-R20, wherein R20is C1-C6alkyl optionally containing one double or triple bond, optionally substituted benzyl or phenyl,

[0099] • -N=N-CO-NR23R24, wherein R23 and R24 are independently selected from H, Ci-Ce alkyl optionally containing one or more double or triple bond, optionally substituted benzyl or phenyl

[0100] • -NH-NH-CS-NR25R26, wherein R25 and R26 are independently selected from H, Ci-Ce alkyl optionally containing one or more double or triple bond, optionally substituted benzyl or phenyl,

[0101] • -NH2, and

[0102] • -NR21R22, wherein R21 and R22 are independently selected from Ci-Ce alkyl, optionally containing one or more double or triple bond, optionally substituted benzyl or phenyl.

[0103] A narrower group of the above compounds of formula (I) or (la) is, wherein

[0104] between the carbons to which R2 and R3are attached is a double bond and the value of n is 0.

[0105] An other narrower group of any of the above compounds of formula (I), (la) or (lb) is wherein X is O; and / or

[0106] R4 is H; and / or

[0107] R3is H.

[0108] A specific group of compounds of formula (la) is, wherein

[0109] are each double bond; and / or

[0110] Rs is OH or Ci-Ce alkoxy, wherein the alkyl group in Ci-Ce alkoxyl optionally contains one double or triple bond; and / or

[0111] R6, R7, R9 and R10 are H; and / or

[0112] Rs is selected from the following:

[0113] • =N-O-Ri6, wherein Rie is Ci-Ce alkyl optionally containing one double or triple bond, or benzyl;

[0114] • =N-NH-CO-NH2, and =N-NH-CS-NH2

[0115] A narrower group of the above specific group of compounds of formula (la) is, wherein R5 is OH, -O-methyl, -O-ethyl, -O-propyl, -O-isopropyl, -O-butyl, -O-tert-butyl, -O-propargyl or -O-allyl; and / or

[0116] R6, R7, R9 and R10 are H; and / or

[0117] Rs is selected from the following:

[0118] • =N-0-Ri6, wherein Rie methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, propargyl, allyl or benzyl,

[0119] • =N-NH-CO-NH2, and

[0120] • =N-NH-CS-NH2

[0121] An even narrower group of compounds is wherein

[0122] Rs is OH or O-allyl and / or

[0123] Rs is =N-NH-CO-NH2, or =N-NH-CS-NH2

[0124] A specific group of compounds of formula (lb) is, wherein

[0125] R11, R12, Ri4 and Ris are independently selected from H and halogen, and / or

[0126] R13 is selected from the following:

[0127] • -NH-O-R20, wherein R20is C1-C6alkyl optionally containing one double or triple bond,

[0128] • -N=N-CO-NH2,

[0129] • -NH-NH-CS-NH2, and

[0130] • -NH2

[0131] A narrower group of the latter specific group of compounds of formula (lb) is, wherein R11, R12, and Ris are H and / or

[0132] R14 is H or Cl, and / or

[0133] R13 is selected from the following:

[0134] • -NH-O-R20, wherein R20is C1-C6alkyl optionally containing one double or triple bond,

[0135] • -N=N-CO-NH2, and

[0136] • -NH2 Another aspect of the present invention is the preparation of the compounds of formula (I). In accordance with the invention the compounds of formula (I) can be prepared by the following processes.

[0137] The compounds of formula (I) wherein X is O can be prepared by using commercially available apigenin as the starting material. The compounds of formula (I) wherein X is S or NRi, can be prepared from commercially available 2-(4-hydroxyphenyl)-4H-thiochromen-4-one (CAS No.

[0138] 205121-94-4) or 5,7-Dihydroxy-2-(4-Hydroxyphenyl)-4(lH)-Quinolinone (CAS No. 884500-86-1).

[0139] The corresponding protoflavone is prepared from apigenin or its N- or S-containing C-ring analogue in a one-step oxidative transformation.

[0140] The preparation of oxime, hydrazone and nitrone functions on the protoflavone 4'-carbonyl group (R8).

[0141] Substitution of the 4'-carbonyl group with an oxime ether moiety can be accomplished in a chemoselective way at neutral pH that leaves the double bond system of the B-ring intact. Scheme 2 depicts our successful semi -synthetic transformations of protoapigenone I'-O-propargyl ether (compound according to Example 3) with the isolated yields of the products indicated.

[0142] 4: R = Me (48.1%) 5: R = Et (40.4%) 6: R = Pr (38.6%) 7: R = / Pr (46.7%) 8: R = Bu (6.3%) 9: R = tBu (29.5%)

[0143]

[0144] 10: R = allyl (43.9%)

[0145] 11: R = propargyl (27%) 12: R = Bn (50.4%) Scheme 2: Semi -synthetic preparation of oxime ether derivatives according to Examples 4-12 from protoapigenone 1'-O-propargyl ether (compound according to example 3).

[0146] The described procedure works well with other protoapigenone derivatives as well. One special exception is the oxime etherification of protoapigenone l'-O-butyl ether (compound according to Example 2) with a tert-butyl functionality. This reaction is accompanied by the rearomatization of the B-ring and the addition of a chlorine atom at C-5'. Upon a closer investigation on the transformation, we found that the planned oxime ether derivative is selectively produced during the synthetic process and is detectable via HPLC-DAD. However, following a work-up featuring liquid-liquid extraction, the product fully decomposes to an aromatic alkoxylamine derivative (compound according to Example 13). (Scheme 3)

[0147]

[0148] Scheme 3: Preparation of compound according to Example 13 from protoapigenone 1'-O-butyl ether (compound according to Example 2). Hydrazone / semicarbazone derivatives can be semi-synthesized from protoapigenone (compound according to Example 1) and its 1 '-( -butyl ether (Compound according to Example 2) analog in a similar manner to oximation. (Scheme 4)

[0149]

[0150] X = 0 (37%) 16 X = 0 (77.5%)

[0151] Scheme 4: Semi -synthetic preparation of compounds according to Examples 14 tol6 from their starting materials.

[0152] Between the two starting materials, protoapigenone 1'-O-butyl ether (2) is a more stable, photochemically less sensitive compound due to the presence of the alkyl moiety at C-l '. In spite of this, while products 14-15 possessed enhanced chemical stabilities compared to their parental compound, product 14 was found to undergo a spontaneous, highly selective, solid-state decomposition during storage (approx. 1 month, 4°C, in the dark), resulting in a flavonoid diazene derivative (compound according to Example 16) with an aromatic B-ring. As shown on Scheme 5 the reaction of protoapigenone l'-O-propargyl ether (compound of Example 3) with A-benzyl hydroxylamine in the solvent mixture of dioxane:water -9:1 yielded the 4'-amino derivative of a natural flavonoid, chrysin (compound according to Example 17) instead of the expected 4 ’-substituted nitrone derivative.

