Chiral beta-hydroxyethylamines and their use in the treatment of hyperglycemia

Abstract

There is herein provided a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein X, R.sup.1, R.sup.2, R.sup.3, R.sup.4 and n have meanings as provided in the description. ##STR00001##

Claims

1. A compound of formula I ##STR00028## or a pharmaceutically acceptable salt thereof, wherein: R.sup.1 represents C.sub.3-11 alkyl, C.sub.3-11 alkenyl, or C.sub.3-11 alkynyl each optionally substituted by one or more halo; R.sup.2 represents C.sub.1-2 alkyl, C.sub.2 alkenyl, or C.sub.2 alkynyl each optionally substituted by one or more halo; R.sup.3 and R.sup.4 each independently represent H or C.sub.1-3 alkyl, C.sub.2-3 alkenyl, or C.sub.2-3 alkynyl each optionally substituted by one or more halo; or R.sup.3 and R.sup.4 may be linked together to form, together with the carbon atom to which they are attached, a 3- to 6-membered ring, which ring optionally is substituted by one or more groups independently selected from halo and C.sub.1 alkyl optionally substituted by one or more halo; each X independently represents halo, R.sup.a, —CN, —N.sub.3, —N(R.sup.b)R.sup.c, —NO.sub.2, —ONO.sub.2, —OR.sup.d, —S(O).sub.pR.sup.e or —S(O).sub.qN(R.sup.f)R.sup.g; R.sup.a represents C.sub.1-6 alkyl, C.sub.2-6 alkenyl, or C.sub.2-6 alkynyl each optionally substituted by one or more groups independently selected from G; each R.sup.b, R.sup.c, R.sup.d, R.sup.e, R.sup.f and R.sup.g independently represents H or C.sub.1-6 alkyl, C.sub.2-6 alkenyl, or C.sub.2-6 alkynyl each optionally substituted by one or more groups independently selected from G; or alternatively any of R.sup.b and R.sup.c and/or R.sup.f and R.sup.g may be linked together to form, together with the nitrogen atom to which they are attached, a 4- to 6-membered ring, which ring optionally contains one further heteroatom and which ring optionally is substituted by one or more groups independently selected from halo, C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and C.sub.2-3 alkynyl each optionally substituted by one or more halo, and ═O; G represents halo, —CN, —N(R.sup.a1)R.sup.b1, OR.sup.c1, —S(O).sub.pR.sup.d1, —S(O).sub.qN(R.sup.e1)R.sup.f1 or ═O; each R.sup.a1, R.sup.b1, R.sup.c1, R.sup.d1, R.sup.e1 and R.sup.f1 independently represents H or C.sub.1-6 alkyl, C.sub.2-6 alkenyl, or C.sub.2-6 alkynyl each optionally substituted by one or more halo; or alternatively any of R.sup.a1 and R.sup.b1 and/or R.sup.e1 and R.sup.f1 may be linked together to form, together with the nitrogen atom to which they are attached, a 4- to 6-membered ring, which ring optionally contains one further heteroatom and which ring optionally is substituted by one or more groups independently selected from halo, C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and C.sub.2-3 alkynyl each optionally substituted by one or more halo, and ═O; n represents 0 to 5; each p independently represents 0, 1 or 2; and each q independently represents 1 or 2; wherein the compound of formula I is present in a diastereomeric excess of at least 90%.

2. The compound according to claim 1, wherein R.sup.1 represents C.sub.3-6 alkyl optionally substituted by one or more F.

3. The compound according to claim 1, wherein R.sup.2 represents C.sub.1-2 alkyl optionally substituted by one or more F.

4. The compound according to claim 1, wherein R.sup.1 represents n-propyl and R.sup.2 represents methyl.

5. The compound according to claim 1, wherein R.sup.3 and R.sup.4 each represent H.

6. The compound according to claim 1, wherein: each X independently represents halo, R.sup.a, —CN, —N.sub.3, —N(R.sup.b)R.sup.c, —NO.sub.2 or OR.sup.d, wherein R.sup.a represents C.sub.1-4 alkyl optionally substituted by one or more F, and wherein R.sup.b, R.sup.c and R.sup.d each independently represent H or C.sub.1-4 alkyl optionally substituted by one or more F or ═O.

