Field of Invention The present invention relates to compounds and compositions useful in modulating the renin-angiotensin system (RAS) in cells, and in particular to compounds which, inter alia, act as angiotensin II antagonists by binding to angiotensin II receptors. The invention also relates to the use of these compounds and compositions in the treatment of conditions responsive to angiotensin II antagonists such as hypeitension, edema, renal failure, benign prostatic hypertrophy glaucoma, atherosclerosis, diabetes, Alzheimer's disease and congestive heart failure.

Background of Invention Blood pressure is regulated by interrelated factors including neural, vascular and volume- related effects, The renin-anigiotensin system (RAS) is an important system for regulating blood pressure. The scheme below diagrammatically illustrates the RAS:

AngiolinSinOgen

j Kidneys Renin Liver

a t ater retent on

Increase in biood pressure Low renal perfusion pressure stimulates the kidneys to produce the proteolytic enzyme renin. This erayme acts on angiotensinogen (a circulating protein) cleaving off the dccapeptidc angiotensin I. Angiotensin I is then cleaved to the octapeptide angiotensin II by the angiotensin coverting enzyme (ACE). This takes place in the lungs, Angiotensin II is the biologically active component of the RAS and exerts its effects by binding to specific receptors located in the plasma membranes of various target organ cells. There are two types of angiotensin receptors known as AT) and AT2. The receptors are a member of the superfamily of seven-transmembrane-domain G protein coupled receptors. Angiotensin II interacts with the transmembrane domains of its ATi receptor, to bring about effects including vasoconstriction, vascular remodelling and salt/water retention, but has negligible or no stimulating action on the heart. The effects however all lead to a rise in blood pressure.

Hypertension is defined as a persistently high arterial blood pressure and is a well- established risk factor for cardiovascular disease, cerebral haemorrhage, stroke and kidney disease. The prevalence of hypertension in the western world in conjunction with its potentially lethal consequences makes treatment of the disease important. Some antihypertensive drugs used in therapy target the enzymes renin and ACE in RAS. For instance, inhibitors of ACE are known whose approved names generally end in "-pril" or in the case of active metabolites "-prilate". Examples such as captopril and enalapril are both commercially available ACE inhibitors which are effective antihypertensive agents, ACE inhibitors, however, are unfortunately known to have adverse side effects such as dizziness and fainting, Skin rash, swelling to the face, mouth and hands, and may also cause a persistent dry cough.

In contrast, antagonists at angiotensin receptors, and more specifically AT| receptors, (angiotensin II antagonists) are also effective at treating hypertensio and congestive heart failure. Such compounds typically have approved names which generally end in "-sartan". Examples include abitesartan, benzyllosartan, elisartan, embusartan, enoltasosartan, fonsartan, forasaitan, glycyllosartan, milfasartan, olmesartan, opomisartan, pratosartan, ripisartan, eprosartan, candesartan (including candersartan cilexetil), irbesartan, saprisartan, tasosartan, telmisartan, valsartan, zolasartan, and losartan (including losartan potassium). It is appreciated in the art however that the terra "sartan" is generally used to refer to any angiotensin II antagonist which blocks the action of angiotensin II at its AT, receptors and includes saralasin and saralasin acetate, CGP-63170, EMD-66397, T3-671 , LR-B/081, A-81282, BlBR-363, BIBS-222, B S-184698, CV-1 1 194, EXP-3174, KW- 3433, L-161 177, L-162154, LR-B/057, LY-235656, PD-150304, U-96849, U-97018, UP, 275-22, WAY-126227, WK-1492.2K. YM-31472, E 177, EMD-73495, HN-65021, L- 159282, ME-3221, SL-91.0102, UP-269-6, YM-358, CGP-49870, GA-00S6, L-1S9689, L- 162234, L-162441 , L-163007, PD-123177, A-81988, BlviS-180560. CGP-38S60A, CGP48369, DA-2079, DE-3489, DuP-167, EXP-063, EXP-6155, EXP-6803, EXP-771 1 , EXP-9270, F -739, HR-720, ICI-D6888, IC1-D7155, IC1 -D8731, isoteoline, KR1 -1 177, L-158809, L-158978, L-159874, LR B087, LY-285434, LY-302289, LY-315995, RG- 13647, RWJ-38970, RWJ-46458, S-8307, S-8308, sarmesin, W -1360, X-6803, 2D-6888, ZD-7155, ZD-8731, BIBS39> CI-996, D P-81 1, DuP^532, EXP-929, L-163017, LY- 301875, XH-148, XR-510 and PD-123319.

Sartans, like angiotensin II itself, interact with the uansmembrane domains of the AT, receptors, Since the sartans selectively bind to ATi receptors, angiotensin II is left with free access to its unblocked AT ₂ receptors. This is beneficial as recent evidence suggests that many of the effects of the interaction between angiotensin II and its AT ₂ receptors reduce blood pressure. Also, the sartans have been shown to lack the side effects demonstrated by other antihypertensives such as the ACE inhibitors. The sartans themselves, while all acting as angiotensin. II antagonists, are characterised with different molecular frameworks. For instance, classes of sartans based on a 5 membered nitrogen containing heterocycle framework (often substituted with a substituted biphenyl group) are described in EP 28,834-A, EP 253,310-A, EP 324,377-A, WO 91/14679, EP 392,317-A, EP 403,159-A, EP 503,162-A, EP 573,271-A, WO 94/03449, WO 94/08989, WO 94/08990, U.S. Pat. No. 4,576,958, U.S. Pat. No. 4,582,847, U.S. Pat. No. 4,207,324 and U.S, Pat. No. 4,340,598. Sartans such as losartan, irbesartan, pratosartan, olmesartan, fonsartan, eprosartan, and saprisartan fall within this class.

Another class of sartans are derived from condensed heterocycles, in particular benzimidazoles and imidazpyridines (often also substituted with a substituted biphenyl group) and are described in EP 399,731 -A, EP 400,974-A, EP 392,317-Α, ΈΡ 260,613-A, EP 412,848-A, EP 420,237-Α,ΈΡ 502,314-A, EP 552,765-A, EP 546,358-A, EP 595,151 - A, EP 598,702-A, EP 245,637-A, U.S. Pat. No. 4,880.804, WO.93/190067, WO 94/01436 and WO 94/204,498. Candesartan and telmisartan fall within this class.

Yet another class of sartans are formed from optionally condensed 6-membered nitrogen containing heterocycles as described in EP 412,848-A, GB 2,234,748^A, WO 91/07404, EP 487,252-A, EP 443,983-A, EP 500,409-A, WO 94/07492, WO 94/1 1379, WO 94/1 1369 and WO 95/002596. Tasosartan falls within this class.

Various other structures are described in EP 566,060-A, EP 434,249-A, WO 94/00450 and EP 604,259. For instance, EP 434,249-A describes the benzofuran class of sartans to which saprisartan' and zolasartan belong. Furthermore, saralasin and sarmesin represent peptide analogues of angiotensin Π.

As of 2000, six orally active sartans have been accepted by the US Food and Drug Administration and are available in the US and various other European countries for the • treatment of hypertension. These compounds include losartan, valsartan, irbesartan, candesartan, telmisartan and eprosartan. While, the mechanism of these compounds is relatively uniform, the pharmacodynamics and pharmacokinetics arc quite different. For instance, irbesartan displays longer activity than losartan and valsartan. Candesartan displays poor oral absorption and is therefore administered as the ester prodrug candesartan ciiexetil. Telmisartan has a very long elimination half-life and causes an increase in serum diogix when used in combination therapy with digoxin. In contrast, eprosartan has a very short half-life (5-7 hr). The above pharmacological variations have both beneficial and negative implications in therapy. It is thus desirable to develop novel sartans with improved and/or new pharmacological profiles.

Summary of Invention

The present invention provides a class of multi-functioning/multipotent biphenyl sartan compounds ('biphenyl sartans') which possess both traditional angiotensin antagonist activity but also antioxidant activity. More specifically the present invention provides biphenyl-sartans which comprise a stable nitroxide moiety which acts as a radical scavenger and in particular a reactive oxygen species (RQS) scavenger:

The invention is based on the discovery that certain nitroxide containin biphenyl sartans ("nitroxide-biphenyl sartans") as herein retain their sartan activity (ie continue to act as angiotensin II antagonists) but also display other unique properties such as elevated antioxidant activity and ma also possess anti- inflammatory activity. Accordingly, the nitroxide-biphenyl sartans of the present invention may function as effective roultipotent (or multi-functioning) antihypertensive agents. Such compounds have significant potential in treating, in addition to hypertension, diseases and conditions linked to, for instance, oxidative stress, such as neurodegenerative diseases (eg Alzheimer's disease, Parkinson's disease, Fredreich's Ataxia, Ataxia Telangiectasia, Wilson's disease, motor neurone disease, etc), mtDNA diseases (eg diabetes mellitus, epilepsy, renal failure, etc), as well as providing treatments for inflammation and ischaemic-reperfusion tissue injury in strokes, heart attacks, organ transplantation and surgery. Accordingly, in an aspect of the invention there is provided a method for the treatment of hypertension and/or conditions associated with oxidative stress by the administration of a nitroxide-biphenyl sartan, or a pharmaceutically acceptable salt thereof, or a composition containing a nitroxide-biphenyl sartan, or a pharmaceutically acceptable salt thereof. In another aspect the invention provides the use of a nitroxide-biphenyl sartan, or a salt thereof, in the manufacture of a medicament for the treatment of hypertension and/or conditions associated with oxidative stress. In another aspect of the invention there is provided a method of intentionally modulating the renin-angiotensin system (RAS) of cells by the application of a nitroxide-biphenyl sartan, or a pharmaceutically acceptable salt thereof, to said cells.