[0153] BnNHOH,

[0154]

[0155] The compounds of formula (lb), can therefore be prepared in a solution of water and 1,4-dioxane, wherein in the formula (lb) X is O from protoapigenone 1'-O-C1-6alkyl ether derivative -wherein the Ci-6 alkyl optionally contains one or more double or triple bonds-, or wherein in the formula (lb) X is S or NRi from the corresponding analogue starting compounds, by using the corresponding reagent Y-RB”, which is an amino derivative of the RB substituent being able to introduce RB as indicated for the different meanings of RB respectively as follows:. when RB is -NH-O-R20, wherein R20is C1-C6alkyl optionally containing one double or triple bond, optionally substituted benzyl or phenyl; the reagent is NH2-O-R20

[0156] . when RB is -N=N-CO-NR23R24, wherein R23 and R24 are independently selected from H, Ci- G> alkyl optionally containing one or more double or triple bond, optionally substituted benzyl or phenyl; the reagent is NH2-NH2-CO-NR23R24

[0157] - when RB is -NH-NH-CS-NR25R26, wherein R25 and R26 are independently selected from H, Ci-Ce alkyl optionally containing one or more double or triple bond, optionally substituted benzyl or phenyl; the reagent is NH2-NH2-CS-NR25R26

[0158] - when RB is -NH2; the reagent is Bn-NH-OH

[0159] - when RB is -NR21R22, wherein R21 and R22 are independently selected from Ci-Ce alkyl, optionally containing one or more double or triple bond, optionally substituted benzyl or phenyl; the reagent is NHR21R22.

[0160] The saturation of ring B in case if B is (a) can be carried out by the method as dislosed in S. B. Ötvös, M. Vágvölgyi, G. Girst, C.-Y. Kuo, H.-C. Wang, F. Fülöp, A. Hunyadi, ChemPlusChem 2018, 83, 71., and the substitution of ring B in formula (I) both in case if B is either (a) or (b) can be carried out by using the appropriately substituted 4'-hydroxyflavone as a starting material for oxidative dearomatization.

[0161] The introduction of R2 substituent can be achieved by using, e.g., commercially available 2,3-Dihydro-5,7-dihydroxy-2-(4-hydroxyphenyl)-2-methyl-4H-l-benzopyran-4-one (CAS No.

[0162] 2524209-65-0), or, e.g., 5,5',7-trihydroxy-2',3,3',4-tetrahydrospiro[l-benzopyran-2, T-inden]-4-one (CAS No. 2598593-22-5) as starting material for oxidative dearomatization. R3 substituent can be achieved by using commercially available kaempferol (CAS No. 520-18-3), or, e.g., 3-methylapigenin (CAS No. 67979-17-3) as starting material for oxidative dearomatization, and R4 substituents can be achieved e.g., by using published selective alkoxylation procedures on apigenin (Kim, Jin- Young, et al. “Synthesis of a Complete Series of O-Methyl Analogues of Naringenin and Apigenin.” Bulletin of the Korean Chemical Society, vol. 28, no. 12, Dec. 2007, pp. 2527-2530) and subsequent oxidative dearomatization, or, e.g., by using commercially available 5-methylapigenin (CAS No. 29376-68-9) as starting material for oxidative dearomatization.

[0163] In a further aspect of the present invention the compounds of formula (I) were tested in biological essays and showed XO inhibitory activity. Thus a further subject of the present invention is the compounds of formula (I), (la) or (lb) for use in the treatment or prevention of a disease of disorder, wherein the disease of disorder is associated with the increased expression of XO enzyme, which disease or disorder can be treated or prevented by the inhibition of XO enzyme. Such diseases are for example gout (P. Pacher, A. Nivorozhkin, Cs. Szabo Therapeutic Effects of Xanthine Oxidase Inhibitors: Renaissance Half a Century after the Discovery of Allopurinol Pharmacol Rev. 2006 March; 58(1): 87-114.), chronic kidney disease (Hunter W. Korsmo,, Ubong S. Ekperikpe and Ilse S. Daehn, Emerging Roles of Xanthine Oxidoreductase in Chronic Kidney Disease Antioxidants 2024, 73(6), 712), chronic stress-induced cerebrovascular dysfunction and cognitive impairment (E. N Burrage, T. Coblentz, S. S Prabhu, R. Childers, R. W Bryner, S. E Lewis, E. DeVallance, E. E Kelley, P. D Chantier, Xanthine oxidase mediates chronic stress-induced cerebrovascular dysfunction and cognitive impairment, J Cereb Blood Flow Metab.. 2023 Jan 18;43(6):905-920), chronic heart failure (Masashi Sakuma, Shigeru Toyoda, Takuo Arikawa, Yota Koyabu, Toru Kato, Taichi Adachi, Hideaki Suwa, Jun-ichi Narita, Koetsu Anraku, Kimihiko Ishimura, Fumitake Yamauchi, Yasunori Sato, Teruo Inoue The effects of xanthine oxidase inhibitor in patients with chronic heart failure complicated with hyperuricemia: a prospective randomized controlled clinical trial of topiroxostat vs allopurinol — study protocol, Clinical and Experimental Nephrology (2018) 22:1379-1386), COPD (L M Heunks1, J Vina, C L van Herwaarden, H T Folgering, A Gimeno, P N Dekhuijzen, Xanthine oxidase is involved in exercise-induced oxidative stress in chronic obstructive pulmonary disease, Am J Physiol. 1999 Dec;277(6): R1697-704.), metabolic syndrome, or metabolic disorders such as diabetes, hypertension, liver dysfunction or cardiovascular disease (Ali, N., Taher, A., Islam, N. et al. Evaluation of the relationship between xanthine oxidase activity and metabolic syndrome in a population group in Bangladesh. Sci Rep 14, 20380 (2024)).

[0164] Examples:

[0165] Preparation of starting materials

[0166] Starting materials were prepared from commercially available apigenin by an easy, economic semi-synthetic method as disclosed in the following prior art: Hunyadi, A.; Chuang, D.-W.; Danko, B.; Chiang, M. Y; Lee, C.-L.; Wang, H.-C.; Wu, C.-C.; Chang, F.-R.; Wu, Y.-C. Direct Semi-Synthesis of the Anticancer Lead-Drug Protoapigenone from Apigenin, and Synthesis of Further New Cytotoxic Protoflavone Derivatives. PLoS ONE 2011, 6 (8), e23922., as shown in Scheme 6, that allows to obtain larger amounts (> 10 g) from protoflavone starting materials through a one-step oxidative transformation of a commercially available flavonoid, apigenin.

