AU9350898A - Bisethers of 1-oxa, aza and thianaphthalen-2-ones as phospholamban inhibitors - Google Patents

Description

WO 99/15523 PCT/FI98/00755 BISETHERS OF 1-OXA, AZA AND THIANAPHTHALEN-2-ONES AS PHOSPHOLAMBAN INHIBITORS The present invention relates to new therapeutically active compounds and salts and esters thereof as well as new intermediates. The 5 invention also relates to pharmaceutical compositions containing these compounds as active ingredients. The compounds of the invention have phospholamban inhibiting properties and are useful in the treatment of heart failure and stunned myocardium. Compounds of the present invention have the structure represented o10 by formulae (I) or (II): R4A 0 R3 R1 I( R5A R2 R5 15 R6 R8 0 ICHn I R9A R7 20 in which R1 is hydrogen, alkyl, alkenyl, aryl, arylalkyl, hydroxyalkyl, halogenalkyl, alkoxy, COR1 0, CONR1 0R 1, OR10, S(O)mR10, NR10COR11 or NR10R11, where R10 is hydrogen, alkyl, alkenyl, aryl, arylalkyl, hydroxyalkyl, halogenalkyl, alkoxy or hydroxy and R11 is hydrogen, 25 alkyl, aryl, arylalkyl, alkoxy, aryloxy, hydroxy or acyl, or in case where X is NR1 1, can R1 also be carboxylalkyl, R6 is hydrogen, alkyl, alkenyl, aryl, arylalkyl, WO 99/15523 PCT/FI98/00755 2 R2 and R7 mean hydrogen, alkyl, aryl, arylalkyl, alkenyl, COR10, CONR10R11, halogen, trifluoromethyl, nitro or cyano, where R10 and R11 are defined as above, R3 is hydrogen, alkyl, aryl or arylalkyl, 5 A means alkyl or substituted alkyl, m is 0-2 and n is 1-3, Y means O, NR11 or S, where R11 is the same as above, X means O, NR11 or S, where R11 is the same as above, R4, R5, R8 and R9 mean independently one of the following groups: N' N , N-N / H or in case where X is NR11, can R4, R5, R8 and R9 also 15 independently mean HOOC-, R1200C-, H2NCO- or HOHNCO- wherein R12 means alkyl, arylalkyl or aryl, and wherein each aryl residue defined above by itself or as part of another group may be substituted, and pharmaceutically acceptable salts and esters thereof. 20 In one class of preferred compounds and pharmaceutically acceptable salts and esters thereof are compounds of formula (I) wherein R2 is hydrogen. In a subclass of this class of compounds and pharmaceutically acceptable salts and esters thereof R1 is hydrogen, C1-6 alkyl, C2-6 alkenyl, C6-10 aryl, C7-12 arylalkyl, C1-6 hydroxyalkyl, C1-6 halogenalkyl or C1-6 25 alkoxy. In a group of this subclass of compounds and pharmaceutically acceptable salts and esters thereof, Y is O or S, preferably O; and X is O. In another group of this subclass of compounds and pharmaceutically acceptable salts and esters thereof, Y is O or S, preferably O; and X is NR1 1, where R1 1 is hydrogen, C1-6 alkyl, C6-10 aryl, C7-12 arylalkyl, C1-6 30 alkoxy, C6-10 aryloxy, hydroxy, C1-6 alkanoyl or C1-6 carboxyalkyl. In a subgroup of these group of compounds and pharmaceutically acceptable salts and esters thereof, R3 is hydrogen, C1-6 alkyl, C6-10 aryl, or C7-12 arylalkyl, preferably C1-6 alkyl, most preferably methyl. In a family of these WO 99/15523 PCT/FI98/00755 3 subgroups of compounds and pharmaceutically acceptable salts and esters thereof, A is preferably straight-chain or branched C1-4 alkylene. In another preferred class of compounds and pharmaceutically acceptable salts and esters thereof, compounds have formula (11) wherein 5 R7 is hydrogen. In a subclass of this class of compounds and pharmaceutically acceptable salts and esters thereof R6 is hydrogen, C1-6 alkyl, C6-10 aryl, C7-12 arylalkyl; and n is 1, 2 or 3, preferably 1 or 2. In a group of this subclass of compounds and pharmaceutically acceptable salts and esters thereof, Y is O or S, preferably O; and X is O. In another group of 10 this subclass of compounds and pharmaceutically acceptable salts and esters thereof, Y is O or S, preferably O; and X is NR11, where R11 is hydrogen, C1-6 alkyl, C6-10 aryl, C7-12 arylalkyl, C1-6 alkoxy, C6-10 aryloxy, hydroxy, C1-6 alkanoyl or C1-6 carboxyalkyl. In a subgroup of these group of compounds and pharmaceutically acceptable salts and esters 15 thereof, A is preferably straight-chain or branched C1-4 alkylene. Each aryl residue in each of these preferred classes of compounds, by itself or as part of another group, may be substituted by 1 to 3, preferably 1 or 2, most preferably one of fluorine, chlorine, bromine, iodine, trifluoromethyl, amino, C1-4 alkyl, C1-4 alkoxy, phenyl, naphthyl, halophenyl, 20 halonaphthyl, benzyl, phenethyl, halobenzyl, halophenethyl, naphthylmethyl, naphthylethyl, C4-7 cycloalkyl, C1-4 alkyl (C4-7)cycloalkyl, hydroxy, mono (C1-4)alkylamino, di-(C1-4)alkylamino, C1-6 alkanoylamino, phenylcarbonylamino, naphthylcarbonylamino nitro, cyano, thiol, or C1-6 alkylthio. 25 Brief description of the drawings FIG. 1A shows the effect of the compound of Example 1c (50 and 100 pM) on the Ca 2 + uptake rate into the cardiac muscle SR vesicles. FIG. 1B shows the effect of the compound of Example 1 c (50 and 100 pM) on the Ca 2 + uptake rate into the fast skeletal muscle SR vesicles. 30 FIG. 2A shows the development of stunned myocardium and the subsequent decrease in the left ventricular systolic pressure. FIG. 2B shows the complete inhibition of the development of stunned myocardium by the compound of Example 8g. The compounds of the invention can be prepared from the 1,3 35 dihydroxy substituted heteroaromatics by alkylation of the dihydroxy WO 99/15523 PCT/FI98/00755 4 compounds by suitable alkylating agents, for example by chloroacetonitrile or bromoacetic ester according to the following Scheme 1, wherein R1 ,R2, R3, X and Y are the same as defined above, R' is a protecting group for the hydroxyl, e.g. methyl, benzyl or tetrahydropyranyl. 5 SCHEME 1 OH R3 OH R3 R 1 R1 + B1", O IN ! + 0 (I/) CK'" N HO O HO X Y H 2R' RR2 R2

K

2

CO

3 , DMF

K

2

CO

3 , DMF 60-120 0 C, 1-5 h 60-120°C, 1-5 h O R3 R1 O R3 O R1 (IV) o x Y (VI) R2 OR2 SNaN 3 , NH 4 CI, DMF O 80-120'C, 1-3 h NaOH or HCI N O R3 H -5O R3 (V) N 1 Ri 0 Ri (VII) O X Y O X Y H # " 0 X R2 HR2 N-N O 10 The cyano compound (IV) described above is used to prepare the 1,2,4 oxadiazole and 1,2,4-thiadiazole derivatives using the methods described in J. Med. Chem. 1996, 39, 5228-5235. The syntheses are shown in Scheme 2, wherein R1, R2, R3, X and Y are the same as defined above.

WO 99/15523 PCT/FI98/00755 5 SCHEME 2 O R3 1

-

~(IV) R2 N\OH X HCI, DMSO H2N O R3 NN R1 HO (VIII) X

H

2 R2 H O Im 2 CS, BFaxEt 2 0 EtCOCI, DBU N O R3 N.O R3 S-N R1 O-N R1(X) (IX(XK (IX) x H O X Y NH R2 R2 S-N 5 The other heterocyclics as groups R4, R5, R8 and R9 are prepared as described in Bioorg. Med. Chem. Lett., 1994, 4, 45-50. The dihydroxyaromatics (111) are made by use of the literature methods. The coumarins (XIV), (XVI) and (XX) are made by the use of the 10 Knoevenagel condensation or von Pechmann reaction as presented in Scheme 3 and 4, where R1,R2 and R3, are the same as defined above, Z is alkyl, aryl, arylalkyl or alkenyl and R' is a protecting group for the hydroxyls e.g. methyl, benzyl or tetrahydropyranyl.

WO 99/15523 PCT/FI98/00755 6 SCHEME 3. 0 'ZN OH ZnCI 2, HCI, Et20 (XI) OH O OH z 0 ZO Zn(Hg)-HCI O R3 O Ri R3COCH(R1)CO2Et (XIII) gI (XII) O O O HCI, BOH OH z z HBr OH R3 N 'N R I H c 0 0 (XIV) 5

Z

WO 99/15523 PCT/FI98/00755 7 SCHEME 4. A OH R3 AO NOH + R1 (XV) 0 HCI, EtOH OH R3 N R1 (XVI) H 0 B R' -O 0 R 1 N X IRl (XVIII) -I R' R2 Piperidine or DBU OR R1 RO (XIX) RO O

R

2 HBr or pyridinex HCI OH R1 (XX) HO 0 0 R2 5 The quinolinones are prepared by the Knorr reaction as described in Scheme 5, wherein R1, R11 and R3 are the same as defined above, X is a halogen.

WO 99/15523 PCT/FI98/00755 8 SCHEME 5. R3 + R1 (XXI) O NH2 0 140-160oC 0 R3 M_.- R1 (XXII) O O 0 R3

H

2

SO

4 M1 (XXIII) I H H NaH, RlX 0 R3 o R1 (XXIV) O) N 0 I IR1 Pyidinex HCI OH R3 H Io (XXV) H N O0 I R11 WO 99/15523 PCT/FI98/00755 9 The cyclic compounds (11) can be prepared correspondingly from compound (XXXI) which can be prepared according to the Scheme 6, wherein R2 and R6 are the same as defined above, R' is a protecting group for the hydroxyls e.g. methyl, benzyl or tetrahydropyranyl. 5 SCHEME 6. A OH [CH n R6 H OH O (XXVI) HCI, EtOH [CH2]_ OH [cH 2 R6 H 1 (XXVIl) B R O [CHn R6 (XXV I) (XXIX) 0 OH 0 R' R2 'K HCI, EtOH RO 0 [CH 2 n R6 (XXX) p 00 R" R2 HBr or pyridinex HOC OH [CH2 R6 H0 (XXXI) H O O0 R2 WO 99/15523 PCT/FI98/00755 10 Cyclic quinolinone compounds (11) can be prepared correspondingly from (XXVI) using Scheme 5. Salts and esters of the compounds, when applicable, may be 5 prepared by known methods. Physiologically acceptable salts are useful as active medicaments, however, preferred are the salts with alkali or alkaline earth metals. Physiologically acceptable esters are also useful as active medicaments. Examples are the esters with aliphatic or aromatic alcohols. The term "alkyl" as employed herein by itself or as part of another 10 group includes both straight, branched and cyclized chain radicals of up to 18 carbon atoms, preferably 1 to 8 carbon atoms, most preferably 1 to 4 carbon atoms. The term "lower alkyl" as employed herein by itself or as part of another group includes straight, branched and cyclized chain radicals of 1 to 7, preferably 1 to 4, most preferably 1 or 2 carbon atoms. Specific 15 examples for the alkyl and lower alkyl residues, respectively, are methyl, ethyl, propyl, isopropyl, butyl, tert. butyl, pentyl, cyclopentyl, hexyl, cyclohexyl, octyl, decyl and dodecyl including the various branched chain isomers thereof. The term "acyl" as employed herein by itself or as part of another 20 group refers to an alkylcarbonyl or alkenylcarbonyl group, the alkyl and alkenyl groups being defined above. The term "aryl" as used herein by itself or as part of another group refers to a monocyclic or bicyclic group containing from 6 to 10 carbon atoms in the ring portion. Specific examples for aryl groups are phenyl, naphtyl and 25 the like. "Aroyl" means in a corresponding way an arylcarbonyl group. The term "alkoxy" as employed herein by itself or as part of another group includes an alkyl group as defined above linked to an oxygen atom. "Aryloxy" means in a corresponding way an aryl group linked to an oxygen atom. 30 The term "substituted" as used herein in connection with various residues refers to halogen substituents, such as fluorine, chlorine, bromine, iodine or trifluoromethyl group, amino, alkyl, alkoxy, aryl, alkyl-aryl, halogen aryl, cycloalkyl, alkylcycloalkyl, hydroxy, alkylamino, alkanoylamino, arylcarbonylamino, nitro, cyano, thiol, or alkylthio substituents.