7. The compound according to claim 1, wherein n represents 0, 1, 2 or 3.

8. The compound according to claim 1, wherein the compound is a compound of formula IA ##STR00029## wherein: X.sup.1, X.sup.2, X.sup.3, X.sup.4 and X.sup.5 each independently represent H or X.

9. The compound according to claim 8, wherein: X.sup.1 and X.sup.5 each represent H; and X.sup.2, X.sup.3 and X.sup.4 each independently represent H, halo, R.sup.a, —CN, —N.sub.3, —N(R.sup.b)R.sup.c, —NO.sub.2 or —OR.sup.d; wherein R.sup.a represents C.sub.1-4 alkyl optionally substituted by one or more F, and wherein R.sup.b, R.sup.c and R.sup.d each independently represent H or C.sub.1-4 alkyl optionally substituted by one or more F or ═O.

10. The compound according to claim 8, wherein the compound of formula I or formula IA is a compound of formula IC ##STR00030##

11. The compound according to claim 10, wherein: X.sup.1, X.sup.2 and X.sup.5 each represent H; X.sup.3 and X.sup.4 each independently represent H, F or —OH; R.sup.1 represents C.sub.3 alkyl; R.sup.2 represents C.sub.1 alkyl; and R.sup.3 and R.sup.4 both represent H.

12. A pharmaceutical composition comprising a compound as defined in claim 1, and optionally one or more pharmaceutically acceptable adjuvant, diluent and/or carrier.

13. A method of treating hyperglycaemia or a disorder characterized by hyperglycaemia comprising administering to a patient in need thereof a therapeutically effective amount of a compound as defined in claim 1, or a pharmaceutically acceptable salt thereof.

14. The method according to claim 13, wherein the hyperglycaemia or disorder characterized by hyperglycaemia is type 2 diabetes.

15. The method according to claim 13, wherein the patient displays severe insulin resistance.

16. The method according to claim 13, wherein the disorder characterised by hyperglycaemia is selected from the group consisting of Rabson-Mendenhall syndrome, Donohue's syndrome (leprechaunism), Type A and Type B syndromes of insulin resistance, the HAIR-AN (hyperandrogenism, insulin resistance, and acanthosis nigricans) syndromes, pseudoacromegaly, and lipodystrophy.

17. A combination product comprising: (a) a compound as defined in claim 1; and (b) one or more other therapeutic agent that is useful in the treatment of hyperglycaemia or a disorder characterized by hyperglycaemia, wherein each of components (a) and (b) is formulated in admixture, optionally with one or more a pharmaceutically-acceptable adjuvant, diluent or carrier.

18. A kit-of-parts comprising: (a) a pharmaceutical composition comprising a compound as defined in claim 1, and optionally one or more pharmaceutically acceptable adjuvant, diluent and/or carrier, and (b) one or more other therapeutic agent that is useful in the treatment of hyperglycaemia or a disorder characterized by hyperglycaemia, optionally in admixture with one or more pharmaceutically-acceptable adjuvant, diluent or carrier, which components (a) and (b) are each provided in a form that is suitable for administration in conjunction with the other.