In a further aspect of the invention there is provided a pharmaceutical composition for use as an antihypertensive and/or antioxidant, the composition comprising an effective amount of a nitroxide-biphenyl sartan, or a pharmaceutically acceptable salt thereof and optionally a carrier or diluent.

In another aspect of the invention there is provided a process for the preparation of nitroxide-biphenyl sartans, or salts thereof.

Brief Description of Figures

Figure .. Bar graph of tissue-based doxorubicin antioxidant assay. Δ Force (g-min) for each of Vehicle control (A), Milfasartan (B, 1 Ομχη), Nitroxyl-Milfasartan (C,

Ι Ομιη) (Compound 7) and Nitroxyl (D, 2 _> 2,5,5-tetramethyl-2,5- dihydropyrrole-l-oxy-3-carboxylic acid) (ΙΟμιη).

Figure 2. Angiotensin II inhibition data. Plot of Δ RFU (Relative Fluorescence Units) as a function of logM angiotensin II concentration for compounds 6 and 5.

Figure 3. Bar graphs of tissue-based lucigenin antioxidant assay. Counts/mg for each of vehicle control, lucigenin, 1% DMSO and DPI (diphenylenciodonium sulphate) at 1 μΜ, 10 μΜ and ΙΟΟμΜ (above); lucigenin, DPI (10 μΜ) and the salt of compound 6 (Compound 7, Example 2) at 1 μΜ, 3 μΜ and 10μΜ

(below). Figure 4a. Competitive inhibition of Angiotensin II-rnediated increases in intracellular calcium by Compound 7. Plot of Δ R tJ (Relative Fluorescence Units) as a function of lo M angiotensin U concentration for compound 7. Figure 4b. Schild plot for Compound 7 with a Κβ of 8.14 determined using the intracellular calcium measurements in CHO cells stably expressing the ATJA rcceptor.

Figure 5. Compound 7 lowers blood pressure over 7 days to a similar level as eprosartan. Plot of blood pressure (mmHg) as a function of time (days).

Figure 6. Persistent inhibition of vascular AT receptors following 7 day treatment with Compound 7 in SHR. Plot of % KPSS + NA (ΙΟμΜ as a function of ATII concentration (log M).

Figure 7. Persistent inhibition of cardiac AT _tA receptors following 7 day treatment with

Compound 7 in SHR. Plot of change of right arterial rate (beats/min) as a function of AT II concentration (log ), Figure 8. Compound 7 prevents intlmal thickening of rubbed carotid arteries in SHR.

Bar graph of arterial thickness (μΜ) when 30 mg/kg day of mtroxide, compound 7, and milfasartan, is administered.

Figure 9. Compound 7 inhibits superoxide levels in rat abdominal aorta. Bar graph of luminescence counts (mg) when 30 mg/kg/day of nitroxide, compound 7, and milfasartan, is administered.

Detailed Description of the Invention Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It will be appreciated that the present invention involves the incorporation of a stable nitroxide moiety unto a biphenyl sartan (i.e., angiotensin II antagonist) to form a nitroxide containing analogue of the angiotensin II antagonist with full or partial retention of the angiotensin antagonist activity.

The term "biphenyl-sartan" as used herein refers to any non-peptide angiotensin II antagonist which displays an in-vitro AT] -receptor affinity lC ₅ o* in the range of about 0.1 nM to about 100 μ (* ICso representing the concentration displacing specifically 50% of The binding of angiotensin) and is additionally characterised with a biphenyl moiety, that for the determination of ICjo values is described

by Keenan et al., J. Med. Chem., 1992, 35, 3858-3872, and by Duncia et al., J. . Med. C/H?m. , l990, JJ, 1312-1329.

Examples of known biphenyl sartans include, but are not limited to, abitesartan, benryllosartan, clisartan, embusartan, enoltasosartan, fonsartan, forasartari, glycyllosartan, milfasartan, olmesartan, opomisartan, piatosartan, ripisartan, candesanan (including candersartan cilexetil), irbesartan, saprisartan, tasosartan, telmisartan, valsartan, zolasartan, and losartan (including losartan potassium), BMS-184698, L-162154, Y -31472, HN- 65021, ME-3221, D-8731, CL-329167, TAK-356, L-162234, L-162441 , L-163017, WJ- 464S8 and BIBS39. It will be appreciated though that the present invention is not limited to the derivatisation of known biphenyl sartans.

Accordingly, the term "nitroxide-biphenyl sartan" refers to any non-peptide angiotensin II antagonist which displays an in-vitro AT _r receptor affinity IC ₅ o* in the range of about

100 μΜ which is characterised with a biphenyl moiety, that is, is also characterised with a stable-nitroxide moiety. The stable-nitroxide moiety may be any chemical moiety which bears a nitroxide free radical, which is stable in the sense that the nitroxyl group does not (to any great extent) decompose under physiological conditions. Non-stable nitroxides are known to decompose to form hydroxylamines and nitrones. Accordingly, based on these decomposition products one of ordinary skill Would be able to assess the suitability of any nitroxide moiety for the purposes of the present invention.

Examples of suitable stable-nitroxide moieties include:

wherein:

Ar represents an aryl group; ¹ to R ⁴ in each occurrence may be independently aryl or G1-C3 alkyl, preferably C-O, alkyl;

n is 1 or 2; and

^r= represents an optional double bond.

More specific suitable stable-nitroxide moieties include:

2,2,5,5'tetramethyI-2,5- dih dropyrrole- 1 -oxy

2,2,0,6 lidinyl-

2,2,4,4-tetramethyl-3- iethyl-3-oxazolidinyloxy oxazolidinyloxy (DOXYL)

The above examples of stable-nitroxide may optionally be substituted with one or more groups which do not (») adversely effect the moieties ability to act as a radical scavenger (i.e., for instance, reduce the stability of the moiety in vivo), or (ii) adversely effect the biphenyl sartans ability to function as an angiotensin antagonist.

The stable nitroxide moiety may be linked to the biphenyl sartan through a single bond or may be "fused" through multiple bonds. Again, the linking of the stable-nitroxide moiety to a biphenyl sartan (or biphenyl sartan precursor molecule) should not adversely interfere with the molecules ability to act as an angiotensin antagonist. To determine whether the linking of any stable-nitroxide moiety does or does not adversely effect this activity the skilled person would conduct comparable IC50 assays using the protocol described previously by Keenan et al., J. Med. Chem., 1992, 35, 3858-3872, and by Duncia et al., J. Med. Chem., 1990, 33, 1312-1329.

In a preferred embodiment the nitroxide-biphenyl sartan compound is represented by general formula (I) ^'

where G represents a substituted heterocyclyl or substituted heteroaryl;

L represents a divalent linker group selected from -0-, -CH2- or -O-CH2-; and

A represents C(O)0R ₃ (where R3 is H or lower alkyl), (where R is selected from NHC(0)0-lower alkyl, NHC(0)-optionally substituted heteroaryl, NHC(0)-optionally substituted heteroaryl, or NHC(0)-optlonally substituted heterocyclyl), optionally substituted heteroaryl, or optionally substituted heterocyclyl; and wherein at least one of the substituent groups on the substituted heterocycle or substituted heteroaryl of G is or comprises a stable-nitroxide moiety.

In an embodiment, and with reference to the biphenyl sartan compounds of formula (1), when L represents -CH ₂ -, G represents a heterocyclyl or heteroaryl selected from the following:

each of which is further substituted and wherein a substituent group is or comprises a stable-nitroxide moiety.

Also in a further embodiment, and with reference to the nitroxide-btphenyl sartan compounds of formula (I), when L represents CH ₂ , -0-CH ₂ - (which includes -CH ₂ -0-) or -0-, G represents a heteroaryl or heterocyclyl selected from the following:

each of which is further substituted and wherein a substituent group is or comprises a stable-nitroxide moiety. In relation to the substituted hetcrocyclyl/heteroaryl groups for 0 the following groups may be selected: where each R5 is independently lower alkyl;

R6 is selected from ~C(lower alkyl) ₂ OH, -S lower alkyl, -N (lower alkyl) C(0)CHC(lower alkyl) _2l or halogen; and

R ₇ is selected from -C0 ₂ H, -C(0)OCH(OC(0)0-lower alkyl) lower alkyl, or -CH ₂ C(0)0-lower alkyl,

wherein the selected group additionally comprises a stable-nitroxide moiety, or a stable nitroxide moiety is present in addition to or in place of the substituent group.

\ In a further embodiment the G group may be selected from

where R' is Ci-C ₈ alkyl, and preferably R* is C ₂ -C ₆ alkyl, Cj-C ₆ alkyl, C ₄ -C ₆ alkyl, n-butyl, pentyl, or hexyl; and wherein the selected group additionally comprises a stable nitroxide moiety, or a stable- nitroxide moiety is present in addition to or in place of the substituent group. Preferably ' is n-butyl. In one embodiment R' is methyl, ethyl, n-propyl or n-butyl.

In a further embodiment the G-stable nitroxide moiety group is represented by :

( )

wherein represents a stable-nitroxide moiety;

X represents a divalent linking group selected from C1-C20 alkyiene, aryJene, - (CH2CH2-O),- (Svhere t is an integer from 2 to 20);

X ^I represents a divalent linking group selected from Ci-Cj alkyiene, O, S; and m is 1 or 2.

In an embodiment X and X ¹ is preferably C 1 -C3 alkyiene, and more preferably CH ₂ .

In another embodiment X Or. is C¾ is selected from:

For each of the above embodiments for G or the G-stab)e-nitroxide moiety group, preferably A is selected from the following groups;

where each R _$ is independently lower alk l, More preferably A is selected from:

In an embodiment the nitroxide biphenyl sartan compounds of formula (1) are as follows:

where R' is Ci-C ₈ alkyl, and preferably R' is C ₂ -Ci alkyl, C3-C alkyi, d-C< _> alkyl, n-butyl, pentyl, or hexyl. Preferably R' is n-butyl. indicates potential attachmen points of the stable-nitroxide moiety group.