WO 99/15523 PCT/FI98/00755 11 The "substituted" groups may contain 1 to 3, preferably 1 or 2, most preferably 1 of the above mentioned substituents. Compounds of the invention may be administered to a patient in therapeutically effective amounts which range usually from about 0.1 to 500 5 mg per day depending on the age, weight, condition of the patient, administration route and the phospholamban inhibitor used. The compounds of the invention can be formulated into dosage forms using the principles known in the art. It can be given to a patient as such or in combination with suitable pharmaceutical excipients in the form of tablets, dragees, capsules, o10 suppositories, emulsions, suspensions or solutions. Choosing suitable ingredients for the composition is a routine for those of ordinary skill in the art. It is evident that suitable carriers, solvents, gel forming ingredients, dispersion forming ingredients, antioxidants, colours, sweeteners, wetting compounds and other ingredients normally used in this field of technology 15 may be also used. The compositions containing the active compound can be given enterally or parenterally, the oral route being the preferred way. The contents of the active compound in the composition is from about 0.5 to 100 %, preferably from about 0.5 to about 20 %, per weight of the total composition. 20 EXPERIMENTS Experiment 1. Effect on calcium uptake into the SR vesicles prepared from cardiac and fast skeletal muscle The inhibitory effect of a given compound on phospholamban can be 25 demonstrated by measuring the effect of the compound on calcium uptake into the SR vesicles prepared from cardiac tissue and into SR vesicles prepared from fast skeletal muscle (psoas m.). Both kind of SR vesicles contain Ca 2 +-ATPase but the vesicles from the fast skeletal muscle do not contain phospholamban (Hoh JFY, "Muscle fiber types and function", 30 Current Opinion in Rheumatology, 4:801-808, 1992). An increase in the calcium uptake into the SR vesicles prepared from cardiac tissue but not into the SR vesicles prepared from fast skeletal muscle indicates that the compound relieves the inhibitory effect of phospholamban on SR Ca 2 + ATPase and thus acts as a phospholamban inhibitor. Since phospholamban 35 represses both the rates of relaxation and contraction in the mammalian heart through its inhibitory effects on the cardiac SR Ca 2 +-ATPase, a WO 99/15523 PCT/FI98/00755 12 compound relieving these effects is potentially useful in the treatment of heart failure. Method Guinea pigs (10-12) were decapited. Their hearts or the psoas 5 muscles were excised, washed in ice-cold 0.9 % NaCI and cut into pieces in a buffer containing 20 mM Tris-maleate, 0.3 M sucrose, pH 7.0. Thereafter tissue pieces were homogenized with Polytron and further with Potter (10 strokes). The homogenate was centrifugated at 1000 g for 15 min at 4 0C. The supernatant was collected and the pellet was resuspended into 5 ml of 10 the buffer (20 mM Tris-maleate, 0.3 M sucrose, pH 7.0) and recentrifugated at 1000 g for 10 min at 4 oC. The obtained supernatant was combined with the earlier collected supernatant and centrifugated once again at 10 000 g for 20 min at 4 oC. The final supernatant was filtered into a bottle equipped with a magnetic stirrer. KCI was added to the filtered supernatant to achieve 15 the final concentration of 0.6 M (at 4 oC). The obtained solution was centrifugated at 100 000 g for 60 min at 4 oC. The pellet was suspended in 5 ml of the buffer containing 20 mM Tris-maleate, 0.3 M sucrose, pH 7.0 and centrifugated at 100 000 g for 60 min at 4 oC. The obtained pellet was suspended in 5 ml of buffer containing 20 mM Tris-maleate, 0.3 M sucrose, 20 0.1 M KCI, pH 7.0 and stored at -80 0C until use. The protein concentration was also measured in order to standardise the separately prepared vesicle preparations. In the calcium uptake assay, the fluorescent indicator, fluo-3 was used to detect the decrease of the extravesicular Ca 2 +-concentration, when 25 the SR Ca 2 +ATPase was transferring Ca 2+ from the extravesicular space into the SR-vesicles. The SR-vesicles obtained above (50 pg protein/ml) were pre incubated with or without the test compound at 37 oC for 5 min in the assay buffer containing 40 mM imidazole, 95 mM KCI, 5 mM NaN3, 5 mM MgCl 2 , 30 0.5 mM EGTA, 5 mM potassium oxalate, 2 pM ruthenium red, 5 pM fluo-3, pH 7.0. The free calcium was adjusted to 0.1 pM or to 0.04 pM by CaC 2 . The reaction was initiated by adding ATP (5 mM). The final reaction volume was 1.5 ml. The fluorescence of reaction mixture was measured for 3 min by using the excitation and emission wavelengths of 510 nm and 530 nm, 35 respectively.

WO 99/15523 PCT/FI98/00755 13 Results Figures 1A and 1B show the effect of the compound of Example 1 c (50 and 100 pM) on the Ca 2+ uptake rate into the cardiac (A) and fast skeletal muscle (B) SR vesicles. It can be seen that the compound of the 5 invention accelerated the calcium uptake into the cardiac SR vesicles but did not change the calcium uptake into the SR vesicle prepared from the fast skeletal muscle. Table 1 shows the effects of various other phospholamban inhibitors of formula (I) or (11) on the Ca 2+ uptake rate into the cardiac (A) and fast 10 skeletal muscle (B) SR vesicles. The experiments were carried out at 0.1 pM and 0.04 pM free calcium concentrations, respectively. TABLE 1. Stimulation (%) of the Ca 2+ uptake into the vesicle preparations obtained from the ventricular myocardium (A) and fast skeletal muscle (B) of 15 the guinea-pig heart. Compound of The stimulation (%) Example No. of Ca 2+ uptake (100 pM) A B 20 3c** 51 0 2c 26 -1 7c 5 -17 8g* 18 0 25 11b 28 nd 12 32 nd 13d*** 23 nd 14c* 18 nd 18e 13 nd 30 21 11 nd 23**** 20 nd * 10 pM, **20 pM, ***50 pM, ****5 pM nd=not determined 35 WO 99/15523 PCT/FI98/00755 14 Experiment 2. The effects on the left ventricular pressure derivatives Method Guinea-pigs of either sex weighing 300-400 g were used in the study. After the guinea-pig was sacrificed by a blow on the skull and decapitated 5 the heart was rapidly excised. The heart was then rinsed in cold oxygenated perfusion buffer. A cannula was inserted into the aorta and secured with a ligature. Retrograde perfusion began as soon as the heart was placed in a thermostatically controlled moist chamber of the Langendorff apparatus. Modified Tyrode solution (37 oC), equilibrated in a thermostatically controlled 10 bulb oxygenator with carbogen (95 % 02 and 5% CO2) was used as a perfusion buffer. The composition of the Tyrode solution was (in mM): NaCI 135; MgCl 2 x 6H 2 0 1; KCI 5; CaCI2 x 2H 2 0 2; NaHCO 3 15; Na 2

HPO

4 X 2H 2 0 1; glucose 10; pH 7.3-7.4. The experiments were carried out under constant pressure condition (50 mmHg). After a short prestabilization (10 15 min) a latex balloon (size 4) was carefully placed into the left ventricle through the left pulmonary vein and the left atrium. The latex balloon was attached to a stainless-steel cannula coupled with a pressure transducer. The latex balloon, the cannula and the chamber of the pressure transducer were filled with ethylene glycol / water (1:1) mixture avoiding any air-bubble. 20 The isovolumetric left ventricular pressure was recorded through the pressure transducer. At the beginning of the experiment, the volume of the balloon was adjusted to obtain a diastolic pressure of approximately 5 mmHg. Before starting the experiment, the heart was allowed to stabilise further for 30 - 50 min with vehicle (0.1% DMSO) in the perfusion buffer. 25 After 15 min baseline recording various concentrations of the test compound were added to the perfusion buffer at 15 min intervals. The concentration range of 0.3 - 30 pM was tested. The vehicle concentration (0.1% DMSO) was kept constant throughout the experiment. Results 30 The ECso 50 values and maximum effects (% change from baseline) of various compounds of the invention on left ventricular systolic pressure are given in Table 2.

WO 99/15523 PCT/FI98/00755 15 TABLE 2. The EC 5 o values and maximum effects (% change from baseline) on left ventricular systolic pressure. Compound of EC 50 maximum 5 Example No. (pM) effect (%) 1c 9 +52 at 30 pM 3c 4 +63 at 10 pM 10 5c >10 +14 at 30 pM 6c 0.5 +25 at 10 pM 7c 2.5 +29 at 10 pM 8g 2 +64 at 10 pM 9d 5 +50 at 30 pM 15 12 5 +22 at 10 pM 13d 10 +48 at 30 pM 14c 1.5 +25 at 10 pM 15c 3 +37 at 10 pM 16c 10 +57 at 30 pM 20 18e 10 +35 at 30 pM 19e 6 +39 at 30 pM Experiment 3. Effect on the development of stunned myocardium in 25 isolated guinea-pig Langendorff heart Method Guinea-pigs of either sex weighing 300-400 g were used in the study. After the guinea-pig was sacrificed by a blow on the skull and decapitated 30 the heart was rapidly excised. The heart was then rinsed in oxygenated perfusion buffer. A cannula was inserted into the aorta and secured with a ligature. Retrograde perfusion began as soon as the heart was placed in a thermostatically controlled moist chamber of the Langendorff apparatus. Modified Tyrode solution (37 oC), equilibrated in a thermostatically controlled 35 bulb oxygenator with carbogen (95 % 02 and 5% CO2) was used as a perfusion buffer. The composition of the Tyrode solution was (in mM): NaCI 135; MgCI 2 x 6H 2 0 1; KCI 5; CaCI2 x 2H 2 0 2; NaHCO 3 15; Na 2

HPO

4 x 2H 2 0 1; glucose 10; pH 7.3-7.4. The experiments were carried out under constant pressure condition (50 mmHg). After a short prestabilization (10 40 min) a latex balloon attached through the stainless-steel cannula to a pressure transducer was carefully placed into the left ventricle through the left pulmonary vein and the left atrium. The latex balloon, the cannula and WO 99/15523 PCT/FI98/00755 16 the chamber of the pressure transducer were filled with ethylene glycol / water (1:1) mixture avoiding any air-bubble. The isovolumetric left ventricular pressure was recorded through the pressure transducer. At the beginning of the experiment, the volume of the balloon was adjusted to obtain the end 5 diastolic pressure of approximately 5 mmHg. Before starting the experiment, the spontaneously beating heart was allowed to stabilise further for 30 - 50 min with vehicle (0.1% DMSO) in the perfusion buffer. After 15 min baseline recordings compound of Example 8g (10 pM) was added to the perfusion buffer. The heart was 15 min later exposed to 10 the 8 minute period of global ischemia followed by reperfusion. This procedure was then repeated twice at 35 min intervals. Another series of experiments was performed with vehicle instead of compound of Example 8g. The vehicle concentration (0.1% DMSO) was kept constant throughout the experiments. The baseline value was the average of the two minute 15 recordings obtained just before compound of Example 8g or vehicle was added to the perfusion buffer. The preischemia values were the average of the two minute recordings obtained just before each ischemia period and the reperfusion values were the average of the two minute recordings obtained at 8 min during each reperfusion period. 20 The results are shown in Figures 2 A and B. Figure 2A shows the development of stunned myocardium and the subsequent decrease in the left ventricular systolic pressure in the control group. Figure 2B shows that the phospholamban inhibitor of Example 8g completely inhibited the development of stunned myocardium. Givens are mean ± SEM of 2-3 25 experiments. EXAMPLES Example 1. Preparation of 3-Benzyl-5,7-bis[(1 H -tetrazol-5-yl) methoxy]-4-methyl-2H -1 -benzopyran-2-one 30 a) 3-Benzyl-5,7-dihydroxy-4-methyl-2H -1 -benzopyran-2-one WO 99/15523 PCT/FI98/00755 17 OH HO O O A solution of phloroglucinol dihydrate (20 g) and ethyl 2-benzylaceto acetate (27.5 ml) in ethanol (320 ml) was treated with dry HCI at 0OC for five hours and the solution was kept at that temperature overnight. The yellow 5 solution was concentrated and triturated with water, the solids filtered, washed with water and dried. The resulting hydrate was thrice evaporated to dryness from toluene, triturated with pethroleum ether (bp. 40-600C) and filtered. Yield 33,4 g (96 %). Melting point 258-260 oC. 1 H-NMR (DMSO-d 6 , 400MHz): 2.525 (s, 3H, CH3), 3.887 (s, 2 H, 10 CH2Ph), 6.171 (d, 1H, J = 2,4 Hz), 6.274 (d, 1H, J = 2,4 Hz), 7.167-7.279 (m, 5H, Ph), 10.2 (s, 1 H, OH), 10.47 (s, 1 H, OH). b) 3-Benzyl-5,7-bis(cyanomethoxy)-4-methyl-2H -1-benzopyran-2-one 0 N/ O O 0 Chloracetonitrile (6.86 g), potassium carbonate (23.9 g) and 12.2 g of 15 the product from example la were stirred in 120 ml of DMF at 100C under nitrogen for two hours. The reaction mixture was cooled and poured into ice water. The solids were filtered and washed with water. Yield 13.8 g (88 %). Melting point 147-154oC. 1 H-NMR (DMSO-d 6 , 400MHz): 2.525 (s, 3H, CH3), 3.969 (s, 2H, 20 CH2Ph), 5.307 (s, 2H, OCH2CN), 5.314 (s, 2H, OCH2CN), 6.814 (d, 1 H, J = 2.5 Hz), 6.940 (d, 1H, J = 2.5 Hz), 7.18-7.292 (m, 5H, Ph).