19. A process for the preparation of a compound as defined in claim 1, or a pharmaceutically acceptable salt thereof, comprising the step of: (i) reaction of a compound of formula II ##STR00031## wherein n, X, R.sup.3, R.sup.4 and n are as defined in claim 1, with a compound of formula III ##STR00032## wherein R.sup.1 and R.sup.2 are as defined in claim 1, optionally in the presence of a suitable solvent known to those skilled in the art; (iia) reaction of a compound of formula IV ##STR00033## wherein n, X, R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are as defined in claim 1 and Y.sup.1 represents H or PG.sup.1 wherein PG.sup.1 is a suitable protecting group as known to those skilled in the art with a suitable reduction agent as known to those skilled in the art; (iib) for compounds of formula IC ##STR00034## wherein X.sup.1, X.sup.2, X.sup.3, X.sup.4 and X.sup.5 each independently represent H or X, wherein X, R.sup.1, R.sup.2, R.sup.3, and R.sup.4 are as defined in claim 1, reaction of a compound of formula IV as defined in step (iia) but wherein Y.sup.1 represents PG.sup.1 wherein PG.sup.1 is a suitable protecting group as known to those skilled in the art in the presence of hydrogen or a suitable hydrogen donor and optionally in the presence of a base and in the presence of a suitable solvent; (iii) for compounds wherein at least one X is present and represents —OH, deprotection of a compound of formula V ##STR00035## wherein n, R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are as defined in claim 1, Y.sup.2 represents H or PG.sup.2, wherein PG.sup.2 represents a suitable protecting group as known to those skilled in the art, and PG.sup.3 represents a suitable protecting group as known to those skilled in the art under conditions known to those skilled in the art; (iv) for compounds wherein at least one X is present and represents NH.sub.2, deprotection of a compound of formula VI ##STR00036## wherein n, X, R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are as defined in claim 1, Y.sup.3 represents H or PG.sup.5, wherein PG.sup.5 represents a suitable protecting group as known to those skilled in the art, Y.sup.4 represents H or PG.sup.6, wherein PG.sup.6 represents a suitable protecting group as known to those skilled in the art, and PG.sup.4 represents a suitable protecting group as known to those skilled in the art, under conditions known to those skilled in the art; (v) for compounds wherein at least one X is present and represents NH.sub.2, reduction of a compound of formula VII ##STR00037## wherein n, X, R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are as defined in claim 1, under conditions known to those skilled in the art.

Description

EXAMPLES

(1) The present invention is illustrated by way of the following examples.

(2) Chemicals and reagents were obtained from commercial suppliers and were used as received unless otherwise stated. All reactions involving moisture sensitive reagents were performed in oven or flame dried glassware under a positive pressure of nitrogen or argon.

(3) Abbreviations

(4) Abbreviations as used herein will be known to those skilled in the art. In particular, the following abbreviations may be used herein.

(5) AcOH acetic acid

(6) aq aqueous

(7) Boc.sub.2O di-tert-butyl dicarbonate

(8) de diastereomeric excess

(9) DIPEA N,N-diisopropylethylamine

(10) EtOAc ethyl acetate

(11) EtOH ethanol

(12) Et.sub.2O diethyl ether

(13) Et.sub.3N triethylamine

(14) h hours

(15) HPLC high-performance liquid chromatography

(16) MeOH methanol

(17) Pd—C palladium on carbon

(18) rt room temperature

(19) Example Compounds

(20) In the event that there is a discrepancy between nomenclature and the structure of compounds as depicted graphically, it is the latter that presides (unless contradicted by any experimental details that may be given and/or unless it is clear from the context).

(21) In the event that there is a discrepancy between nomenclature and the structure of compounds as depicted graphically, it is the latter that presides (unless contradicted by any experimental details that may be given and/or unless it is clear from the context).

Example 1: 4-((R)-1-Hydroxy-2-(((R)-pentan-2-yl)amino)ethyl)phenol Acetate

(22) ##STR00014##

(a) 4-Benzyloxyacetophenone

(23) ##STR00015##

(24) K.sub.2CO.sub.3 (7.5 g, 54.4 mmol) was added to a solution of 4-hydroxyacetophenone (3.7 g, 27.2 mmol) in acetone (180 mL) at rt. The mixture was stirred at rt for 1 h and benzylbromide (3.2 mL, 27.2 mmol) was added. The mixture was heated at reflux for 12 h, allowed to cool and filtered. Concentration of the filtrate gave the sub-title compound (6.1 g, 27.1 mmol, ˜100%) which was used in the next step without further purification.

(b) 4-Benzyloxyphenacyl Bromide

(25) ##STR00016##

(26) Pyridinium tribromide (9.5 g, 29.8 mmol) was added to a solution of 4-benzyloxyacetophenone (6.1 g, 27.1 mmol) in CH.sub.2Cl.sub.2 (275 mL) and MeOH (100 mL) at rt. After 3 h at rt the mixture was concentrated and the residue partitioned between water and EtOAc. The layers were separated and the aqueous layer was extracted with EtOAc. The combined organic phases were washed with brine, dried over Na.sub.2SO.sub.4 and filtered. Concentration of the filtrate and crystallization of the residue from hexane/EtOAc gave the sub-title compound (6.4 g, 21.1 mmol, 78%).