In a further embodiment the nitroxide-biphcny] sartan compounds are selected from:

11 000495

where R' is C)-Cg alkyl, for example, R' is C2-C6 alkyl, C3~C<s alkyl, C4- ; alkyl, n-butyl, pentyl, or hexyl. In an embodiment R' is methyl, ethyl, n-propyl, or n-butyl. In another embodiment R' is butyl, In a further embodiment the nitroxide-biphenyl sartan compounds are represented by formula (II):

wherein R' is d-Ce alkyl, preferably methyl, ethyl, n-propyl, or n-butyl; and

R ^1' is selected from:

In an embodiment, and with reference to formula (II), R ¹ ' is selected from:

Thc term "substituted" means that the group includes one or more substituents. One or more hydrogen atoms on the group may be replaced by substituent groups independently selected from halogens, alkyl, <_¼. ₆ alkenyl, Cu alkynyl, -(CH ₂ ) _P C3.7 cycloalkyl, - (CH ₂ ) _P C ₄ .7 cycJoalkenyl, -(CH ₂ ) _P aryl, -(CH ₂ ) _P heterocyclyl, -(CH ₂ ) _P heteroaryl, -C ₆ H ₄ S(0) _< ,C|. ₆ ^' alkyl, -C(Ph) _3> -CN, -OR, -0-(CH ₂ ),-6-R, -0-(CH ₂ )|.<;-OR, -OC(0)R, - C(0)R, -C(0)OR, -OC(0)NR'R", -NR'R", -NRC(0)R ^, ₎ -NRC(0)NR'R", -NRC(S)NR'R", -NRS(0) ₂ R', -NRC(0)OR', -C( R)NR'R", -C(=NOR*)R, -C(=N0H)NR'R", -qOJ R' "* -C(=NCN)-NR'R", -C(=NR)NR'R", -C(=NR')SR", -NR'C('=NGN)SR'! ₎ -CONRS0 ₂ R', -C(S)NR'R", -S(0) _q R, ~S0 ₂ NR'R", -S0 ₂ NRC(0)R', -OS(0) ₂ R, -PO(OR) ₂ and -N0 ₂ ;

·

where p is 0-6, q is 0-2 and each R, R' aritt R" is independently selected from H, Ci.g alkyl, C ₂ .6 alkcnyl, C _& alkynyl, C3.7 cycloalkyl, Co. _? cycloalkeny , aryl, heterocyclyl, heteroaryl, aryl C,-4 alkyl, hetero C|. alkyl, and heterocyclyl C alkyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, heteroaryl, aryl C _\ . _f , alkyl, heteroaryl C] _6 alkyl, or heterocyclyl Ci.i alkyl, may be optionall substituted with one to six of same or different groups selected from halogen, hydroxy, lower alkyl, lower alkoxy, -C0 ₂ H, CF ₃₎ CN, phenyl NH ₂ and -N0 ₂ ; or when R' and R" are attached to the same nitrogen atom, they may, together with the atom to which they are attached, form a 5 to 7 membered nitrogen containing heterocyclic ring,

'

Unless otherwise defined and only in respect of the ring atoms of non-aromatic carbocyclic or heterocyclic compounds, the ring atoms of Such compounds may also be optionally substituted with one or two =0 groups, instead of or in addition to the above described optional substituents.

When the substituent is or contains an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkcnyl, aryl, heteroaryl or heterocyclyl group, the group may itself be optionally substituted with one to six of the same or different substituents selected from halogens, C|_6 alkyl, C|.e alkoxy, C2.6 lkenyl, C ₂ -6 alkynyl, C|.6 haloalkyl (in particular -CF3), d.e haloalkoxy (such as -OCF3), -OH, phenyl, benzyl, furanyl, thiofuranyl, benzofuranyl, benzothiofuranyl, phenoxy, benzyloxy, benzoyl, -NH ₂₍ -NHC1-4 alkyl, -N(C alkyfh, -CN, -N0 ₂ , -S C)^ alkyl, -S ary] (in particular -SPh), mercapto, C ₍ . ₆ alkylcarbonyl, C|. _$ alkoxycarbonyl and COiH.

The term " ^■ alkyl". as used alone or in combination herein refers to a straight or branched chain saturated hydrocarbon group. The term "C1-12 alkyl" refers to such a group containing from one to twelve carbon atoms and the terms "C^ alkyl" and "lower alkyl" refer to such groups containing from one to six carbon atoms, such as methyl ("Me"), ethyl ("Et"), n-propy.l ("n-Pr"), isopropyl ("i-Pr"), n-butyl ("n-Bu"), isobutyl ("i-Bu"), sec-butyl ("s-Bu"), tert-butyl ("t-Bu") and the like.

The term "aryl" refers to carbocyclic (non-heterocyclic) aromatic rings Or ring systems. The aromatic rings may be mono- or bi-cyclic ring systems. The aromatic rings or ring systems are generally composed of 5 to 10 carbon atoms. Examples of suitable aryl groups include but are not limited to phenyl, biphenyl, naphthyl, tetrahydronaphthyl, and the ^' like.

Preferred aryl groups include phenyl, naphthyl, indenyl, azulenyl, fluorenyl or anthracenyl.

The term "arylalky!" refers to carbocyclic aromatic rings as previously described substituted by an alkyl group also as previously described. Examples of arylalkyl groups include benzyl and the like.

"Alkenyl" refers to a monovalent alkenyl group which may be straight chained or branched and preferably have from 2 to TO carbon atoms and more .preferably 2 to 6 carbon atoms and have at least 1 and preferably from 1-2, carbon to carbon, double bonds. Examples include ethenyl (-CH=CH ₂ ), κ-propenyl (-CH ₂ CH=CH ₂ ), iso-propenyl (-C(CH ₃ )~CH ₂ ), but-2-enyl (-CH ₂ CH=GHCH ₃ ), and the like.

"Alkynyl" refers to alkynyl groups preferably having from 2 to 10 carbon atoms and more preferably 2 to 6 carbon atoms and having at least 1 , and preferably from 1 -2, carbon to carbon, triple bonds. Examples of alkynyl groups include ethynyl (-O CH), propargyl (-CH ₂ C≡ CH), pent-2-ynyl (-CH ₂ CsCCH ₂ -C¾), and the like. "Alkylene" refers to divalent alkyl groups preferably having from 1 to 10 carbon atoms and more preferably 1 to 6 carbon atoms, Examples of such alkylene groups include methylene (-CH2-), ethylene (-CH ₂ CH ₂ -), and the propylene isomers (e.g., -CH2CH2CH2- and -CH(CH ₃ )CH ₂ -), and the like.

"Aryl" ^' refers to an unsaturated aromatic carbocyclic group having a single ring (eg. phenyl) or multiple condensed rings (eg. naphthyl or anthryl), preferably having from 6 to 14 carbon atoms. Examples of aryl groups include phenyl, naphthyl and the like.

"Arylene" refers to a divalent aryl group wherein the aryl group is as described above.

"Cycloalkyl" refers to cyclic alkyl groups having a single cyclic ring or multiple condensed rings, preferably incorporating 3 to 8 carbon atoms. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooetyl, and the like, or multiple ring structures such as adamantanyl, indanyl, and the like.

"Cycloalkenyl" refers to cyclic alkenyl groups having a single cyclic ring and at least one point of internal unsaturation, preferably incorporating 4 to 8 carbon atoms. Examples of suitable cycloalkenyl groups include, for instance, cyclobut-2-enyl, cyclopent-3-enyl, cyclohex-4»enyl, cyclooct-3-enyl and the like.

"Halo" or "halogen" refers to fluoro, chloro, brorno and iodo.

The term "heteroaryl" refers to a monovalent aromatic carbocyclic group, preferably of from 2 to 10 carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfur within the ring. The term "heterocyolyl" refers to a monovalent saturated or unsaturated grou having a single ring or multiple condensed rings, preferably from 1 to 8 carbon atoms and from 1 to 4 hetero atoms selected from nitrogen, sulfur and oxygen within the ring. The heterocycle or heteroaryl may be fused to a carbocyclic ring such as phenyl, naphthyl, indenyl, aiulcnyl, fluorenyli and anthracenyl.

Examples of heterocyclyl and heteroaryl groups include, but are not limited to, oxazolyl, pyrrolyl, iraidazolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyi, indolizinyl, isoindolyl, indolyl, jndazolyl, purinyl, quinolizinyl, isoquinolinyl, quinolinyl, phthalazinyl, naphthylpyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, carba2olyl, carbolinyl, phenanthridiny], acridinyl, phenanthrolinyl, isothiazolyl, phenazinyl, isoxazolyl, isothiazolyl, phenoxazinyl, phenothiazinyl, imidazolidiny], imidazolinyl, piperidinyl, piperazirty!, indolinyl, phthalimidyl, 1 ,2,3,4-tetrahydroisoquinolinyl, thiadiazoly], oxadiazolyl, oxatria2olyl, tetrazoiyl, thiazolidinyl, morpholinyl, piperidinyl, pyrrolidinyl, tetrahydrofuranyl, triazolyl, benzothiofuranyl, thiofuranyl, thiazolyl, isothiazolyl, tetrahydrothiophenenyl, napthottiienyl, thianthrenyl, thioxanyl, dithianyl, thiomorpholinyl, tetrahydrothienyl, dihydrothienyl, dithiolyl, oxathiazolyl, oxathiazinyl, and the like.

