BACKGROUND OF THE INVENTION Field of the Invention This invention relates to novel therapeutic agents that bind to mammalian receptors and modulate their activity. More particularly, the invention relates to novel therapeutic multi-binding compounds (agents) that bind to and modulate the in vivo activity of endothelin receptors in mammals and to pharmaceutical compositions comprising such compounds. These multi-binding compounds are particularly useful medications for the prophylaxis and treatment of various mammalian conditions that are mediated by the endothelin receptors, such as diseases of the cardiovascular system and renal system.

References The following publications may be cited in this application as superscript numbers:

1. J. March, Advanced Organic Chemistry, 4"Edition, Wiley-Interscience New York (1992).

2. Remington's Pharmaceutical Sciences, Publishing Publishing Company, Philadelphia, PA, 17th ed. (1985).

3. Green, Protective Groups in Organic Synthesis, 2"d Edition, John Wiley & Sons, New York, New York (199 ).

4. G. A. Gray and D. J. Webb,"The Endothelin System and Its Potential As A Therapeutic Target In Cardiovascular Disease", Pharmacol.

Ther. 72: 109-148 (1996).

5. G. Noll et al.,"Endothelin and Endothelin Antagonists: Potential Role In Cardiovascular and Renal Disease", Molecular and Cellular Biochemistry, 157 : 259-267 (1996).

6. D. J. Webb and F. E. Strachan,"Clinical Experience With Endothelin Antagonists", Amer. J. Hypertension 11: 71 S-79S (1998).

7. H. R. Brunner,"Endothelin Inhibition As A Biologic Target For Treating Hypertension", Amer. J. Hypertensios 103S-109S (I 998).

8. U. S. Patent No. 5,414,010 issued to Downing et al. on May 9, 1995, entitled"Dimeric Benzimidazoles as Central Nervous System Agents".

9. Volker Breu et al.,"Separable Binding Sites For the Natural Agonist Endothelin-I and the Non-Peptide Antagonist Bosentan On Human Endothelin-A Receptors", Eur. J. Biochem., 231,266-270 (1995).

10. S. Laurent et al.,"The Arterial Wall: A New Pharmacological And Therapeutic Target", Fundam. Clin. Pharmacol., 10: 243-257 (1996).

11. Oie, E., et al."ET-receptor Antagonism, Myocardial Gene Expression, And Ventricular Remodeling During CHF in Rats", Am. J. P} siol., (1998) 275: H868.

12. Herizi, A., et al.,"Prevention of the Cardiovascular and Renal Effects of Angiotensin II by Endothelin Blockade", Hvpertenslon, 1998 ; 31 [part 1]: 10-14.

13. Sogni et al.,"Beneficial Hemodynamic Effects of Bosentan, A Mixed ETA and ETB Receptor Antagonist, in Portal Hypertensive Rats" Hepatology, (1998) 28 (3): 655-659.

14. Haleen, S., et al.,"Efficacy of CI-1020, an Endothelin-A Receptor Antagonist, in Hypoxic Pulmonary Hypertension,"J. of Cardiovasc.

Pharmacol., (1998) 31 (suppl. 1) : S331-S335.

15. Piovezan, A., et al.,"Effects of Endothelin-1 On Capsaicin-Induced Nociception in Mice"Eur. J. Pharmacol., 351 (1998) 15-22.

16. Zuccarello, M., et al.,"Prevention of Subarachnoid Hemorrhage-induced Cerebral Vasospasm By Oral Administration of Endothelin Receptor Antagonist"J. Neurosurg. (1996) 84 : 503-507.

17. J. D. De-Melo, et al.,"Articular nociception Induced by Endothelin-l.

Carrageenan and LPS in Naïve and Previously Inflamed Knee-Joints In the Rate: Inhibition By Endothelin Receptor Antagonists"Puin, 77 (1998) 261-269.

18. R. Choussat, et al.,"Acute Effects Of An Endothelin-1 Receptor Antagonist Bosentan At Different Stages Of Heart Failure In Conscious Dogs"Cardiovascular Research, 39 (1998) 580-588.

19. K. M. McCulloch, et al.,"Endothelin Receptors Mediating Contraction of Rat and Human Pulmonary Resistance Arteries; Effect of Chronic Hypoxia in the Rat"ETReceptors in Pulmonan. Resistance,-Irteries, (1998) 1621-1630.

20. P-E. Massart, et al.,"Angiotensin II and Endothelin-1 Receptor Antagonists Have Cumulative Hypotensive Effects in Canine Page Hypertension"Journal of Hypertension, 1998, 16: 835-841.

21. A. Oldner, et al.,"The Endothelin Receptor Antagonist Bosentan Restores Gut Oxygen Delivery and Reverses Intestinal Mucors Acidosis in Porcine Endotoxin Shock"Gut, 1998 ; 42: 696-702.

22. B. Nhi T. Nguyen, et al.,"The Role of Endothelin in Heart Failure and Hypertension"Pharmacotherapv, 18 (4): 706-719 (1998).

23. T. D. Warner,"Characterization of Endothelin Synthetic Pathwas and Receptor Subtypes: Physiological and Pathophysiological Implications"European Heart Journal (1993) 14 (Supple 1) 49-17.

24. P. Nambi,"Endothelin Receptors In Normal and Diseased Kidneys" Clinical and Experimental Pharmacology and Physioloy (1996) 23, 326-330.

25. D. E. Kohan,"Endothelins: Renal Tubule Synthesis and Actions" Clinical and Experimental Pharmacology and Physiologo (1996) 23, 337-344.

26. E. P. Nord,"Signaling Pathways Activated By Endothelin Stimulation of Renal Cells"Clinical and E. rperimental Pharmacologv and Physiology (1996) 23,331-336.

27. I. Bruzzi et al.,"Endothelin Is A Key Modulator of Progressive Renal Injury : Experimental Data and Novel Therapeutic Strategies"Clinical and Experimental Pharmacology and Physiologl! (1996) 23, 349-353.

28. D. P. Brooks,"Role of Endothelin In Renal Function and Dysfunction" Clinical and Experimental Pharmacolog) ld Ph1siologS (1996) 23, 345-348.

29. A. May, et al.,"Endothelin Antagonists Bosentan Blocks Neurogenic Inflammation, But is not effective in aborting migraine attacks"Pcrin, 67 (1996) 375-378.

30. M. Busso, et al.,"Nucleotide Dimers Suppress HIV Expression In Vitro"Aids Res. Hum. Retrovirzses, (1988) 4 (6) 449-455.

31. European Patent No. 0 526 708 Al, issued Feb. 10, 1993 to F.

Hoffmann-La Roche AG, entitled"Sulfonamide, Ihre Herstellung L'nd Verwendung Als Heilmittel Und Zwischenprodukte" (Inventors : K.

Burri et al.).

32. PCT Application No. WO 93/08799.

All of the above publications are herein incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety.

State of the Art A receptor is a biological structure with one or more binding domains that reversibly complexes with one or more ligands, where that complexation has biological consequences.

Receptors can exist entirely outside the cell (extracellular receptors), within- the cell membrane (but presenting sections of the receptor to the extracellular milieu and cytosol), or entirely within the cell (intracellular receptors). They may also function independently of a cell (e. g., clot formation). Receptors within the cell membrane allow a cell to communicate with the space outside of its boundaries (i. e., signaling) as well as to function in the transport of molecules and ions into and out of the cell.

A ligand is a binding partner for a specific receptor or family of receptors. A ligand may be the endogenous ligand for the receptor or alternatively may be a synthetic ligand for the receptor such as a drug, a drug candidate or a pharmacological tool.

The super family of seven transmembrane proteins (7-TMs), also called G-protein coupled receptors (GPCRs), represents one of the most significant classes of membrane bound receptors that communicates changes that occur outside of the cell's boundaries to its interior, triggering a cellular response when appropriate. The G-proteins, when activated, affect a wide range of downstream effector systems both positively and negatively (e. g., ion channels, protein kinase cascades, transcription. transmigration of adhesion proteins, and the like).

The ligands that bind to G-protein cellular receptors may be specifically classified as follows: 1. Full agonists-ligands that when bound trigger the maximum activity seen by natural ligands; 2. Partial agonists-ligands that when bound trigger sub-maximal activity : 3. Antagonist-ligands that when bound inhibit or prevent the activity arising from a natural ligand binding to the rezeptor. Antagonists may be of the surmountable class (results in the parallel displacement of the dose-response curve of the agonist to the right in a dose dependent fashion without reducing the maximal response for the agonist) or insurmountable class (results in depression of the maximal response for a given agonist with or without the parallel shift);

4. Inverse antagonist-ligands that when bound decrease the basal activity of the unbound receptor (if There are four fundamental measurable properties that pertain to the interaction of a ligand with its receptor including G-protein cellular receptors: 1) the affinity of the ligand for the receptor, which relates to the energetics of the binding; 2) the efficacy of the ligand for the receptor, which relates to the functional downstream activity of the ligand ; 3) the kinetics of the ligand for the receptor, which defines the onset of action and the duration of action ; and 4) the desensitization of the receptor for the ligand.

With regard to the ligand, it is the combination of these properties that provides the foundation for defining the nature of the functional response. Thus, an activating ligand (or agonist) has affinity for the receptor and downstream efficacy, in contrast, an inhibiting ligand (antagonist) has affinity for the receptor but no efficacy.

Selectivity defines the ratios of affinities or the ratios of efficacies of a given ligand compared across two receptors. It is the selectivity of a specific drug that provides the required biological profile. For example, in certain therapeutic settings. it is currently thought that a highly selective drug may be preferred (e. g., Losartan (Cozaar), an antihypertensive, is a highly selective antagonist for the AT I receptor).

In contrast, it is considered that a drug with a broad spectrum of receptor activity may be preferred in other therapeutic settings.

Current drugs (ligands) targeting receptors, including G-protein receptors, have clinical shortcomings identified by one or more of low efficacy, low affinity, poor safety profile, lack of selectivity or overselectivity for the intended receptor, and suboptimal duration of action and onset of action. Accordingly, it would be beneficial to develop ligands that have improved affinity, efficacy, selectivity, onset of action and duration of action.

Affinitv of ligand for target receptor An increase in ligand affinity to the target receptor may contribute to reducing the dose of ligand required to induce the desired therapeutic effect. A reduction in ligand affinity will remove activity and may contribute to the selectivity profile for a ligand.

Efficacy of ligand at a target receptor (functional effect) An increased ligand efficacy at a target receptor can lead to a reduction in the dose required to mediate the desired therapeutic effect. This increase in efficacy may arise from an improved positive functional response of the ligand or a change from a partial to full agonist profile. Reduced efficacy of a full agonist to a partial agonist or antagonist may provide clinical benefit by modulating the biological response.

Selectivitv of ligand compared across receptor subtypes An increase in the selectivity of the ligand across receptor subtypes requires that the affinity or efficacy of the ligand at other receptors is reduced relative to the desired receptor.

A decrease in the selectivity of the ligand may also be desired. For example. the angiotensin II endogenous ligand activates both the AT 1 and AT'receptor subtypes. However, Losartan is a selective AT I receptor antagonist.

Onset of Action More rapid onset of action of the ligand to effect a biological response is often preferred.

Duration of Action An increased duration of action of the ligand to effect a biological response may be preferred. For example) adrenergic agonists such as albuterol have a relatively short duration of action of approximately 3-4 hours and an increase in

duration of action would simplify the dosing regimen required to administer this drug (ligand).

Desensitization of the receptor for the liand Desensitization is best defined as the variety of processes by which the functional interaction of the receptor with its G-protein are influenced. These processes lead ultimately to a reduction in cellular response to the activating agonist.

Such phenomena are most often observed during prolonged stimulation of the receptor. The two main pathways for receptor desensitization are reduction in receptor density or changes in receptor structure by phosphorylation mechanisms.

Receptor density is altered by receptor sequestration. This is a reversible process that is observable within minutes and is a dynamic sorting of receptors with receptors being cycled Lo and from the membrane. On the other hand, receptor down- regulation is generally slower, in the order of hours, and is irreversible, involving, destruction of the receptor. Finally, receptor density may be affected by an alteration in the rate of synthesis. For example, the rate of ß2 mRNA synthesis and degradation are controlled by levels of c-AMP within the cell.

Alternatively, receptor desensitization may occur through changes in receptor structure, such as receptor phosphorylation. For example, agonist induced activation of the P,-adreneruic receptor, which is positively coupled to adenylate cyclase through Gs, results in an elevation in an increase in the levels of c-AMP and an increase in the activity of protein kinase A. This kinase can readily phosphorylate the consensus site in the third intracellular loop. The phosphorylated, 32-adrenergic receptor exhibits significantly reduced coupling to Gs. Besides PKA, the G-protein coupled receptor kinases (GRK) are also involved in the desensitization of GPCRs. For the 02- adrenergic receptor, there are two of these kinases bARKI and bARK2. These GRKs are more specific and will only phosphorylate an agonist activated receptor.

Furthermore this GRK desensitization requires an arrestin protein.

Receptor oligomerization also plays a role in receptor function. This is best exemplified in the area of growth receptors that are known to act functionally and structurally as dimers, e. g., EGF-R and interferon receptor. It is also known that dimerization is involved in the functioning of the steroid receptor. Preliminary evidence is beginning to appear on the importance of oligomerization in G-protein coupling and signaling. It is proposed that receptor oligomerization may play a role in different receptor functions such as mediating coupling of the G-protein or receptor internalization.

U. S. Patent No. 5,414,010 issued to Downing et al. Sdiscloses dimeric benzimidazoles for the treatment of disorders that respond to dopaminergic blockade.

The dimeric benzimidazoles selectively bind to dopamine D3 receptors. Downing et al. disclose that these compounds are useful antipsychotic agents for treating psychoses such as schizophrenia. Downing et al. are silent on whether monomeric benzimidazoles have any effect on dopamine D3 receptors and whether the dimeric forms produce enhanced effects over its monomeric forms.

M. Busso et al. 30 discloses a series of nucleotide homo-and heterodimers that were synthesized and compared to their monomers for their anti-HIV and cytotoxic properties in vitro. Both were reported to inhibit HIV-induced syncytia formation. reverse transcriptase production, and the expression of HIV p24 antigens. Greater anti-HIV potency and enhanced cytotherapeutic indices were reported with the heterodimers relative to their monomers on an equimolar basis.