(c) tert-Butyl (R)-(2-(4-(benzyloxy)phenyl)-2-oxoethyl)(pentan-2-yl)carbamate

(27) ##STR00017##

(28) DIPEA (1.9 mL, 10.8 mmol) was added dropwise to a solution of (R)-pentyl-2-amine hydrobromide (1.2 g, 7.21 mmol) in CH.sub.2Cl.sub.2 (70 mL) at −20° C. 4-Benzyloxyphenacyl bromide (1.1 g, 3.60 mmol) in CH.sub.2Cl.sub.2 (50 mL) was added over 15 min at −20° C. The mixture was stirred at −20° C. for 2 h and kept at that temperature for 60 h. Boc.sub.2O (4.1 mL, 18.0 mmol) was added and the mixture was stirred at rt for 2 h. The mixture was washed with water and brine, dried over Na.sub.2SO.sub.4, concentrated and purified by chromatography to give the sub-title compound (0.60 g, 1.46 mmol, 40%).

(d) tert-Butyl ((R)-2-(4-(benzyloxy)phenyl)-2-hydroxyethyl)((R)-pentan-2-yl)carbamate

(29) ##STR00018##

(30) A mixture of tert-butyl (S)-(2-(4-(benzyloxy)phenyl)-2-oxoethyl)(pentan-2-yl)carbamate (300 mg, 0.73 mmol), (S,S)—N-(p-toluenesulfonyl)-1,2-diphenylethanediamine(chloro)-(p-cymene)ruthenium(II) (20 mg, 0.03 mmol) and formic acid/Et.sub.3N (5:2, 2 mL) was stirred at rt for 24 h and additional portions of catalyst (20 mg) and formic acid/Et.sub.3N (5:2, 0.25 mL) were added. This procedure was repeated at 24 h intervals five more times (total amount of catalyst 120 mg (0.30 mmol), total reaction time 7 d). Water (15 mL) and CH.sub.2Cl.sub.2 (30 mL) were added and the aq layer was extracted with CH.sub.2Cl.sub.2. The combined organic phases were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated. The residue was purified by chromatography and HPLC to give the sub-title compound (0.25 g, 0.60 mmol, de>99%, 83%).

(e) (R)-1-(4-(Benzyloxy)phenyl)-2-(((R)-pentan-2-yl)amino)ethan-1-ol

(31) ##STR00019##

(32) A solution of NaOH (0.43 g, 10.6 mmol) in water (2 mL) was added to a solution of tert-butyl ((R)-2-(4-(benzyloxy)phenyl)-2-hydroxyethyl)((R)-pentan-2-yl)carbamate (0.22 g, 0.53 mmol) in EtOH (3 mL). The mixture was heated at 100° C. in a sealed tube for 40 h and allowed to cool. Water and EtOAc was added and the layers were separated. The aq layer was extracted with EtOAc and the combined organic phases were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated to give the sub-title compound which was used in the next step without further purification (0.13 mg, 0.41 mmol, 78%).

(f) 4-((R)-1-Hydroxy-2-(((R)-pentan-2-yl)amino)ethyl)phenol Acetate

(33) ##STR00020##

(34) A mixture of (R)-1-(4-(benzyloxy)phenyl)-2-(((R)-pentan-2-yl)amino)ethan-1-ol (125 mg, 0.40 mmol), 10% Pd—C (85 mg, 0.08 mmol) and AcOH (3 mL) was hydrogenated at 9 atm at rt for 1.5 h, filtered through Celite and concentrated. The residue was taken up into a small amount of water, filtered through Celite and the filtrate was concentrated to about 1 mL. The material was purified by reverse phase chromatography to give the title compound (60 mg, 0.21 mmol, de>99%, 53%).

(35) .sup.1H NMR (400 MHz, D.sub.2O) δ 7.38-7.30 (m, 2H), 6.98-6.92 (m, 2H), 4.96 (t, J=6.6 Hz, 1H), 3.43-3.33 (m, 1H), 3.29 (d, J=6.6 Hz, 2H), 1.92 (s, 3H), 1.78-1.67 (m, 1H), 1.64-1.52 (m, 1H), 1.51-1.34 (m, 2H), 1.32 (d, J=6.6 Hz, 3H), 0.93 (t, J=7.3 Hz, 3H).