The compounds of the invention may be in crystalline form either as the free compounds or as solvates (e.g. hydrates) and it is intended that both forms are within the scope of the present invention _. . Methods of solvation are generally known within the art. It will also be recognised that compounds of the invention may possess asymmetric centres and are therefore capable of existing in more than one stereoisomeric form. The invention thus also relates to compounds in substantially pure isomeric form at one or more asymmetric centres eg., greater than about 90% ee, such as about 95% or 97% ee or greater than 99% ee, as well as mixtures, including racemic mixtures, thereof. Such isomers may be prepared by asymmetric synthesis, for example using chiral intermediates, or mixtures may be resolved by conventional methods, eg., chromatography, or use of a resolving agent.

Furthermore, depending on the substitution pattern the compounds of the present invention may be capable of undergoing tautomerism. Accordingly, all possible tautomers of a compound of the present invention fall within the scope and spirit of the invention.

The biphenyl sartan compounds of the present invention can be prepared based on modification of the synthetic procedures described in, for example, US 6,573,288, US 5,889,020, US 6,335,451, US 5,087,634, US 6,004,989, EP 0475206, EP 0443983, EP 0500409, EP 0487252, EP 0412 848, WO 94/08989, EP 0502314 and EP 0392317.

In respect of compounds of formula (I) an example of a proposed , synthetic approach is depicted below in scheme 1.

Scheme 1

In the above scheme the biphenyl compound (I)(c) may be prepared by reacting a compound of formula (I)(a) with a compound of formula (I)(b) under the palladium- catalysed coupling conditions described in Littke, A.F., and Fu, G.C., Angew. Chem. , Int. Ed., 2002, 1, 4176-421 1.

The preparation of variously substituted compounds of formulae (I)(a) and (I)(b) to introduce L and A moieties (or protected derivatives thereof) may involve commonly known electrophilic aromatic substitution chemistry and optionally, further functional group inter-conversions of the resultant substituted aryls.

In an example where L' is methyl (and therefore L= -CH ₂ -) and G is a substituted heteroaryl or substituted heterocyclyi, the preparation of compounds of formula (I) may take place by initiall halogcnating the L' group (eg with N-brornosuccinimide) and reacting the resultant alkylbromide with a suitably activated nucleophilic substituted heteroaromatic or heterocyclic compound. For example, the nucleophilic substituted heteroaromatie or heterocyclic compound may be a nucleophilic N-containmg substituted heteroaromatic or heterocyclic compound such as tertra2ole, piperidine, morpholine and the like, activated in the presence of a base, Alternatively the GH group may be a nucleophilic substituted amine which can be subsequently cyclised to form a substituted heteroaryl or heterocyclyi. In a further process, the GH group may be a nucleophilic carbon moiety (e.g, an enolate) which can be subsequently cyclised to form the substituted heteroaryl or heterocyclyi.

• A more specific example of a process for preparing compounds of formula (I) is depicted below in scheme 2 with reference to the preparation of nitroxide group containing biphenyl-sartans based on milfasartan.

Scheme 2

wherein R ¹ is Cj-Ca alkyl, preferably CiC ₆ alkyl.

Other compounds of formula I can be prepared by the addition, removal or modification of existing $ubstituents. This could be achieved by using standard techniques for functional group inter-conversion that are well known in the industry, such as those described in "Comprehensive organic transformations: a guide to functional group preparations" by Larock R. C, New York, VCH Publishers, Inc. 1989. Examples of functional group inter-conversions are: -C(0)NR*R** from -C0 ₂ CHj by heating with or without catalytic metal cyanide, e.g. NaCN, and HNR*R** in CH3OH; - OC(0)R from -OH with e.g., C1C(0)R in pyridine; -NC(S)NR*R** from -NHR with an alkylisothiocyanate or thiocyamc acid; -NRC(0)OR* from -NHR with alkyl chloroformate; -NRC(0)NR*R** from -NHR by treatment with an isocyanate, e.g. HN-C-0 or RN=C=0; -NRC(0)R* from -NHR by treatment with C1C(0)R* in pyridine; -C(=NR)NR*R** from -CCN ^R^S with H ₃ NR ⁺ OAc ^" by heating in alcohol; - C(NR*R**)SR from -C(S)NR*R** with R-I in an inert solvent, e.g. acetone; - C(S)NR*R** (where R* or R** is not hydrogen) from -C(S)NH ₂ with HNR*R**; - C(=NCN)-NR*R** - from -C(=NR*R**)-SR with NH ₂ CN by heating in anhydrous alcohol, alternatively from ~C(=NH)-NR*R** by treatment with BrCN and NaOEt i EtOH; -NR-C(=NCN)SR from -NHR* by treatment with (RS) ₂ C=NCN; -NR**S0 ₂ R from -NHR* by 'treatment with C1S0 ₂ R by heating in pyridine; -NR*C(S)R from -NR*C(0)R by treatment with Lawesson's reagent [2,4-bis(4-methoxyphenyl)-l , 3,2,4- dithiadiphosphetane-2,4-disulfide}; -NRSO2CF3 from -NHR with triflic anhydride and base, -CH(NH ₂ )CHO from -CH(NH ₂ )C(0)OR+ with Na(Hg) and HCl/EtOH; - CH ₂ C(0)OH from -C(0)OH by treatment with SOC1. then CH ₂ N ₂ then H ₂ 0/Ag ₂ 0; - C(0)OH from -CH ₂ C(0)OCH ₃ by treatment with Ph gX/HX then acetic anhydride then Cr0 ₃ ; R-OC(0)R* from RC(0)R* by R* *C0 ₃ H; -CCH ₂ OH from -C(0)OR* with Na / R*OH; -CHCH ₂ from -C¾CH ₂ OH by the Chugaev reaction; -NH ₂ from -Ό(0)ΟΗ by the Curtius reaction; -NH ₂ from -C(0)NHOH with TsCl/base then H ₂ 0; -CHC(0)CHR from - CHCHOHCHR by using the Dess-Martin Periodinane regent or CrOs / aqH SO<t / acetone; -C6H5CHO from -QH ₅ CH ₃ with Cr0 ₂ Cl ₂ ; -CHO from -CN with SnCl ₂ / HC1; -CN from - C(0)NHR with PCl _5i -CH ₂ from -C(0)R With N ₂ H ₄ / OH.

During the reactions a number of the moieties may need to be protected. Suitable protecting groups are well known in industry and have been described in many references such as Protecting Groups in Organic Synthesis, Greene T , Wiley-lnteiscience, New York, 1981.

In another aspect, the present invention provides pharmaceutical compositions for use as RAS modulators, more particularly as an antihypertensive, the composition comprising an effective amount of a nitroxide-biphenyl sartan of the present invention or a pharmaceutically acceptable salt thereof, including a pharmaceutically acceptable derivative thereof, and optionally a pharmaceutically acceptable carrier or diluent. The term "composition" is intended to include the formulation of an active ingredient with encapsulating material as earner, to give a capsule in which the active ingredient (with or without other carrier) is surrounded by carriers.

The pharmaceutical compositions or formulations include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral (including intramuscular, sub-cutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation.

The biphenyl-sartans of the invention, together with a conventional adjuvant, carrier, or diluent, may thus be placed into the fonn of pharmaceutical compositions and unit dosages thereof, and in such form may be employed as solids, such as tablets or filled capsules, or liquids such as solutions, suspensions, emulsions, elixirs, or capsules filled with the same, all for oral use, in the form of suppositories for rectal administration; or in the form of sterile injectable solutions for parenteral (including subcutaneous) use. Such pharmaceutical compositions and unit dosage forms thereof may comprise conventional ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effectiv amount of the active ingredient commensurate with the intended daily dosage range to be employed. Formulations containing ten (10) milligrams of active ingredient or, more broadly, 0.1 to one hundred (100) milligrams, per tablet, are accordingly suitable representative unit dosage forms,

The biphenyl-sartans of the present invention can be administered in a wide variety of oral and parenteral dosage forms. It will be obvious to those skilled in the art that the following dosage forms may comprise, as the active component, either a compound of the invention or a pharmaceutically acceptable salt of a compound of the invention.

Preferably, the compounds of the present invention may be administered to a subject as a pharmaceutically acceptable salt. It will be appreciated however that non- pharmaceutically acceptable salts also fall within the scope of the present invention since these may be useful as intermediates in the preparation of pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts include, but are not limited to salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, maleic, citric, lactic, mucic, gluconic, bena ic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benezenesulphonic, salicyclic sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.

Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium. In particular, the present invention includes within its scope cationic salts eg sodium or potassium salts, or alky] esters (eg methyl, ethyl) of the phosphate group. Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others,

It will be appreciated that any compound that is a prodrug of biphenyl-sartans of the present invention is also within the scope and spirit of the invention. The term "pro-drug" is used in its broadest sense and encompasses those derivatives that are converted in vivo to the compounds of the invention. Such derivatives would readily occur to those skilled in the art, and include, for example, compounds where a free hydroxy group is converted into an ester, such as an acetate or phosphate ester, or where a free amino group is converted into an amide (eg. a-aminoacid amide). Procedures for esterifying, eg. acylating, the compounds of the invention are well known in the art and may include treatment of the compound with an appropriate carboxylic acid, anhydride or chloride in the presence of a suitable catalyst or base.

For preparing pharmaceutical compositions from the compounds of the present invention, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispensable granules. A solid carrier can be one or more substances which may also act as diluents, flavouring agents, solubilisers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material,

In powders, the carrier is a finely divided solid that is in a mixture with the finely divided active component.

In tablets, the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain from five or ten to about seventy percent of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term "preparation" is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid forms suitable for oval administration.

For preparing suppositories, a low melting wax, such as an admixture of fatty acid glycerides or cocoa butter, is first melted and the active component is dispersed homogeneously therein, as by stirring. The molten homogenous mixture is then poured into convenient sized moulds, allowed to cool, and thereby to solidify.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water-propylene glycol solutions. For example, parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution.