One important class of GPCRs is the endothelin receptors including the endothelin (ET,, ETB) receptors, which are characterized by seven (7)-hydrophobic transmembrane domains. ETA receptors are found in vascular smooth muscles and mediate vasoconstriction and proliferation. ETB receptors are found in vascular endothelium cells and mediate transient vasodilatation. The biology, pharmacology and role of the endothelin system has been the subject of a number of recent reviews. 4 5. 6, 7, 9, 10. 22, 23 The cDNAs of endothelin receptors ETA and ETB have been cloned and studied. The receptors share similarities in structural organization, and the mature

proteins have an overall identity of between 55 and 64%, with the 7-transmembrane domains and cytoplasmic loops being highly conserved, and the extracellular domains (including the N-terminus) differing in sequence and length, as shown in Figure 3 (taken from G. Gray et al.'). It is believed that the transmembrane domains 1, II III and VII are important for ligand binding, and domains IV, V and VI for isopeptide selectivity. The transmembrane domain III, particularly the C-terminal end, is implicated in coupling of human ET, receptors to G proteins and subsequent liberation of intracellular Ca2+. 4, 9 The endothelin receptors mediate actions of endothelin isopeptide ligand agonists. The receptor subtypes differ in their isopeptide selectivity, ligand binding specificity, tissue distribution and physiological actions.

Endothelins are a family of endogenous 21 amino acid isopeptide ligand agonists (ET-l, ET-2 and ET-3) with potent effects on the cardiovascular system and the kidney. Encoded by separate genes, these isopeptides are generated by proteolytic cleavage of their corresponding biologically inactive prepropolypeptides. As shown in Figure 1 (taken from G. Gray et the endothelins are closely related structurally and are characterized by the presence of two intrachain disulfide bonds and a conserved carboxy terminus amino acid sequence, which is required for biologic activity. 4 6 The endothelins are found in a variety of mammalian species and are closely related as well to the sarafotoxin peptides found in the venom of the snake Actractaspis engaddensis.

Endothelin-1 (i. e., ET-I), the major isopeptide in vascular endothelial cells. may play an important role in cardiovascular and renal disease.'-''°---'Increased levels of ET-1 appear in the circulation in pathologic conditions such as pulmonary hypertension, atherosclerosis, coronary vasospasm, acute myocardial infarction. congestive heart failure and renal failure.

Endothelin-1 synthesis by endothelial cells is primarily under transcriptional regulation, which is responsive to a variety of factors, both positive and negative, as

shown in Figure 2 (taken from D. Webb et al 6). Post-transcriptional regulation is thought to involve destabilization of the preproendothelin-1 mRNA.

Evidence from in vitro mutagenesis studies and molecular modeling studies suggests that different transmembrane domains are involved in ligand binding to ET, and ET, receptor subtypes4 These ligand binding sites are also distinguishable by their affinities for ET isopeptides. The ET, receptor, for example, exhibits very high (subnanomolar) affinity for ET-1 and ET-2 and approximately two orders of magnitude lower affinity for ET-3. By contrast, ETB binds all 3 isopeptides with equivalently high affinity. Differences in binding affinity reflect potency differences of the endothelins.

The ETA receptor is primarily localized to vascular smooth muscle cells, where it mediates ET-1 vasoconstriction of long duration It is also the major receptor subtype in cardiac muscle. The prolonged exposure of ET-1 to cardiac muscle ET, may result in cardiac hypertrophy.

The ET, receptor comprises two subtypes, ETB, and ETB2. ETB, is a high affinity receptor found on vascular endothelial cells, where, in response to ET-1, it mediates the release of vasodilatory factors, e. g., EDHF (hyperpoianzing factor), prostacyclin and nitric oxide, and thereby produces a transient vasodilation. ET,,. receptors are present on vascular smooth muscle cells and mediate vasoconstriction, particularly in small resistance vessels and veins. Although both ETA and ETB, mediate endothelin-1 vasoconstriction actions in human blood vessels, their tissue distributions differ (e. g., ET112 is present in renal vessels, ET, in pulmonarv arteries; ETB2 in the proximal arteries and ET in the distal arteries of the coronary bed).

The endothelin receptors activate phospholipase C to cause hydrolysis of phosphatidyl inositol and generation of cytosolic inositol trisphosphate and membrane-bound diacylglycerol (DAG). Inositol trisphosphate causes an early rapid rise in calcium [Ca213 through its release from intracellular stores. Diacylglycerol

activates protein kinase C, increasing the sensitivity of the contractile apparatus to Ca2 ;, which activates nuclear signaling mechanisms6 Antagonists have been developed to prevent the binding of the endothelin ligands to their respective receptors. The antagonist binds to the receptor and thereby either blocks the agonist from binding to that site, or alters the binding site, so that the agonist can not bind to the receptor. If the agonist does not bond to the receptor, the affects of the endothelin are not expressed and the diseased condition is ameliorated.

The binding of the antagonist to the receptor prevents transmission of impulses that initiate the symptoms of the disease.

There have been numerous studies to determine the binding sites of the agonists and peptide and non-peptide antagonists to the endothelin receptors. ' It is suggested that the C terminal part of ET-1 interacts preferentially with the transmembrane domain regions I, II, III and VII with secondary interaction sites at transmembrane domains IV, V and VI, which may recognize the amino terminal loop of ET-1. The binding site of non-peptide antagonists is believed to be different from the ET-I binding site, but might overlap with the secondary interaction sites. The binding sites on the endothelin receptors for synthetic antagonists have been studied as well in an effort to develop therapeutic drugs for the treatment of diseases in which the endothelin receptors play a role.

Endothelin receptor antagonists provide novel agents for the treatment of conditions involving vasoconstriction or vasospasm in which ET-1 is implicated as a contributing factor (e. g. congestive heart failure, hypertension, atherosclerosis, cerebral vasospasm following subarachnoid hemorrhage, to name a few). There is considerable interest in developing new therapies for these conditions. In congestive heart failure, endothelin antagonists have been shown to decrease pulmonary pressure and systemic blood pressure through vasodilation. While other classes of therapeutic agents (e. g., angiotensin-converting enzyme (ACE) and angiotensin II (All) inhibitors) are used for this purpose, such agents have side effects that compromise renal function and cause hypotensive episodes. There is a continuing need for new

agents to counter the significant morbidity and mortality from congestive heart failure (CHF), for example.

Endothelin receptor antagonists are also useful for the treatment of pulmonary hypertension. Unlike ACE inhibitors, nonselective endothelin receptor antagonists may counteract NO-mediated pulmonary vascular contraction, reduce pulmonary wedge pressures and reduce pulmonary remodeling associated with pulmonary hypertension. Selective ETA receptor antagonists may also be useful in managing hypertension, as indicated by their ability to improve endothelial function and blood pressure in All-induced hypertension. In addition to their antihypertensive effects, endothelin antagonists are potentially useful for treating other pathological conditions in which ET-1 plays a role (e. g., atherosclerosis, cardiac and vascular hypertrophy and progression of renal impairment, to name a few). Conditions involving intermittent vasospasm, e. g., subarachnoid hemorrhage4 16 and acute renal failure, may also benefit from treatment with endothelin receptor antagonists. There is no current treatment for subarachnoid hemorrhage, for example, which causes cerebrospinal fluid (CSF) levels of ET-1 to rise during a period of hours to days, and, secondarily, cerebral spasm, which leads to further ischemia and significant brain damage.

A large number of ET receptor antagonists (peptide and nonpeptide. selective and nonselective) have been developed. Table 1 below (information taken from G.

Gray et al. 4) is a list of known ET receptor antagonists. A number of drugs are currently in clinical trials. Table 2 below provides a list of drugs currently in some phase of clinical trials. The drug, Bosentan (Ro 47-0203), whose chemical structure" is illustrated herein, appeared to be a particularly promising drug'"'''*"''''"''. but clinical trials were discontinued because of toxicity. Thus, there continues to be a need for endothelin receptor antagonists with improved efficacy, increased selectivity and reduced side effects.

Table I

Known Antagonists for Endothelin Receptors Categorv Ligand Selectivitv Peptide Antagonist BQ-123 ET, FR 139317 ETA TTA-386 ETA BQ 518 ETA BQ-788 ETB Res 701-1 ETB BQ-017 ETB IRL 2500 ETB PD 145065 ETA B TAK-044 ETAB Nonpeptide Antagonists 97-139 ET, BMS 182874 ET, LU 127043 ETA PD 155080 ET, Ro 46-8443 ETB Ro 47-0203 ETA, B CGS 27830 ETAB SB 209670 ET PD 160672 ET, H SB-217242 ETAB

Table 2 Examples of Endothelin Antagonists In Clinical Trials Agent Clinical Phase Indications K., LApM) TAK-044 Phase II Hypertension, myocardial infarction, renal failure, brain hemorrhage, ischemia 240 PD 145065 Phase I Hypertension, ** BQ-123 Phase I HTN, Peripheral ** (Suspended) disease Ro 47-0203 Dropped CHF, CVA, HTN, ischemia 20,000 (Bosentan) J-104132 Phase IIa * 34 SB 209670 Phase II Restenosis, renal failure, 200 migraine SB-217242 Phase II HTN, COPD 1100 TBC-11251 Phase II CHF, HTN, COPD 1400 Ro-48-5695 Phase II CHF 300 LU-123252 Phase II* CHF, CAD 1500 BMS-193884 Phase I CHF 4700 Ro-61-0612 Phase I CVD, HTN-1000 ZD-1611 Phase I CHF, HTN ** S-0139 Phase I HTN, CVD, Cerebral ischemia/stroke ** PD-180988 Pre-clin. Hypertension, CHF and others 460 *Actual clinical phase unclear from literature **Information unknown at the time of this writing CHF-congestive heart failure HTN-hypertension CVD-cardiovascular disease CAD-coronary artery disease COPD-c. o. pulmonary disease Accordingly, novel ligand antagonists having desired potency and therapeutic effect for the endothelin receptors would be particularly desirable in order to eliminate or treat a variety of diseases of the heart, kidney, endocrine and nervous systems (and including vascular smooth muscle and non-vascular smooth muscle) in mammalian

patients. Such novel ligand antagonists would preferably achieve the desired potency and therapeutic effect by modulating one or more of the ligand's properties as to efficacy, affinity, safety profile, selectivity, duration of action and/or onset of action.

SUMMARY OF THE INVENTION This invention is directed, in part, to novel multi-binding compounds (agents) that bind to endothelin receptors and have an antagonistic affect. The multi-binding compounds of this invention are useful in the treatment and prevention of diseases or conditions mediated by endothelin receptors This invention is further directed to pharmaceutical compositions comprising the novel multi-binding compounds, methods of preparing the novel multi-binding compounds and methods of treating diseases or conditions mediated by endothelin receptors using such novel multi-binding compounds.

In particular, the multi-binding compounds of this invention can be used to treat vascular disorders and endothelial and myocardial dysfunction, such as congestive heart failure, pulmonary hypertension, essential hypertension, cerebral vasospasm following subarachnoid hemorrhage, renal failure of ischemia origin. portal hypertension, cardiac hypertrophy, myocardial infarction (ischemia, unstable angina), restenosis, pre-eclampsia, and migraine and as a prophylaxis for atherosclerosis.

Accordingly, in one of its composition aspects, this invention is directed to a multi-binding compound and salts thereof comprising 2 to 10 ligands, which may be the same or different and which are covalently attached to a linker or linkers which may be the same of different, wherein each of said ligands comprises a ligand domain capable of binding to a endothelin receptor. Preferably, at least two, and more preferably, each of the ligands comprises a ligand domain capable of binding to a endothelin receptor. Most preferably, the ligand comprises a ligand domain capable

of binding to the endothelin receptors to block the effects of ligand agonists endothelin-1, endothelin-2 and endothelin-3.

In another of its composition aspects, the invention provides a multi-binding compound represented by Formula I : (L) p (X) q wherein each L is a ligand that may be the same or different at each occurrence and is independently selected from ligands comprising a ligand domain capable of binding to a endothelin receptor; X is a linker that may be the same or different at each occurrence; p is an integer of from 2 to 10 ; q is an integer of from 1 to 20 ; and pharmaceutically acceptable salts thereof. Preferably, g is less than p.

Preferably, eacti ligands, L, in the multi-binding compound of Formula I (L, X, ;) is independently selected from a compound of Formulae II, IIa, III or IV described below.

In its most general form, ligand L has the structure of (a) Formula II : RA-S (O) r-N (H)-RB 11 wherein : RA is a group selected from an aryl, optionally substituted aryl, lieteroarvl, auii optionally substituted heteroaryl; and R"is a group selected from a heteroaryl and an optionally substituted heteroaryl.

Preferably, RA is independently a five or six membered aromatic ring with a substitution in a meta or para position relative to the S (O), group ; and RB is independently a five or six membered aromatic ring optionally with a plurality of substitutions.

More preferably, when RA is substituted, the substituent is a group selected from alkyl, substituted alkyl, alkylene, substituted alkylene, alkoxy, substituted alkoxy, alkylalkoxy, alkylthioalkoxy, acyl, acylamino, aminoacyl, aminoacyloxy, acyloxy, aryl, optionally substituted aryl, aryloxy, amino, substituted amino, carboxyalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, halo, heteroaryl, optionally substituted heteroaryl, heteroaryloxy, heteroarylene, heterocyclyl, substituted heterocyclyl, heterocyclooxy, thioheterocyclooxy, heterocyclene, oxyacylamino, thiol, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, a covalent bond to a linker or another ligand, a functional group FG for providing a covalent bond to a linker or another ligand, or the substituent includes a functional group FG for providing a covalent bond to a linker or another ligand.

Also more preferably, when RB is substituted, the substituent groups may be independently one or more of amino, substituted amino; alkyl, alkoxy, and alkylene. and the substituted versions thereof ; aryl and heteroaryl and the optionally substituted aryl or heteroaryl ; aryloxy, acyl, acylamino, aminoacyl, aminoacyloxy, acyloxy, heterocyclyl, optionally substituted heterocyclyl, carboxyalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, a covalent bond to a linker to another ligand, a functional group FG for providing a covalent bond to a linker or another ligand, or the substituent includes a functional group for providing a covalent bond to a linker or another ligand.

In either R'or R'above, FG is selected from halo, oxy, hydroxy, amino, substituted amino, thiol, acyl and carboxy group, or any combination thereof, for reacting with a complementary group on the linker or another ligand to form a covalent bond. Preferably, FG is an amino, thiol or hydroxy group.