[0167] 1: R = H (34.1%) 2: R = Bu (35.2%) 3: R = propargyl (31%)

[0168]

[0169] Scheme 6

[0170] Examples 1-3: Preparation of protoflavone starting materials: protoapigenone (1), protoapigenone 1'-O-butyl ether (2) and protoapigenone 1'-O-propargyl ether (3) Apigenin was purchased from Biosynth Carbosynth (Staad, Switzerland) in an RP-HPLC purity of 94% and was used as a starting material for semi -synthetic transformations without any further purification. Typically, a 1 g aliquot of apigenin (3.70 mmol) was dissolved in a concentration of 1 mg / ml in a 9:1 (v / v) mixture of acetonitrile and either water or the alcohol to be coupled at C-l'. Two equivalents of (bis(trifluoroacetoxy)iodo)benzene (PIFA; 3.18 g, 7.40 mmol) were added to the solution, and the mixture was stirred at 80 °C for an hour. Later, the solution was cooled to room temperature, and the solvent was evaporated under reduced pressure on a rotary evaporator. The obtained residue was re-dissolved in acetone and subjected to flash chromatographic purification using our previously published methods (Hunyadi, A.; Chuang, D.-W.; Danko, B.; Chiang, M. Y; Lee, C.-L.; Wang, H.-C.; Wu, C.-C.; Chang, F.-R.; Wu, Y.-C. Direct Semi-Synthesis of the Anticancer Lead-Drug Protoapigenone from Apigenin, and Synthesis of Further New Cytotoxic Protoflavone Derivatives. PLoS ONE 2011, 6 (8), e23922.) that afforded protoapigenone analogs 1-3 with the following yields: protoapigenone (1, 34.1%), protoapigenone 1'-O-butyl ether (2, 35.2%), protoapigenone 1'-O-propargyl ether (3, 31%).

[0171] Preparation of compounds of formula (I)

[0172] Examples 4-12:

[0173] A 150 mg aliquot of protoapigenone 1'-O-propargyl ether (3; 0.462 mmol) was selected for transformation and, at first, three molar equivalents of the corresponding (9-substituted hydroxylamine hydrochloride derivative was measured out in a round-bottom flask. Then, 19.28 ml of 1,4-di oxane was added to the flask. Subsequently, an equimolar amount of potassium hydroxide with respect to the hydroxylamine salt was dissolved in 2.14 ml of water, and the obtained aqueous solution was added under stirring to the formerly prepared dioxanic solution. Following this, the 150 mg aliquot of compound 3 was added to the prepared solution and the reaction mixture was stirred for 24 h at room temperature. Following the transformation, the solution was evaporated to dryness, water (50 ml) was added, and extraction was performed with ethyl-acetate (3x50 ml). Subsequently, the collected organic phases were combined, dried over Na2SO4, filtered, and the solvent was evaporated under reduced pressure. Later, the obtained products were purified via preparative-scale RP-HPLC that allowed us the isolation of the corresponding protoflavone 4'-oxime ether derivatives in the following yields: compound 4 (63.44 mg; 48.12 %); compound 5 (60.57 mg; 40.38 %); compound 6 (57.92mg; 38.61%); compound 7 (70.08 mg; 46.72%); compound 8 (9.5 mg; 6.33%); compound 9 (44.24 mg; 29.49%); compound 10 (65.95 mg; 43.97 %); compound 11 (40.54 mg; 27.02%); compound 12 (75.56 mg; 50.37%). Structure elucidation

[0174] Example 4

[0175]

[0176] Pale yellow solid; Isolated yield: 63.44 mg (48.12 %); for1H and13C NMR data, see Tables 1 and 2, respectively; HR-MS C19H16NO6, calculated: 354.09721, found: 354.09656.

[0177] Example 5

[0178]

[0179] Pale yellow solid; Isolated yield: 40.38% (60.57 mg); for 'H and13C NMR data, see Tables 1 and 2, respectively; HR-MS C20H18NO6, calculated: 368.11286, found: 368.11229.

[0180] Example 6

[0181]

[0182] Pale yellow solid; Isolated yield: 38.61% (57.92 mg); for1H and13C NMR data, see Tables 1 and 2, respectively. HR-MS C21H20NO6, calculated: 382.12851, found: 382.12789. Example 7

[0183]

[0184] Pale yellow solid; Isolated yield: 46.72% (70.08 mg); for1H and13C NMR data, see Tables 1 and 2, respectively. HR-MS C21H20NO6, calculated: 382.12851, found: 382.12803.

[0185] Example 8

[0186]

[0187] Pale yellow solid; Isolated yield: 6.33% (9.5 mg); for 'H and13C NMR data, see Tables 1 and 2, respectively. HR-MS C22H22NO6, calculated: 396.14416, found: 396.14363.

[0188] Example 9

[0189]

[0190] Pale yellow solid; Isolated yield: 29.49% (44.24 mg); for1H and13C NMR data, see Tables 1 and 2, respectively. HR-MS C22H22NO6, calculated: 396.14416, found: 396.14357. Example 10

[0191]

[0192] Pale yellow solid; Isolated yield: 43.97% (65.95 mg); for 'H and13C NMR data, see Tables 1 and 2, respectively. HR-MS C21H18NO6, calculated: 380.11286, found: 380.11229.

[0193] Example 11

[0194]

[0195] Pale yellow solid; Isolated yield: 27.02% (40.54 mg); for 'H and13C NMR data, see Tables 1 and 2, respectively. HR-MS C21H16NO6, calculated: 378.09721, found: 378.09656.

[0196] Example 12

[0197]

[0198] Pale yellow solid; Isolated yield: 50.37% (75.56 mg); for 'H and13C NMR data, see Tables 1 and 2, respectively. HR-MS C25H20NO6, calculated: 430.12851, found: 430.12768.

[0199] Example 13:

[0200] A 70 mg aliquot of protoapigenone l'-O-butyl ether (2; 0.204 mmol) was selected for transformation and, at first, three molar equivalents of O-tert-butylhydroxylamine hydrochloride (77.05 m, 0.612 mmol) was measured out in a round-bottom flask. Following this, 9.45 ml of 1,4-di oxane was added to the flask. Subsequently, an equimolar amount of potassium hydroxide with respect to the hydroxylamine salt (34.41 mg, 0.612 mmol) was dissolved in 1.05 ml of water, and the obtained aqueous solution was added under stirring to the formerly prepared dioxanic solution. Following this, the 70 mg aliquot of compound 2 was added to the prepared solution and the reaction mixture was stirred for 24 h at room temperature. Following the transformation, the solution was evaporated to dryness, water (50 ml) was added, and extraction was performed with ethyl-acetate (3x50 ml). Subsequently, the collected organic phases were combined, dried over Na2SO4, filtered, and the solvent was evaporated under reduced pressure. The obtained crude residue was subjected to preparative RP-HPLC purification that eventually afforded the chlorinated flavonoid allyl amine derivative 13 in a yield of 35.2% (25.89 mg).