Example 2: 4-((R)-1-Hydroxy-2-(((R)-pentan-2-yl)amino)ethyl)phenol Hemisulphate

(36) ##STR00021##

(37) Sulphuric acid (aq, 0.1 M, 0.85 mL) was added to a solution of the acetate in Example 1 (48 mg, 0.17 mmol) in H.sub.2O (1 mL). The mixture was concentrated and dried over P.sub.2O.sub.5. The residue was treated with toluene (2 mL) and concentrated. The procedure was repeated and the residue was treated with Et.sub.2O, concentrated and dried over P.sub.2O.sub.5 to give the title compound (34 mg, 0.12 mmol, 74%)

(38) .sup.1H NMR (400 MHz, D.sub.2O) δ 7.37-7.31 (m, 2H), 6.98-6.92 (m, 2H), 4.96 (t, J=6.6 Hz, 1H), 3.45-3.34 (m, 1H), 3.29 (d, J=6.7 Hz, 2H), 1.79-1.68 (m, 1H), 1.65-1.53 (m, 1H), 1.52-1.34 (m, 2H), 1.33 (d, J=6.6 Hz, 3H), 0.93 (t, J=7.3 Hz, 3H).

Example 3: 3-((R)-1-Hydroxy-2-(((R)-pentan-2-yl)amino)ethyl)phenol Acetate

(39) ##STR00022##

(40) The title compound was prepared in accordance with Example 1, using 3-benzyloxyacetophenone in Step (b).

(41) .sup.1H NMR (400 MHz, D.sub.2O) δ 7.37-7.32 (m, 1H), 7.01-6.96 (m, 1H), 6.95-6.88 (m, 2H), 4.98 (dd, J=9.1, 3.9 Hz, 1H), 3.42-3.34 (m, 1H), 3.31 (dd, J=13.0, 3.9 Hz, 1H), 3.26 (dd, J=13.0, 9.1 Hz, 1H), 1.91 (s, 3H), 1.77-1.67 (m, 1H), 1.64-1.52 (m, 1H), 1.51-1.33 (m, 2H), 1.32 (d, J=6.6 Hz, 3H), 0.93 (t, J=7.3 Hz, 3H).

Example 4: 3-((R)-1-Hydroxy-2-(((R)-pentan-2-yl)amino)ethyl)phenol Hemisulphate

(42) ##STR00023##

(43) The title compound was prepared in accordance with Example 3, using the acetate from Example 2.

(44) .sup.1H NMR (400 MHz, D.sub.2O) δ 7.35 (t, J=7.9 Hz, 1H), 7.00 (d, J=7.7 Hz, 1H), 6.97-6.88 (m, 2H), 4.99 (dd, J=9.1, 3.7 Hz, 1H), 3.45-3.22 (m, 3H), 1.79-1.66 (m, 1H), 1.65-1.52 (m, 1H), 1.52-1.35 (m, 2H), 1.32 (d, J=6.6 Hz, 3H), 0.93 (t, J=7.3 Hz, 3H).

Example 5: (R)-1-(4-Fluorophenyl)-2-(((R)-pentan-2-yl)amino)ethan-1-ol

(45) ##STR00024##

(a) tert-Butyl (2-(4-fluorophenyl)-2-oxoethyl)(pentan-2-yl)carbamate

(46) ##STR00025##

(47) A solution of 4-fluorophenacyl bromide (500 mg, 2.31 mmol) in CH.sub.2Cl.sub.2 (6.6 mL) was added over 10 min to a solution of 2-aminopentane (301 mg, 3.46 mmol) and DIPEA (298 mg, 2.31 mmol) in CH.sub.2Cl.sub.2 (1.6 mL) at 0° C. The cooling bath was removed and the mixture was stirred at rt for 2 h. A solution of Boc.sub.2O (1.26 g, 5.76 mmol) in CH.sub.2Cl.sub.2 (6 mL) was added and the mixture was stirred at rt for 2 h, washed with H.sub.2O, brine and dried over Na.sub.2SO.sub.4. Concentration and purification by chromatography gave the sub-title compound (520 mg, 1.61 mmol, 70%).