In a most preferred embodiment, RA is one of the following groups:

and R'A is an alkyl or substituted alkyl, or a substituted aryl, acyl, acyloxy, preferably as illustrated below:

or a covalent bond to a linker X or another ligand L, a functional group FG, as defined above, or the substituent on the alkyl or aryl, acyl or acyloxy includes a functional group FG to provide a covalent bond with a linker or another ligand.

Further to the most preferred embodiment, RB is one of the following groups :

wherein: R'B, R-s and R'B are independently halo, hydrogen, cycloalkyl, cycloalkenyl. heterocyclyl, heteroaryl, aryloxy, alkaryl, alkoxy, alkylalkoxy, acyloxy, acylaminoo amino, aminoacyl, or an optionally substituted version of applicable ones of the above, a covalent bond to a linker X or another ligand L, a functional group FG, as defined above, for providing a covalent bond to a linker X or another ligand L, or the hereinabove substituent includes a functional group FG for providing a covalent bond to a linker X or another ligand L.

Preferably, R'B, R'B and RUZ are selected from hydrogen, halo, heterocyclyl, heteroaryl, alkyl, hydroxy, alkoxy, acyloxy, alkaryl, aryloxy, alkylalkoxy, or substituted versions of applicable ones above.

In another preferred embodiment thereof, the ligand L has the structure of Formula IIa below : (b) Formula IIa: Ila

wherein : R'is a substituent at any position of an optionally substituted aryl or heteroaryl group (represented herein by a benzene ring), preferably a position a or para to the sulfonamide group, and R'is selected from H, alkyl, substituted alkyl. alkoxy, substituted alkoxy, halo, cycloalkyl, alkylthio. aralkyl. substituted aralkyl. a covalent bond attaching the ligand to a linker, or a group of formula R''-FG, where R"is a lipophilic group, preferably selected from a lower alkyl, aromatic or fatty acid derivative group, and FG is an functional group for the covalent attachment of the hgand to a linker or another ligand, as defined above; R2 is a group represented by the formula W-R2a or W-R2a-Q-FG, where WisO, SorNH, R'a is alkyl, substituted alkyl, alkylene, substituted alkylene, alkoxy,

substituted alkoxy, all as defined herein, and Q is a heterocyc ; yl group (preferably pyridine, pyran, furan or morpholine) or a substituted alkylene interrupted by a H-bond acceptor, preferably O or N, and FG is defined as above; R'is a group selected from H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, alkoxyalkyl, substituted alkoxyalkyl, amino, halo, thio, substituted amino, heteroaryl, substituted heteroaryl, or a group of formula R3a-FG, where R"is a group selected from H, alkyl, substituted alkyl, cycloalkyl. substituted cycloalkyl, alkoxy, substituted alkoxy, alkoxyalkyl, substituted alkoxyalkyl, amino, halo, thio, substituted amino, heteroaryl, substituted heteroaryl, and FG is as defined above; and Ra is substituted aryl, preferably aryloxy-, thioaryloxy, aralkenyl, or aryl-CO-. most preferably alkoxy-phenyloxy.

In still another preferred embodiment, the ligand L has the structure of Formula III as described below: (c) Formula III: III wherein: one of R 5or R'is a functional group FG for the covalent attachment of the ligand to a linker, and the other of R'or R6 is H or an optional substituent as defined herein for aryl, preferably OH, alkoxy, halogen, O, amino, substituted amino,-NH-

C (O)-CH3,-(CH2) nCOOH,-(CH2) nCOOR,-(CH) nCOO (CH) nAr,-NRCOOH, NRCOOR,-NRCOO (CH) Ar, where R is H or an alkyl ; Ar is a symbol representing an aryl or heteroaryl group; n is an integer from 1 to 10; and FG is as defined above. Preferably, FG is an amino. thiol, or hydroxy group; R7 and Rg are independently aryl or heteroaryl, and preferably a substituted aryl or heteroaryl. More preferably, R7 and R8 are independently either a group of the formula IIIa : Ifla wherein: A is (CH,) m or substituted (CH2) m, and m is an integer from I to 3, B is O or-CH,-, and preferably O, and R"is an optional substituent at any position of the optionally substituted aryl or heteroaryl group and is selected from alkoxy or substituted alkoxy, any of the substituents defined above for optionally substituted aryl or heteroaryl, or a covalent bond attaching the ligand to a linker X or another ligand, or a functional group FG, as defined above. Moreover, any of the substituents defined hereinabove may contain a functional group FG for attaching the ligand to a linker X or another ligand L; or a group of Formula IIIb :

wherein R"is as defined above; and Me is methyl, other alkyl, or alkylene, alkenyl, alkynyl, alkenylene, alkynylene, or substituted versions thereof.

In still another preferred embodiment, the ligand L has the structure of Formula IV, described below : (d) Formula IV:

(IV) wherein: RC, R°, RE and R"are independently a group selected from alkyl, substituted alkyl, alkylene, substituted alkylene, alkaryl, alkoxy, substituted alkoxy ! alkylalliox. alkylthioalkoxy, alkenyl, alkenylene, alkynyl and alkynylene and/or substituted versions thereof, acyl, acylamino, aminoacyl, aminoacyloxy, acyloxy, aryl, optionally substituted aryl, aryloxy, arylene, amino, substituted amino, carboxyalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, optionally substituted heteroaryl, heteroaryloxy, heteroarylene, heterocyclyl, optionally

substituted heterocyclyl, heterocyclooxy, thioheterocyclooxy, heterocyclene, oxyacylamino, spire-attached cycloalkyl, thiol, thioalkoxy, substituted thioalkoxy, thioaryloxy, and thioheteroaryloxy, wherein any of the above may optionally contain an attaching group FG for attaching to a linker or another ligand ; or halo, hydrogen, a covalent bond to a linker or another ligand, or an attaching group FG for attaching to a linker or another ligand ; and R'and R'are independently a group selected from aryl, heteroaryl, heterocyclyl, cycloalkyl, or optionally substituted versions thereof, wherein any of the above may optionally contain an attaching group FG for attaching to a linker or another ligand ; or halo, hydrogen, a covalent bond to a linker or another ligand. or an attaching group FG for attaching to a linker or another ligand, wherein FG is as defined above.

In a preferred embodiment of ligands having the structure of Formula IV, RF and RG are independently an aryl, optionally substituted aryl, heteroaryl or optionally substituted heteroaryl. In a more preferred embodiment, ligands L of Formula IV are modeled after the structural formulae for ligand LU-135252, illustrated in Figure 4.

In another preferred embodiment, each ligand L is independently selected from a compound of Formula II/Ila, or III/IIIa defined above, which are modeled after the structural formulae for Bosentan''and SB-90967032, respectively, for example, also illustrated in Figure 4: Ligands, which are modeled after a known ligand, are analogs thereof or a"Synthon".

In still another preferred embodiment, each ligand L is independently selected from a compound listed in Table 1, and analogs thereof.

The following formulas show an example of an analog of SB 209670 (Formula III/IIIa), named"Synthon A", and an example of an analog of Bosentan (Formula II/IIa), named"Synthon B". The structures below are illustrative of the ligands that can be used to prepare the multi-binding compounds of the present invention.

SB 209670 Analog-Synthon A Bosentan Analog-Synthon B In still another of its composition aspects, the linker X is either a covalent bond or a diacyl compound independently selected from a structure of Formula (\) : X'-C (=O)- (R9) n-C (=O)-X' (V) wherein: R9 is independently alkyl, substituted alkyl, alkylene, substituted alkylene, alkaryl, alkoxy, substituted alkoxy ; alkylalkoxy, alkylthioalkoxy, alkenyl. alkenylene. alkynyl and alkynylene and/or substituted versions thereof, acyl. acylamino. aminoacyl, aminoacyloxy, acyloxy, aryl, optionally substituted aryl, aryloxy, arylene. amino, substituted amino, carboxyalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, optionally substituted heteroaryl heteroaryloxy, heteroarylene, heterocyclyl, optionally substituted heterocyclvl, heterocyciooxy, thioheterocyclooxy, heterocyctene, oxyacytamino, spiro-anachcd cycloalkyl, thiol, thioalkoxy, substituted thioalkoxy, thioaryloxy, and thioheteroaryloxy. n is an integer from 1 to 20 X'and X1 are end groups that will react with either an amino, hydroxy. halo, alkyl, alkoxy, thiol, thioalkoxy containing groups on the ligands or on precursors thereof, to form a linkage. Representative examples of the X'and X-end groups include, but are not limited to hydrogen, hydroxy, alkoxy, halo, or haloalkyl, amino, substituted amino, SH, and SO,, for example, or a covalent bond to the ligand.

Alternately, the linker X may be represented by the following formula (VI) : -X'-Z- (Y'-Z) m-Y"-Z-X'- (VI) in which: m is an integer of from 0 to 20; X'at each separate occurrence is-O-,-S-,-S (O)-,-S (O) 2-,-NR-,-N'R R-,- C (O)-,-C (O) O-,-C (O) NH-,-C (S),-C (S) O-,-C (S) NH- or a covalent bond, where R and R at each separate occurrence are as defined below for R'and R" ; Z is at each separate occurrence selected from alkylene, substituted alkylene, alkylalkoxy, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, cycloalkenylene, substituted alkenylene, arylene, substituted arylene, heteroarylene, heterocyclene, substituted heterocyclene, crown compounds, or a covalent bond; Y'and Y'at each separate occurrence are selected from:

-S-S-or a covalent bond; in which: n is 0, 1 or 2 ; and R'and R"at each separate occurrence are selected from hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl or heterocyclic.

In still another preferred embodiment, the linker X is a covalent bond between complementary functional groups on respective ligands L to be joined.

In yet another of its composition aspects, this invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a therapeutically effective amount of one or more multi-binding compounds (or pharmaceutically acceptable salts thereof) comprising 2 to 10 ligands which may be the same or different and which are covalently attached to a linker or linkers, which may be the same or different. Each of said ligands comprises a ligand domain capable of binding to an endothelin receptor of a cell mediating mammalian diseases or conditions, thereby modulating the diseases or conditions.

In one of the pharmaceutical composition aspects, the multi-binding compounds are represented by Formula I defined above. Each ligand L comprises a ligand domain capable of binding to a endothelin receptor; thereby inhibiting the action of endothelin agonists, such as ET-1, ET-2 and ET-3 at the endothelin receptors. Such pharmaceutical compositions are useful for modulating diseases of the heart, kidney and endocrine and nervous systems in mammals, which are modulated by endothelin receptors.

Preferably, the pharmaceutical compositions of this invention comprise ligands L having the structure of Formula (II), (IIa), (III) or (IV) and linkers X having the structure of Formula (V) or (VI), where q is less than p, or the linker is a

covalent bond between ligands. More preferably, the ligands of the multi-binding compounds are selected from the group consisting of known endothelin ligand antagonists, for example, SB 20967032, SB-217242, Bosentan", Ro-48-5695, TBC- 11251, ZD-1611, and LU-135252 and others listed in Table 1, for example, and analogs thereof. More preferably, each of the ligands L have a ligand domain capable of selectively binding to the endothelin receptor.

According to one of its method aspects, this invention provides a method of modulating the activity of an endothelin receptor in a biologic tissue, which method comprises contacting a tissue having an endothelin receptor with a multi-binding compound (or pharmaceutically acceptable salts thereof) under conditions sufficient to produce a change in the activity of the receptor in said tissue, wherein the multi- binding compound comprises 2 to 10 ligands which may be the same or different and which are covalently attached to a linker or linkers, which may be the same or different, each of said ligands comprising a ligand domain capable of binding to an endothelin receptor.

In another method of modulating aspects, the multi-binding compounds are represented by Formula I as defined above, wherein each ligand is covalently attached to the linker and each ligand comprises a ligand domain capable of binding to a endothelin receptor; thereby inhibiting the action of endothelin agonists, such as ET-1, ET-2 and ET-3 at the endothelin receptors. The binding of the multi-binding compounds to the endothelin receptor modulates the diseases and conditions mediated by such receptors. In particular, the method is useful for modulating diseases of the heart, kidney and endocrine and nervous systems in mammals.

Preferably, the multi-binding compounds of the method of modulating comprise ligands L having the structure of Formulas (II), (IIa), (III) or (IV) and linkers X having the structure of Formula (V) or (VI), where q is less than p, or the linker is a covalent bond between respective ligands. More preferably, the ligands L of the multi-binding compounds are selected from the group consisting of known

ligand antagonists, such as those listed in Table 1, analogs thereof and the ligand precursors thereof. In another most preferred embodiment, each of the ligands have a ligand domain capable of selectively binding to the endothelin receptor.

In another of the invention's method aspects, this invention provides a method of preparing a multi-binding compound represented by Formula I, as defined above, which comprises the steps of : (a) providing at leastp equivalents of a ligand L or precursors thereof and at least q equivalents of linker or linkers X; and (b) covalently attaching said ligands to said linkers te produce a multi-binding compound; or (b') covalently attaching said ligand precursors to said linkers and completing the synthesis of said ligands thereupon, thereby to produce a multi-binding compound. Preferably, the ligands L have the structure of Formula (II), (IIa), (III) or (IV) and the linkers X have the structure of Formula (V) or (VI), where q is less than p, or the linker is a covalent bond between respective ligands.

In another one of its method aspects, this invention is directed to a method for treating a disease or condition in a mammal resulting from an activity of an endothelin receptor, which method comprises administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable excipient and one or more multi-binding compounds (or pharmaceutically acceptable salts thereof) comprising 2 to 10 ligands which may be the same or different and which are covalently attached to a linker or linkers, which may be the same or different, each of said ligands comprising a ligand domain capable of binding to an endothelin receptor of a cell mediating mammalian diseases or conditions.

Accordingly, in one of its method of treating aspects, the multi-binding compounds, or pharmaceutically acceptable salts thereof, are represented by Formula I, as defined above. The action of the multi-binding compound effectively inhibits

the action of endothelin agonists, such as ET-1, ET-2, and ET-3 at the endothelin receptors and modulating the diseases and conditions resulting therefrom.

A preferred embodiment is the use of pharmaceutical compositions comprising ligands L having the structure of Formula (II), (IIa), (III) or (IV) and linkers X having the structure of Formula (V) or (VI), where q is less than p, or the linker is a covalent bond between respective ligands. Most preferably, the multi-binding compounds comprise ligands with ligand binding domains capable of selectively binding to the endothelin receptors in mammals.