WO 99/15523 PCT/FI98/00755 18 c) 3-Benzyl-5,7-bis[(1 H -tetrazol-5-yl)methoxy]-4-methyl-2H -1 benzopyran-2-one N, N-N H 00 ,IN N-N H The product from example 1 b (1 g), sodium azide (0.42 g) and 5 ammonium chloride (0.34 g) were stirred in DMF (5 ml) under nitrogen at 100 oC for 5 hours. The reaction mixture was allowed to cool down and then poured into ice water. The pH of the solution was adjusted to 10-11 and then the solution either extracted once with ethyl acetate or filtered through CELITE. The solution was acidified to pH 2 with hydrochloric acid, kept at 10 50C and filtered. Yield 0.96 g (81 %). Melting point 229-233oC. 1 H-NMR (DMSO-d 6 , 400MHz): 2.468 (s, 3H, CH3), 3.937 (s, 2H, CH2Ph), 5.596 (s, 2H, OCH2Tet), 5.602 (s, 2H, OCH2Tet), 6.832 (d, 1H, J =2.4 Hz), 6.851 (d, 1H, J = 2.4 Hz), 7.171-7.283 (m, 5H, Ph). Example 2. Preparation of 7,8,9,10-Tetrahydro-1,3-bis[(1 H -tetrazol-5 15 yl)methoxy]-7-phenyl-6H-dibenzo[b,d]pyran-6-one a) 7,8,9,10-Tetrahydro-1,3-dihydroxy-7-phenyl-6H-dibenzo[b,d]pyran 6-one OH HO &O O A solution of phloroglucinol (0.7 g) and 2-ethoxycarbonyl-3 20 phenylcyclohexanone (1,5 g) in ethanol was treated with dry HCI as described in example 1 a. The product was first recrystallized from ethanol water (1:1) and then triturated with ether. Yield 0.61 g.

WO 99/15523 PCT/FI98/00755 19 1 H-NMR (DMSO-d 6 , 400MHz): 1.38-1.52 (m, 1H), 1.57-1.66 (m, 1H), 1.69-1.78 (m, 1H), 1.86-1.96 (m, 1H), 2.9-3.02 (m, 1H), 3.3-3.4 (m,1H), 4.050 (b, 1H), 6.157 (d, 1H, J = 2.4 Hz), 6.297 (d, 1H, J = 2.4 Hz), 7.076 7.265 (m, 5H), 10.153 (s, 1H), 10.456 (s, 1H). 5 b) 7,8,9,10-Tetrahydro-1,3-bis(cyanomethoxy)-7-phenyl-6H dibenzo[b,d]pyran-6-one N The product from example 2a (0.5 g) was treated with chloroaceto nitrile (0.25 g) and potassium carbonate (1.12g) in DMF (5 ml) as described 10 in example lb. Yield 0.6 g. 1 H-NMR (DMSO-d 6 , 400MHz): 1.38-1.58 (m, 1H), 1.6-1.7 (m, 1H), 1.7-1.76 (m, 1 H), 1.89-1.99 (m, 1 H), 2.9-3.03 (m, 1 H), 3.2-3.28 (m 1 H), 4.111(b, 1H), 5.314 (s, 2H), 5.349 (s, 2H), 6.840 (d, 1H, J =2.5 Hz), 6.925 (d, 1H, J = 2.5 Hz), 7.108-7.274 (m, 5H). 15 c) 7,8,9,10-Tetrahydro-1,3-bis[(1 H -tetrazol-5-yl)methoxy]- 7-phenyl 6H-dibenzo[b,d]pyran-6-one r , N--N Nz'NH N The product from example 2b (0.6 g) was treated with sodium azide (0.2 g) and ammonium chloride (0.17 g) in DMF (5 ml) as in example 1c. The 20 product was recrystallized from a mixture of DMF, ethanol and water (approximately 1:2:3). Yield 0.41 g. Melting point: 153-1540C.

WO 99/15523 PCT/FI98/00755 20 1 H-NMR (DMSO-d 6 , 400MHz): 1.38-1.5 (m, 1H), 1.5-1.6 (m, 1H), 1.69-1.76 (m, 1H), 1.87-1.96 (m, 1H), 2.9-3.05 (m, 1H), 3.2-3.3 (m, 1H), 4.094 (b, 1H), 5.602 (s, 2H), 5.643 (s, 2H), 6.832 (d, 1 H, J = 2.3 Hz), 6.851 (d, 1H, J = 2.3.Hz), 7.089-7.212 (m, 5H). 5 Example 3. Preparation of 3-Benzyl-5,7-bis[( 2,5-dihydro-5-oxo-4H 1,2,4-oxadiazol-3-yl)- methoxy]-4-methyl-2H -1-benzopyran-2-one a) 3-Benzyl-5,7-bis[(hydroxyamidino)methoxy]-4-methyl-2H -1 benzopyran-2-one

H

2 N HON 00 0 0

H

2 N" N' OH 10 Triethylamine (1.94 ml) was added to a suspension of hydroxylamine hydrochloride (0.97 g) in DMSO (2 ml) and the resulting mixture stirred at room temperature for thirty minutes. The crystals were filtered and washed with THF. The filtrate was concentrated and the product from example lb (0.5 g) added. This solution was kept at 75 oC overnight. The reaction 15 mixture was treated with ice water, the pH adjusted to 11 and the solids filtered, washed with water, and dried. Yield 0.5 g. Melting point: 155-160oC. 1 H-NMR (DMSO-d 6 , 400MHz): 2.56 (s, 3H, CH3), 3.938 (s, 2H), 4.466 (s, 2H), 4.486 (s, 2H), 5.565 (s, H, NH2), 5.709 (s, 2H, NH2), 6.658 (d, 1H, J =2.4 Hz), 6.692 (d, 1H, J =2.4 Hz), 7.168-7.284 (m, 5H, Ph), 9.346 20 (s, 1H, OH), 9.362 (s, 1H, OH). b) 3-Benzyl-5,7-bis[(ethoxycarbonyloxyamidino)methoxy]-4-methyl 2H -1 -benzopyran-2-one WO 99/15523 PCT/FI98/00755 21 00 0 0 0

H

2 N 0 O. -N Ethyl chloroformiate (0.45 ml) was added to a solution of the product from example 3a (1 g) and pyridin (0.38 ml) in DMF (5 ml) at OC. The reaction mixture was kept at that temperature for an additional 30 minutes 5 and then ice water added. The solids were filtered and washed with water. Yield 1.63 g. Melting point 87-92o0C. 1 H-NMR (DMSO-d 6 , 400MHz): 1.215-1.256 (m, 6H), 2.553 (s, 3H), 3.947 (s, 2H), 4.140-4.198 (m, 4H), 4.566 (s, 2H), 4.599 (s, 2H), 6.688 (d, 1H, J = 2.4 Hz), 6.718 (d, 1H, J = 2.4 Hz), 6.792 (b, 2H, NH2), 6.818 (b, 2H, 10 NH2), 7.171-7.285 (m, 5H). c) 3-Benzyl-5,7-bis[( 2,5-dihydro-5-oxo-4H-1,2,4-oxadiazol-3-yl) methoxy]-4-methyl-2H -1 -benzopyran-2-one H N-, O NO H 0 0 0 O-N The product from the previous example (1.5 g) and DBU (0.8 ml) in 15 DMF (5 ml) was stirred at room temperature overnight. The reaction mixture was treated with ice water and acidified. The solids were filtered and washed with water. The resulting solid mass was taken in 0.1 N sodium hydroxide WO 99/15523 PCT/FI98/00755 22 solution, treated with activated carbon and finally acidified. Yield 0.64 g. Melting point: 130-1360C. 1 H-NMR (DMSO-d 6 , 400MHz): 2.524 (s, 3H), 3.954 (s, 2H), 5.187 (s, 2H), 5.215 (s, 2H), 6.748 (d, 1 H, J = 2.4 Hz), 6.834 (d, 1 H, J = 2.4 Hz), 5 7.158-7.289 (m, 5H), 12.8 (b, 2H). Example 4. Preparation of 7,8,9,10-Tetrahydro-bis[(1 H -tetrazol-5 yl)methoxy] -1,3-dihydroxy-6H-dibenzo[b,d]pyran-6-one a) 7,8,9,10-Tetrahydro-1,3-dihydroxy-6H-dibenzo[b,d]pyran-6-one OH HO 0 O 10 Phloroglucinol (1 g) and ethyl 2-oxocyclohexane carboxylate (1.32 g) were stirred in 75 % sulfuric acid (10 ml) overnight, the mixture poured into ice water and filtered. Yield: 1.55 g. 1 H-NMR (DMSO-d 6 , 400MHz): 1.65 (b, 4H), 2.345 (b, 2H), 3.037 (b, 2H), 6.138 (d, 1 H, J = 2.4 Hz), 6.245 (d, 1 H, J = 2.4 Hz), 10.069 (b, 1 H, OH), 15 10.322 (s, 1 H, OH). b) 7, 8, 9, 10-Tetrahydro-bis(cyanomethoxy)-1,3-dihydroxy-6H dibenzo[b,d]pyran-6-one 0 NZ The product from the previous example (0.5 g), chloroacetonitrile 20 (0.34 g) and potassium carbonate (1.5 g) in DMF (5 ml) were reacted as in example lb. Yield: 0.44 g. 1 H-NMR (DMSO-d 6 , 400MHz): 1.68 (b, 4H), 2.41 (b, 2H), 3.00 (b, 2H), 5.297 (s, 2H), 5.309 (s, 2H), 6.797 (d, 1 H, J = 2.4 Hz), 6.899 (d, 1H, J = 2.4 Hz).