(b) (R)-1-(4-Fluorophenyl)-2-(((R)-pentan-2-yl)amino)ethan-1-ol

(48) ##STR00026##

(49) The title compound was obtained using the procedures in Example 1, Step (d) followed by chromatographic separation and hydrolysis in accordance with the procedure in Example 1, step (e).

(50) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.34 (dd, J=8.4, 5.6 Hz, 2H), 7.03 (t, J=8.4 Hz, 2H), 4.61 (dd, J=9.4, 3.4 Hz, 1H), 2.91 (dd, J=12.0, 3.6 Hz, 1H), 2.69-2.59 (m, 2H), 1.47-1.28 (m, 4H), 1.06 (d, J=6.0 Hz, 3H), 0.91 (t, J=6.8 Hz, 3H).

Example 6: (R)-1-(3-fluorophenyl)-2-(((R)-pentan-2-yl)amino)ethan-1-ol

(51) ##STR00027##

(52) The title compounds were prepared in accordance with Example 5 from 3-fluorophenacyl bromide.

(53) .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.33-7.26 (m, 1H), 7.14-7.09 (m, 2H), 7.00-6.90 (m, 1H), 4.66 (dd, J=8.8, 3.6 Hz, 1H), 2.97-2.92 (m, 1H), 2.72-2.62 (m, 3H), 1.47-1.26 (m, 4H), 1.07 (dd, J=6.4, 1.2 Hz, 3H), 0.93-0.89 (m, 3H).

BIOLOGICAL EXAMPLES

(54) L6-myoblasts were grown in Dulbecco's Modified Eagle's Medium (DMEM) containing 4.5 g/l glucose supplemented with 10% fetal bovine serum, 2 mM L-Glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin and 10 mM HEPES. Cells were plated at 1×10.sup.5 cells per ml in 24-well plates. After reaching 90% confluence the cells were grown in medium containing 2% FBS for 7 days where upon cells differentiated into myotubes.

Biological Example 1: Glucose Uptake

(55) Differentiated L6-myotubes were serum-starved over night in medium containing 0.5% fatty-acid free BSA and stimulated with agonist, final concentration 1×10.sup.−5. After 1 h 40 min cells were washed with warm, glucose free medium or PBS and another portion of agonist was added to glucose free medium. After 20 min the cells were exposed to 50 nM .sup.3H-2-deoxy-glucose for another 10 min before washed in ice cold glucose free medium or PBS and lysed in 0.2 M NaOH for 1 h in 60° C. Cell lysate was mixed with scintillation buffer (Emulsifier Safe, Perkin Elmer and radioactivity detected in a β-counter (Tri-Carb 2800TR, Perkin Elmer). The activity for each compound is compared to that of isoproterenol. If a compound shows activity of more than 75% of that of isoproterenol, the activity is denoted with +++, if it is between 75 and 50% it is denoted with ++; if it is between 50 and 25% it is denoted with +; if it less than 25% it is denoted with −.

Biological Example 2: Measurement of Intracellular cAMP Levels

(56) Differentiated cells were serum-starved over night and stimulated with agonist, final concentration 1×10.sup.−5, for 15 min in stimulation buffer (HBSS supplemented with 1% BSA, 5 mM HEPES and 1 mM IBMX, pH 7.4) The medium was then aspirated and to end the reaction 100 μL of 95% EtOH was added to each well of a 24-well plate and cells were kept in −20° C. over night. The EtOH was let to evaporate and 500 μL of lysis buffer (1% BSA, 5 mM HEPES and 0.3% Tween-20, pH 7.4) was added to each well before put in −80° C. for 30 min and then kept in −20° C. Intracellular cAMP levels were detected using an alpha screen cAMP kit (6760635D from Perkin Elmer). The activity for each compound is compared to that of isoproterenol. If a compound shows activity of more than 75% of that of isoproterenol, the activity is denoted with +++, if it is between 75 and 50% it is denoted with ++; if it is between 50 and 25% it is denoted with +; if it less than 25% it is denoted with −.

(57) Using the assays described in Biological Examples 1 and 2 the following results were obtained.

(58) TABLE-US-00001 Compound Biological Biological example no. example 1 example 2 2 ++ + 4 +++ ++ 5 ++ − 6 ++ −