The multi-binding compounds, pharmaceutical compositions and methods of treating and modulating in accordance with the invention target endothelin receptors, which mediate diseases or conditions associated with the heart, kidney. endocrine glands and nervous system in mammals. Conditions, such as congestive heart failure, pulmonary hypertension, cerebral vasospasm following subarachnoid hemorrhage, essential hypertension, myocardial infarction, myocardial ischemia, unstable angina, restenosis, renal failure of ischemic origin, portal hypertension cardiac hypertrophy, atherosclerosis, eclampsia, cerebrovascular disease, vascular disease, migraines, and auto-immune diseases, such as Morbus Wegener and Morbus Raynaud, to name a few, may be tratable with the novel multi-binding compounds of this invention.

In still another aspect, this invention is directed to general synthetic methods for generating large libraries of diverse multimeric compounds which multimeric compounds are candidates for possessing multibinding properties. The diverse multimeric compound libraries provided by this invention are synthesized by combining a linker or linkers with a ligand or ligands to provide for a library of multimeric compounds wherein the linker and ligand each have complementary functional groups permitting covalent linkage. The library of linkers is preferably selected to have diverse properties such as valency, linker length, linker geometry and rigidity, hydrophilicity or hydrophobicity, amphiphilicity, acidity, basicity and polarization. The library of ligands is preferably selected to have diverse attachment points on the same ligand, different functional groups at the same site of otherwise the same ligand, and the like.

This invention is also directed to libraries of diverse multimeric compounds which multimeric compounds are candidates for possessing multibinding properties.

These libraries are prepared via the methods described above and permit the rapid and efficient evaluation of what molecular constraints impart multibinding properties to a ligand or a class of ligands targeting a receptor.

Accordingly, in one of its method aspects, this invention is directed to a method for identifying multimeric ligand compounds possessing multibinding properties which method comprises: (a) identifying a ligand or a mixture of ligands wherein each ligand contains at least one reactive functionality; (b) identifying a library of linkers wherein each linker in said library comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand; (c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the ligand or mixture of ligands identified in (a) with the library of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker

and at least two of said ligands; and (d) assaying the multimeric ligand compounds produced in (c) above to identify multimeric ligand compounds possessing multibinding properties.

In another of its method aspects, this invention is directed to a method for identifying multimeric ligand compounds possessing multibinding properties which method comprises: (a) identifying a library of ligands wherein each ligand contains at least one reactive functionality; (b) identifying a linker or mixture of linkers wherein each linker comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand ; (c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the library of ligands identified in (a) with the linker or mixture of linkers idei. ified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands; and (d) assaying the multimeric ligand compounds produced in (c) above to identify multimeric ligand compounds possessing multibinding properties.

The preparation of the multimeric ligand compound library is achieved by either the sequential or concurrent combination of the two or more stoichiometric equivalents of the ligands identified in (a) with the linkers identified in (b).

Sequential addition is preferred when a mixture of different ligands is employed to ensure heterodimeric or multimeric compounds are prepared. Concurrent addition of the ligands occurs when at least a portion of the multimer comounds prepared are homomultimeric compounds.

The assay protocols recited in (d) can be conducted on the multimeric ligand compound library produced in (c) above, or preferably, each member of the library is isolated by preparative liquid chromatography mass spectrometry (LCMS).

In one of its composition aspects, this invention is directed to a library of

multimeric ligand compounds which may possess multivalent properties which library is prepared by the method comprising: (a) identifying a ligand or a mixture of ligands wherein each ligand contains at least one reactive functionality; (b) identifying a library of linkers wherein each linker in said library comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand ; and (c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the ligand or mixture of ligands identified in (a) with the library of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands.

In another of its composition aspects, this invention is directed to a library of multimeric ligand compounds which may possess multivalent properties which library is prepared by the method comprising: (a) identifying a library of ligands wherein each ligand contains at least one reactive functionality; (b) identifying a linker or mixture of linkers wherein each linker comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand ; and (c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the library of ligands identified in (a) with the linker or mixture of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands.

In a preferred embodiment, the library of linkers employed in either the methods or the library aspects of this invention is selected from the group comprising flexible linkers, rigid linkers, hydrophobic linkers, hydrophilic linkers, linkers of different geometry, acidic linkers, basic linkers, linkers of different polarization and amphiphilic linkers. For example, in one embodiment, each of the linkers in the

linker library may comprise linkers of different chain length and/or having different complementary reactive groups. Such linker lengths can preferably range from about 2 to 100.

In another preferred embodiment, the ligand or mixture of ligands is selected to have reactive functionality at different sites on said ligands in order to provide for a range of orientations of said ligand on said multimeric ligand compounds. Such reactive functionality includes, by way of example, carboxylic acids, carboxylic acid halides, carboxyl esters, amines, halides, isocyanates, vinyl unsaturation, ketones, aldehydes, thiols, alcohols, anhydrides, and precursors thereof. It is understood, of course, that the reactive functionality on the ligand is selected to be complementary to at least one of the reactive groups on the linker so that a covalent linkage can be formed between the linker and the ligand.

In other embodiments, the multimeric ligand compound is homomeric (i. e., each of the ligands is the same, although it may be attached at different points) or heterodimeric (i. e., at least one of the ligands is different from the other ligands).

In addition to the combinatorial methods described herein, this invention provides for an interative process for rationally evaluating what molecular constraints impart multibinding properties to a class of multimeric compounds or ligands targeting a receptor. Specifically, this method aspect is directed to a method for identifying multimeric ligand compounds possessing multibinding properties which method comprises: (a) preparing a first collection or iteration of multimeric compounds which is prepared by contacting at least two stoichiometric equivalents of the ligand or mixture of ligands which target a receptor with a linker or mixture of linkers wherein said ligand or mixture of ligands comprises at least one reactive functionality and said linker or mixture of linkers comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand wherein said contacting is conducted under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two

of said ligands; (b) assaying said first collection or iteration of multimeric compounds to assess which if any of said multimeric compounds possess multibinding properties; (c) repeating the process of (a) and (b) above until at least one multimeric compound is found to possess multibinding properties; (d) evaluating what molecular constraints imparted multibinding properties to the multimeric compound or compounds found in the first iteration recited in (a)- (c) above; (e) creating a second collection or iteration of multimeric compounds which elaborates upon the particular molecular constraints imparting multibinding properties to the multimeric compound or compounds found in said first iteration; (f) evaluating what molecular constraints imparted enhanced multibinding properties to the multimeric compound or compounds found in the second collection or iteration recited in (e) above ; (g) optionally repeating steps (e) and (f) to further elaborate upon said molecular constraints.

Preferably, steps (e) and (f) are repeated at least two times, more preferably at from 2-50 times, even more preferably from 3 to 50 times, and still more preterablv at least 5-50 times.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the structure of endothelin agonists ET-1, ET-2 and ET-3; Figure 2 illustrates the synthesis of ET-1 in mammalian cells ; Figure 3 illustrates the seven transmembrane domain of ETA receptor; Figure 4 illustrates the structures of known monovalent ligand antagonists useful for the invention; Figure 5 illustrates a method of optimizing linker geometry for the multi- binding compounds of the invention;

Figures 6A and 6B illustrates representative linker cores and linking mechanism, respectively, useful for the invention; and Figures 7A-7B illustrate reaction schemes for preparing specific analogs of preferred ligands in accordance with the invention, while Figure 7B additionally illustrates the reaction scheme for preparing a multi-binding compound of one of the preferred embodiments; Figures 8A-8D illustrate representative multi-binding compounds that are prepared in accordance with the invention; Figure 9 illustrates the reaction scheme for the preparation of a multi-binding compound of Formula (B) in accordance with a preferred embodiment; Figure 10 illustrates the reaction scheme for the preparation of a multi-binding compound of Formula (C) in accordance with a preferred embodiment; and Figure 11 illustrates the reaction scheme for the preparation of a multi-binding compound of Formula (D) in accordance with a preferred embodiment.

DETAILED DESCRIPTION OF THE INVENTION This invention is directed to multi-binding compounds that are antagonists of the endothelin receptors, ET,, ET,, and their applicable subtypes. The invention is further directed to pharmaceutical compositions containing the multi-binding compounds. The invention is still further directed to methods of making the multi- binding compounds and pharmaceutical compositions thereof, and methods for treating disorders mediated by endothelin receptors. When discussing such compounds, compositions or methods, the following terms have the following meanings unless otherwise indicated. Any undefined terms have the meaning recognized in the art.

The term"alkyl"refers to a monoradical branched or unbranched saturated hydrocarbon chain preferably having from 1 to 40 carbon atoms, more preferably 1 to 10 carbon atoms, and even more preferably 1 to 6 carbon atoms. This term is exemplified by groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, n-hexyl, n-decyl, tetradecyl, and the like.

The term"substituted alkyl"refers to an alkyl group as defined above, having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro,-SO- alkyl,-SO-substituted alkyl,-SO-aryl, -SO-heteroaryl,-SO,-alkyl,-SO,-substituted alkyl,-SO,-aryl and-SO,-heteroaryl.

The term"alkylene"refers to a diradical of a branched or unbranched saturated hydrocarbon chain, preferably having from I to 40 carbon atoms, more preferably 1 to 10 carbon atoms and even more preferably 1 to 6 carbon atoms. This term is exemplified by groups such as methylene (-CH,-), ethylene (-CH, CH,-), the propylene isomers (e. g.,-CH, CH, CH,- and-CH (CH,) CH,-) and the like.

The term"substituted alkylene"refers to an alkylene group, as defined above. having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino. substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl. heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro,-SO- alkyl,-SO-substituted alkyl,-SO-aryl,-Sf)-heteroaryl,-SO2-alkyl,-SO2-substituted alkyl,-SO2-aryl and-SO,-heteroaryl. Additionally, such substituted alkylene groups include those where 2 substituents on the alkylene group are fused to form one or more cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heterocyclic or heteroaryl groups fused to the alkylene group. Preferably such fused groups contain from 1 to 3 fused ring structures.

The term"alkaryl"refers to the groups-alkylene-aryl and-substituted alkylene-aryl where alkylene, substituted alkylene and aryl are defined herein. Such alkaryl groups are exemplified by benzyl, phenethyl and the like.

The term"alkoxy"refers to the groups alkyl-O-, alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O-, where alkyl, alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein. Preferred alkoxy groups are alkyl-O-and include, by way of example, methoxy, ethoxy, rt-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1, 2-dimethylbutoxy, and the like.

The term"substituted alkoxy"refers to the groups substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O-where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein.

The term"alkylalkoxy"refers to the groups-alkylene-O-alkyl, alkylene-O-substituted alkyl, substituted alkylene-O-alkyl and substituted alkylene-O- substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted atkytene are as defined herein. Preferred alkylalkoxy groups are alkylene-O-alkyl and include, by way of example, methylenemethoxy (-CH2OCH3), ethylenemethoxy (-CH2CH2OCH3), n-propylene-iso-propoxy (-CH2CH2CH2OCH (CH3) 2), methylene-t-butoxy (-CH2-O-C (CH3) 3) and the like.

The term"alkylthioalkoxy"refers to the group-alkylene-S-alkyl, alkylene-S-substituted alkyl, substituted alkylene-S-alkyl and substituted alkylene-S- substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein. Preferred alkylthioalkoxy groups are alkylene-S-alkyl and include, by way of example, methylenethiomethoxy (-CH, SCH3), ethylenethiomethoxy(-CH, CH, SCH3), n-propylene-iso-thiopropoxy (-CH2CH, CH, SCH (CH3),), methylene-t-thiobutoxy (-CH, SC (CH,),) and the like.

The term"alkenyl"refers to a monoradical of a branched or unbranched unsaturated hydrocarbon group preferably having from 2 to 40 carbon atoms, more preferably 2 to 10 carbon atoms and even more preferably 2 to 6 carbon atoms and having at least I and preferably from 1-6 sites of vinyl unsaturation. Preferred alkenyl groups include ethenyl (-CH=CH2), n-propenyl (-CH, CH=CH,), iso-propenyl C (CH,) =CH2), and the like.

The term"substituted alkenyl"refers to an alkenyl group as defined above having from I to 5 substituents, and preferably 1 to 3 subst : tuents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl. cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro,-SO- alkyl,-SO-substituted alkyl,-SO-aryl,-SO-heteroaryl,-SO,-alkyl,-SO,-substituted alkyl,-SO,-aryl and-SO,-heteroaryl.

The term"alkenylene"refers to a diradical of a branched or unbranched unsaturated hydrocarbon group preferably having from 2 to 40 carbon atoms, more preferably 2 to 10 carbon atoms and even more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-6 sites of vinyl unsaturation. This term is exemplified by groups such as ethenylene (-CH=CH-), the propenylene isomers (e. g.

-CH, CH=CH- and-C (CH,) =CH-) and the like.

The term"substituted alkenylene"refers to an alkenylene group as defined above having from 1 to 5 substituents, and preferably from 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy,

thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro,-SO-alkyl,-SO-substituted alkyl,-SO-aryl,-SO-heteroaryl,-SO,- alkyl,-SO2-substituted alkyl,-SO2-aryl and-SO,-heteroaryl. Additionally, such substituted alkenylene groups include those where 2 substituents on the alkenylene group are fused to form one or more cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heterocyclic or heteroaryl groups fused to the alkenylene group.

The term"alkynyl"refers to a monoradical of an unsaturated hydrocarbon preferably having from 2 to 40 carbon atoms, more preferably 2 to 20 carbon atoms and even more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-6 sites of acetylene (triple bond) unsaturation. Preferred alkynyl groups include ethynyl (-C---Ch), propargyl (-CH2C-CH) and the like.

The term"substituted alkynyl"refers to an alkynyl group as defined above having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro,-SO- alkyl,-SO-substituted alkyl,-SO-aryl,-SO-heteroaryl,-SO,-alkyl,-SO,-substituted alkyl,-SO2-aryl and-SO,-heteroaryl.

The term"alkynylene"refers to a diradical of an unsaturated hydrocarbon preferably having from 2 to 40 carbon atoms, more preferably 2 to 10 carbon atoms and even more preferably 2 to 6 carbon atoms and having at least 1 and preferably from 1-6 sites of acetylene (triple bond) unsaturation. Preferred alkynylene groups include ethynylene (-C=-C-), propargylene (-CH, C=-C-) and the like.