[0201]

[0202] OH O

[0203] Pale brown solid; Isolated yield: 35.2% (25.89 mg); for 'H and13C NMR data, see Tables 1 and 2, respectively. HR-MS [M+NH4]+C19H22ClN2O4, calculated: 377.12681, found: 376.09417.

[0204] Example 14:

[0205] A 200 mg aliquot of protoapigenone (1; 0.699 mmol) was selected for transformation and, at first, three molar equivalents of semicarbazide hydrochloride (233.78 mg, 2.096 mmol) was measured out in a round-bottom flask. Later, 27 ml of 1,4-di oxane was added to the flask. Subsequently, an equimolar amount of potassium hydroxide with respect to the hydroxylamine salt (117.61 mg, 2.096 mmol) was dissolved in 3 ml of water, and the obtained aqueous solution was added under stirring to the formerly prepared dioxanic solution to liberate the reagent from its salt. Following this, the 200 mg aliquot of protoapigenone (1) was added to the prepared solution and the reaction mixture was stirred for 48 h at room temperature. During this time, the round-bottom flask was fully covered with aluminium foil to protect the photosensitive protoapigenone starting material from a light-related decomposition. Following the transformation, the solution was evaporated to dryness, water (50 ml) was added, and extraction was performed with ethyl-acetate (3x50 ml). Next, the collected organic phases were combined, dried over Na2SO4, filtered, and the solvent was evaporated on a rotary evaporator. Eventually, the obtained residue was subjected to preparative RP-HPLC purification that afforded the desired protoapigenone 4'-semicarbazone derivative (14) in a yield of 64.1% (153.75 mg). 0

[0206]

[0207] OH O

[0208] Pale yellow solid; Isolated yield: 64.1% (153.75 mg); for 'H and13C NMR data, see Tables 1 and 2, respectively. HR-MS C16H14N3O6, calculated: 344.08826, found: 344.08852.

[0209] Example 15:

[0210] A200 mg aliquot of protoapigenone (1; 0.699 mmol) was measured out in a round-bottom flask.

[0211] 30 ml of 1,4-di oxane: water - 9:1 (v / v) was added to the flask. Subsequently, three molar equivalents of thiosemicarbazide (191.04 mg, 2.096 mmol) was added to the solution, and the reaction mixture was stirred at room temperature for 5 days. During this time, the round-bottom flask was fully covered with aluminium foil to protect the photosensitive protoapigenone starting material from a light-related decomposition. Following the transformation, the solution was evaporated to dryness, and the obtained residue was re-dissolved in acetone, and polyamide (1.5 g) was added to the solution. The solvent was evaporated to prepare the sample for dry loading flash chromatographic purification on a 15 g manually filled polyamide column. The purification was performed in gradient mode by linearly changing the mobile phase composition of dichloromethane (A) and methanol (B) - 100:0 to 80:20 (v / v; A: B) in 60 minutes. The applied flow rate was 15 ml / min. The method allowed us to isolate the desired protoapigenone 4'-thiosemicarbazone derivative (15) in a yield of 47.2% (118.51 mg).

[0212] s

[0213]

[0214] OH O

[0215] Pale brown solid; Isolated yield: 47.2% (118.51 mg); for

[0216]

[0217] and13C NMR data, see Tables 1 and 2, respectively. HR-MS C16H14N3O5S, calculated: 360.06542, found: 360.06574.

[0218] Example 16:

[0219] A 200 mg aliquot of protoapigenone l'-O-butyl ether (2; 0.584 mmol) was selected for transformation. At first, three molar equivalents of semicarbazide hydrochloride (195.47 mg, 1.752 mmol) was measured out in a round-bottom flask. Later, 27 ml of 1,4-dioxane was added to the flask. Subsequently, an equimolar amount of potassium hydroxide with respect to the hydroxylamine salt (98.33 mg, 1.752 mmol) was dissolved in 3 ml of water, and the obtained aqueous solution was added under stirring to the formerly prepared dioxanic solution to liberate the reagent from its salt. Following this, the 200 mg aliquot of protoapigenone l'-O-butyl ether (2) was added to the prepared solution and the reaction mixture was stirred for 48 h at room temperature. During this time, the round-bottom flask was fully covered with aluminium foil to protect the starting material from a light-related decomposition. Following the transformation, the solution was evaporated to dryness, water (50 ml) was added, and extraction was performed with ethyl-acetate (3x50 ml). Next, the collected organic phases were combined, dried over Na2SO4, filtered, and the solvent was evaporated on a rotary evaporator. This step was followed by the immediate preparative RP-HPLC purification of the product that is theorized to afford the 4'-semicarbazone derivative of compound 2 in a yield of 37% (86.33 mg). The product immediately starts to selectively decompose to a flavonoid type diazene derivative (compound 16; complete solid-state decomposition upon storage in the fridge at 4°C takes approx. 3-4 weeks), which we further purified with preparative HPLC that afforded compound 16 in a yield of 77.5% (54.49 mg).

[0220]

[0221] OH O

[0222] Pale yellow solid; Isolated yield: 77.5% (54.49 mg); for

[0223]

[0224] and13C NMR data, see Tables 1 and 2, respectively. HR-MS C16H12N3O5, calculated: 326.07769, found: 326.07681.

[0225] Example 17:

[0226] A 200 mg aliquot of protoapigenone l'-O-propargyl ether (3; 0.617 mmol) was selected for transformation. In step one, three molar equivalents of A-benzylhydroxylamine hydrochloride (295.31 mg, 1.85 mmol) were measured out in a round-bottom flask. Following this, 27 ml of 1,4-di oxane was added to the flask. An equimolar amount of potassium hydroxide with respect to the hydroxylamine salt (103.81 mg, 1.85 mmol) was dissolved in 3 mL of water, and the obtained solution was added under stirring to the formerly prepared dioxanic solution. Subsequently, the 200 mg aliquot of compound 3 was added to the mixture. The reaction mixture was stirred for 24 h at room temperature. Following this, the reaction solution was evaporated to dryness, water (50 ml) was added, and extraction was performed with ethylacetate (3x50 ml). Subsequently, the organic phases were combined, dried over Na2SO4, filtered, and the solvent was evaporated on a rotary vacuum evaporator. Chromatographic purification of the product was performed with preparative RP-HPLC that afforded 4'-aminochrysin (17) in a yield of 16.75% (23.02 mg).

[0227]

[0228] OH 0

[0229] Pale yellow solid; Isolated yield: 16.75% (23.02 mg); for 'H and13C NMR data, see Tables 1 and 2, respectively. HR-MS C15H12NO4, calculated: 270.07608, found: 270.07554.