WO 99/15523 PCT/FI98/00755 23 c) 7,8,9,10-Tetrahydro-bis[(1 H -tetrazol-5-yl)methoxy] -1,3-dihydroxy 6H-dibenzo[b,d]pyran-6-one N, N O 10 N-N \ H Tr 0 00O N-N H The product from the previous example (0.4 g) was treated with 5 sodium azide (0.18 g) and ammonium chloride (0.14 g) in DMF (2.5 ml) as in example 1c. The product was recrystallized from ethanol-DMF (1:1). Yield 0.17 g. Melting point 283-286oC. 1 H-NMR (DMSO-d 6 , 400MHz): 1.626 (b, 4H), 2.393 (b, 2H), 2.971 (b, 2H), 5.583 (s, 2H), 5.599 (s, 2H), 6.811 (s, 2H). 10 Example 5. Preparation of 5,7-Bis[(1 H-tetrazol-5-yl)methoxy]-4 phenyl-2H-1 -benzopyran-2-one a) 5,7-Dihydroxy-4-phenyl-2H-1-benzopyran-2-one OH HO O 0 A solution of phloroglucinol (2.00 g) and ethyl benzoylacetate (3.05 g) 15 in ethanol (30 ml) was treated with dry HCI as described in example la. The product was recrystallized from ethanol-water (1:1). Yield 3.0 g (75 %). 1 H-NMR (DMSO-d6, 300 MHz): 5.739 (s, 1H, CH=C), 6.155 (d, 1H, J =2.3 Hz), 6.263 (d, 1H, J =2.3 Hz), 7.305-7.381 (m, 5H, Ph), 10.084 (s, 1H, OH), 10.368 (s, 1H, OH). 20 b) 5,7-Bis(cyanomethoxy)-4-phenyl-2H-1-benzopyran-2-one WO 99/15523 PCT/FI98/00755 24 N K0 0 0 The product from previous example (1.00 g) was treated with chloroaceto nitrile (0.62 g) and potassium carbonate (2.72 g) in DMF (5 ml) as described in example 1 b. The reaction mixture was poured into ice water and the 5 mixture extracted with ethyl acetate. Ethyl acetate was washed with 1 M NaOH, dried with sodium sulfate and evaporated. The product was recrystallized from isopropanol. Yield 0.41 g (31 %). 1 H-NMR (DMSO-d6, 300 MHz): 4.845 (s, 2H, OCH2CN), 5.344 (s, 2H, OCH2CN), 6.086 (s, 1 H, CH=C), 6.770 (d, 1H, J = 2.4 Hz), 7.040 (d, 1 H, 10 J = 2.4 Hz), 7.320-7.443 (m, 5H, Ph). c) 5,7-Bis[(1 H-tetrazol-5-yl)methoxy]-4-phenyl-2H-1 -benzopyran-2 one HNIN O 0 HN O O N=N The product from previous example (0.40g) was treated with sodium 15 azide (0.16 g) and ammonium chloride (0.14 g) in DMF (2 ml) at 100 oC for 2 hours. The product was isolated as described in example l c. Yield: 0.40 g (79 %). Melting point 222-224 oC. 1 H-NMR (DMSO-d6, 400 MHz): 5.148 (s, 2H, OCH2Tet), 5.649 (s, 2H, OCH2Tet), 5.968 (s, 1 H, CH=C), 6.811 (d, 1 H, J = 2.3 Hz), 6.962 (d, 20 1H, J = 2.3 Hz), 6.994-7.185 (m, 5H, Ph). Example 6. Preparation of 7,8,9,10-Tetrahydro-1,3-bis[(1 H-tetrazol-5 yl)methoxy]-8-phenyl-6H- dibenzo[b,d]pyran-6-one WO 99/15523 PCT/FI98/00755 25 a) 7,8,9,10-Tetrahydro-1,3-dihydroxy-8-phenyl-6H-dibenzo[b,d]pyran 6-one H A solution of phloroglucinol (1.56 g) and ethyl 2-oxo-5-phenylcyclo 5 hexane-carboxylate (2.52 g) in ethanol (25 ml) was treated with dry HCI as described in example la. The precipitate was filtered and washed with water and EtOH. Yield 1.0 g (32 %). 1 H-NMR (DMSO-d6, 400 MHz): 1.72-1.82 (m, 1H), 2.01 (b, 1H), 2.317-2.387 (m, 1H), 2,707-2,763 (m, 1H), 2,830 (b, 1H), 3,041 (b, 1H), 3.35 10 and 3.40 (b, 1H), 6.174 (d, 1H, J = 2.3 Hz), 6.277 (d, 1H, J = 2.3 Hz), 7.200 7.350 (m, 5H, Ph), 10.131 (s, 1H, OH), 10.401 (s, 1H, OH). b) 7,8,9,10-Tetrahydro-1,3-bis(cyanomethoxy)-8-phenyl-6H dibenzo[b,d]pyran-6-one N N 15 The product from previous example (1.0 g) was treated with chloro acetonitrile (0.57 g) and potassium carbonate (1.0 g) in DMF (5 ml) as described in example 1 b. DMF was evaporated and residue dissolved in EtOAc. Ethyl acetate was washed with 1 M NaOH, dried with sodium sulfate and evaporated. The product was recrystallized from acetone-isopropanol 20 (1:3). Yield 0.50 g (40 %). 1 H-NMR (DMSO-d6, 300 MHz): 1.75-1.88 (m, 1H), 2.05 (b, 1H), 2.38 2.48 (m, 1H), 2.77-2.85 (m, 1H), 2.90 (b, 1H), 3.07 (b, 1H), 3.22 and 3.28 (b, 1H), 5.316 (s, 2H, OCH2CN), 5.331 (s, 2H, OCH2CN), 6.829 (d, 1 H, J = 2.5 Hz), 6.939 (d, 1 H, J = 2.5 Hz), 7.210-7.380 (m, 5H, Ph).

WO 99/15523 PCT/FI98/00755 26 c) 7,8,9,10-Tetrahydro-1,3-bis[(1 H-tetrazol-5-yl)methoxy]-8-phenyl 6H- dibenzo[b,d]pyran-6-one H NN N N II I H/ N-N O NN The product from previous example (0.30 g) was treated with sodium 5 azide (0.10 g) and ammonium chloride (0.09 g) in DMF (2 ml) at 100 0C for 3.5 hours. The product was isolated in the same manner as in example ic. Yield 0.30 g (82 %). Melting point 235-245 oC. 1 H-NMR (DMSO-d6, 400 MHz): 1.70-1.80 (m, 1H), 1.96 (b, 1H), 2.38 2.446 (m, 1 H), 2.836 (m, 2H), 3.052 (b, 1H), 3.252 and 3.301 (b, 1H), 5.604 10 (s, 2H, OCH2CN), 5.632 (s, 2H, OCH2CN), 6.827 (d, 1 H, J = 2.5 Hz), 6.858 (d, 1 H, J = 2.5 Hz), 7.209-7.351 (m, 5H, Ph). Example 7. Preparation of 5,7-Bis[(1 H-tetrazol-5-yl)methoxy]-4 methyl-3-(2-phenylethyl)-2H-1 benzopyran-2-one a) 5,7-Dihydroxy-4-methyl-3-(2-phenylethyl)-2H-1-benzopyran-2-one H 15 HO O A solution of phloroglucinol (0.87 g) and ethyl 2-(2-phenylethyl) acetoacetate (1.62 g) in ethanol (30 ml) was treated with dry HCI as described in example 1 a. Yield: 1.77 g (87 %). Melting point 248-252 C. 1 H-NMR (DMSO-d6, 300 MHz): 2.413 (s, 3H, CH3), 2.652-2.782 (m, 20 4H, CH2CH2), 6.151 (d, 1H, J = 2.4 Hz), 6.256 (d, 1H, J = 2.4 Hz), 7.183 7.304 (m, 5H, Ph), 10.137 (s, 1H, OH), 10.369 (s, 1H, OH). b) 5,7-Bis(cyanomethoxy)-4-methyl-3-(2-phenylethyl)-2H-1 benzopyran-2-one WO 99/15523 PCT/FI98/00755 27 0 o 0 0 The product from previous example (0.90 g) was treated with chloroacetonitrile (0.48 g) and potassium carbonate (2.1 g) in DMF (5 ml) at 100 oC for 0.5 hours. The product was isolated as described in example lb. 5 Yield 1.00 g (88 %). Melting point 179-183 0C. 1 H-NMR (DMSO-d6, 300 MHz): 2,384 (s, 3H, CH3), 2.699-2,754 (m, 2H, CH2CH2), 2.805-2.841 (m, 2H, CH2CH2), 5,302 (s, 4H, OCH2CN), 6,790 (d, 1 H, J = 2.5 Hz), 6.909 (d, 1 H, J = 2.5 Hz), 7.190-7.307 (m, 5H, Ph). c) 5,7-Bis[(1 H-tetrazol-5-yl)methoxy]-4-methyl-3-(2-phenylethyl)-2H 10 1 benzopyran-2-one H H N The product from previous example (0.40g) was treated with sodium azide (0.15 g) and ammonium chloride (0.12 g) in DMF (2 ml) at 100 oC for 2.5 hours. The product was isolated as described in example i c. Yield 0.385 15 g (78 %). Melting point 248-250 oC. 1 H-NMR (DMSO-d6, 400 MHz): 2.368 (s, 3H, CH3), 2.668-2.707 (m, 2H, CH2CH2), 2.783-2.822 (m, 2H, CH2CH2), 5.593 (s, 2H, OCH2Tet), 5.604 (s, 2H, OCH2Tet), 6.819 (d, 1 H, J = 2.3 Hz), 6.834 (d, 1 H, J = 2.3 Hz), 7.161-7.291 (m, 5H, Ph). 20 Example 8. Preparation of 5,7-Bis[(1 H-tetrazol-5-yl)methoxy]-1,3 dibenzyl-4-methyl-2(1 H)-quinolinone a) 2-Benzyl-3-oxobutanoic acid 3,5-dimethoxyanilid WO 99/15523 PCT/FI98/00755 28 0 0 0 NO I H 3,5-Dimethoxyaniline (5 g) was added in portions to a preheated (160 0C) ethyl 2-benzyl acetoacetate (15 ml) under nitrogen and kept at that temperature for 60 minutes. The cooled solution was diluted with heptane 5 ethyl ether and filtered. Yield 5.2 g (49 %). 1 -H-NMR (DMSO-d6, 300 MHz): 2.183 (s, 3H), 3.069 (d, 2H, J = 7.2 Hz), 3.923 (t, 1 H, J = 7.2 Hz), 6.616 (dd. 1 H, J = 2.3 Hz), 6.765 (d, 2H, J = 2.3 Hz), 7.13- 7.3 (m, 5H), 10.123 (s, 1H). b) 3-Benzyl-5,7-dimethoxy-4-methyl-2(1 H)-quinolinone 0 I IN NO 10H 10 I H The product from the previous example (1.2 g) was added to a preheated (85 oC) methanesulfonic acid (3.5 ml) and kept at that temperature for 15 minutes. The solution was allowed to cool and then treated with ice water. The product was filtered, washed with sodium 15 bicarbonate and water. Yield 1.08 g (95 %). 1-H-NMR (300 MHz):2.486 (s, 3H), 3.785 (s, 3H), 3.808 (s, 3H), 3.985 (s, 2H), 6.315 (d, 1H, J = 2.4 Hz), 6.472 (d, 1H, J = 2.4 Hz), 7.1-7.3 (m, 5 H), 11.52 (s, 1 H). c) 3-Benzyl-5,7-dihydroxy-4-methyl-2(1 H)-quinolinone OH H N 0 20 H The product from the previous example (1 g) was refluxed under nitrogen in pyridine hydrochloride (5 g) for twenty minutes. The reaction WO 99/15523 PCT/FI98/00755 29 mixture was treated with water and the product filtered. Yield 0.9 g (100 %). Melting point: 307 - 312 oC. 1 -H-NMR (300 MHz):2.503 (s, 3H), 3.942 (s, 2H), 6.102 (d, 1H, J= 2.3 Hz), 6.187 (d, 1H, J =2.3 Hz), 7.1-7.25 (m, 5H), 9.725 (s, 1H), 9.984 (s, 5 1H), 11.285 (s, 1H). d) 1,3-Dibenzyl-5,7-dimethoxy-4-methyl-2(1 H)-quinolinone O ' N I 0 The product from the example 8b (1 g), potassium t-butoxide (0.62 g) and benzyl bromide (0.68 ml) were stirred in DMSO (10 ml) at 60 oC for 4 10 hours. The reaction mixture was treated with water, extracted with toluene and evaporated. The product was triturated with ethyl ether and filtered. Yield 0.5 g (39 %). 1 -H-NMR (400 MHz):2.537 (s, 3H), 3.708 (s, 3H), 3.826 (s, 3H), 4.124 (s, 2H), 5.56 (b, 2H), 6.413-6.434 (m, 2H), 7.154- 7.332 (m, 10H). 15 e) 1,3-Dibenzyl-5,7-dihydroxy-4-methyl-2(1 H)-quinolinone. OH H NO The product from the previous example (2 g) was treated with pyridine hydrochloride (10 g) as described in example 8c. The product was extracted with ethyl acetate and evaporated. Yield 1,4 g (75 %). 20 1 -H-NMR (400 MHz):2.570 (s, 3H), 4.076 (s, 2H), 5.450 (b, 2H), 6.135 (d, 1H, J = 2.2 Hz), 6.199 (d, 1 H, J = 2.2 Hz), 7.128 - 7.333 (m, 10 H), 9.83 (b, 1H), 10.166 (s, 1H). f) 5,7-Bis(cyanomethoxy)-1,3-dibenzyl-4-methyl-2(1 H)-quinolinone.