Sterile liquid form compositions include sterile solutions, suspensions, emulsions, syrups and elixirs. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable carrier, such as sterile water, sterile organic solvent or a mixture of both.

The biphenyl-sartans according to the present invention may thus be formulated for parenteral administration (e.g. by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily Or aqueous vehicles, and may contain formulation agents such as suspending, stabilising and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution, for constitution with a suitable vehicle, eg. sterile, pyrogen-free water, before use. Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavours, stabilising and thickening agents, as desired.

Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcelJulose, sodium carboxymethylcellulose, or other well known suspending agents.

Also included are solid form preparations that are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations ma contain, in addition to the active component, colorants, flavours, stabilisers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilising agents, and the like.

For topical administration to the epidermis the compounds according to the invention may be formulated as ointments, creams or lotions, or as a transdermal patch. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilising agents, dispersing agents, suspending agents, thickening agents, or colouring agents.

Formulations suitable for topical administration in the mouth include lozenges comprising active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier. Solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray, The formulations may be provided in single or multidose form. In the latter case of a dropper or pipette, this may be achieved by the patient administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray, this may be achieved for example by means of a metering atomising ; spray pump. To improve nasal delivery and retention the compounds according to the invention may be encapsulated with cyclodextrins, or formulated with other agents expected to enhance delivery and retention in the nasal mucosa. Administration to the respiratory tract may also be achieved by means of an aerosol formulation in which the active ingredient is provided in a pressurised pack with a suitable propellant such as a chlorofluorocarbon (CFC) for example dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. The aerosol may conveniently also contain a surfactant such as lecithin. The dose of drug may be controlled by provision of a metered vaive.

Alternatively the active ingredients may be provided in the form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropyiroethyl cellulose and polyvinylpyrrolidone (PVP). Conveniently the powder carrier will form a gel in the nasal cavity, The powder composition may be presented in unit dose form for example in capsules or cartridges of, e.g., gelatin, or blister packs from which the powder may be administered by means of an inhaler, In formulations intended for administration to the respiratory tract, including intranasal formulations, the compound will generally have a small particle size for example of the order of 5 to 10 microns or less. Such a particle size may be obtained by means known in the art, for example by micronisation. When desired, formulations adapted to give sustained release of the active ingredient may be employed. The pharmaceutical preparations are preferably in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

The invention also includes the compounds in the absence of carrier where the compounds are in unit dosage form.

The amount of the biphenyl-sartans administered may be in the range from about 10 mg to 2000 mg per day, depending on the activity of the compound and the disease to be treated. Liquids or powders for intranasal administration, tablets or capsules for oral administration and liquids for intravenous administration are the preferred compositions.

The compositions may furtlier contain one or more other antihypertensive compounds. For example the compositions may contain a second antihypertensive agent such as a non- nitroxide containing antihypertensive sartan.

As discussed above the present inventors have found that nitroxide containing biphenyl- sartan derivatives of saltans retain their ability to effectively act as AH antagonists. Accordingly, the biphenyl-sartans of the present invention may be used in therapies where existing sartans prove effective, and specifically, in the Treatment of hypertension. Yet the compounds of the present invention are predicted to also have added benefits due to the incorporation of the nitroxide moiety. One of the main advantage lies in the ability of the nitroxide group to act as an antioxidant. For instance, the present inventors have found that the nitroxide-sartans possess favourable antioxidant properties. The implications of this means that the biphenyl sartan of the present invention, in addition to retaining All antagonist activity may also serve to treat conditions associated with oxidative stress where antioxidant treatment may be beneficial.

Humans consume approximately 250 grams of oxygen per day and a typical human cell metabolises about 10 ¹² molecules of oxygen per day. An inevitable consequence of our dependence on oxygen is that small amounts of highly reactive radical and non-radical derivatives of diatomic oxygen, such as 0 ₂ '\ H ₃ 0 ₂ , ΌΗ K0 ₂ ROOH and ONOO ^" , are generated in vivo. Such entities are known as reactive oxygen species (ROS). The main source of ROS within the arterial wall is a form of the enzyme NAD(P)H oxidase. This enzyme generated superoxide radicals by catalysing the reduction of O2 (see scheme 3). Superoxide radicals can subsequently be converted to more potent ROS. For example, dismutation provides hydrogen peroxide and reaction with nitric oxide affords peroxynitrite (see scheme 3).

Scheme 3

NAD(P)H oxidase ,

20 ₂ + NAD(P)H i-i™ ^ 20 ₂ + NAD(P) i- H ⁺

NO ^' + 0 ₂ ^" " ONOO- Living organisms utilise ROS as inter- and intracellular mediators of signal transduction. However, ROS can oxidise all major classes of biomolecules and are harmful at high concentrations. Living organisms are protected against ROS by a group of antioxidant compounds and enzymes. Notable antioxidant en2ymes are the enzymes glutathione peroxidase (GPx) and thioredoxin reductase which both contain selenium. Antioxidants prevent the formation of ROS or intercept ROS and exclude them from further activity. In healthy aerobic organisms, ROS production is counterbalanced by antioxidant defence networks and ROS levels are tightly regulated. However, sometimes the endogenous antioxidant defence network becomes overwhelmed by excess ROS. This imbalance between ROS and antioxidants in favour of ROS is referred to as oxidative and it has been implicated in the pathology of a vast array of diseases including, hyperlipidcmia, diabetes rhelHtus, ischemic heart disease, atherosclerosis and chronic heart failure. There is a growing body of evidence which suggests that oxidative stress is also involved in the pathogenesis of hypertension. This is because one of the many effects of angiotensin II is to stimulate NAD(P)H oxidase and thereby increase the amount of NAD(P)H oxidase derived ROS present in the vasculature. The numerous mechanisms via which these ROS proceed to bring about hypertension are yet to be fully elucidated. It is thought that hydrogen peroxide may increase the concentration of calcium cations in vascular cells and calcium cations are known to induce vasoconstriction. Alternatively, ROS may activate genes and transcription factors mediated oxidation of arachidonic acid to F ₂ -isoprostanes, which are prostaglandin-like compounds that are potent vasoconstrictors.

Accordingly, administering synthetic antioxidants or supplements of dietary antioxidants has proved to be an effective strategy for the treatment of diseases linked with oxidative stress.- ^' In particular, empirical evidence suggests that antioxidants constitute potential weapons against hypertension. In one study a group of mildly hypertensive patients received 200IU of vitamin E (a dietary antioxidant) per day (see Touyz, R. M. Expert Rev, Cardiovascular. Ther. , 1, 91., 2003). Over twenty-seven weeks the average systolic blood pressure of the group decreased by 24%. Other antioxidants, such as vitamin C and allopurinol, also reduce blood pressure in hypertensive humans.

From this it has been postulated that the biphenyl-sartan compounds of the present invention may be effective multipotent drugs for the treatment of hypertension as well as other conditions and diseases linked to oxidative stress. Multipotent drugs may be obtained by combining two different pharmacophore groups in a single molecule. Such drugs would be desirable for the treatment of hypertension, because hypertension has a complex pathogenesis and the treatment with one single-action drug often fails to sufficiently control the illness. Multipotent drugs such as the biphenyi sartans of the present invention may provide similar benefits to combinations of single- action drugs without pharmacokinetic drug interactions and many of the other disadvantages associated with combination therapy.

Since both sartans and antioxidants have antihypertensive effects, it would seem that drugs capable of functioning as both sartans and antioxidants would be useful multipotent antihypertensives/antioxidants,

Accordingly, the biphenyl-sartans of the present invention may be useful in the treatment of conditions associates with oxidative stress. For instance, the biphenyl-sartans of the present invention may be useful in the treatment of neurodegenerative diseases and conditions such as Alzheimer's disease, Parkinson's disease, parkinsonian syndrome (multiple system atrophy and progressive supernuclear palsy), amyotrophic lateral sclerosis, dementia (including Lewy body dementia), Friedrich's ataxia, Wilson's disease, Ataxia Telangiectasia, Motor neurone disease, Alexander disease, Alper's disease, Batten disease (also known as Spielmeyer-Vogt-Sjogren-Batten disease), Canavan disease, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease, Huntington disease, Kennedy's disease, rabbe disease, Machado-Joseph disease (Spinocerebellar ataxia type 3), Multiple sclerosis, Multiple System Atrophyl, Pelizaeus-Merzbacher Disease, Pick's disease, Primary lateral sclerosis, Refsum's disease, Sandhoff disease, Schiider's disease, Spinocerebellar ataxia (multiple types with varying characteristics), Spinal muscular atrophy, Steele-Richardson-Olszcwski disease, and Tabes dorsalis.

Furthermore, mtDNA diseases such as cardiomyopathy, heart failure, heart block, arrhythmia, diabetes, pancreatitis, retinopathy, optic neuropathy, renal failure, Kearns Sayre Syndrome, Sudden Infant Death Syndrome, dementia and epilepsy, stroke may also be effectively treated using the biphenyl-sartans of the present invention. Other conditions such as inflammation, ischaemic-reperfusion tissue injury in strokes, heart attacks, organ transplantation and surgery, edema, atherosclerosis, may- also be beneficially treated with the biphenyl-sartans of the present invention.