[0230] NMR spectra were recorded at 25 °C on a Bruker Ascend 500 MHz spectrometer equipped with a Prodigy BBO 5 mm CryoProbe (Bruker, Billerica, MA, USA) at 500 MHz (1H) and 126 MHz (13C). In general, 5-6 mg of the compounds were dissolved in DMSO-6and transferred to 5 mm NMR tubes for recording spectra. Chemical structures of the derivatives were determined by employing comprehensive one- and two-dimensional NMR methods. The HR-MS flow injection analysis was performed with a Thermo Scientific Orbitrap Exploris 240 hybrid quadrupole-Orbitrap (Thermo Fischer Scientific, Waltham, MA, USA) mass spectrometer coupled to a Waters Acquity I-Class UPLC TM (Waters, Manchester, UK). Table 1:1H chemical shifts of examples 4–17; in DMSO-6.

[0231] No. Example 4 Example 5 Example 6 Example 7 Example 8

[0232] lH J (Hz) *H J (Hz)lH J (Hz) H J (Hz)lH J (Hz)

[0233]

[0234] 2 3 6.34 s 6.34 s 6.34 s 6.33 s 6.34 s

[0235] 4

[0236] 5- 12.52 s 12.52 s 12.52 s 12.53 s 12.52 s

[0237] OH

[0238] 6 6.20 d; 2.1 6.19 d; 2.0 6.20 d; 2.0 6.20 d; 2.1 6.20 d; 2.0

[0239] 7- 10.91 br s 10.94 br s 10.93 br s 10.93 br s 10.90 br s OH

[0240] 8 6.24 d; 2.1 6.24 d; 2.0 6.24 d; 2.0 6.24 d; 2.1 6.24 d; 2.0

[0241] 9

[0242] 10

[0243] V

[0244] 2' 6.41 dd; 10.2, 6.39 dd; 10.3, 6.40 dd; 10.3, 6.38 dd; 10.3, 6.40 dd; 10.3

[0245] 2.6 2.5 2.5 2.5 2.5

[0246] 3' 7.17 dd; 10.2, 7.18 dd; 10.3, 7.19 dd; 10.3, 7.17 dd; 10.2, 7.18 dd; 10.3

[0247] 2.0 1.9 1.8 1.9 1.9 5' 6.67 dd; 10.1, 6.68 dd; 10.1, 6.68 dd; 10.0, 6.69 dd; 10.1, 6.68 dd; 10.1,

[0248] 2.0 1.8 1.8 1.8 1.9

[0249] 6' 6.26 dd; 10.0, 6.25 dd; 10.1, 6.25 dd; 10.0, 6.23 dd; 10.1, 6.25 dd; 10.1,

[0250] 2.5 2.5 2.4 2.5 2.5

[0251] 1" 4.08 4.07 t; 2.9 4.08 t; 2.7 4.07 t; 2.9 4.07 t; 2.7

[0252] 2"

[0253] 3" 3.49 t; 2.4 3.49 t; 2.4 3.48 t; 2.4 3.48 t; 2.4 3.48 t; 2.5

[0254] I'" 3.97 s 4.22 q; 7.0 4.13 t; 6.6 4.42 hept; 6.3 4.18 t; 6.6

[0255] 2"' 1.28 t; 7.1 1.69 1.27 dd; 6.3, 1.6 1.65

[0256] 3'" 0.93 t; 7.4 1.38

[0257] 4'" 0.92 t; 7.4

[0258] O

[0259] 5'"

[0260] No. Example 9 Example 10 Example 11 Example 12

[0261]

[0262] *H J (Hz) *H J (Hz) *H J (Hz) *H J (Hz)

[0263] 2

[0264] 3 6.33 s 6.34 s 6.35 s 6.35 s

[0265] 4

[0266] 5- 12.53 s 12.52 s 12.52 s 12.53 s

[0267] OH

[0268] 6 6.19 d; 2.1 6.20 d; 2.0 6.20 d; 2.0 6.21 d; 2. I 7- 10.93 br s 10.93 br s 10.93 br s 10.93 br s OH

[0269] 8 6.25 d; 2.1 6.24 d; 2. I 6.24 d; 2.1 6.25 d; 2. 1 9

[0270] 10

[0271] 1'

[0272] 2' 6.36 dd; 10.3, 6.42 dd; 10.3, 2.5 6.47 dd; 10.3, 6.43 dd; 10.3

[0273] 2.5 2.5 2.5 3' 7.18 dd; 10.3, 7.21 dd; 10.3, 1.9 7.17 dd; 10.3, 7.24 dd; 10.3

[0274] 1.9 1.9 1.9 4'

[0275] 5' 6.70 dd; 10.1, 6.68 dd; 10.1, 1.9 6.70 dd; 10.1, 6.69 dd; 10.1

[0276] 1.9 1.9 1.9 6' 6.21 dd; 10.1, 6.27 dd; 10.1, 2.5 6.32 dd; 10.1, 6.28 dd; 10.2

[0277] 2.5 2.5 2.5 1" 4.07 t; 2.9 4.08 t; 2.7 4.09 t; 2.7 4.08 t; 2.5 2”

[0278] 3" 3.48 t; 2.4 3.49 t; 2.4 3.49 t; 2.4 3.49 t; 2.4 1'" 4.71 dd; 5.5, 1.9 4.85 d; 2.3 5.25 s 2'" 1.32 s 6.03 ddt; 16.3, 10.8,

[0279] 5.6 E dd; 17.4, 1.8 3.56 t; 2.4 7.40

[0280] 5.34 dd; 10.3, 1.8

[0281] Z

[0282] 5.26

[0283] 4'" 7.40

[0284] 5'" 7.34

[0285] No. Example 13 Example 14 Example 15 Example 16 Example 17

[0286]

[0287] *H J (Hz) *H J (Hz) *H J (Hz) 'H J (Hz) *H J (Hz) 2

[0288] 3 6.84 s 6.36 s 6.37 s 7.09 s 6.60 s

[0289] 4

[0290] 5- 12.89 s 12.64 s 12.62 s 12.72 s 13.10 s OH

[0291] 6 6.20 d; 1.7 6.17 d; 1.9 6.17 d; 1.6 6.24 s 6.16 d; 2. 1 7- 10.71 br s 10.85 br s 10.86 br s 10.91 br s 10.66 br s OH

[0292] 8 6.51 d; 1.7 6.20 d; 2.0 6.20 d; 1.7 6.55 s 6.44 d; 2.1 9

[0293] 10

[0294] r OH 6.57 br s OH 6.66 br s 2' 7.92 dd; 8.7, 6.25 dd; 10.3, 6.34 dd; 10.2, 8.30 d; 8.5 7.75 d; 8.9

[0295] 2.4 dd; 10.3, 7.96 d; 8.4 6.67 d; 8.7 1.6

[0296]

[0297] NH2 br s 8.99 6.01

[0298] 5' 6.41 dd; 10.0, 6.45 dd; 10.2, 7.96 d; 8.4 6.67 d; 8.7

[0299] 1.7 1.5

[0300] 6' 7.99 d; 1.5 6.08 dd; 10.0, 6.22 dd; 10.1, 8.30 d; 8.5 7.75 d; 8.9

[0301] 2.5 2.5

[0302] NH s NH 11.18 s NH27.89, br s, br

[0303] 10.28 br s NH2 8. OO, br s, br s 7.79 s

[0304] NH28.41

[0305] 6.54

[0306] 3"

[0307]

[0308] Table 2:13C chemical shifts of compounds 4–17; in DMSO-6.