The term"substituted alkynylene"refers to an alkynylene group as defined above having from I to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro,-SO- alkyl,-SO-substituted alkyl,-SO-aryl,-SO-heteroaryl,-SO,-alkyl,-SO,-substituted alkyl,-SO,-aryland-SO2-heteroaryl.

The term"acyl"refers to the groups HC (O)-, alkyl-C (O)-, substituted alkyl- C (O)-, cycloalkyl-C (O)-, substituted cycloalkyl-C (O)-, cycloalkenyl-C (O)-, substituted cycloalkenyl-C (O)-, aryl-C (O)-, heteroaryl-C (O)- and heterocyclic-C (O)- where alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl and heterocyclic are as defined herein.

The term"acylamino"or"aminocarbonyl"refers to the group-C (O) NRR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, heterocyclic or where both R groups are joined to form a heterocyclic group (e. g. morpholino) wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.

The term"aminoacyl"refers to the group-NRC (O) R where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.

The term"aminoacyloxy"or"alkoxycarbonylamino"refers to the group -NRC (O) OR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and

heterocyclic are as defined herein.

The term"acyloxy"refers to the groups alkyl-C (0) 0-, substituted alkyl- C (O) O-, cycloalkyl-C (0) 0-, substituted cycloalkyl-C (O) O-, aryl-C (O) O-, heteroaryl- C (O) O-, and heterocyclic-C (O) 0- wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclic are as defined herein.

The term"aryl"refers to an unsaturated aromatic carbocyclic group of from 6 to 20 carbon atoms having a single ring (e. g., phenyl) or multiple condensed (fused) rings (e. g., naphthyl or anthryl). Preferred aryls include phenyl, naphthyl and the like.

Unless otherwise constrained by the definition for the aryl substituent, such aryl groups can optionally be substituted with from 1 to 5 substituents, preferably 1 to 3 substituents, selected from the group consisting of acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy,-SO-alkyl,-SO-substituted alkyl,-SO- aryl,-SO-heteroaryl,-SO2-alkyl,-SO2-substituted alkyl,-SO,-aryl,-SO=-heteroaryl and trihalomethyl. Preferred aryl substituents include alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy.

The term"aryloxy"refers to the group aryl-O-wherein the aryl group is as defined above including optionally substituted aryl groups as also defined above.

The term"arylene"refers to the diradical derived from aryl (including substituted aryl) as defined above and is exemplified by 1,2-phenylene, 1,3- phenylene, 1,4-phenylene, 1,2-naphthylene and the like.

The term"amino"refers to the group-H2.

The term"substituted an. lino refers to the group-NRR where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl and heterocyclic provided that both R's are not hydrogen.

The term"carboxyalkyl"or"alkoxycarbonyl"refers to the groups "-C (O) O-alkyl","-C (O) O-substituted alkyl","-C (O) O-cycloalkyl","-C (O) O- substituted cycloalkyl","-C (O) O-alkenyl","-C (O) O-substituted alkenyl", "-C (O) O-alkynyl"and"-C (O) O-substituted alkynyl"where alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl and substituted alkynyl are as defined herein.

The term"cycloalkyl"refers to cyclic alkyl groups of from 3 to 20 carbon atoms having a single cyclic ring or multiple condensed rings. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.

The term"substituted cycloalkyl"refers to cycloalkyl groups having from 1 to 5 substituents, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro,-SO-alkyl,-SO- substituted alkyl,-SO-aryl,-SO-heteroaryl,-SO2-alkyl,-SO,-substituted alkyl,-SO,- aryl and-SO2-heteroaryl.

The term"cycloalkenyl"refers to cyclic alkenyl groups of from 4 to 20 carbon

atoms having a single cyclic ring and at least one point of internal unsaturation.

Examples of suitable cycloalkenyl groups include, for instance, cyclobut-2-enyl, cyclopent-3-enyl, cyclooct-3-enyl and the like.

The term"substituted cycloalkenyl"refers to cycloalkenyl groups having from I to 5 substituents, and preferably I to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thioi, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl. heteroaryioxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro,-SO- alkyl,-SO-substituted alkyl,-SO-aryl,-SO-heteroaryl,-SO2-alkyl,-SO-substituted< BR> alkyl,-SO2-aryl and-SO,-heteroaryl.

The term"halo"or"halogen"refers to fluoro, chloro, bromo and iodo.

The term"heteroaryl"refers to an aromatic group of from 1 to 15 carbon atoms and I to 4 heteroatoms selected from oxygen, nitrogen and sulfur within at least one ring (if there is more than one ring).

Unless otherwise constrained by the definition for the heteroaryl substituent, such heteroaryl groups can be optionally substituted with 1 to 5 substituents, preferably 1 to 3 substituents, selected from the group consisting of acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy,- SO-alkyl,-SO-substituted alkyl,-SO-aryl,-SO-heteroaryl,-SO2-alkyl,-SO,- substituted alkyl,-SO,-aryl,-SO,-heteroaryl and trihalomethyl. Preferred aryl

substituents include alkyl, alkoxy, halo, cyano, nitro, trihalomethyl, and thioalkoxy.

Such heteroaryl groups can have a single ring (e. g., pyridyl or furyl) or multiple condensed rings (e. g., indolizinyl or benzothienyl). Preferred heteroaryls include pyridyl, pyrrolyl and furyl.

The term"heteroaryloxy"refers to the group heteroaryl-O-.

The term"heteroarylene"refers to the diradical group derived from heteroaryl (including substituted heteroaryl), as defined above, and is exemplified by the groups 2,6-pyridylene, 2,4-pyridiylene, 1,2-quinolinylene, 1,8-quinolinylene, 1, 4- benzofuranylene, 2,5-pyridnylene, 2,5-indolenyl and the like.

The term"heterocycle"or"heterocyclic"refers to a monoradical saturated or unsaturated group having a single ring or multiple condensed rings, from 1 to 40 carbon atoms and from 1 to 10 hetero atoms, preferably I to 4 heteroatoms, selected from nitrogen, sulfur, phosphorus, and/or oxygen within the ring.

Unless otherwise constrained by the definition for the heterocyclic substituent, such heterocyclic groups can be optionally substituted with 1 to 5, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro,-SO-alkyl,-SO-substituted alkvl.

-SO-aryl,-SO-heteroaryl,-SO-alkyl,-SO,-substitutedalkyl,- SO,-aryl and-SO,- heteroaryl. Such heterocyclic groups can have a single ring or multiple condensed rings. Preferred heterocyclics include morpholino, piperidinyl, and the like.

Examples of nitrogen heterocycles and heteroaryls include, but are not limited to, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine,

indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, morpholino, piperidinyl, tetrahydrofuranyl, and the like as well as N-alkoxy-nitrogen containing heterocycles.

A preferred class of heterocyclics include"crown compounds"which refers to a specific class of heterocyclic compounds having one or more repeating units of the formula [- (CH,-) mY-] where m is equal to or greater than 2, and Y at each separate occurrence can be O, N, S or P. Examples of crown compounds include, by way of example only, [- 3-NH-] 3, [-((CH2) 2-0) 4-((CH2) 2-NH) J and the like. Typically such crown compounds can have from 4 to 10 heteroatoms and 8 to 40 carbon atoms.

The term"heterocyclooxy"refers to the group heterocyclic-O-.

The term"thioheterocyclooxy"refers to the group heterocyclic-S-.

The term"heterocyclene"refers to the diradical group formed from a heterocycle, as defined herein, and is exemplified by the groups 2. 6-morphoHno. 2. 5- morpholino and the like.

The term"oxyacylamino"or"aminocarbonyloxy"refers to the group -OC (O) NRR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.

The term"spiro-attached cycloalkyl group"refers to a cycloalkyl group attached to another ring via one carbon atom common to both rings.

The term"thiol"refers to the group-SH.

The term"thioalkoxy"refers to the group-S-alkyl.

The term"substituted thioalkoxy"refers to the group-S-substituted alkyl.

The term"thioaryloxy"refers to the group aryl-S-wherein the aryl group is as defined above including optionally substituted aryl groups also defined above.

The term"thioheteroaryloxy"refers to the group heteroaryl-S-wherein the heteroaryl group is as defined above including optionally substituted aryl groups as also defined above.

As to any of the above groups that contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible. In addition, the compounds of this invention include all stereochemical isomers arising from the substitution of these compounds, whether the isomers are those arising in the ligands, the linkers, or the multivalent constructs including the ligands and linkers.

The term"pharmaceutically-acceptable salt"refers to salts which retain the biological effectiveness and properties of the multi-binding compounds of this invention and which are not biologically or otherwise undesirable. In many cases, the multi-binding compounds of this invention are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.

Pharmaceutically-acceptable base addition salts can be prepared from inorganic and organic bases. Salts derived from inorganic bases, include by way of example only, sodium, potassium, lithium, ammonium, calcium and magnesium salts.

Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, such as alkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines, di (substituted alkyl) amines, tri (substituted alkyl) amines. alkenyl amines, alkenyl amines, trialkenyl amines, substituted alkenyl amines,

di (substituted alkenyl) amines, tri (substituted alkenyl) amines, cycloalkyl amines, di (cycloalkyl) amines, tri (cycloalkyl) amines, substituted cycloalkyl amines, disubstituted cycloalkyl amine, trisubstituted cycloalkyl amines, cycloalkenyl amines, di (cycloalkenyl) amines, tri (cycloalkenyl) amines, substituted cycloalkenyl amines, disubstituted cycloalkenyl amine, trisubstituted cycloalkenyl amines, aryl amines, diaryl amines, triaryl amines, heteroaryl amines, diheteroaryl amines, triheteroaryl amines, heterocyclic amines, diheterocyclic amines, triheterocyclic amines, mixed di- and tri-amines where at least two of the substituents on the amine are different and are selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic, and the like. Also included are amines where the two or three substituents, together with the amino nitrogen, form a heterocyclic or heteroaryl group.

Examples of suitable amines include, by way of example only, isopropylamine, trimethyl amine, diethyl amine, tri (iso-propyl) amine, tri (n-propyl) amine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine, purines, piperazine, piperidine, morpholine, N-ethylpiperidine, and the like. It should also be understood that other carboxylic acid derivatives would be useful in the practice of this invention, for example, carboxylic acid amides, including carboxamides, lower alkyl carboxamides. dialkyl carboxamides, and the like.

Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids. Salts derived from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Salts derived from organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid, salicylic acid, and the like.

The term"pharmaceutically-acceptable cation"refers to the cation of a pharmaceutically-acceptable salt.

The term"protecting group"or"blocking group"refers to any group which when bound to one or more hydroxyl, thiol, amino or carboxyl groups of the compounds (including intermediates thereof) prevents reactions from occurring at these groups and which protecting group can be removed by conventional chemical or enzymatic steps to reestablish the hydroxyl, thiol, amino or carboxyl group. The particular removable blocking group employed is not critical and preferred removable hydroxyl blocking groups include conventional substituents such as allyl, benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidine, phenacyl, t-butyl-diphenylsilyl and any other group that can be introduced chemically onto a hydroxyl functionality and later selectively removed either by chemical or enzymatic methods in mild conditions compatible with the nature of the product.

Preferred removable thiol blocking groups include disulfide groups, acyi groups, benzyl groups, and the like.

Preferred removable amino blocking groups include conventional substituents such as t-butyoxycarbonyl (t-BOC), benzyloxycarbonyl (CBZ), fluorenylmethoxycarbonyl (FMOC), allyloxycarbonyl (ALOC), and the like which can be removed by conventional conditions compatible with the nature of the product.

Preferred carboxyl protecting groups include esters such as methyl, ethyl, propyl, t-butyl etc. which can be removed by mild conditions compatible with the nature of the product.

The term"pharmaceutically-acceptable cation"refers to the cation of a pharmaceutically-acceptable salt.

The term"library"refers to at least 3, preferably from 10-to 109 and more preferably from 102 to 104 multimeric compounds. Preferably, these compounds are

prepared as a multiplicity of compounds in a single solution or reaction mixture which permits facile synthesis thereof. In one embodiment, the library of multimeric compounds can be directly assayed for multibinding properties. In another embodiment, each member of the library of multimeric compounds is first isolated and, optionally, characterized. This member is then assayed for multibinding properties.

The term"collection"refers to a set of multimeric compounds which are prepared either sequentially or concurrently (e. g., combinatorially). The collection comprises at least 2 members; preferably from 2 to 109 members and still more preferably from 10 to 104 members.

The term"multimeric compound"refers to compounds comprising from 2 to 10 ligands covalently connected through at least one linker which compounds may or may not possess multibinding properties (as defined herein).

The term"pseudohalide"refers to functional groups which react in displacement reactions in a manner similar to a halogen. Such functional groups include, by way of example, mesyl, tosyl, azido and cyano groups.

The term"optional"or"optionally"means that the subsequently described event, circumstance or substituent may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

The term"inert organic solvent"means a solvent which is inert under the conditions of the reaction being described in conjunction therewith including, by way of example only, benzene, toluene, acetonitrile, tetrahydrofuran, dimethylformamide, chloroform, methylene chloride, diethyl ether, ethyl acetate, acetone, methylethyl ketone, methanol, ethanol, propanol, isopropanol, t-butanol, dioxane, pyridine, and the like. Unless specified to the contrary, the solvents used in the reactions described herein are inert solvents.

The term"endothelin receptor"refers to a member of the family of guanine- nucleotide-binding regulatory (G)-protein-coupled receptors which is found in many tissues (e. g., lung, brain, CV system, placenta, lung, kidney, adrenal cortex, brain).

The interaction of endothelin peptides with the endothelin receptor results in effects on vascular and nonvascular smooth muscle, heart, nervous tissue, kidney and adrenal glands. The endothelin system (receptors and endothelins) are implicated in various pathological conditions involving vasospasm and vasoconstriction (e. g., congestive heart failure, pulmonary hypertension, cerebral vasospasm following subarachnoid hemorrhage, myocardial ischemia, restenosis, renal failure of ischemic origin, atherosclerosis, and others).

The term"ligand"or"endothelin ligand"as used herein denotes a compound that is a binding partner for the endothelin receptor and is bound thereto by complementarity. The specific region or regions of the ligand that is (are) recognized by the endothelin receptor is designated as the"ligand domain". A ligand may be either capable of binding to a receptor by itself, or may require the presence of one or more non-ligand components for binding (e. g., ions, a lipid molecule, a solvent molecule, a water molecule, or the like).