From the abov discussion it would be evident that one of the main advantages of the multipotent biphenyl-sartan derivatives of the present invention will be their ability to provide cardioprotective qualities in addition to the antihypertensive effects. It has been identified (see, for instance, Daugherty A, et al; Trends Cardiovasc. Med., 2004, 14, Π7- 120) that angiotensin II is responsible (at least in part) for the progression of tissue damage in cardiovascular disease. The angiotensin inhibition and subsequent positive antihypertension effects of known sartans do not solely account for the cardiovascular morbidity and mortality attributable to this action. Accordingly, the present modifications (nitroxide addition) to known sartans are seen to be beneficial in the context of increasing the sartans ability to prevent (or enhance the prevention of) tissue damage in the cardiovascular system and hence enhance any cardioprotective qualities which may be observable in the corresponding unmodified ^' sartan.

In order to ensure maximisation of this dual activity in vivo it is preferred that the compounds of the present invention are appropriately "matched" in the context of both the antihypertensive and antioxidant effects which are observed for any individual molecule. As such, each of the pharmacophoric subunits of the conjugated nitroxide-biphenyl sartan compounds of the present invention is preferably designed or arranged to ensure that each of the intended molecular targets is modulated to an appropriate degree in vivo. This "matching" of activities is preferable as compounds with large differences in in viiro affinity for respective targets will only be able to act at both targets (in vivo) at high doses based on the lowest affinity target. This would increase the risk of side effects associated with the higher affinity target. Therefore, molecules with in vitro activities that are within an order of magnitude of each other are preferred as they would achieve similar levels of receptor occupancy/biological activity in vivo. Accordingly, in a further preferred embodiment the novel nitroxide-biphenyl sartan compounds of the present invention are characterised with in vitro antioxidant activity that is matched with the in vitro angiotensin II antagonist activity. In this regard, it will also be appreciated that In order to facilitate such matching the compounds disclosed herein may be chemically modified. In particular, and as an example, matching may be facilitated through the elongation or reduction of the ' alkyl substituent in the embodiments discussed above. In this way, it will be appreciated that the compounds of the present invention can be "tuned" in order to ensure maximum in vivo efficacy in relation to both antioxidant and antihypertensive activities.

The invention will now be described in the following Examples. The Examples are not to be construed as limiting the invention in any way. Examples

Synthetic examples

Example 3

To a stirred solution of sodium hydride (50 % dispersion in mineral oil) (0.21 g, 5.34 mmol) in anhydrous DMF (12 rtiL) at 0 °C was added a solution of 6-butyl-2-methyl-5- [^'-[l-itriphenylmethy -lH-tetrazol-S-yitl , biphenyl]-4-yl]methyl]pyrimidin-4(l f)-one (1) ( 2.00 g, 3.10 mmol) in anhydrous DMF (12 mL). After stirring for 10 min., lithium bromide ( 0.91 g, 5.34 mmol) in anhydrous DMF (20 mL) was added. After another 10 min., crude 3¾omomethyl-2,2^5-tetrame y]-2,S-dihydropyrrole-l-oxyl (2) (1 ,00 g, 4.28 mmol) in DMF (20 mL) was added to the reaction mixture. The ice-bath was then removed and the mixture was stirred for 2 hr at r.t. The mixture was then poured into a vigorously stirred ice-water slurry. The pH was adjusted to with 5 % aqueous AcOH and the precipitate was filtered off and washed with ¾0. Further purification of the crude via column chromatography (EtOAc; Pet. spirit, 3:1) yielded the crude trirylated product ( 1.23 g, 49,8 %) which was then dissolved in MeOH ( 70 mL) and refluxed overnight. The MeOH was removed in vacuo and the residue was purified by column chromatography ( EtOAc:MeOH, 3:1) to give 5 as pale yellow foam ( 0.56 g, 33 ¼).

I -Vma (neat): 1554, 1643, 2972, 3330 cm/'

MS (ESI) m/z 575.3[M+Na]' ^4'

HRMS calcd. for C ₃ 2H ₃ 8N ₇ 0 ₂ [M+Na]' ⁺ 575.29792; found.575.29790

HPLC purity analysis showed that compound S is 98.5 % pure.

Example 2

To a stirred solution of NaH (60 % dispersion in mineral oil) (0.052 g, 0.777 mmol) in anhydrous DMF (3.1 mL) at 0°C was added a solution of 6-butyl-2-methyl-5-[[2'-(l- (triphenylmethyl)-lH-tetraz^ (1) (0.500 g, 0.777 mmol) In anhydrous DMP (6.2 mL). After 10 min, LiBr (0.230 g, 2.64 mmol) ^' dissolved in anhydrous DMF (6.2 mL) was added. After stirring for 10 min, nitroxide 3 (0,310 g, 1.08 mmol) in DMF (6.2 mL) was added and the cold bath was removed -and the mixture stirred at r.t. for 2 hr. The mixture was then poured into a vigorously stirred ice-water slurry. The pH was adjusted to 5 with 5 % aq. AcOH. The precipitate was filtered off and washed with ¾0. The crude material was purified by flash chromatography (Pet. spirit:EOAc, 2:1, followed by Pet. spirit:EtOAc, 1 : 1) to furnish the title compound as slight pale yellow foam which was dissolved in MeOH (35 mL) and refluxed overnight. The MeOH was then removed in vacuo and the residue was purified- by flash chromatography (straight £t ₂ 0, followed b EtOAc:MeOH, 6: 1) to afford compound 6 ( 0.31 g, 42 %) as pate yellow foam.

IR v _m9x (neat): 747, 1542, 1651, 2972 cm ^"1

The ES spectrum displayed a triplet with hyperfme coupling constant AN = 15.0805 and g = 2.00375.

HRMS (ESI) calcd. for Ο,4¾ ₈ Ν ₇ 0 ₂ [M+ H 631.26999, found, 631.26996.

Anal, calcd. for C^U _3S ^ ₇ 0 ₂ S: C 67.08; H 6.29; N 16.1 1 ; O 5.26; S 5.27 %, found: C 67.03 ; H 6.39; N 16.23; O 5.3; S 5.05 %. HPLC purity analysis showed that compound 6 is 98.4 % pure. Example 3

NaH (60% in dispersion oil) (5.26 mg, 0.13 nunol) was washed once with dry hexane (1.50 mL) before anhydrous THF (0.50 mL) was added. Thienopyrolyoxyl derivative of milfasartan (6) (80 mg, 0.13 mmol) in anhydrous THF (0.5 mL) was added slowly to the stirred mixture. The reaction was stirred at r.t. for 2 hr. THF was then removed in vacuo and the crude residue dissolved in HjO. The aqueous layer was extracted with CHClj to remove any unreacted starting material, then filtered through celite. Following the removal of ¾0 via fre z drying, 7 was obtained as a pale yellow powder (56 mg, 84 %),

Ex mr^eJ,

Compound 11 was isolated as pale yellow foam (200mg, 49 % over two steps) from 2,6- dimethyl-5-[[2 ^, -[1 -(triphenylmethyl)- 1 toetrazol-5-y] [1 , 1 'biphenyl]-4- yl]methyl]pyrimidin-4(lH)-one (8) (298 mg, 1.28 mrool) and 3-bromomethyl-2,2,5,5- tetramethyl-2,5-dihydropyrroIe-l-oxyl (2) (555 mg, 0,93 mmol).

IR _Vme , (neat): 756, 1246, 1538, 1658, 1733, 2976 cm ^"1

MS (ESI) m/z 511.3 4 [M+H]' ⁺

HRMS (ESI) calcd.for [M+Na] ^,+ 533.25097; found, 533.25073

HPLC purity analysis showed that compound 11 is over 99 % pure.

Example 5

Compound 12 was prepared as pale yellow foam (200mg, 46 % oyer two steps) from 2,6- dimethyl-S [2 ^, -[l -(triphenylmethyi)-lH-tetrazol"5-y]tl ,rbiphenyl]-4- yl]methylJpyrirnidin-4(l#)-one (8) (0.52 g, 0.874 mmol) and 2-(bromomethy])-4,4,6,6- tetramethyl-4 _> 6-dihydro-5Hthienot2 _> 3-c]pyrrol-5-yloxyl (3) (0.350 mg, 1.21 mmol). H V™** (neat): 761, 1241 , 1541, 1651, 2976 cm ^'1

MS (ESI) m z 567.2 [M+H] ^'+

HRMS(ESI) calcd. for C31H32N7O2S [M+Na]" ⁺ , 589.22304; found, 589.22272

HPLC purity analysis showed that compound 12 is over 99 % pure.

Example 6

Compound 13 was prepared as pale yellow foam (168 mg, 38 % over two steps) from 6- cthyl-a-mtthyl-S-CP'-i I -(triphenylmethyl)- 1 H-tetrazol-5-y][ 1 , 1 'bipheny l]-4- yl]methyl]pyrimidin-4(lH)-one (9) (640 mg, 1.04 rnmol) and 3-bromomethyl-2,2,5,5- tetramethyl-2,5-dihydropyrrole-l-oxyl (2) (360 mg, 1.56mmol).

i v _mfl)s (neat): 1544, 1652, 2976 cm ^"1

MS (ESI) m/z 525.3 [ +H] ^* *

HRMScalcd. for C30H34N7O2 [M+Na] ^'+ , 547.26662; found, 547.26648

HPLC purity analysis showed that compound 13 is over 99 % pure.

Example 7

Compound 14 was prepared as pale yellow foam (212 mg, 39 % over two steps) from 6- ethyl-2-methyl-5-[[2>[Htriphenylme ^

yl}roethyl]pyrimidin-4(lH)-onc (9){640 mg, 1.04 mmol) arid 2-(bromomethy])-4,4,6,6- terramethyl-4,6-dihycko-5H-thierio[2,3-c]pyrrol-5-yloxyl (3) (360 mg, 1.56mmol), IR v _max (neat): 761 , 1542, 1648, 2977 cm ^"1

MS (ESI) /z 581.3 [M+H] ^'+

HRMS oalcd. for 603.23869; found, 603.23859

HPLC purity analysis showed that compound 14 is over 99 % pure.