WO 99/15523 PCT/FI98/00755 30 NO 0 N 00 N!:N The product from the previous example (1.4 g) was treated with chloroacetonitrile (0.76 g) and K2CO3 (2.5 g) in DMF (20 ml) as described in example 1 b. Yield 1.5 g (89 %). 5 1 -H-NMR (400 MHz):2.555 (s, 3H), 4.146 (s, 2H), 5.214 (s, 2H), 5.275 (s, 2H), 5.578 (s, 2H), 6.735 (s, 2H), 7.13-7.33 (m, 10H). g) 5,7-Bis[(1 H-tetrazol-5-yl)methoxy]-1,3-dibenzyl-4-methyl-2(1 H) quinolinone. HNN O &N 0 N' NN 10 The product from the previous example (1.3 g) was treated with sodium azide (0.41 g) and ammonium chloride (0.34 g) as described in example l c. Yield: 0.69 g (45 %). 1 -H-NMR (400 MHz):2.471 (s, 3H), 4.113 (s, 2H), 5.477 (s, 2H), 5.55 (b, 2H), 5.574 (s, 2H), 6.670 (d, 1 H, J = 2.1 Hz), 6.775 (d, 1 H, J = 2.1 Hz), 15 7.13-7.32 (m, 10 H). Example 9. Preparation of 5,7-Bis[(1 H-tetrazol-5-yl)methoxy] -3 benzyl-1,4-dimethyl-2(1 H)-quinolinone. a) 3-Benzyl-5,7-dimethoxy-1,4-dimethyl-2(1 H)-quinolinone. 0 I I WO 99/15523 PCT/FI98/00755 31 The product from example 8b (0.5 g), t-BuOK (0.2 g) and methyl iodide (0.4 ml) were stirred in DMSO (5 ml) at 35 oC for two days. The reaction mixture was treated with water and extracted with toluene. The product was purified by column chromatography using toluene-ethyl acetate 5 acetic acid 8 : 2: 1 as the eluent. Yield 0.24 g (46 %). 1 -H-NMR (300 MHz):2.51 (s, 3H), 3.632 (s, 2H), 3.846 (s, 3H), 3.896 (s, 3H), 4.047 (s, 2H), 6.468 (d, 1 H, J = 2.3 Hz), 6.558 (d, 1 H, J = 2.3 Hz), 7.1-7.26 (m, 5H). b) 3-Benzyl-5,7-dihydroxy-1,4-dimethyl-2(1 H)-quinolinone. OH HO N 0 10 The product from the previous example (0.2 g) was treated with pyridine hydrochloride (2 g) as described in example 8c and the product extracted with ethyl acetate. Yield 0.16 g (89 %). 1-H-NMR (400 MHz):2.567 (s, 3H), 3.515 (s, 3H), 4.005 (s, 2H), 6.244 15 (d, 1 H, J = 2.3 Hz), 6.268 (d, 1 H, J = 2.3 Hz), 7.08-7.25 (m. 5H), 9.879 (s, 1H), 10.113 (s,1H). c) 5,7-Bis(cyanomethoxy)-3-benzyl-1,4-dimethyl-2(1 H)-quinolinone. N 0 JO N The product from the previous example (0.15 g), chloroacetonitrile 20 0.08 g) and K2CO3 (0.28 g) were reacted in DMF (2 ml) as described in example lb. Yield 0.16 g (84 %). 1 -H-NMR (400 MHz): 2.524 (s, 3H), 3.658 (s, 3H), 4.079 (s, 2H), 5.292 (s, 2H), 5.379 (s, 2H), 6.766 (d, 1 H, J = 2.3 Hz), 6.855 (d, 1 H, J = 2.3 Hz), 7.13-7.24 (m 5H).

WO 99/15523 PCT/FI98/00755 32 d) 5,7-Bis[(1 H-tetrazol-5-yl)methoxy] -3-benzyl-1,4-dimethyl-2(1 H) quinolinone. HN~Y~ N 0 NY I NN N NH The product from the previous example (0.15 g) was treated with 5 NaN3 (57 mg) and NH4CI (47 mg) in DMF (2 ml) as described in example lc. Yield 0.115 g. Melting point: 250-253oC. 1-H.-NMR (400 MHz): 2.451 (s, 3H), 3.649 (s, 3H), 4.042 (s, 2H), 6.792 (d, 1H, J = 2.2 Hz), 6.833 (d, 1H, J = Hz), 7.1-7.25 (m, 5H). Example 10. Preparation of 3-Benzyl-5,7-bis[(2-methyl-1 H-tetrazol-5 10 yl)methoxy]-4-methyl-2H-1 -benzopyran-2-one and the three isomers.

N

0 O N, N 0.07 ml of methyl iodide was added to a solution of 0.2 g of the product from example 1c and 0.31 g of K2CO3 in 2 ml of DMF and the mixture stirred at room temperature for 4 hours. The reaction mixture was 15 poured into ice water and filtered. Yield 0.2 g as a mixture of four regioisomers, melting point 71-76oC. 1 H-NMR (DMSO-d 6 , 400MHz): 2.47 (s, CH3), 2.48 (s, CH3), 3.93 (s, CH2Ph), 4.11 (s, NCH3), 4.12 (s, NCH3), 4.15 (s, NCH3), 4.38 (s, NCH3), 4.40 (s, NCH3), 5.51 (s, OCH2), 5.52 (s, OCH2), 5.62 (s, OCH2), 5.67 (s, 20 OCH2), 6.84-6.91 (m, 2H), 7.16-7.28 (m,5H, Ph).

WO 99/15523 PCT/FI98/00755 33 Example 11. Preparation of 3-Benzyl-5,7-bis[1-(1 H -tetrazol-5 yl)ethoxy]4-methyl-2 H -1 -benzopyran-2-one, mixture of stereoisomers a) 3-Benzyl-5,7-bis-[( 1 -cyano)ethoxy)-4-methyl-2 H -1 -benzopyran- 2-one i.. 0 O O 5 The product from example 1 a (1 g), 2-chlorpropionitrile (0.7 g) and potassium carbonate (2 g) were heated in DMF (15 ml) under nitrogen at 110oC for sixty minutes. The mixture was treated with water, filtered and washed with 1 N NaOH and water. Yield 1.2 g. 1 H-NMR (DMSO-d 6 , 300MHz): 1.74-1.78 (t + t, 6 H, CH-CH3), 2.53 10 (s, 3 H), 3.97 (s, 2H), 5.58-5.66 (m, 2H, CH-CH3), 6.87 (m, 1H), 6.99 (d, 1H), 7.18-7.31 (m, 5H). b) 3-Benzyl-5,7-bis[1-(1 H -tetrazol-5-yl)ethoxy]4-methyl-2 H -1 benzopyran-2-one, mixture of stereoisomers. HN "-N NN .INy/ \N

-

15 The product from the previous example (0.5 g), sodium azide (0.18 g) and ammonium chloride (0.15 g) were heated in DMF (7 ml) at 100 oC for 90 minutes. The product was treated with water, extracted with ethyl acetate and evaporated. Yield 0.57 g. Melting point 91-1040C.

WO 99/15523 PCT/FI98/00755 34 1 H-NMR (DMSO-d 6 , 300MHz): 1.69-1.77 (m, 6 H, CH-CH3), 2.54 (s, 3H), 3.94 (s, 2H), 6.10-6.17 ((m, 2H, CH-CH3), 6.65 (dd, 1H), 6.74 (dd, 1H), 7.13-7.30 (m, 5H). Example 12. Preparation of 5,7-Bis(carboxymethoxy)-1,3-dibenzyl-4 5 methyl-2(1H)-quinolinone HO N 0 O The product from example 8f (0.2 g) was refluxed in a solution of concentrated hydrochloric acid (3 ml) and acetic acid (2 ml) for one hour. The product was filtered at 25 0C. Yield 0.14 g. 10 1 H-NMR (300 Mhz, DMSO-d6): 2.63 (s, CH3), 4.14 (s, 2H, CH2Ph), 4.66 (s, 2 H, OCH2COOH), 4.79 (s, 2H, OCH2COOH), 5.53 (s, 2H, NCH2Ph), 6.41 (d, 1H, J = 2.2 Hz), 6.45 (d, 1H, J = 2.2 Hz), 7.13-7.34 (m, 10 H, Ph). Example 13. Preparation of 3-Benzyl-5,7-bis[(1H-tetrazol-5 yl)methoxy]-1 -(4-fluorobenzyl)-4-methyl-2(1 H)-quinolinone 15 a) 1-Benzyl-5,7-dimethoxy-3-(4-fluorobenzyl)-4-methyl-2(1 H) quinolinone 0 N O N 0 F The product from example 8b (2 g), potassium -tert-butoxide (0.87 g) and 4-fluorobenzylchloride (1.12 g) were heated in DMSO (20 ml) at 60 oC 20 for three hours as in example 8d. Yield 1.28 g.

WO 99/15523 PCT/FI98/00755 35 1 H-NMR (400 Mhz, DMSO-d6): 2.53 (s, 3H), 3.73 (s, 3H), 3.83 (s, 3H), 5.55 (s, 2H), 6.43 (s, 2H), 7.12-7.2 (m, 5 H), 7.26-7.28 (m, 4H). b) 3-Benzyl-5,7-dihydroxy-1 -(4-fluorobenzyl)-4-methyl-2(1 H) quinolinone OH HO N 0 F 5F The product from previous example (1.25 g) were heated in pyridine hydrochloride (12.5 g) at about 225 0C for 9 minutes. Yield 1 g. 1 H-NMR (300 Mhz, DMSO-d6): 2.56 (s, 3H), 4.07 (s, 2H), 5.4 (b, 2H), 6.13 (d, 1H, J = 2.1 Hz), 6.20 (d, 1H, J = 2.1 Hz), 7.12-7.28 (m, 9H), 9.88 (s, 10 1H), 10.22 (s, 1H). c) 3-Benzyl-5,7-Bis(cyanomethoxy)-1-(4-fluorobenzyl) -4-methyl 2(1 H)-quinolinone 0I O NO O ~ N 0 F The product from the previous example (1 g), CICH2CN (0.43 g) and K2CO3 15 (1.42 g) were heated in DMF (8 ml) at 120 oC for one hour. Yield 0.94 g. 1 H-NMR (300 Mhz, DMSO-d6): 2.55 (s, 3H), 4.14 (s, 2H), 5.25 (s, 2H), 5.28 (s, 2H), 5.57 (s, 2H), 6.74 (s, 2H, ArH), 7.1 -7.3 (m, 9H).