While it is contemplated that many endothelin receptor ligand antagonists that are currently known can be used in the preparation of multi-binding compounds of this invention, it should be understood that portions of the ligand structure that are not essential for specific molecular recognition and binding activity may be varied substantially, replaced with unrelated structures and, in some cases, omitted entirely without affecting the binding interaction. It should be further understood that the term "ligand"or"endothelin ligand"is not intended to be limited to compounds known to be useful as endothelin receptor-binding compounds (e. g., known drugs, such as those listed in Table 1). Those skilled in the art will understand that the term ligand can equally apply to a molecule that is not normally associated with endothelin cellular receptor binding properties. In addition, it should be noted that ligands that exhibit marginal activity or lack useful activity as monomers can be highly active as

multivalent compounds because of the benefits conferred by multi-valency. The primary requirement for a ligand as defined herein is that it has a ligand domain, as defined above, which is available for binding to a recognition site on an endothelin receptor.

Accordingly, examples of ligand useful for this invention include, but are not limited to, endothelin antagonists in preclinical and clinical trials (see Figure 4 and Tables 1 and 2). Endothelin agonists that act selectively on ETB receptor subtypes are also contemplated for use in the multi-binding compounds of this invention. Each of these ligands are being tested for their usefulness in the treatment of one of more of the following diseases or conditions : congestive heart failure, pulmonary hypertension, cerebral vasospasm following subarachnoid hemorrhage, myocardial ischemia, restenosis, renal failure of ischemic origin, atherosclerosis, and others.

The term"ligand"or"ligands"as used herein is intended to include the racemic forms of the ligands as well as individual stereoisomers of the ligands, including enantiomers and diastereomers and non-racemic mixtures thereof. Thus, the scope of the invention, as described and claimed, encompasses the racemic forms of the ligands as well as the individual stereoisomers and non-racemic mixtures thereof.

The term"ligand precursor"refers to a compound that is a starting material or an intermediate in the synthesis of a completed ligand. The ligand precursor may be coupled to a linker with completion of ligand synthesis being carried out in a separate step.

The term"analog"or"synthon"refers to a variation to one or more"R" groups on a ligand. In a multi-binding compound of the invention, the synthon may be coupled to another synthon, or to its counterpart ligand (see for example, Figures 7B and 8A-8D).

The term"ligand binding site"as used herein denotes the site on a receptor, such as an endothelin receptor, that recognizes a ligand domain and provides a

binding partner for that ligand. The ligand binding site may be defined by monomeric or multimeric structures. This interaction may be capable of producing a unique biological effect, for example agonism, antagonism, modulation, or may maintain an ongoing biological event, and the like.

It should be recognized that the ligand binding sites of endothelin receptors that participate in biological multivalent binding interactions are constrained to varying degrees by their intra-and intermolecular associations. For example, endothelin receptor ligand binding sites may be covalently joined in a single structure, noncovalently associated in one or more multimeric structures, embedded in a membrane or biopolymer matrix, and so on, and therefore have less translational and rotational freedom than if the same sites were present as monomers in solution.

A"multi-binding agenC"or"multi-binding compound"refers to a compound that is capable of multivalency as defined below, and which has 2 to 10 ligands, which may be the same or different, covalently bound to one or more linkers, which may be the same or different, wherein the ligands comprise a ligand domain capable of binding to one or more endothelin receptors. It may be preferable in some instances that the ligand domain is selective for one endothelin receptor, ETA vs. ET,, receptor, for example (i. e., to more effectively treat a particular disease mediated by only one endothelin receptor) and in other instances that the ligand domain is equally selective for both ETA and ETB receptors. The multi-binding compound provides a biological and/or therapeutic effect greater than the aggregate of unlinked monovalent ligands equivalent thereto. That is to say that the biological and/or therapeutic effect of the ligands attached to the multi-binding compound is greater than that achieved by the same number of unlinked ligands made available for binding to the ligand binding sites on the receptor or receptors.

The multi-binding compounds of this invention are capable of acting as multi- binding agents with surprisingly enhanced activity over their monovalent counterparts. Without intending to be bound by theory, the enhanced activity of these compounds is believed to arise at least in part from their ability to bind in a

multivalent manner with multiple ligand binding sites on one or more endothelin receptors, which gives rise to a more favorable net free energy of binding.

Multivalent binding interactions are characterized by the concurrent interaction of multiple ligands with multiple ligand binding sites on one or more endothelin receptors. Multivalent interactions differ from collections of individual monovalent interactions by imparting greater biological and/or therapeutic effect. Just as multivalent binding can amplify binding affinities, it can also amplify differences in binding affinities, resulting in enhanced binding specificity as well as affinity.

The phrase"greater biologic and/or therapeutic effect"or"increased biologic and/or therapeutic effect"includes for example increased ligand-receptor binding interactions (e. g., increased affinity for a target, increased ability to elicit a functional change in the target, improved kinetics), increased specificity for a target, increased selectivity for the target, increased potency, increased efficacy, decreased toxicity. decreased side effects, increased duration of action, improved bioavailability, improved pharmacokinetics, improved activity spectrum, increased therapeutic index, and the like. The multi-binding compounds of this invention will exhibit at least one. and preferably more than one, of the above-mentioned effects.

The term"potency"as used herein refers to the minimum concentration at which a ligand is able to achieve a desirable biological or therapeutic effect. The potency of a ligand is typically proportional to its affinity for its ligand binding site.

In some cases the potency may be non-linearly correlated with its affinity. In comparing the potency of two drugs, e. g., a multi-binding agent and the aggregate of its unlinked ligand, the dose-response curve of each is determined under identical test conditions (e. g. an in vitro or in vivo assay, in an appropriate animal model). The finding that the multi-binding agent produces an equivalent biological or therapeutic effect at a lower concentration than the aggregate unlinked ligand (e. g. on a per weight, per mole or per ligand basis) is indicative of enhanced potency.

The term"uni-valency"as used herein refers to a single binding interaction between one ligand as defined herein with one ligand binding site as defined herein.

It should be noted that a molecule having multiple copies of a ligand (or ligands) exhibits uni-valency when only one ligand is interacting with a ligand binding site.

Examples of a univalent interaction are depicted above.

The term"multivalency"as used herein refers to the concurrent binding of from 2 to 10 linked ligands (which may be the same or different) and two or more corresponding ligand binding sites on one or more receptors which receptors may be the same or different.

For example, two ligands connected by a linker that bind concurrently to two ligand binding sites would be considered as bi-valency; three ligands thus connected would be an example of tri-valency. An example of tri-valency illustrating a multi- binding agent bearing three ligands versus a monovalent binding interaction is shown below: univalent interaction

trivalent interaction It should be understood that all compounds that contain multiple copies of a ligand attached to a linker do not necessarily exhibit the phenomena of multi-valency, i. e., that the biological and/or therapeutic effect of the multi-binding agent is greater man the sum of the aggregate of unlinked ligands made available to the ligand binding site. For multivalency to occur, the ligands that are connected by a linker have to be presented to their receptors by the linker in a specific manner in order to bring about the desired ligand-orienting result, and thus produce a multi-binding agent.

The term"selectivity"or"specificity"is a measure of the binding preferences of a ligand for different ligand binding sites (receptors). The selectivity of a ligand with respect to its target ligand binding site relative to another ligand binding site is given by the ratio of the respective values of Kd (i. e., the dissociation constants for each ligand-receptor complex) or in cases where a biological effect is observed below the Kd, the ratio of the respective Ex,, s (i. e., the concentrations that produce 50% of the maximum response for the ligand interacting with the two distinct ligand binding sites (receptors)).

The terms"agonism"and"antagonism"are well known in the art. Ligands which are full agonists are ligands which when bound trigger the maximum activity seen by the natural ligands. Ligands which are partial agonists are ligands which when bound trigger sub-maximum activity. Ligands which are antagonists are ligands that when bound, inhibit or prevent the activity arising from a natural ligand binding to the receptor. Antagonists may be of the surmountable class (results in the parallel displacement of the dose-response curve of the agonist to the right in a dose dependent fashion without reducing the maximal response for the agonist) or insurmountable class (results in depression of the maximal response for a given agonist with or without the parallel shift). Ligands which are inverse agonists are ligands that, when bound, decrease the basal activity of the unbound receptor or which provide an activity opposite of the natural agonist.

Ligands have measurable properties that relate to the interaction of the ligand and the receptor. These include the affinity of the ligand for the receptor, which relates to the energetics of the binding, the efficacy of the ligand for the receptor, which relates to the functional downstream activity of the ligand, the kinetics of the ligand for the receptor, which defines the onset of action and the duration of action, and the desensitization of the receptor for the ligand. Selectivity defines the ratio of the affinity and/or efficacy of a ligand across two receptors. The term"modulatoy effect"refers to the ability of the ligand to change the activity of an agonist or antagonist through binding to a ligand binding site. It is a combination of these properties which provides the foundation for defining the nature of the functional response.

The term"treatment"refers to any treatment of a pathologic condition in a mammal, particularly a human, and includes: (i) preventing the pathologic condition from occurring in a subject which may be predisposed to the condition but has not yet been diagnosed with the condition and, accordingly, the treatment constitutes prophylactic treatment for the pathologic condition ; (ii) inhibiting the pathologic condition, i. e., arresting its development ; (iii) relieving the pathologic condition, i. e., causing regression of the disease or condition; or (iv) relieving the symptoms mediated by the pathologic condition..

The phrase"pathologic condition, which is modulated by treatment with a ligand"covers all disease states and/or conditions that are generally acknowledged in the art to be usefully treated with a ligand for an endothelin receptor in general, and those disease states that have been found to be usefully treated by a specific multi-

binding compound of the invention. Such disease states include, by way of example only, congestive heart failure, pulmonary hypertension, cerebral vasospasm following subarachnoid hemorrhage, essential hypertension, myocardial ischemia, unstable angina, restenosis, renal failure of ischemic origin, portal hypertension, cardiac hypertrophy, prophylaxis for atherosclerosis, pre-eclampsia, and migraine.

The term"therapeutically effective amount"refers to that amount of a multi- binding compound that is sufficient to effect treatment, as defined above, when administered to a mammal in need of such treatment. The therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.

The term"pharmaceutically acceptable excipient"is intended to include vehicles and carriers capable of being co-administered with a multi-binding compound to facilitate the performance of its intended function. The use of such media for pharmaceutically active substances is well known in the art. Examples of such vehicles and carriers include solutions, solvents, dispersion media, delay agents. emulsions and the like. Any other conventional carrier suitable for use with the multi- binding compounds also falls within the scope of the present invention.

The term"linker", identified where appropriate by the symbol"X", refers to a group or groups that covalently link (s) from 2 to 10 ligands (as identified herein) in a manner that provides for a compound capable of multi-valency. Among other features, the linker is a ligand-orienting entity that permits attachment of multiple copies of a ligand (which may be the same or different) thereto. In some cases the linker may be biologically active. The term linker does not, however, extend to cover solid inert supports such as beads, glass particles, fibers and the like. But it is to be understood that the multi-binding compounds of this invention

can be attached to a solid support if desired, for example, for use in separation and purification processes and foi similar applications.

The term"multimeric compound"refers to compounds comprising from 2 to 10 ligands covalently connected through at least one linker which compounds may or may not possess multi-binding properties (as defined herein).

The extent to which multivalent binding is realized depends upon the efficiency with which the linker or linkers that joins the ligands presents the joined ligands to the array of available ligand binding sites. Beyond presenting ligands for multivalent interactions with ligand binding sites, the linker (s) spatially constrains these interactions to occur within dimensions defined by the linker (s). Thus the structural features of the linker (valency, geometry, orientation, size, flexibility, chemical composition) are features of multi-binding compounds that play an important role in determining their activities.

The linkers used in this invention are selected to allow multivalent binding of ligands to any desired ligand binding sites of a endothelin receptor, whether such sites are located interiorly, both interiorly and on the periphery of the receptor, at the boundary region between the lipid bilayer and the receptor, or at any intermediate position thereof. The preferred linker length will vary depending on the distance between adjacent ligand binding sites, and the geometry, flexibility and composition of the linker. The length of the linker will preferably be in the range of about 2A to about I OOA, more preferably from about 2A to about 5 OA and even more preferably from about 7A to about 20A.

The ligands are covalently attached to the linker or linkers using conventional chemical techniques. The reaction chemistry resulting in such linkage are well known in the art and involve the use of complementary reactive functional groups (FG) on the linker and ligand. Preferably, the complementary reactive functional groups on the linker are selected relative to the functional groups

available on the ligand for binding or which can be introduced onto the ligand for binding. Again, such complementary reactive functional groups are well known in the art. For example, a reaction between a carboxylic acid of either the linker or the ligand and a primary or secondary amine of the ligand or the linker in the presence of suitable well-known activating agents results in formation of an amide bond covalently linking the ligand to the linker; reaction between an amine group of either the linker or the ligand and a sulfonyl halide of the ligand or the linker results in formation of a sulfonamide bond covalently linking the ligand to the linker; and reaction between an alcohol or phenol group of either the linker or the ligand and an alkyl or aryl halide of the ligand or the linker results in formation of an ether bond covalently linking the ligand to the linker.

Table 3 illustrates numerous complementary reactive groups and the resulting bonds formed by reaction there between. Where functional groups are lacking, they can be created by suitable chemistries that are described in standard organic chemistry texts, such as J. March'.

Table 3: Complementary Binding Chemistries First Reactive Second Reactive Linkage Group Group hydroxyl isocyanate urethane amine epoxide amine/alcohol tosyl halide amine sulfonamide carboxyl amine amide hydroxyl alkyl/aryl halide ether The linker is attached to the ligand at a position that retains ligand domain- receptor binding and specifically permits the ligand domain of the ligand to orient

itself to bind to the ligand binding site. Such positions and synthetic protocols for linkage are well known in the art. The term linker embraces everything that is not considered to be part of the ligand, e. g., ancillary groups such as solubilizing groups, lipophilic groups, groups that alter pharmacodynamics or pharmacokinetics, groups that modify the diffusability of the multi-binding compound, groups that attach the ligand to the linker, groups that aid the ligand- orienting function of the linker, for example, by imparting flexibility or rigidity to the linker as a whole, or to a portion thereof, and so on. Suitable linkers for the present invention are discussed below.