Example 8

Compound IS was prepared as pale yellow foam (1 5 mg,47 % overtwo steps) from 6- propyl- 2-methy]-5-[[2 '-[ 1 -(triphenylmethyl)- 1 H etrazo]-5 _" y][l, ] 'biphenyl]-4.

yl3methylJpyrimidin-4(lH)-one (10) (402 mg, 0,64 mmol) and 3-bromomcthyl-2,2,5,5- tetramethyl-2,5^ihydropyrrole-l -oxyl (2) (224 mg, 0,96mmo]).

MS (ESI) m/z 539.3 [Μ+Ή] ' ⁺

HRMScalcd, for C31H3.N7O2 [M+Na] ^'+ , 561.28227; found, 561.2821 1

HPLC purity analysis showed that compound 15 is over 99 % pure.

Example 9

Compound 16 was prepared as pale yellow foam, (262 mg, 56 % over two steps) from 6- propyl-2-methyl-5-[[2 H 1 -(triphenylmethyJ 1 H-tetrazol-5-y] [ 1 , l 'biphenyl]-4- yi]methyl]pyrimidin-4(lH)'One (10) (550 mg, 0.88mmol) and 2-(bromomethyl)-4,4,6,6- tetxamethyM.e-dihydro-SH-thienop^-cjpyrrol^-yloxyl (3) (450 mg, 3.00mmol);

IR VM* (neat): 735, 1542, 1650, 2974 cm ⁰

MS (ESI) /z 595.3 [M+H] ^{'+ "}

HRMS calcd. for C ₃₃ H _i6 N ₇ 0 ₂ S[M+Na] ^'+ , 617.25434; found, 617.25402

HPLC purity analysis showed that compound 16 is over 99 % pure.

Biological data

I) AT H binding data/protocol

Chinese hamster ovary cells stably expressing the rat ATi a receptor (as per Thomas, W et al, Mol. Endocrinol., 1998, 12, 1513) were used to assess the potency of the milfasartart analogues. Compound 5 (Example 1) and compounds (Example 2) where subjected to the assay to determine if this derivatives inhibit angiotensin H-evoked increases in intracellular calcium in CHO cells stably expressing the A receptor. CHO cells were loaded with Fluo4-AM and then incubated with 100 nM of compound. The results are displayed in 2011/000495

- 61 -

Figure 2. Changes in intracellular [Ca2+] evoked by increasing concentrations of angiotensin II in the absence and presence of the antagonist were measured over a 3 min period using the Flexstation Π (Molecular Devices). RFU = Relative Fluorescence Units. AT) _a binding constants (KB) were determined and are expressed as ρΚβ values. An estimate of 9.1 for the ρΛ¾ of the nitroxyl-milfasartan derivative (Compound 5, Example

I) , and 8.4 for the pKo of Compound 6 (Example 2) compares favourably with previous estimates obtained for milfasaitan (9.9). These K^ estimates confirm that the milfasartan derivatives of the present invention remain potent AT) receptor antagonists.

II) Antioxidant effects of novel angiotensin II receptor antagonists using doxorubicin- induced negative inotropy

Animals

Adult male and female C57B1/6 mice (28 ± 0,5 g, «-l 10) were used in this study. Mice were housed in the Department of Pharmacology Biological Research Facility and maintained at 22°C with a 12 h light/dark cycle. Animals were given free access to food and water. Experiments using animal tissues were undertaken with the approval of the University of Melbourne Animal Ethics Committee in accordance with the Code of practice of the National Health and Medical Research Council of Australia. Tissue preparation

Mice , were anaesthetised using spontaneous inhalation of halothane (5% in (Vroom air) and their hearts quickly excised. Left atria were dissected in gassed Krebs solution (in mM, Na* 144, K ⁺ 5.9, Mg ²⁴ 1.2, Ca ²⁺ 2.5, ¾PCV 1.2, CT 128.7, S0 ₄ ^2" 1.2, HCOj 25, glucose II, EDTA 0.04).

The left auia were each mounted onto two 3QG S shaped hooks and set u in 10 ml organ baths that were positioned in a fume hood to scavenge aerated doxorubicin particles, rebs solution in the jacketed organ baths was kept at approximately 37°C with continuous bubbling of 95% Oz/5% C0 ₂ . Left atria were stretched to 1 g of tension using an adjustable micrometer (Mitutoyo Manufacturing) and stimulated at 1 Hz, 0.3 ms at 120% of threshold voltage using a stimulator (S88, Grass Instruments, Warwick, RI, USA).

Contractions of the preparation were recorded using an isometric force transducer (FTO3C, Grass Instruments) connected to a PowerLab 8SP (AD Instruments) through a 6 channel transducer amplifier (model 108, Baker Medical Research Institute). Stimulated inotropic responses of the left atria were recorded using Chart software (version 5.5.3 AD Instruments, NSW, Australia) and measured as changes in force of contraction (g).

Experimental protocols Doxorubicin-mediated negative inotropy

After the tissue was left to equilibrate for 1 h, it was contracted to approximately 80% maximal force with 0.1 μΜ of isoprenaline. Once the response reached a plateau, 30 μ of doxorubicin was added to the organ bath and left for 90 min. In separate atria, to establish the effects of time on the tissue and the contractile response to isoprenaline, the same experiment was carried out, without the addition of doxorubicin.

Antioxidant moieties and nitroxlde-sartans

Following isoprenaline contraction, the tissue was incubated with 10 μΜ of milfasartan, compound 7 or sodium 2,2,5,5-tetramethyl-2,5-dihydropyriOle- l-oxyl-3-carboxylate (nitroxidc) for 30 min, Following this incubation period, doxorubicin (30 μ ) was added to the bath and left for 90 min,

AT] receptor binding of analogues

To investigate the effects of AT _t receptor occupancy on doxorubicm-induced damage, tissues were incubated with 10 μ of milfasartan prior to the addition of isoprenaline. Tissues were then contracted with isoprenaline and once the response reached plateau, phenol-milfasartan or phenol (10 μ ) was added to the organ bath. Drugs

Drugs used and suppliers were: doxorubicin (Sigma, St. Louis, MO, USA), isoprenaline (Sigma), quercefin (Sigma), diphenyleneiodonium sulphate (DPI) (Sigma) and ebselen (Sigma). Analysis

Data are presented as the mean (± SEM) change in left atrial force over 90 min from the baseline reading prior to the addition of doxorubicin (after contraction with isoprenaline). The effects of the compounds on doxorubicin-mediated negative inotropy were expressed as the mean (± SEM) area under the curve for the change in force from baseline. One-way ANOVA with Dunnett's post hoc test (GraphPad Prism 5, La Jblla, CA, USA) was used for comparison of values between drug treatments, Differences were considered significant if <0.05.

The results of this analysis in respect of Compound 5 (example 1) is presented in Figure 1. This figure indicates that Compound 5 has the ability to prevent the reduction in contractility in mouse left atria induced by doxorubicin, -indicating that it has cardioprotective properties.

HI) Protocol for determining antioxidant effects of novel angiotensin II receptor antagonists using lucigeriin enhanced chemiluminescence

Theoretical basis of assay

The lucigenin probe is reduced by superoxide, (produced by a tissue sample stimulated with NADPH) resulting in the production of a cation radical and an unstable dioxetane. The unstable dioxetane decomposes and emits a photon. Photon emission can be measured using a scintillator or photometer (Janiszewski et al, "Overestimation of NADH-driven vascular oxidase activity due to hicigen artifacts", Free Radical Biol. Med 32(5):446-453, 2002). The antioxidant status of a compound can be measured by quantifying the reduction ίη photon emission as compared to the control. DPI (diphenyleneiodonium sulphate), an NADPH oxidase inhibitor, is used as a positive control for the assay.

Animals

Male Sprague Dawley rats (250-350 g body weight) were used in this study. Rats were housed in the Department of Pharmacology Biological Research Facility and maintained at 22°C with a 12 light/dark cycle, Animals were given free access to food and water. Experiments using animal tissues were undertaken with the approval of the University of Melbourne Animal Ethics Committee in accordance with the Code of practice of the National Health and Medical Research Council of Australia.

Tissue preparation

Rats were anaesthetised using spontaneous inhalation of halothane (5% in O^/room air) and killed using cervical dislocation. Thoracic aorta were dissected out in rebs-Hepes buffer solution buffer (in mM NaCI, 99-01; KCl, 4.69; CaClj, 1.87; MgS0 ₄ , 1.20; ₂ ΗΡ0 ₄ , 1.03; NaHC0 ₃ , 25.0; HEPES (4-(2-hydroxyethylH -piperazineeftancsu]fonic acid 20.0, and glucose, 1 1.1 , pB 7.4) and gassed with carbogen. Aorta were cleared of fat and connective tissue and cut into approximately 3 mm segments. Experimental protocols

Tissues were incubated at 37°C in a 6 well plates containing rebs-HEPES buffer solution, NADPH (nicotinamide adenine dinucleotide phosphate-oxidase 100 μΜ) and DETCA (diethylthiocarbamate 300 μΜ) for 45 min in 37°C oven (Forma Scientific; OH, USA). Control tissues were also incubated with the NADPH oxidase in inhibitor DPI (diphenyleneiodonium 0.1, 1 or 10 μΜ see Tabic below for drug actions). After a 45 min incubation period, aorta segments were placed in a 96 well Optiplate (Perkin Elmer; Downers Grove, IL, USA) containing NADPH (100 μΜ), lucigenin (5 μΜ) and DPI (0.1 ,1 or 10 μΜ). Photon emission was detected by a luminescence counter (Perkin Elmer; Downers Grove, IL, USA) which read each well 12 times over 25 min. Background photon emission counts of the 96 well Optiplate were also taken prior to addition of the tissue segments. Vessel segments were then dried in a 65°C oven (Daihan Scientific; Seoul, Korea) overnight in order to determine the dry weight of the vessel. Counts were expressed as average counts per mg of dry tissue weight.