WO 99/15523 PCT/FI98/00755 36 d) 3-Benzyl-5,7-bis[(1 H-tetrazol-5-yl)methoxy]-1-(4-fluorobenzyl)-4 methyl-2(1 H)-quinolinone N=N 0 N 0 HN F The product from the previous example (0.5 g), sodium azide (0.14 g) 5 and ammonium chloride (0.12 g) were heated in DMF (5 ml) at 120 oC for 90 min. The product was triturated with acetonitrile. Yield 0.28 g. Melting point: 126-132 oC. 1 H-NMR (300 Mhz, DMSO-d6): 2.48 (s, 3H), 4.11 (s, 2H), 5.51 (s, 2H), 5.55 (s, 2H), 5.58 (s, 2H), 6.67 (d, 1H, J = 2.1 Hz), 6.78 (d, 1H, J = 2.1 Hz). 10 Example 14. Preparation of 5,7-Bis[(1 H-tetrazol-5-yl)methoxy]-3-(4 chlorobenzyl)-4-methyl-2H-1 -benzopyran-2-one a) 3-(4-Chlorobenzyl)-5,7-dihydroxy-4-methyl-2H-1-benzopyran-2-one OH HO 0 0 CI A solution of phloroglucinol (1.57 g) and ethyl 2-(4-chlorobenzyl) 15 acetoacetate (3.18 g) in ethanol (25 ml) was treated with dry HCI at 0 0C for 1.5 hours and the solution was kept at that temperature overnight. Solvent was evaporated and the precipitate triturated with water. Yield 3.87 g (98 %). Melting point 270-278 oC. 1 H-NMR (DMSO-d6, 300 MHz): 2.52 (s, 3H, CH 3 ), 3.87 (s, 2H, CH2), 20 6.17 (d, 1H, J = 2.4 Hz), 6.28 (d, 1H, J = 2.4 Hz), 7.18-7.34 (m, 4H, Ph), 10.21 (s, 1 H, OH), 10.48 (s, 1 H, OH). b) 5,7-Bis(cyanomethoxy)-3-(4-chlorobenzyl)-4-methyl-2H-1 benzopyran-2-one WO 99/15523 PCT/FI98/00755 37 N N toi O O O Cl The product from the previous example (1.00 g), chloroacetonitrile (0.50 g) and potassium carbonate (2.18 g) were heated in DMF (5 ml) at 100 oC for 30 minutes. The product was isolated as described in example 1 b. 5 Yield 0.90 g (72 %). 1 H-NMR (DMSO-d6, 300 MHz): 2.52 (s, 3H, CH3), 3.95 (s, 2H, CH2), 5.308 (s, 2H, OCH2CN), 5.312 (s, 2H, OCH2CN), 6.81 (d, 1H, J = 2.5 Hz), 6.94 (d, 1 H, J = 2.5 Hz), 7.22-7.33 (m, 4H, Ph). c) 5,7-Bis[(1 H-tetrazol-5-yl)methoxy]-3-(4-chlorobenzyl)-4-methyl-2H 10 1 -benzopyran-2-one H N W-N \N 0 S O CI The product from the previous example (0.40 g), sodium azide (0.14 g) and ammonium chloride (0.11 g) were heated in DMF (2 ml) at 100 oC for 2 hours. The product was isolated as in example ic. Yield 0.40 g (82 %). 15 1 H-NMR (DMSO-d6, 300 MHz): 2.46 (s, 3H, CH3), 3.92 (s, 2H, CH2), 5.602 (s, 2H, OCH2Tet), 5.609 (s, 2H, OCH2Tet), 6.83 (d, 1 H, J = 2.5 Hz), 6.85 (d, 1 H, J = 2.5 Hz), 7.20-7.33 (m, 4H, Ph). Example 15. Preparation of 5,7-Bis[(1 H-tetrazol-5-yl)methoxy]-3-(4 20 nitrobenzyl)-4-methyl-2H-1 -benzopyran-2-one a) 5,7-Dihydroxy-4-methyl-3-(4-nitrobenzyl)-2H-1-benzopyran-2-one WO 99/15523 PCT/FI98/00755 38 HO I I O HO 0 0 N 0 A solution of phloroglucinol (0.48 g) and ethyl 2-(4-nitrobenzyl)aceto acetate (1.00 g) in ethanol (150 ml) was treated with dry HCI at 0 oC for 7.5 hours and the solution was kept at that temperature overnight. Solvent was 5 evaporated and the precipitate triturated with water. Yield 0.63 g (51%). Melting point 280-285 oC. 1 H-NMR (DMSO-d6, 300 MHz): 2.53 (s, 3H, CH3), 4.03 (s, 2H, CH2), 6.19 (d, 1H, J = 2.4 Hz), 6.29 (d, 1H, J = 2.4 Hz), 7.40-7.51 and 8.11-8.17 (m, 4 H, Ph), 10.25 (s, 1 H, OH), 10.52 (s, 1H, OH). 10 b) 5,7-Bis(cyanomethoxy)-3-(4-nitrobenzyl)-4-methyl-2H-1 benzopyran-2-one N IL_ O 0 0 0 N~ 0 The product from the previous example (0.57 g), chloroacetonitrile (0.27 g) and potassium carbonate (1.20 g) were heated in DMF (2 ml) at 100 15 OC for 50 minutes. The product was isolated as described in example lb. Yield 0.47 g (67 %). Melting point 178-185 OC. 1 H-NMR (DMSO-d6, 400 MHz): 2.53 (s, 3H, CH3), 4.11 (s, 2H, CH2), 5.319 (s, 2H, OCH2CN), 5.323 (s, 2H, OCH2CN), 6.83 (d, 1 H, J = 2.4 Hz), 6.96 (d, 1H, J = 2.4 Hz), 7.48-7.53 and 8.12-8.16 (m, 4H, Ph).

WO 99/15523 PCT/FI98/00755 39 c) 5,7-Bis[(1 H-tetrazol-5-yl)methoxy]-3-(4-nitrobenzyl)-4-methyl-2H-1 benzopyran-2-one XNN \NI \NN N -- N H 5 /N +,O / Ir O O O N N 0 H The product from the previous example (0.38 g), sodium azide (0.12 g) and ammonium chloride (0.11 g) were heated in DMF (3 ml) at 100 oC for 2 hours. The product was isolated as described in example ic. Yield 0.25 g 10 (54 %). Melting point 240-244 oC. 1 H-NMR (DMSO-d6, 400 MHz): 2.47 (s, 3H, CH3), 4.08 (s, 2H, CH2), 5.611 (s, 2H, OCH2Tet), 5.623 (s, 2H, OCH2Tet), 6.85 (d, 1 H, J = 2.4 Hz), 6.87 (d, 1H, J = 2.4 Hz), 7.46-7.50 and 8.12-8.16 (m, 4H, Ph). Example 16. Preparation of 5,7-Bis[(1 H-tetrazol-5-yl)methoxy]-3 15 cyclopentyl-4-methyl-2H-1 -benzopyran-2-one a) 3-Cyclopentyl-5,7-dihydroxy-4-methyl-2H-1-benzopyran-2-one HO HO 0 O A solution of phloroglucinol (2.00 g) and ethyl 2-cyclopentylaceto acetate (3.14 g) in ethanol (40 ml) was treated with dry HCI at 0 oC for 2.5 20 hours and the solution kept at that temperature overnight. Solvent was evaporated and the precipitate purified with flash chromatography eluting with toluene-EtOAc-AcOH (8:1:1). Yield 1.22 g (29 %). 1 H-NMR (DMSO-d6, 300 MHz): 1.50-1.88 (m, 8H, -(CH2)4-), 2.57 (s, 3H, CH3), 3.25 (m, 1H, CH), 6.11 (d, 1H, J = 2.4 Hz), 6.25 (d, 1H, J = 2.4 25 Hz), 10.25 (b, 2H, OH). b) 5,7-Bis(cyanomethoxy)-3-cyclopentyl-4-methyl-2H-1-benzopyran-2 one WO 99/15523 PCT/FI98/00755 40 0 0 0 The product from the previous example (0.50 g), chloroacetonitrile (0.31 g) and potassium carbonate (0.61 g) were heated in DMF (2 ml) at 80 oC for 40 minutes. The product was isolated as described in example 1 b. 5 Yield 0.56 g (86 %). 1 H-NMR (DMSO-d6, 300 MHz): 1.55-1.90 (m, 8H, -(CH2)4-), 2.56 (s, 3H, CH3), 3.37 (m, 1H, CH), 5.29 (s, 2H, OCH2CN), 5.31 (s, 2H, OCH2CN), 6.75 (d, 1 H, J = 2.5 Hz), 6.88 (d, 1 H, J = 2.5 Hz). c) 5,7-Bis[(1 H-tetrazol-5-yl)methoxy]-3-cyclopentyl-4-methyl-2H-1 10 benzopyran-2-one NN H -N N NN H N H 15 The product from the previous example (0.30 g), sodium azide (0.13 g) and ammonium chloride (0.11 g) were heated in DMF (1 ml) at 100 oC for 1.5 hours. The product was isolated as described in example 1 c. Yield 0.30 g (80 %). Melting point 248-252 oC. 1 H-NMR (DMSO-d6, 400 MHz): 1.53-1.89 (m, 8H, -(CH2)4-), 2.51 (s, 20 3H, CH3), 3.34 (m, 1 H, CH), 5.59 (s, 2H, OCH2Tet), 5.61 (s, 2H, OCH2Tet), 6.80 (s, 2H). Example 17. Preparation of 5,7-Bis[(1 H-tetrazol-5-yl)methoxy]-4 methyl-3-(1 -naphtylmethyl)-2H-1 -benzopyran-2-one a) 5,7-dihydroxy-4-methyl-3-(1-naphtylmethyl)-2H-1-benzopyran-2 25 one WO 99/15523 PCT/FI98/00755 41 HO HO O O A solution of phloroglucinol (0.47 g) and ethyl 2-(1-naphtylmethyl) acetoacetate (1.00 g) in ethanol (20 ml) was treated with dry HCI at 0 oC for 3 hours and the solution kept at that temperature overnight. Solvent was 5 evaporated and the precipitate triturated with water and recrystallized from isopropanol-water (1:1). Yield 0,96 g (78 %). Melting point 275-280 °C. 1 H-NMR (DMSO-d6, 400 MHz): 2.45 (s, 3H, CH3), 4.32 (s, 2H, CH2), 6.23 (d, 1 H, J = 2.5 Hz), 6.32 (d, 1 H, J = 2.5 Hz), 6.97-8.25 (m, 7H, Naph), 10.26 (s, 1 H, OH), 10.53 (s, 1 H, OH). 10 b) 5,7-Bis(cyanomethoxy)-4-methyl-3-(1-naphtylmethyl)-2H-1 benzopyran-2-one O The product from the previous example (0.80 g), chloroacetonitrile (0.36 g) and potassium carbonate (0.66 g) were heated in DMF (4 ml) at 100 0C for 1 15 hour. The product was isolated as in example 1 b. Yield 0.30 g (30 %). 1 H-NMR (DMSO-d6, 300 MHz): 2.45 (s, 3H, CH3), 4.40 (s, 2H, CH2), 5.34 (s, 2H, OCH2CN), 5.36 (s, 2H, OCH2CN), 6.86 (d, 1 H, J = 2.5 Hz), 7.010 (d, 1 H, J = 2.5 Hz), 7.016-8.27 (m, 7H, Naph). c) 5,7-Bis[(1 H-tetrazol-5-yl)methoxy]-4-methyl-3-(1-naphtylmethyl)-2H 20 1 -benzopyran-2-one N O N-NH O OO

N--NH

WO 99/15523 PCT/FI98/00755 42 The product from the previous example (0.25 g), sodium azide (0.080 g) and ammonium chloride (0.072 g) were heated in DMF (2 ml) at 100 oC for 2.5 hours. The product was isolated as described in example 1 c. Yield 0.11 g (36 %). Melting point 164-174 oC. 5 1H-NMR (DMSO-d6, 300 MHz): 2.40 (s, 3H, CH3), 4.37 (s, 2H, CH2), 5.63 (s, 2H, OCH2Tet), 5.65 (s, 2H, OCH2Tet), 6.87 (d, 1 H, J = 2.5 Hz), 6.92 (d, 1 H, J = 2.5 Hz), 6.98-8.26 (m, 7H, Naph). Example 18. Preparation of 1-Benzyl-5,7-bis-[(1 H-tetrazol-5-yl) methoxy]-4-methyl-2(1 H)-quinolinone 10 a) 5,7-Dimethoxy-4-methyl-2(1 H)-quinolinone O 0 N 0 H tert-Butyl acetoacetate (1.58 g) was heated to 120 oC and 3,5 dimethoxyaniline (1.53 g) dissolved in xylene (4 ml) was added. The mixture was heated at 120-130 oC for 20 minutes and then cooled to room 15 temperature. Methanesulfonic acid (2 ml) was added and the mixture was stirred at ambient temperature for 10 minutes. Water (40 ml) was added and the precipitate filtered and dried. Yield 1.31 g (60 %). 1H-NMR (DMSO-d6, 300 MHz): 2.50 (s, 3H, CH3), 3.79 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 6.03 (s, 1 H, CH=C), 6.31 (d, 1 H, J = 2.3 Hz), 20 6.45 (d, 1H, J= 2.3 Hz), 11.4 (b, 1H, NH). b) 1-Benzyl-5,7-dimethoxy-4-methyl-2(1 H)-quinolinone 0 N 0 WO 99/15523 PCT/FI98/00755 43 The product from the previous example (1.20 g) was suspended to DMSO (15 ml) and t-BuOK (0.68 g) and benzylbromide (1.03 g) were added. Reaction mixture was stirred at ambient temperature overnight. Water was added and the product extracted to EtOAc. EtOAc was dried and evaporated 5 to dryness. The product was recrystallized from toluene. Yield 0.80 g (47 %). 1 H-NMR (DMSO-d6, 300 MHz): 2.55 (d, 3H, J = 1.1 Hz, CH3), 3.71 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 5.48 (b, 2H, NCH2), 6.29 (d, 1 H, J = 1.1 Hz, CH=C), 6.4 (s, 2H), 7.18-7.33 (m, 5H, Ph). c) 1-Benzyl-5,7-dihydroxy-4-methyl-2(1 H)-quinolinone OH I H N 0 10 The product from the previous example (0.69 g) was dissolved to