[0309] Ex. 4 5 6 7 8 9 10 11 12 13 14 15 16 17 No.

[0310] 165.9 165.9 165.9 166.0 166.0 166.1 165.9 165.7 165.8 162.3 169.8 169.4 161.6 164.5 106.7 106.7 106.7 106.7 106.7 106.7 106.7 106.8 106.7 103.3 105.4 105.6 106.5 100.7 181.5 181.5 181.5 181.5 181.5 181.5 181.5 181.5 181.5 181.5 181.9 181.8 181.6 181.4 161.4 161.4 161.4 161.4 161.4 161.4 161.4 161.4 161.4 162.3 161.5 161.4 161.4 161.4 99.2 99.2 99.2 99.2 99.2 99.2 99.2 99.2 99.2 98.8 99.0 99.0 99.1 98.5 164.6 164.7 164.7 164.7 164.6 164.7 164.7 164.7 164.6 161.3 164.5 164.5 164.6 163.7 8 93.9 93.9 93.9 93.9 93.9 93.9 93.9 93.9 93.9 94.0 93.8 93.8 94.1 93.7 9 157.4 157.4 157.4 157.4 157.4 157.4 157.4 157.4 157.4 157.2 157.5 157.5 157.4 157.1 10 103.8 103.8 103.8 103.8 103.8 103.8 103.8 103.8 103.8 103.7 103.6 103.6 104.0 103.5 1' 75.3 75.3 75.3 75.4 75.3 75.4 75.3 75.2 75.3 122.0 69.3 69.2 134.3 116.5 2' 134.4 134.1 134.1 133.8 134.1 133.4 134.5 135.2 134.5 126.2 136.0 137.6 127.7 128.0 3' 119.6 119.8 119.8 119.9 119.8 120.0 119.7 119.6 119.8 114.1 117.1 117.6 123.0 113.4 4' 145.8 145.7 145.7 145.4 145.7 145.1 146.1 146.6 146.2 149.6 136.3 138.0 152.5 152.8 5' 128.3 128.5 128.5 128.7 128.5 129.1 128.3 128.0 128.3 117.1 128.1 127.9 123.0 113.4 6' 130.2 129.9 129.9 129.6 129.9 129.1 130.3 131.1 130.4 126.9 131.3 133.7 127.7 128.0 1" 52.1 52.1 52.1 52.1 52.1 52.1 52.2 52.3 52.2 79.6 156.7 179.2 164.4

[0311] 2" 80.4 80.4 80.4 80.4 80.4 80.5 80.4 80.3 80.4 26.3

[0312] 3" 77.7 77.7 77.7 77.6 77.7 77.6 77.7 77.8 77.7 V" 62.5 70.2 76.0 76.4 74.3 80.0 75.2 62.2 76.3 2"' 14.4 21.8 21.3 30.6 27.2 133.9 79.7 137.1 3'" 10.2 18.6 118.1 78.1 128.1 4"' 13.7 128.4 5'" 128.0 In vitro bioactivity assays

[0313] Compounds according to Examples 4-17 were tested for their XO-inhibitory effect on an in vitro bioassay using the anti-gout drug allopurinol as a positive control (Hunyadi, A.; Martins, A.; Danko, B.; Chuang, D.-W.; Trouillas, P.; Chang, F.-R.; Wu, Y.-C.; Falkay, G. Discovery of the First Non-Planar Flavonoid That Can Strongly Inhibit Xanthine Oxidase: Protoapigenone l'-O-Propargyl Ether. Tetrahedron Letters 2013, 54 (48), 6529-6532. https: / / doi. Org / 10.1016 / j.tetlet.2013.09.087.). Determination of XO-inhibitory effect was performed by using commercially available XO activity assay kit (Sigma- Aldrich Ltd., USA), following the provided protocol.

[0314] Based on the above disclosed assays the in vitro xanthine oxidase inhibitory effects of compounds according to Examples 4 to 17 are shown in Table 3.

[0315] It is to be noted that each of the compounds showed XO inhibitory effect, and the compounds according to Examples 13, 14, and 16 exerted an outstandingly robust inhibitory effect on the bioassay with IC50 values below 1 pM.

[0316] 95% CI XO-inhibition % (at 100 IC50

[0317] # average SD (Confidence gM) (Al)

[0318] interval) Allopurinol

[0319] 97.6 99.0 100.4 99.0 1.4 4.06 3.554 to 4.603 (+)

[0320] Example:

[0321] 4 n.a. 85.8 85.5 85.6 0.21 13.19 10.54 to 16.74 5 56.2 56.2 54.8 55.8 0.8 n.a. n.a.

[0322] 6 48.8 47.7 48.4 48.3 0.6 n.a. n.a.

[0323] 7 63.6 62.4 62.6 62.9 0.6 n.a. n.a.

[0324] 8 51.4 53.8 54.6 53.3 1.7 n.a. n.a.

[0325] 9 20.1 21.3 32.5 24.6 6.9 n.a. n.a.

[0326] 10 49.3 49.6 51.3 50.1 1.1 n.a. n.a.

[0327] 11 40.9 44.7 42.7 42.8 1.9 n.a. n.a.