Antioxidant effect of antioxidant moieties and novel sartaos

To determine the antioxidant properties of the antioxidant moieties and novel saltans, vessel segments were incubated in a 6 well plates containing NADPHj DETCA, Krebs- HEPES buffer, vehicle (1% methanol or DMSO) and antioxidant moieties or novel biphenyl sartan (0.1 , 1 and 10 μΜ) for 45 min at 37 °C. Following an incubation period, aorta segments were transferred to a 96 well Optiplate containing NADPH (100 μΜ), lucigenin (5 'μ ) and antioxidant moieties or novel biphenyl sartans (0.1, 1 or 10 μ ). Photon emission of the vessel segments were detected using a luminescence counter. Drug action of compounds used in the lucigenin enhanced chemiluminescence assay.

Drug Drug action Concentration/Volume

Krebs-HEPES Physiological buffering 2 ml

solution solution

DETCA Superoxide dismutase 300 μ

inhibitor

Lucigenin Fluorescent probe to detect 5 Μ

superoxide

NADPH+ Stimulates NADPH oxidase 100 μΜ

complex

DPI NADPH oxidase inhibitor 5 μΜ

Drugs

Drugs used and suppliers were: nicotinamide adenine dinucleotide phosphate-oxidase (NADPH, Sigma, St. Louis, MO, USA), diethylthiocarbamate (DETCA, Sigma), 4-2- hydroxyethyl-l-piperazineethanesulfonic acid (HEPES, Sigma), diphenyleneiodonium (DPI, Sigma) and lucigenin (Sigma). ^ϊν ) Determination of the potency of Compound 7 at ATj _,A receptors.

Method

CHO cells stably expressing the AT _)A receptor were loaded with Fluo4-AM and then incubated with 10, 30 or 100 nM Compound 7 for 30 min. Changes in intracellular [Ca ²⁺ ] (RFU = Relative Fluorescence Units) evoked by increasing concentrations of angiotensin II in the absence and presence of Compound 7 were measured over a 3 min period using the Flexstation I! (Molecular Devices) (Staples, MK, Grange, L, Angus, JA, et al. (201 1 ) Organic BioMolecular Chem 9: 473-479). The results are shown in Figures 4a and 4b. In the presence of increasing concentrations of Compound 7 there was a progressive right- ward shift of the angiotensin Il-evoked increases in intracellular calcium in CHO cells stably expressing the AT I A receptor. w = 5 for each group.

V) Determination of the stability of Compound 7 in the presence of CHO cells

The stability of Compound 7 was evaluated using HPLC analysis. Compound 7 was incubated with CHO cells and buffered saline for 30 min or for 2 hrs. The 30 min incubations yielded a single peak that corresponded to the Compound 7 standards suggesting the antagonist activity in the CHO cell assay was attributable to Compound 7.

The 2 hr incubations yielded a second peak of an, as yet, unidentified compound. In buffered saline this corresponded to about 1% of the original Compound 7 and this, increased to 9% in the presence of the CHO cells.

Determination of the potency of Compounds 13-16 at ATJA receptors.

For these compounds a single concentration of 1 μΜ was tested for the ability to shift the angiotensin II concentration-responses curves in CHO cells, Methods as for Figure 1 , Table 1. pKn estimates for Compounds 13-16, Estimates derived using the equation - logKe = log(r-l) - log[B]; where r = dose ratio, [B] is concentration of compound tested,

VI) Determination of the effects of Compound 7 on blood pressure in spontaneously hypertensive rats (SHR1

Method

Blood pressure was monitored in conscious SHR via radiotransmission. A telemetry transmitter (TL1 1M2-C50-PXT; Data Sciences International, St Paul, MN, USA) was implanted into the abdominal aorta under general anaesthesia and sterile surgical conditions (Huetteman. DA, Bogie, H (2009) Methods Mol Biol 573: 57-73). The- animals were allowed to recover and stabilise for 7 days. A baseline blood pressur measurement was recorded prior to the initiation of drug or vehicle treatment (Day 0).

General anaesthesia was then induced on Day 0, and an osmotic mini pump (delivery rate 10 μ1/ ι; Alzet osmotic pump Model 2ML1; DURECT Corporation, CA, USA) implanted subcutaneously (s.c.) between the shoulder blades to deliver drug or vehicle continuously for 7 days. The treatment groups were eprosartan 30 mg/kg in 50% DMSO, Compound 7 30 mg/kg or 60 mg kg in 50% DMSO and Compound 7 30 mg/kg in water.

Blood pressure measurements in conscious rats were taken daily for 7 days following implantation. The animals were then euthanased and cardiac and vascular tissues removed for further investigation in vitro (Figure 5). On completion of the telemetiy measurements rats were anaesthetised with a mixture of isoflurane/100% oxygen and a section of the main right carotid artery was removed for determination of the responsiveness to angiotensin Π. Vessels were cut into 2 mm ring segments, then mounted in a multi chamber ulvany-Halpem style isometric myograph as described by Wright, CE, Angus JA, ei al. (2000) Br J Pharmacol 131 : 1325-1336. The time frame from removing the arteries to testing the angiotensin II responsiveness was 2- 2.5 hrs. In animals treated with eprosartan or Compound 7 dissolved in water, responsiveness to angiotensin II was not different to the 50% DMSO solvent-treated animals. In contrast, animals treated with Compound 7 dissolved in 50% DMSO demonstrated persistent inhibition of angiotensin receptors.

On completion of the telemetiy measurements rats were anaesthetised with a mixture of isoflurane/100% oxygen and the heart rapidly removed for determination of th responsiveness to angiotensin Π. The spontaneously beating right atrium was dissected free from the left atrium and ventricles, pierced by two hooks and mounted in an acrylic organ bath filled with Krebs' solution as described by Winkel, D, TibbaUs, J, Mo!enaar, P, Lambert, O, Coles, P, Ross-Smith, M, Wiltshire, C, Fenncr, PJ, Gershwin, LA, Hawdon, GM, Wright, CE, Angus, JA (2005) Clin Exp Pharmacol Physiol ' 32: 777-788. The time frame from removing the arteries to testing the angiotensin H responsiveness was 1-1.5 hrs. In this case all animals treated with the sartans demonstrated persistent inhibition of angiotensin receptors (Figure 7). VII) Determination of the effects of Compound 7 on carotid arter intimal thickening in male spontaneously hypertensive rats (SHR),

Method

Rubbing of the carotid artery is known to cause damage to the lining of blood vessels which develops over 14 days in SHR into a lesion of intimal thickening (Dalle Lucca, S, Dalle Lucca, J, Borges, A, Ihara, S, Paiva, T (2000) Brazilian J Med Biol Res 33: 1 - 927). SH were anaesthetized and an osmotic mini-pum (model 2MD, Alzet, Cupertino, CA, USA) containing a 14 day drug treatment was implanted subcutaneous ly (s.c,). During the same surgery vascular injury was induced by passing an inflated balloon catheter (2F Fogarty; Edward Lifesciences, Irvine, CA, USA) through the common carotid artery three times (Clowes, A, Reidy, .M, Clowes, M (1983a) Lab Invest 49: 327-333; Clowes, AW, Reidy, MA, Clowes, MM (1983b) Lab Invest 49: 208-215; Fingerle, J, Au, Y, Clowes, A, Reidy, M (l 990) Arterioscler Thromb Vase Biol 10: 1082-1087).

The osmotic mini pump was filled'with either Compound 7, milfasartan, sodium 2,2,5, 5- tetramethyl-2,5-dihydropyrrole-l -oxyl-3-carboxylate (nitroxide) or saline solvent; the pump delivered a dose of 30 mg kg day. This dose was chosen based on previous reports where 30 mg kg/day losarfan was able to decrease blood pressure and the extent of vascular injury (De Blois, D, Tea, B-S, Dam, T-V, Tremblay, J, Harriet, P (1997) Hypertension 29: 340-344).

On completion of the 14 day treatment, rats were perfusion-fixed (6% paraformaldehyde) at 150 mmHg and both injured and uninjured carotid arteries were excised and cut into 5 μιη thick sections. Sections were stained with haematoxylin and eosin and analysed to determine the extent of intimal thickening (Dalle Lucca, S, Dalle Lucca, J, Borgcs, A, Ihara, S, Paiva, T (2000) Brazilian J Med Biol Res 33: 919-927). A section of the abdominal aorta was removed prior to fixation to assess the effect of treatment on superoxide levels.

The effects of saline (120 μΐ/day), nitroxide, Compound 7 or milfasartan (each 30 mg/kg/day s.c.) for 14 days on change in intimal thickness (μπι) in SHR injured carotid arteries compared to contralateral uninjured ^, control vessels (Δ intimal thickness). Data are mean + SEM. */ ^> <0.05 compared to saline; # <0,05 compared to milfasartan using oneway ANOVA with Dunnett's post test. There were 6 rats in each treatment group (Figure 8). Superoxide counts of abdominal aortae taken from SHR in different 14 day treatment groups (n**5 each). Saline (120 μΐ/d), nitroxide, Compound 7 or milfasartan (each 30 mg/kg/d) were delivered via an osmotic mini-pump (s.c). Values are average + SE luminescent counts per mg of dry tissue weight (counts/mg) of abdominal aortae stimulated with NADPH (100 μΜ) to generate superoxide. This was detected using lucigenin (5 μM)-enhanced chemiluminescence. * <0<05 vs, saline, #P<Q,Q5 vs, milfasartan; one-way ANOVA with Dunnett's post hoc analysis (Figure 9).