CH

2

CI

2 (14 ml) and the reaction mixture cooled to -20 oC. BBr 3 (2.4 g) in

CH

2

CI

2 (1 M solution) was added and the mixture was allowed to warm to ambient temperature during the night. The precipitate was filtered, washed 15 with CH 2

CI

2 and dissolved to EtOAc. EtOAc was washed with dilute HCI, dryed and evapotated to dryness. Yield 0.34 g (54 %). 1 H-NMR (DMSO-d6, 300 MHz): 2.56 (d, 3H, J= 1.0 Hz, CH3), 5.33 (b, 2H, NCH2), 6.11 (d, 1H, J = 2.1 Hz), 6.13 (d, 1H, J= 1.0 Hz, CH=C), 6.17 (d, 1H, J =2.1 Hz), 7.12-7.34 (m, 5H, Ph), 9.90 (b, 1H, OH), 10.22 (s, 1H, OH). 20 d) 1-Benzyl-5,7-bis(cyanomethoxy)-4-methyl-2(1 H)-quinolinone N/ O N0

NN

WO 99/15523 PCT/FI98/00755 44 The product from the previous example (0.34 g), chloroacetonitrile (0.13 g) and potassium carbonate (0.34 g) were heated in DMF (2 ml) at 100 oC for 1.5 hours. Water was added and the precipitate filtered and dried. The product was recrystallized from isopropanol. Yield 0.20 g (46 %). 5 1 H-NMR (DMSO-d6, 400 MHz): 2.57 (s, 3H, CH3), 5.22 (s, 2H, OCH2CN), 5.30 (s, 2H, OCH2CN), 5.50 (b, 2H, NCH2), 6.42 (s, 1 H, CH=C), 6.70 (d, 1 H, J = 2.1 Hz), 6.73 (d, 1 H, J = 2.1 Hz), 7.21-7.32 (m, 5H, Ph). e) 1 -Benzyl-5,7-bis-[(1 H-tetrazol-5-yl)methoxy]-4-methyl-2(1 H) quinolinone N O 0 N " v "NH" N \ \ I-I N ," NH 10 N The product from the previous example (0.20 g), sodium azide (0.072 g) and ammonium chloride (0.060 g) were heated in DMF (2 ml) at 100 oC for 3 hours. The product was isolated as described in example 1 c. Yield 0.21 g (85 %). Melting point 246-249 oC. 15 1 H-NMR (DMSO-d6, 400 MHz): 2.50 (s, 3H, CH3), 5.48 (b, 4H, OCH2Tet, NCH2), 5.60 (s, 2H, OCH2Tet), 6.34 (s, 1H, CH=C), 6.64 (d, 1H, J = 1.9 Hz), 6.77 (d, 1H, J = 1.9 Hz), 7.18-7.32 (m, 5H, Ph). Example 19. Preparation of 1-Benzyl-5,7-bis[1 H-tetrazol-5 yl)methoxy]-3-(2-fluorobenzyl)-4-methyl-2(1 H)-quinolinone 20 a) 5,7-Dimethoxy-3-(2-fluorobenzyl)-4-methyl-2(1 H)-quinolinone O F ON H 0

H

WO 99/15523 PCT/FI98/00755 45 Ethyl 2-(2-fluorobenzyl)acetoacetate (2.5 g) in xylene (1 ml) was heated to 150 oC and 3,5- dimethoxyaniline (1.46 g) in xylene (4 ml) was added in small portions during 30 minutes. The reaction mixture was heated at 160 oC for 3 hours and then cooled to room temperature. Methanesulfonic 5 acid (1.7 ml) was added and the mixture was stirred at ambient temperature for 30 minutes. Water was added and the precipitate filtered and dried. The product was triturated with warm ethanol. Yield 0.64 g (21 %). 1 H-NMR (DMSO-d6, 300 MHz): 2.45 (s, 3H), 3.79 (s, 3H), 3.82 (s, 3H), 3.97 (s, 2H), 6.33 (d, 1H, J = 2.4 Hz), 6.48 (d, 1 H, J = 2.4 Hz), 6.90-7.25 1o (m, 4H), 11.61 (s, 1 H). b) 1-Benzyl-5,7-dimethoxy-3-(2-fluorobenzyl)-4-methyl-2(1 H) quinolinone O F 0 I, The product from the previous example (0.62 g) was treated with t-BuOK 15 (0.23 g) and benzylbromide (0.36 g) in DMSO (12 ml) at 60 oC for 2.5 hours. The product was isolated as described in example 18b. Yield 0.39 g (49 %). 1 H-NMR (DMSO-d6, 400 MHz): 2.51 (s, 3H), 3.72 (s, 3H), 3.84 (s, 3H), 4.11 (s, 2H), 5.55 (b, 2H), 6.433 (d, 1H, J= 2.1 Hz), 6.443 (d, 1H, J = 2.1 Hz), 6.97-7.33 (m, 9H). 20 c) 1-Benzyl-5,7-dihydroxy-3-(2-fluorobenzyl)-4-methyl-2(

H)

quinolinone OH F H N 0 WO 99/15523 PCT/FI98/00755 46 The product from the previous example (0.34 g) was treated with BBr3 (8.48 g) in CH2CI2 (7 ml) as described in example 18c. Yield 0.30 g (82 %). 1 H-NMR (DMSO-d6, 400 MHz): 2.55 (s, 3H), 4.06 (s, 2H), 5.40 (b, 2H), 6.13 (d, 1 H, J = 2.1 Hz), 6.22 (d, 1 H, J = 2.1 Hz), 6.97-7.33 (m, 9H), 10.3 (b, 2H). 5 d) 1-Benzyl-5,7-bis(cyanomethoxy)-3-(2-fluorobenzyl)-4-methyl-2(1 H) quinolinone W,:NO F N O F NI 1 The product from the previous example (0.21 g), chloroacetonitrile (0.086 g) and potassium carbonate (0.37 g) were heated in DMF (2 ml) at 10 100 oC for 2 hours. The product was isolated as described in example 1 b. Yield 0.18 g (71 %). 1 H-NMR (DMSO-d6, 400 MHz): 2.53 (s, 3H), 4.13 (s, 2H), 5.23 (s, 2H), 5.29 (s, 2H), 5.57 (b, 2H), 6.746 (d, 1 H, J = 2.3 Hz), 6.756 (d, 1 H, J = 2.3 Hz), 7.00-7.32 (m, 9H). 15 e) 1-Benzyl-5,7-bis[1 H-tetrazol-5-yl)methoxy]-3-(2-fluorobenzyl)-4 methyl-2(1 H)-quinolinone N 0O F N-NH H N IH 0 N 0 N-NH The product from the previous example (0.17 g), sodium azide (0.051 g) and ammonium chloride (0.042 g) were heated in DMF at 100 0C for 3 20 hours. The product was isolated as described in example c. Yield 0.17 g (85 %). Melting point 135-140 oC.

WO 99/15523 PCT/FI98/00755 47 1 H-NMR (DMSO-d6, 400 MHz): 2.46 (s, 3H), 4.10 (s, 2H), 5.48 (s, 2H), 5.51 (b, 2H), 5.59 (s, 2H), 6.68 (d, 1 H, J = 2.2 Hz), 6.79 (d, 1 H, J = 2.2 Hz), 6.99-7.32 (m, 9H). Example 20. Preparation of 1-Benzyl-5,7-bis[1 H-tetrazol-5-yl) 5 methoxy]-4-methyl-3-(2-phenylethyl)-2(1 H)-quinolinone a) 5,7-Dimethoxy-4-methyl-3-(2-phenylethyl)-2(1 H)-quinolinone 0 N H Ethyl 2-(2-phenylethyl)acetoacetate (2.70 g) in xylene (5 ml) was treated with 3,5-dimethoxyaniline (1.60 g) at 150 oC as described in example 10 19a. Methanesulfonic acid (4.0 ml) was added at room temperature and the mixture heated at 80 oC for 1 hour. The product was isolated as described in example 19a. Yield 1.38 g (41 %). 1 H-NMR (DMSO-d6, 400 MHz): 2.45 (s, 3H), 2.64-2.68 (m, 2H), 2.82 2.86 (m, 2H), 3.78 (s, 3H), 3.81 (s, 3H), 6.30 (d, 1H, J = 2.3 Hz), 6.45 (d, 1 H, 15 J =2.3 Hz), 7.18-7.30 (m, 5H), 11.45 (s, 1H). b) 1 -Benzyl-5,7-dimethoxy-4-methyl-3-(2-phenylethyl)-2(1 H) quinolinone The product from the previous example (0.61 g), t-BuOK (0.24 g) and 20 benzylbromide (0.36 g) were heated in DMSO (12 ml) at 60 oC for 2 hours. The product was isolated as described in example 18b. Yield 0.31 g (40 %).

WO 99/15523 PCT/FI98/00755 48 1H-NMR (DMSO-d6, 400 MHz): 2.51 (s, 3H), 2.73-2.77 (m, 2H), 2.96 3.00 (m, 2H), 3.70 (s, 3H), 3.83 (s, 3H), 5.55 (b, 2H), 6.40 (s, 2H), 7.17-7.33 (m, 10 H). c) 1-Benzyl-5,7-dihydroxy-4-methyl-3-(2-phenylethyl)-2(1 H) 5 quinolinone HO0 HO O The product from the previous example (0.31 g) was treated with BBr3 (0.75 g) in CH2CI2 (5 ml) as in example 18c. Yield 0.26 g (89 %). 1 H-NMR (DMSO-d6, 300 MHz): 2.56 (s, 3H), 2.69-2.75 (m, 2H), 2.90 10 2.95 (m, 2H), 5.39 (b, 2H), 6.08 (d, 1H, J = 2.0 Hz), 6.19 (d, 1H, J = 2.0 Hz), 7.11-7.33 (m, 10H), 10.2 (b, 2H). d) 1-Benzyl-5,7-bis(cyanomethoxy)-4-methyl-3-(2-phenylethyl)-2(1 H) quinolinone N I O O 15 The product from the previous example (0.22 g), chloroacetonitrile (0.091 g) and potassium carbonate (0.39 g) were heated at 100 oC for 2 hours. The product was isolated as in example 1 b. Yield 0.20 g (76 %).

WO 99/15523 PCT/FI98/00755 49 1H-NMR (DMSO-d6, 400 MHz): 2.50 (s, 3H), 2.73-2.77 (m, 2H), 2.98 3.02 (m, 2H), 5.21 (s, 2H), 5.29 (s, 2H), 5.56 (b, 2H), 6.70 (d, 1 H, J =2.1 Hz), 6.72 (d, 1H, J = 2.1 Hz), 7.18-7.33 (m, 10H). e) 1-Benzyl-5,7-bis[1 H-tetrazol-5-yl)methoxy]-4-methyl-3-(2 5 phenylethyl)- 2(1 H)-quinolinone /N= N N=N 0 I N, NH O O The product from the previous example (0.19 g), sodium azide (0.057 g) and ammonium chloride (0.047 g) were heated in DMF at 100 oC for 3 hours. The product was isolated as described in example lc. Yield 0.18 g 10 (78 %). Melting point 215-218 0C. 1 H-NMR (DMSO-d6, 400 MHz): 2.46 (s, 3H), 2.70-2.74 (m, 2H), 2.95 2.99 (m, 2H), 5.47 (s, 2H), 5.54 (b, 2H), 5.57 (s, 2H), 6.64 (d, 1 H, J = 2.0 Hz), 6.77 (d, 1H, J = 2.0 Hz), 7.16-7.33 (m, 10H). Example 21. Preparation of 5,7-Bis(aminocarbonylmethoxy)-1,3 15 dibenzyl-4-methyl-2(1 H)-quinolinone.