[0328] 12 71.6 72.7 73.5 72.6 1.0 8.10 7.068 to 9.093 13 n.a. 102.2 105.9 104.1 2.65 0.44 0.4013 to 0.4894 14 97.0 98.4 98.6 98.0 0.8 0.35 0.2564 to 0.4469 15 97.7 96.5 95.6 96.6 1.0 7.30 6.068 to 8.714 0.06206 to 16 n.a. 97.2 107.6 102.4 7.36 0.066

[0329] 0.07005 17 84.6 82.7 83.4 83.6 1.0 3.86 2.051 to 6.356

[0330] Table 3:

[0331] Some of the compounds were also tested as to their selectivity of their antioxidant effect, thus the compounds according to examples 4, 13 and 16 were further evaluated for their cytotoxicity on NIH / 3T3 mouse fibroblast cell line by using MTT colorimetric assay as published before: Cs. Bus, A. Kulmany, N. Kusz, T. Gonda, I. Zupko, A. Mandi, T. Kurtan, B. Toth, J. Hohmann, A. Hunyadi, A. Vasas Oxidized Juncuenin B Analogues with Increased Antiproliferative Activity on Human Adherent Cell Lines: Semisynthesis and Biological Evaluation Journal of Natural Products 202083 (11), 3250-3261., DOI: 10.1021 / acs.jnatprod.0c00499The results are shown in Table 4. We identified a multiple magnitude selectivity (approx. 615-times higher) of the antioxidant activity of the compound of Example 16 over its’ cytotoxicity. The other tested compounds of Example 4 and 13 also showed selectivity.).

[0332] NIH / 3T3 mouse In vitro xanthine oxidase inhibition

[0333] fibroblast compound c (pM) inhibition (%) average SD IC50 (pM) IC50 (pM) SD 4 100 85.76 85.46 85.61 0.21 13.19 45.22 4.13 13 100 102.19 105.93 104.059 2.65 0.44 22.48 0.31 16 100 97.16 107.57 102.4 7.36 0.066 40.63 7.27

[0334]

[0335] Table 4:

[0336] Conclusion: Compounds according to Examples 4-16 are new compounds. The compounds of Examples 4 to 17 according to the above results possess XO-inhibitory activity, and the tested compounds were found also to be selective due to their decreased cytotoxic side-effect.

[0337] The novel compounds according to the present invention represent new chemical scaffolds among the natural product family of flavonoids and are therefore core structures of a broad range of related compounds that can be similarly synthetised as described here. This opens door to a new chemical space related to bioactivities similar or better than those published for flavonoids and therefore of potential use to prepare new antioxidant, cardio- and cerebrovascular protective, anti-inflammatory, antiviral, and antitumor agents. The present invention demonstrates for the first time to use protoflavones as chemical intermediates to prepare i) nitrogen-containing flavonoids with a non-aromatic B-ring, and derivatives of classical flavones with unique B-ring substitution pattern processing XO-inhibitory effect. Based on the previously published SAR available for flavonoids, i.e., the ability to adopt a planar A-C-B ring structure (as in flavones or flavonols) is a must for flavonoids to act as potent XO inhibitors, and the sole exception to this was our compound 3, the high XO inhibitory potential observed for our presented compounds represents a high novelty and unexpected results.

Claims

Claims1. Compound of formula Iwhereinis a simple or double bond;if between the carbons to which R2 and R3 are attached is a simple bond the value of n is 1,if between the carbons to which R2 and R3 are attached is a double bond the value of n is 0;X is O, S or NRi, wherein, Ri is H or Ci-Ce alkyl;R2 is H, OH, Ci-Ce alkyl, or Ci-Ce alkoxy;Rj is H, OH, Ci-Ce alkyl, Ci-Ce alkoxy or -OC(O)Ci-Ce alkyl;R4 is H, Ci-Ce alkyl or-C(O)Ci-Ce alkyl, wherein the alkyl group in Ci-Ce alkyl or -C(O)Ci-Ce alkyl optionally contains one or more double or triple bond(s);ring B is selected from:wherein in (a)are independently from each other a double or simple bond,R5 is H, OH, Ci-Ce alkyl, Ci-Ce alkoxy or -C(O)Ci-Ce alkyl, wherein the alkyl group in Ci-Ce alkyl or Ci-Ce alkoxyl or -C(O)Ci-Ce alkyl optionally contains one or more double or triple bond(s), or Rs is phenyl, phenoxyl, benzyl, benzyloxy, or a halogen; Re, R7, R9 and Rio are independently selected from H, OH, halogen, Ci-Ce alkyl, Ci-Ce alkoxy and -OC(O) Ci-Ce alkyl, wherein the alkyl in Ci-Ce alkyl, Ci-Ce alkoxy or - OC(O) Ci-Ce alkyl optionally contains one or more double or triple bond(s);Rs is selected from the following:• =N-0-Ri6, wherein Rie is Ci-Ce alkyl optionally containing one double or triple bond, optionally substituted benzyl or phenyl,• -NH-O-R17, wherein R17 is Ci-Ce alkyl optionally containing one double or triple bond, optionally substituted benzyl or phenyl,• =N-NH-CO-NR27R28, wherein R27 and R28are independently selected from H, Ci-Ce alkyl optionally containing one double or triple bond, optionally substituted benzyl or phenyl,• =N-NH-CS-NR29R3O, wherein R29 and R30 are independently selected from H, Ci-Ce alkyl optionally containing one double or triple bond, optionally substituted benzyl or phenyl,• -NH2, and• -NR18R19, wherein Ris and R19 are independently selected from Ci-Ce alkyl, optionally containing one or more double or triple bond, optionally substituted benzyl or phenyl;wherein in (b)R11, R12, Ri4 and Ris are independently selected from H, OH, halogen, Ci-Ce alkyl, Ci- Ce alkoxy and -OC(O) Ci-Ce alkyl, wherein the alkyl group in Ci-Ce alkyl, Ci-Ce alkoxy or -OC(O) Ci-Ce alkyl optionally contains one or more double or triple bond(s);R13 is selected from the following:• -NH-O-R20, wherein R20is C1-C6alkyl optionally containing one double or triple bond, optionally substituted benzyl or phenyl,• -N=N-CO-NR23R24, wherein R23 and R24 are independently selected from H, Ci-Ce alkyl optionally containing one or more double or triple bond, optionally substituted benzyl or phenyl• -NH-NH-CS-NR25R26, wherein R25 and R26 are independently selected from H, Ci-Ce alkyl optionally containing one or more double or triple bond, optionally substituted benzyl or phenyl,• -NH2, and• -NR21R22, wherein R21 and R22 are independently selected from Ci-Ce alkyl, optionally containing one or more double or triple bond, optionally substituted benzyl or phenyl;provided that the compound of formula (I) is not one of the following:OH OOH Oor the salt thereof.