The relative orientation in which the ligand domains are displayed derives from the particular point or points of attachment of the ligand to the linker, and on the framework geometry. The determination of where acceptable substitutions can be made on a ligand is typically based on prior knowledge of structure-activity relationships (SAR) of the ligand and/or congeners and/or structural information about ligand-receptor complexes (e. g., X-ray crystallography, NMR, and the like).

Such positions and synthetic protocols for linkage are well known in the art.

Following attachment to the linker or a significant portion thereof (e. g. 2-10 atoms of linker), the linker-ligand conjugate is tested for retention of activity in a relevant assay system. For example, if a linker-ligand conjugate shows activity at a concentration of less than I mM, it is considered to be acceptable for use in constructing a multi-binding compound.

At present, it is preferred that the multi-binding agent is a bivalent compound, in which two ligands L are covalently linked, or a trivalent compound, in which three ligands are covalently linked. In some embodiments, the) ligands are linked through functional groups on the ligand, such that such functional groups are the linker moieties.

Methodology Linkers

The linker, when covalently attached to multiple copies of the ligand, provides a biocompatible, substantially non-immunogenic multi-binding compound of this invention. The biological activity of the multi-binding compound is highly sensitive to the valency, geometry, composition, size, flexibility or rigidity, etc. of the linker as well as the presence or absence of anionic or cationic charge, the relative hydrophobicity/hydrophilicity of the linker, and the like on the linker. Accordingly, the linker is preferably chosen to maximize the biological activity of the multi-binding compound. The linker may be biologically "neutral", i. e., not itself contribute any biological activity to the multi-binding compound or it may be chosen to enhance the biological activity of the molecule.

In general, the linker may be chosen from any organic molecule construct that orients two or more ligands to the receptors to permit multi-valency. In this regard. the linker can be considered as a"framework"on which the ligands are arranged in order to bring about the desired ligand-orienting result, and thus produce a multi- binding compound.

For example, different orientations can be achieved by including in the framework groups mono-or polycyclic groups, aryl and/or heteroaryl groups, or structures incorporating one or more carbon-carbon multiple bonds (alkenyl, alkenylene, alkynyl or alkynylene groups). The optimal geometry and composition of frameworks (linkers) used in the multi-binding compounds of this invention are based upon the properties of their intended receptors. For example, it may be preferred in some cases to use rigid cyclic groups (e. g., aryl, heteroaryl, etc.), or in other cases less-rigid cyclic groups (e. g., cycloalkyl, heterocyclyl or crown groups) to reduce conformational entropy when such may be necessary to achieve energetically coupled binding.

The presentation of different hydrophobic/hydrophilic characteristics of the linker as well as the presence or absence of charged moieties can readily be achieved and controlled by the skilled artisan. For example, the hydrophobic nature of a linker derived from hexamethylene diamine (H2N (CH2) 8NH,) or related polyamines can be modified to be substantially more hydrophilic by replacing the

alkylene group with a poly (oxyalkylene) group such as found in the commercially available"Jeffamines" (class of surfactants). By controlling the hydrophilicity/hydrophobicity, the ability of the compounds to cross the blood/brain barrier can be controlled. This can be important when one wishes to maximize or minimize CNS effects.

Different frameworks can be designed to provide preferred orientations of the ligands. The identification of an appropriate framework geometry for ligand domain presentation is an important first step in the construction of a multivalent binding agent with enhanced activity. Systematic spatial searching strategies can be used to aid in the identification of preferred frameworks through an iterative process. Figure 5 illustrates a useful strategy for determining an optimal framework display orientation for ligand domains and can be used for preparing the bivalent compounds of this invention. Various alternative strategies known to those skilled in the art of molecular design can be substituted for the one described here.

As shown in Figure 5, the ligands (shown as filled circles) are attached to a central core structure such as phenyldiacetylene (Panel A) or cyclohexane dicarboxylic acid (Panel B). The ligands are spaced apart from the core by an attaching moiety of variable lengths m and ii. If the ligand possesses multiple attachment sites (see discussion below), the orientation of the ligand on the attaching moiety may be varied as well. The positions of the display vectors around the central core structures are varied, thereby generating a collection of compounds. Assay of each of the individual compounds of a collection generated as described will lead to a subset of compounds with the desired enhanced activities (e. g., potency, selectivity). The analysis of this subset using a technique such as Ensemble Molecular Dynamics will suggest a framework orientation that favors the properties desired.

A wide variety of linkers is commercially available (see, e. g., Chem Sources USA and Chem Sources International; the ACD electronic database; and

Chemical Abstracts). Many of the linkers that are suitable for use in this invention fall into this category. Others can be readily synthesized by methods known in the art, and as described below. Examples of linkers include aliphatic moieties, aromatic moieties, steroidal moieties, peptides, and the like. Specific examples are peptides or polyamides, hydrocarbons, aromatics, heterocyclics, ethers, lipids, cationic or anionic groups, or a combination thereof. Figure 6A illustrates representative linker cores useful for the invention.

The process may require the use of multiple copies of the same central core structure or combinations of different types of display cores. It is to be noted that core structures other than those shown here can be used for determining the optimal framework display orientation of the ligands. The above-described technique can be extended to trivalent compounds and compounds of higher-order valency.

One skilled in the art would be able to identify bonding patterns that would produce multivalent compounds. Methods for producing these bonding arrangements are described in March'.

Examples of molecular structures in which the above bonding pattern could be employed as components of the linker are shown herein.

It can therefore be seen that there is a plethora of possibilities for the composition of a linker. Examples of linkers include aliphatic moieties, aromatic moieties, steroidal moieties, peptides, and the like. Specific examples are peptides or polyamides, hydrocarbons, aromatic groups, ethers, lipids, cationic or anionic groups, or a combination thereof.

Having selected a preferred framework geometry, the physical properties of the linker can be optimized by varying the chemical composition. The composition of a linker can be varied in numerous ways to achieve the desired physical properties.

Examples are given below, but it should be understood that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. For example, properties of the linker can be modified by the addition or insertion of ancillary groups into the linker, for example, to change the solubility of the multi-binding compound (in water, fats, lipids, biological fluids, etc.), hydrophobicity, hydrophilicity, linker flexibility, antigenicity, stability, and the like. For example, the introduction of one or more poly (ethylene glycol) (PEG) groups onto the linker enhances the hydrophilicity and water solubility of the multi-binding compound, increases both molecular weight and molecular size and, depending on the nature of the unPEGylated linker, may increase the in vivo retention time. Further PEG decreases antigenicity and potentially enhances the overall rigidity of the linker.

Ancillary groups that enhance the water solubility/hydrophilicity of the linker, and accordingly, the resulting multi-binding compounds, are useful in practicing this invention. Thus, it is within the scope of the present invention to use ancillary groups such as, for example, small repeating units of ethylene glycols, alcohols, polyols, (e. g., glycerin, glycerol propoxylate, saccharides, including mono-, oligosaccharides, etc.) carboxylates (e. g., small repeating units of glutamic acid, acrylic acid, etc.), amines (e. g., tetraethyienepentamine), and the like to enhance the water solubility and/or hydrophilicity of the multi-binding compounds of this invention. In preferred embodiments, the ancillary group used to improve water solubility/hydrophilicity will be a polyether. In particular, preferred embodiments, the ancillary group will contain a small number of repeating ethylene oxide (-CH, CH, O-) units.

The incorporation of lipophilic ancillary groups within the structure of the linker to enhance the lipophilicity and/or hydrophobicity of the multi-binding compounds described herein is within the scope of this invention. Lipophilic groups useful with the linkers of this invention include, but are not limited to, lower alkyl, aromatic groups and polycyclic aromatic groups. The aromatic groups may be either unsubstituted or substituted with other groups, but are at least substituted with a group that allows their covalent attachment to the linker. As used herein the term"aromatic groups"incorporates both aromatic hydrocarbons

and heterocyclic aromatics. Other lipophilic groups useful with the linkers of this invention include fatty acid derivatives, which may or may not form micelles in aqueous medium and other specific lipophilic groups, which modulate interactions between the multi-binding compound and biological membranes.

Also within the scope of this invention is the use of ancillary groups which result in the multi-binding compound of Formula I being incorporated into a vesicle, such as a liposome or a micelle. The term"lipid"refers to any fatty acid derivative that is capable of forming a bilayer or micelle, such that a hydrophobic portion of the lipid material orients toward the bilayer while a hydrophilic portion orients toward the aqueous phase. Hydrophilic characteristics derive from the presence of phosphato, carboxylic, sulfat, amino, sulfhydryl, nitro and other like groups well known in the art. Hydrophobicity could be conferred by the inclusion of groups that include, but are not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups of up to 20 carbon atoms and such groups substituted by one or more aryl, heteroaryl, cycloalkyl, and/or heterocyclic group (s). Preferred lipids are phosphoglycerides and sphingolipids, representative examples of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine. phosphatidylinositol, phosphatidic acid, palmitoyleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidyl-ethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoyl- phosphatidylcholine or dilinoleoylphosphatidylcholine. Other compounds lacking phosphorus, such as sphingolipid and glycosphingolipid families are also within the group designated as lipid. Additionally, the amphipathic lipids described above may be mixed with other lipids including triglycerides and sterols.

The flexibility of the linker can be manipulated by the inclusion of ancillary groups that are bulky and/or rigid. The presence of bulky or rigid groups can hinder free rotation about bonds in the linker or bonds between the linker and the ancillary group (s) or bonds between the linker and the functional groups. Rigid groups can include, for example, those groups whose conformational freedom is restrained by the presence of rings and/or multiple bonds, for example, aryl,

heteroaryl, cycloalkyl, cycloalkenyl and heterocyclic groups. Other groups, which can impart rigidity, include polypeptide groups such as oligo-or polyproline chains.

Rigidity may also be imparted by internal hydrogen bonding or by hydrophobic collapse.

Bulky groups can include, for example, large atoms, ions (e. g., iodine, sulfur, metal ions, etc.) or groups containing large atoms, polycyclic groups, including aromatic groups, non-aromatic groups and structures incorporating one or more carbon-carbon multiple bonds (i. e., alkenes and alkynes). Bulky groups can also include oligomers and polymers that are branched-or straight-chain species. Species that are branched are expected to increase the rigidity of the structure more per unit molecular weight gain than are straight-chain species.

Rigidity can also be imparted electrostatically. Thus, if the ancillary groups are either positively or negatively charged, the similarly charged ancillary groups will force the linker into a configuration affording the maximum distance between each of the like charges. The energetic cost of bringing the like-charged groups closer to each other will tend to hold the linker in a configuration that maintains the separation between the like-charged ancillary groups. Further ancillary groups bearing opposite charges will tend to be attracted to their oppositely charged counterparts and potentially may enter into both inter-and intramolecular ionic bonds. This non-covalent mechanism will tend to hold the linker into a conformation that allows bonding between the oppositely charged groups. The addition of ancillary groups which are charged, or alternatively, bear a latent charge which is unmasked, following the addition to the linker, by deprotection, a change in pH, oxidation, reduction or other mechanisms known to those skilled in the art, is within the scope of this invention.

In preferred embodiments, rigidity (entropic control) is imparted by the presence of alicyclic (e. g., cycloalkyl), aromatic and heterocyclic groups. In other preferred embodiments, the linker comprises one or more six-membered rings. In still further preferred embodiments, the ring is an aryl group such as, for example, phenyl or naphthyl, or a macrocyclic ring such as, for example, a crown compound.

In view of the above, it is apparent that the appropriate selection of a linker group providing suitable orientation, entropy and physico-chemical properties is well within the skill of the art. Eliminating or reducing antigenicity of the multi- binding compounds described herein is also within the scope of this invention. In certain cases, the antigenicity of a multi-binding compound may be eliminated or reduced by use of groups such as, for example, poly (ethylene glycol).

As explained above, the multi-binding compounds described herein comprise 2-10 ligands of the same or different type attached covalently to a linker, wherein the linker links the ligands in such a manner that they are presented to the receptor for multivalent interactions with ligand binding sites thereon/therein. The linker spatially constrains these interactions to occur within dimensions defined by the linker. This and other factors increases the biological activity and/or therapeutic effect of the multi-binding compound as compared to the same number of ligands used in mono-binding form.

The multi-binding compounds of this invention are preferably represented by the empirical formula (L) p (X) q where L, X, p and q are as defined above. This is intended to include the several ways in which the ligands can be linked together in order to achieve the objective of multi-valency, and a more detailed explanation is provided below.

As noted previously, the linker may be considered as a framework to which ligands are attached. Thus, it should be recognized that the ligands can be

attached at any suitable position on this framework, for example, at the termini of a linear chain or at any intermediate position.

The simplest and most preferred multi-binding compound is a bivalent compound which can be represented as L-X-L, where each L is independently a ligand which may be the same or different and X is independently the linker.

Examples of such bivalent compounds are provided in Figure 5, where each shaded circle represents a ligand. A trivalent compound could also be represented in a linear fashion, i. e., as a sequence of repeated units L-X-L-X-L, in which L is a ligand and is the same or different at each occurrence, as is X. However, a trivalent compound (or trimer) can also be a multi-binding compound comprising three ligands attached to a central core, and thus represented as (L) X, where the linker X could include, for example, an aryl or cycloalkyl group. Tetravalent compounds can be represented as, for example, in a linear array, e. g., L-X-L-X-L-X-L, a branched array, e. g., (i. e., a branched construct analogous to the isomers of butane (n-butyl, iso-butyl. sec-butyl, and t-butyl), or in a tetrahedral array, e. g., where X and L are as defined herein. Alternatively, it could be represented as an aryl or cycloalkyl derivative as described above with four (4) ligands attached to the core linker.

The same considerations apply to higher multi-binding compounds of this invention containing 5-10 ligands. However, for multi-binding agents attached to a central linker such as aryl, cycloalkyl, or heterocyclyl group, or even a crown compound, there is a self-evident constraint that there must be sufficient attachment sites on the linker to accommodate the number of ligands present.

For example, a benzene ring could not directly accommodate more than 6 ligands, whereas a multi-ring linker (e. g., biphenyl) could accommodate a larger number of ligands.

Certain of the above described compounds may alternatively be represented as cyclic chains of the form:

and variants thereof, wherein the formula (L) p (X) q is also intended to be inclusive of a cyclic compound of formula (-L-X-) n, wherein n is 2-10.

All of the above variations are intended to be within the scope of the invention defined by the formula (L) p (X) q.