H

2 NyO 0 O N 0

H

2 N The mixture of 5,7-dihydroxy-1,3-dibenzyl-4-methyl-2(1 H)-quinolinone (0.5 g), potassium carbonate (0.9 g) and 2-chloroacetamide ( 0.25 g) in DMF (6.5 ml) were reacted at 100 oC for two hours. The reaction mixture was WO 99/15523 PCT/FI98/00755 50 treated with ice water and filtered. The product was triturated with hot ethanol. Yield: 0.32 g. Melting point 252-2530C. 1 H-NMR (400 MHz, DMSO-d6): 2.63 (s, 3H, CH3), 4.13 (s, 2H, PhCH2), 4.37 (s, 2H, OCH2), 4.55 (s, 2H, OCH2), 5.54 (s, 2H, NCH2Ph), 5 6.40 (d, 1H, J = 2 Hz, ArH), 6.53 (d, 1H, J = 2 Hz, ArH), 7.13-7.33 (m, 10 H, Ph), 7.44 (d, 2H, J = 65 Hz, CONH2), 7.47 (d, 2H, J = 68 Hz, CONH2). Example 22. Preparation of 5,7-Bis(ethoxycarbonylmethoxy)-1,3 dibenzyl-4-methyl-2(1 H)-quinolinone. o o O N 0 10 The mixture of 5,7-dihydroxy-1,3-dibenzyl-4-methyl-2(1 H)-quinolinone (1 g), ethyl 2-bromoacetate (0.63 ml) and potassium carbonate (1.49 g) in DMF (5 ml) was heated under nitrogen at 110 oC for three hours, poured into ice water and filtered. The resulting solid material was triturated with ether and filtered again. Yield: 1.03 g, melting point 113-116 oC. 15 1 H-NMR (400 MHz, DMSO-d6): 1.15 (t, 3H, CH3CH2, J = 7.1 Hz), 1.20 (t, 3H, CH3CH2, J = 7.1 Hz), 2.63 (s, 3H, CH3), 4.03 (q, 2H, CH2CH3, J = 7.1 Hz), 4.13 (s, 2H, CH2Ph), 4.17 (q, 2H, CH2CH3, J = 7.1 Hz), 4.78 (s, 2H, OCH2), 4.90 (s, 2H, OCH2), 6.41 (d, 1 H, J = 2.2 Hz), 6.44 (d, 1 H, J = 2.2 Hz), 7.13-7.33 (m, 10 H, Ph).

WO 99/15523 PCT/FI98/00755 51 Example 23. Preparation of 5,7-Bis(hydroxyaminocarbonylmethoxy) 1,3-dibenzyl-4-methyl-2(1 H)-quinolinone OH I 0 HN-o O N 0 QY) HOH The product from the previous example (0.3 g), hydroxylamine 5 hydrochloride (0.32 g) and 5 N NaOH (1.05 ml) were reacted in ethanol (8 ml) at 50 oC for six hours. The reaction mixture was treated with water and made basic (pH 10) and filtered. The filtrate was acidified to pH 2 and filtered. Yield: 0.2 g, melting point 121-127C. 1 H-NMR (400 MHz, DMSO-d6): the tautomeric forms of hydroxamic 10 acid are seen in OCH2-signals: 2.63 (s,3H, CH3), 4.13 (S, 2H, CH2Ph), 4.41 (s, 2H, OCH2), 4.54 (s, 2H,OCH2), 4.64 (s, 2H, HON=C(OH)CH20), 4.65 (s, 2H, HON=C(OH)CH20), 4.77 (s, 2H, HON=C(OH)CH20), 4.78 ((s, 2H, HON=C(OH)CH20), 5.54 (s, 2H, NCH2Ph), 6.38-6.54 (m, 2H, ArH), 7.14 7.34 (m, 10 H, Ph), 9.05 (b, 2H, NOH), 10.84 (s, 1 H, HONHCO), 10.88 (s, 15 1 H, HONHCO). Example 24. Preparation of 5,7-Bis -[1-(6-hydroxypyridazinyl)]oxy-1,3 dibenzyl-4-methyl-2(1 H)-quinolinone a) 5,7-Bis -[1 -(6-chloropyridazinyl)]oxy-1,3-dibenzyl-4-methyl-2(1 H) quinolinone C

N

N 0 C N' N 0 20 WO 99/15523 PCT/FI98/00755 52 A mixture of 1,3-dibenzyl-5,7-dihydroxy-4-methyl-2(1 H)-quinolinone (0.5 g), 3,6-dichloropyridazine (0.83 g) and potassium carbonate (0.75 g) in DMF (12,5 ml) was stirred at 80 0C for 4 hours. The reaction mixture was treated with water at pH 8 and filtered. The solids were recrystallized from 5 ethanol-DMF (2:1). Yield 0.5 g. Melting point 208-218o0C. 1 H-NMR (DMSO-d6, 300 MHz): 2.43 (s, 3H,CH3), 4.16 (s, 2H, CH2Ph), 5.58 (s, 2H, NCH2Ph), 7.09-7.33 (m, 12H, ArH + Ph), 7.55 (d, 1H, PyridH, J = 9,2 Hz), 7.70 (d, 1 H, PyridH, J = 9,2 Hz),7.93 (d, 1H, PyridH, J = 9,2 Hz), 7.98 (d, 1H, PyridH, J = 9,2 Hz). 10 b) 5,7-Bis -[1 -(6-hydroxypyridazinyl)]oxy-1,3-dibenzyl-4-methyl-2(1 H) quinolinone H

N

N 00 N0 O N The product from the previous example (0.2 g) and potassium acetate (0.13 g) in acetic acid (5 ml) were refluxed for 4 hours. The mixture was 15 evaporated, treated with water at pH 10 and filtered. The filtrate was acidified to pH 6 and filtered. Yield 70 mg. 1 H-NMR (DMSO-d6, 300 MHz): 2.47 (s, 3H, CH3), 4.15 (s, 2H, CH2Ph), 5.55 (s, 2H, NCH2), 6.93- 7.34 (m, 15 H, PyridH + ArH + Ph), 7.47 (d, 1H, J = 10 Hz), 12.25 (s, 1H, NH), 12.38 (s, 1H NH).

Claims (18)

1. Compounds of formula (I) or (11): R4A 0 R3 5 R1 [[ R" (I) 0 X Y I R5A R2 R6 10 R8 ,A0 [CH 2 n I( 0 x Y 15 R9/A R7 R9 in which R1 is hydrogen, alkyl, alkenyl, aryl, arylalkyl, hydroxyalkyl, 20 halogenalkyl, alkoxy, COR10, CONR10R11, OR10, S(O)mR10, NRO10COR11 orNR10R11, where R10 is hydrogen, alkyl, alkenyl, aryl, arylalkyl, hydroxyalkyl, halogenalkyl, alkoxy or hydroxy and R11 is hydrogen, alkyl, aryl, arylalkyl, alkoxy, aryloxy, hydroxy or acyl, or in case where X is NR1 1, can R1 also be carboxylalkyl, 25 R6 is hydrogen, alkyl, alkenyl, aryl, arylalkyl, R2 and R7 mean hydrogen, alkyl, aryl, arylalkyl, alkenyl, COR10, CONR 10R11, halogen, trifluoromethyl, nitro or cyano, where R10 and R11 are defined as above, R3 is hydrogen, alkyl, aryl or arylalkyl, 30 A means alkyl or substituted alkyl, m is 0-2 and n is 1-3, Y means O, NR11 or S, where R11 is the same as above, X means O, NR1 1 or S, where R11 is the same as above, R4, R5, R8 and R9 mean independently one of the following groups: 35 WO 99/15523 PCT/FI98/00755 54 N" N So oN H--O, H3 I \ N'N /N-N H 5 or in case where X is NRi 1, can R4, R5, R8 and R9 also independently mean HOOC-, R1200C-, H2NCO- or HOHNCO-, wherein R12 means alkyl, arylalkyl or aryl, and wherein each aryl residue defined above by itself or as part of another 10 group may be substituted, and pharmaceutically acceptable salts and esters thereof, provided that the compound of formula (I) is not 3-benzyl-5,7-bis((l H tetrazol-5-yl)-methyloxy)-4-methyl-2H-1 -benzopyran-2-one. 15

2. A compound of claim 1 wherein said compound has formula (I) and R2 is hydrogen.

3. A compound of claim 2 wherein R1 is hydrogen, C 1 .6 alkyl, C2-.6 alkenyl, C6.-10 aryl, C7-12 arylalkyl, C 1- 6 hydroxyalkyl, C 1- 6 halogenalkyl or 20 C1.-6 alkoxy.

4. A compound of claim 3 wherein Y is O or S, and X is O.

5. A compound of claim 3 wherein Y is O or S; and X is NR1 1, where 25 R11 is hydrogen, C 1- 6 alkyl, C6- 10 aryl, C7-12 arylalkyl, C 1 .6 alkoxy, C6.-10 aryloxy, hydroxy, C 1 -6 alkanoyl or C 1 -6 carboxyalkyl.

6. A compound of claim 5, wherein R3 is hydrogen, C1-6 alkyl, C6.-10 aryl, or C7-12 arylalkyl. 30

7. A compound of claim 6, wherein R3 is C 1 .6 alkyl.

8. A compound of claim 7, wherein A is straight-chain or branched C 1 -. 4 alkylene and R4 and R5 are each WO 99/15523 PCT/F198/00755 55 N N N O N OH H3C\N. .N H3 \> O=M N-N , N-N / H 5 ; or where X is NR11, then R4 and R5 can also be HOOC-, R1200C-, H2NCO- or HOHNCO-, where R12 is C1-6 alkyl, C6-10 aryl, or C7-12 arylalkyl.

9. A compound of claim 1, wherein said compound has formula (11) and 10 R7 is hydrogen.

10. A compound of claim 9, wherein R6 is hydrogen, C1-6 alkyl, C6.-10 aryl, C7-12 arylalkyl; and n is 1,2 or 3. 15

11. A compound of claim 10, wherein Y is O or S, and X is O.

12. A compound of claim 10, wherein Y is O or S; and X is NR11, where R11 is hydrogen, C1.-6 alkyl, C6-10 aryl, C7-12 arylalkyl, C1-6 alkoxy, C6-10 aryloxy, hydroxy, C1-6 alkanoyl or C 1 -6 carboxyalkyl. 20

13. A compound of claim 11, wherein A is straight-chain or branched C1-4 alkylene.

14. A compound of claim 13, wherein R4 and R5 are each 25 N's- N 0_HN - H3C N NN NN , N-N / H WO 99/15523 PCTIFI98/00755 56 or where X is NR11, then R4 and R5 can also be HOOC-, R120OC-, H2NCO- or HOHNCO-, where R12 is C1-6 alkyl, C6-10 aryl, or C7.-12 arylalkyl. 5

15. A pharmaceutical composition comprising a compound of claim 1 as an active ingredient together with pharmaceutically acceptable carrier.

16. A method of treating heart failure comprising administering to a subject in need thereof a therapeutically effective amount of a compound of 10 claim 1.

17. A method for the treatment and prevention of stunned myocardium comprising administering to a subject in need thereof a therapeutically effective amount of a compound of claim 1. 15

18. A compound of formula (XXV): OH R3 R1 20 (XXV) HO N O R11 wherein R1 is hydrogen, alkyl, alkenyl, aryl, arylalkyl, hydroxyalkyl, 25 halogenalkyl, alkoxy, COR10, CONR10R11, OR10, S(O)mR10, NR10 OCOR11 or NR10 R11, where R10 is hydrogen, alkyl, alkenyl, aryl, arylalkyl, hydroxyalkyl, halogenalkyl, alkoxy or hydroxy and R1 1 is hydrogen, alkyl, aryl, arylalkyl, alkoxy, aryloxy, hydroxy or acyl, or carboxylalkyl, R3 is hydrogen, alkyl, aryl or arylalkyl, 30 R11 is hydrogen, alkyl, aryl, arylalkyl, alkoxy, aryloxy, hydroxy or acyl, or carboxylalkyl, and wherein each aryl residue defined above by itself or as part of another group may be substituted.