2. A compound according to claim 1 which has the formula (la)whereinare independently from each other a simple or double bond;if between the carbons to which R2 and R3 are attached is a simple bond the value of n is 1,if between the carbons to which R2 and R3 are attached is a double bond the value of n is 0;X is O, S or NRi, wherein, Ri is H or Ci-Ce alkyl;R2 is H, OH, Ci-Ce alkyl, or Ci-Ce alkoxy;Rj is H, OH, Ci-Ce alkyl, Ci-Ce alkoxy or -OC(O)Ci-Ce alkyl;R4 is H, Ci-Ce alkyl or-C(O)Ci-Ce alkyl, wherein the alkyl group in Ci-Ce alkyl or -C(O)Ci-Ce alkyl optionally contains one or more double or triple bond(s);Rs is H, OH, Ci-Ce alkyl, Ci-Ce alkoxy or -C(O)Ci-Ce alkyl, wherein the alkyl group in Ci-Ce alkyl or Ci-Ce alkoxyl or -C(O)Ci-Ce alkyl optionally contains one or more double or triple bond(s), or Rs is phenyl, phenoxyl, benzyl, benzyloxy, or halogen;Re, R7, R9 and Rio are independently selected from H, OH, halogen, Ci-Ce alkyl, Ci-Ce alkoxy and -OC(O) Ci-Ce alkyl, wherein the alkyl in Ci-Ce alkyl, Ci-Ce alkoxy or -OC(O) Ci-Ce alkyl optionally contains one or more double or triple bond(s);Rs is selected from the following:• =N-0-Ri6, wherein Rie is Ci-Ce alkyl optionally containing one double or triple bond, optionally substituted benzyl or phenyl,• -NH-O-R17, wherein R17 is Ci-Ce alkyl optionally containing one double or triple bond, optionally substituted benzyl or phenyl,• =N-NH-CO-NR27R28, wherein R27 and R28are independently selected from H, Ci-Ce alkyl optionally containing one double or triple bond, optionally substituted benzyl or phenyl,• =N-NH-CS-NR29R3O, wherein R29 and R30 are independently selected from H, Ci-Ce alkyl optionally containing one double or triple bond, optionally substituted benzyl or phenyl,• -NH2, and• -NR18R19, wherein Ris and R19 are independently selected from Ci-Ce alkyl, optionally containing one or more double or triple bond, optionally substituted benzyl or phenyl.

3. A compound according to claim 1 which has the formula (lb)X is O, S or NRi, wherein, Ri is H or Ci-Ce alkyl;Rj is H, OH, Ci-Ce alkyl, Ci-Ce alkoxy or -OC(O)Ci-Ce alkyl;R4 is H, Ci-Ce alkyl or-C(O)Ci-Ce alkyl, wherein the alkyl group in Ci-Ce alkyl or -C(O)Ci-Ce alkyl optionally contains one or more double or triple bond(s);Rn, R12, Ri4 and Ris are independently selected from H, OH, halogen, Ci-Ce alkyl, Ci-Ce alkoxy and -OC(O) Ci-Ce alkyl, wherein the alkyl group in Ci-Ce alkyl, Ci-Ce alkoxy or -OC(O) Ci-Ce alkyl optionally contains one or more double or triple bond(s);R13 is selected from the following:• -NH-O-R20, wherein R20is C1-C6alkyl optionally containing one double or triple bond, optionally substituted benzyl or phenyl,• -N=N-CO-NR23R24, wherein R23 and R24 are independently selected from H, Ci-Ce alkyl optionally containing one or more double or triple bond, optionally substituted benzyl or phenyl• -NH-NH-CS-NR25R26, wherein R25 and R26 are independently selected from H, Ci-Ce alkyl optionally containing one or more double or triple bond, optionally substituted benzyl or phenyl,• -NH2, and• -NR21R22, wherein R21 and R22 are independently selected from Ci-Ce alkyl, optionally containing one or more double or triple bond, optionally substituted benzyl or phenyl.

4. A compound according to any of claims 1 or 2, whereinbetween the carbons to which R2 and R3are attached is a double bond and the value of n is 0.

5. A compound according to any of claims 1 to 4, whereinX is O; and / orR4 is H; and / orR3is H.

6. A compound of formula (la) according to any of claims 2, 4 or 5, whereinare each double bond; and / orRs is OH or Ci-Ce alkoxy, wherein the alkyl group in Ci-Ce alkoxyl optionally contains one double or triple bond; and / orR6, R7, R9 and R10 are H; and / orRs is selected from the following:• =N-0-R16, wherein R16is C1-C6alkyl optionally containing one double or triple bond, or benzyl;• =N-NH-CO-NH2, and• =N-NH-CS-NH27. A compound of formula (la) according to any of claims 2, 4, 5 or 6, whereinR5 is OH, -O-methyl, -O-ethyl, -O-propyl, -O-isopropyl, -O-butyl, -O-tert-butyl, -O-propargyl or -O-allyl; and / orR6, R7, R9and R10are H; and / orRs is selected from the following:• =N-0-Ri6, wherein Rie methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, propargyl, allyl or benzyl,• =N-NH-CO-NH2, and• =N-NH-CS-NH28. Compound of formula (la) according to any of claims 2, 4 to 7, whereinR5 is OH or O-allyl and / orRs is =N-NH-CO-NH2, or =N-NH-CS-NH29. A compound of formula (lb) according to claim 3 or 5, whereinR11, R12, R14and R15are independently selected from H and halogen, and / orR13 is selected from the following:• -NH-O-R20, wherein R20is C1-C6alkyl optionally containing one double or triple bond,• -N=N-CO-NH2,• -NH-NH-CS-NH2, and• -NH210. A compound of formula (lb) according to any of claim 3, 5 or 9, whereinR11, R12, and R15are H and / orR14is H or Cl, and / orR13 is selected from the following:• -NH-O-R20, wherein R20is C1-C6alkyl optionally containing one double or triple bond,• -N=N-CO-NH2, and• -NH211. A compound according to claim 1 selected from the following:and12. A compound of any of claims 1 to 11 for use as a medicament.

13. A compound of formula (I)wherein ring B, R2, R3, R4, n and X are as defined in claim 1 or a salt thereof, or a compound according to any of claims 1 to 11 for use in the treatment or prevention of a disease or disorder associated with the increased expression of xanthine oxidase enzyme, such as gout, chronic kidney disease, chronic stress-induced cerebrovascular dysfunction, cognitive impairment, chronic heart failure, COPD, metabolic syndrome, metabolic disorders such as diabetes, hypertension, liver dysfunction and cardiovascular disease.

14. Process for the preparation of a compound of formula (lb),R12wherein X, R3, R4, R11, R12, R13, R14, R15are as defined in claim 1,characterized in that, the process includeswherein X is O, reacting the corresponding protoapigenone 1'-O-C1-6alkyl ether derivative, wherein the Ci-6 alkyl optionally contains one or more double or triple bonds, or wherein X is S or NRi reacting the corresponding analoguewith a compound of formula Y-R13 ”, which is an amino derivative of the R13 substituent being able to introduce R13 through the formation of an intermediate that is a corresponding analog of formula (la), followed by a subsequent elimination of the I'-O-Ci-e alkyl ether group and the re-aromatization of the B-ring.

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