With the foregoing in mind, a preferred linker may be represented by either a covalent bond between respective ligands or the structure of Formula (V): X'-C (=O)- (R9 ; n-C (=O)-X (V) wherein: R9 is independently alkyl, substituted alkyl, alkylene, substituted alkylene, alkaryl, alkoxy, substituted alkoxy; alkylalkoxy, alkylthioalkoxy, alkenyl, alkenylene, alkynyl and alkynylene and/or substituted versions thereof, acyl, acylamino, aminoacyl, aminoacyloxy, acyloxy, aryl, optionally substituted aryl, aryloxy, arylene, amino, substituted amino, carboxyalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, optionally substituted heteroaryl, heteroaryloxy, heteroarylene, heterocyclyl, optionally substituted heterocyclyl, heterocyclooxy, thioheterocyclooxy, heterocyclene, oxyacylamino, spiro-attached cycloalkyl, thiol, thioalkoxy, substituted thioalkoxy, thioaryloxy, and thioheteroaryloxy. n is an integer from 1 to 20 X'and X2 are end groups that will react with either an amino, hydroxy, halo, alkyl, alkoxy, thiol, thioalkoxy containing groups on the ligands, or on precursors thereof, to form a linkage. Representative examples of the X'and rA'2 end groups include, but are not limited to hydrogen, hydroxy, alkoxy, halo, or haloalkyl, amino, substituted amino, SH, and SO,, for example, or a covalent bond to the ligand.

In another preferred embodiment, the linker X may be represented by either a covalent bond between respective ligands or the following formula (Vl) : -X'-Z- (Y-Z-Y"-Z-X'- (V !) in which: m is an integer of from 0 to 20; X'at each separate occurrence is-O-,-S-.-S (O)-,-S (O),-,-NR-,-N R R-, -C (O)-,-C (O) O-,-C (O) NH-,-C (S),-C (S) O-,-C (S) NH- or a covalent bond. where R and R at each separate occurrence are as defined below for R'and R" ; Z is at each separate occurrence selected from alkylene, substituted alkylene, alkylalkoxy, cycloalkylene, substituted cycloalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, cycloalkenylene, substituted alkenylene, arylene, substituted arylene, heteroarylene, heterocyclene. substituted heterocyclene, crown compounds, or a covalent bond; Y'and Y'at each separate occurrence are selected from:

-S-S-or a covalent bond; in which: n is 0, 1 or 2 ; and R'and R"at each separate occurrence are selected from hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl or heterocyclic.

Examples of representative linker cores, are illustrated in Figure 6. The examples of linker cores in Figure 6 are illustrative only, and it is not the intent of the inventors to be limited to any of those depicted therein. The linker cores useful for the invention are as defined above for Formulas (V) and (VI).

In view of the above description of the linker, it is understood that the term"linker"when used in combination with the term"multi-binding compound"includes both a covalently contiguous single linker (e. g., L-X-L) or multiple covalently non-contiguous linkers (L-X-L-X-L).

Ligands Any compound which is an antagonist of one or more of the endothelin receptors (e. g., ETA, ETB, and their respective subtypes) and which can be covalently linked to a linker, or to each other with appropriate functional groups, can be used as a ligand to prepare the compounds described herein. The antagonistic affects of the ligand at the receptor may be used to treat pathologic conditions mediated by the endothelin receptors. A number of endothelin ligand antagonists are know and are listed in Table 1, some of which are illustrated in Figure 4. Any of these antagonists may be used to prepare the multi-binding compounds of the present invention. Moreover, analogs of any of the aforementioned ligand antagonists may be used to prepare the multi-binding compounds of the present invention. Two analogs, Synthon A and Synthon B, are preferred examples of analogs for the invention.

Preferred ligands L of the present invention are independently selected from a compound of Formulas (II), (IIa), (III) and (IV) illustrated below: (a) Formula II : RA-S (O) -N (H)-RB II wherein: RA is a group selected from an aryl, optionally substituted aryl, heteroaryl, and optionally substituted heteroaryl; and RB is a group selected from a heteroaryl and an optionally substituted heteroaryl.

Preferably, RA is independently a five or six membered aromatic ring with a substitution in a meta or para position relative to the S (O) Z group ; and RB is independently a five or six membered aromatic ring optionally with a plurality of substitutions.

More preferably, when RA is substituted, the substituent is a group selected from alkyl, substituted alkyl, alkylene, substituted alkylene, alkoxy, substituted alkoxy, alkylalkoxy, alkylthioalkoxy, acyl, acylamino, aminoacyl, aminoacyloxy, acyloxy, aryl, optionally substituted aryl, aryloxy, amino, substituted amino, carboxyalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, halo, heteroaryl, optionally substituted heteroaryl, heteroaryloxy, heteroarylene, heterocyclyl, substituted heterocyclyl, heterocyclooxy, thioheterocyclooxy, heterocyclene, oxyacylamino, thiol, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, a covalent bond to a linker or another ligand, a functional group FG for providing a covalent bond to a linker or another ligand, or the substituent includes a functional group FG for providing a covalent bond to a linker or another ligand.

Also more preferably, when RB is substituted, the substituent groups may be independently one or more of amino, substituted amino; alkyl, alkoxy, and alkylene, and the substituted versions thereof ; aryl and heteroaryl and the optionally substituted aryl or heteroaryl ; aryloxy, acyl, acylamino, aminoacyl, aminoacyloxy, acyloxy, heterocyclyl, optionally substituted heterocyclyl, carboxyalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, a covalent bond to a linker to another ligand, a functional group FG for providing a covalent bond to a linker or another ligand, or the substituent includes a functional group for providing a covalent bond to a linker or another ligand.

In either RA or RB above, FG is selected from halo, oxy, hydroxy, amino, substituted amino, thiol, acyl and carboxy group, or any combination thereof, for

reacting with a complementary group on the linker or another ligand to form a covalent bond. Preferably, FG is an amino, thiol or hydroxy group.

In a most preferred embodiment, RA is one of the following groups: (a) (b) (c) (d) and R'A is an alkyl or substituted alkyl, or a substituted aryl, acyl, acyloxy, preferably as illustrated below: or a covalent bond to a linker X or another ligand L, a functional group FG, as defined above, or the substituent on the alkyl or aryl, acyl or acyloxy includes a functional group FG to provide a covalent bond with a linker or another ligand.

Further to the most preferred embodiment, RB is one of the following groups: (a) (b) (c) wherein:

R'B, R"and R"are independently halo, hydrogen, cycloalkyl, cycloalkenyl, heterocyclyl, heteroaryl, aryloxy, alkaryl, alkoxy, alkylalkoxy, acyloxy, acylamino, amino, aminoacyl, or an optionally substituted version of applicable ones of the above, a covalent bond to a linker X or another ligand L, a functional group FG, as defined above, for providing a covalent bond to a linker X or another ligand L, or the hereinabove substituent includes a functional group FG for providing a covalent bond to a linker X or another ligand L.

Preferably, R'B, R2B and R'B are selected from hydrogen, halo, heterocyclyl, heteroaryl, alkyl, hydroxy, alkoxy, acyloxy, alkaryl, aryloxy, alkylalkoxy, or substituted versions of applicable ones above.

In another preferred embodiment thereof, the ligand L has the structure of Formula IIa below: (b) Formula IIa : IIa wherein: R'is a substituent at any position of an optionally substituted aryl or heteroaryl group (represented herein by a benzene ring), preferably a position ssletcz orpara to the sulfonamide group, and R'is selected from H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, halo, cycloalkyl, alkylthio, aralkyl, substituted aralkyl, a covalent bond attaching the ligand to a linker, or a group of formula R"- FG, where

R'a is a lipophilic group, preferably selected from a lower alkyl, aromatic or fatty acid derivative group, and FG is an functional group for the covalent attachment of the ligand to a linker or another ligand, as defined above; R2 is a group represented by the formula W-R2a or W-R2a-Q-FG, where W is O, S or NH, R2a is alkyl, substituted alkyl, alkylene, substituted alkylene, alkoxy, substituted alkoxy, all as defined herein, and Q is a heterocyclyl group (preferably pyridine, pyran, furan or morpholine) or a substituted alkylene interrupted by a H-bond acceptor, preferably O or N, and FG is defined as above; R3 is a group selected from H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, alkoxyalkyl, substituted alkoxyalkyl, amino, halo, thiol, substituted amino, heteroaryl, substituted heteroaryl, or a group of formula R3a-FG, where R3a is a group selected from H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, alkoxyalkyl, substituted alkoxyalkyl, amino, halo, thiol, substituted amino, heteroaryl, substituted heteroaryl, and FG is as defined above; and R4 is substituted aryl, preferably aryloxy-, thioaryloxy, aralkenyl, or aryl- CO-, most preferably alkoxy-phenyloxy.

In still another preferred embodiment, the ligand L has the structure of Formula III as described below: (c) Formula III: (III)

wherein: one of R'or R'is a functional group FG for the covalent attachment of the ligand to a linker, and the other of R'or R'is H or an optional substituent as defined herein for aryl, preferably OH, alkoxy, halogen, O, amino, substituted amino,-NH-C (O)-CH3,-(CH2) nCOOH,-(CH2) nCOOR,- (CH2) nCOO (CH2) nAr, NRCOOH,-NRCOOR,-NRCOO (CH,) nAr, where R is H or an alkyl ; Ar is a symbol representing an aryl or heteroaryl group ; n is an integer from 1 to 10 ; and FG is as defined above. Preferably, FG is an amino. thiol, or hydroxy group; R'and R'are independently aryl or heteroaryl, and preferably a substituted aryl or heteroaryl. More preferably, R'and R3 are independently either a group of the formula IIIa : (Illa) wherein: A is (CH,) n, or substituted (CH,) n"and m is an integer from 1 3, B is O or-CH,-, and preferably O, and R"is an optional substituent at any position of the optionally substituted aryl or heteroaryl group and is selected from alkoxy or substituted alkoxy, any of the substituents defined above for optionally substituted aryl or heteroaryl, or a covalent bond attaching the ligand to a linker X or another ligand, or a functional group FG, as defined above. Moreover, any of the substituents defined hereinabove may contain a functional group FG for attaching the ligand to a linker X or another ligand L; or a group of Formula IIIb :

wherein R"is as defined above; and Me is methyl, other alkyl, or alkylene, alkenyl, alkynyl, alkenylene, alkynylene, or substituted versions thereof.

In still another preferred embodiment, the ligand L has the structure of Formula IV, described below: (d) Formula IV: (IV) wherein: RC, R°, RE and RH are independently a group selected from alkyl, substituted alkyl, alkylene, substituted alkylene, alkaryl, alkoxy, substituted alkoxy; alkylalkoxy, alkylthioalkoxy, alkenyl, alkenylene, alkynyl and alkynylene and/or substituted versions thereof, acyl, acylamino, aminoacyl, aminoacyloxy, acyloxy, aryl, optionally substituted aryl, aryloxy, arylene, amino, substituted amino, carboxyalkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, optionally substituted heteroaryl, heteroaryloxy, heteroarylene, heterocyclyl, optionally substituted heterocyclyl, heterocyclooxy, thioheterocyclooxy, heterocyclene, oxyacylamino, spiro-attached cycloalkyl, thiol, thioalkoxy, substituted thioalkoxy, thioaryloxy, and thioheteroaryloxy, wherein any of the above may optionally contain an attaching group FG for attaching to a linker or another ligand ; or halo, hydrogen, a covalent bond to a linker or another ligand, or an attaching group FG for attaching to a linker or another ligand ; and RF and R° are independently a group selected from aryl, heteroaryl, heterocyclyl, cycloalkyl, or optionally substituted versions thereof, wherein any of the above may optionally contain an attaching group FG for attaching to a linker or another ligand ; or halo, hydrogen, a covalent bond to a linker or another ligand, or an attaching group FG for attaching to a linker or another ligand, wherein FG is as defined above.

In a preferred embodiment of ligands having the structure of Formula IV, RF and RG are independently an aryl, optionally substituted aryl, heteroaryl or optionally substituted heteroaryl. In a more preferred embodiment, ligands L of Formula IV are modeled after the structural formulae for ligand LU-135252. illustrated in Figure 4.

In another preferred embodiment, each ligand L is independently selected from a compound of Formula 11/IIa, or III/IIIa defined above, which is modeled after the structural formulae for Bosentan and SB-209670, respectively. for example, also illustrated in Figure 4.

The following formulas show an example of an analog of SB 209670 (Formula III/IIIa), named"Synthon A", and an example of an analog of Bosentan (Formula II/IIa), named"Synthon B". The structures below are illustrative of the ligands that can be used to prepare the multi-binding compounds of the present invention.

SE 209670 Analog-Synthon A Bosentan Analog-Synthon B Preparation of Multi-binding Compounds The multi-binding compounds of this invention can be prepared from readily available starting materials using the following general methods and procedures. It will he appreciated that where typical or preferred process conditions (i. e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Further, unless otherwise specified, the reaction times and conditions are intended to be approximate. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.

Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. The choice of a suitable protecting group for a particular functional group as well as suitable conditions for protection and deprotection are well known in the art. For example, numerous protecting groups, and their introduction and removal, are described in T. W. Green'and references cited therein.

Any compound that inhibits a endothelin agonist ligand (e. g., ET-1, ET- 2, or ET-3) can be used as a ligand in this invention. As discussed in further detail below, numerous such endothelin inhibitors are known in the art and any of these known compounds or analogs thereof may be employed as ligands in this invention. Some of the known inhibitors are listed in Table 1 and are illustrated in Figure 4. Typically, such multi-binding compounds selected for use as a ligand will have at least one functional group, such as an amino, thiol, hydroxyl, halo or carboxyl group and the like, which allows the compound to be readily coupled to another ligand via a suitable linker. Compounds having such functionality are either known in the art or can be prepared by routine modification of known compounds using conventional reagents and procedures.

The ligand can be covalently attached to the linker through any available position on the ligand, provided that when the ligand is attached to the linker, the ligand retains its ability to inhibit endothelin agonists. Preferably, the linker is attached to a site on the ligand where structure-activity studies (SAR) show that a wide variety of substituents are tolerated without loss of receptor activity.

As was previously discussed, the linker or linkers can be attached to different positions on the ligand molecule to achieve different orientations of the ligand domains and thereby facilitate multivalency. By way of illustration, the positions that are potentially available for linking an arylsulfonamide ligand of formula (II) or (IIa) (e. g., Bosentan, where R'= 4-tert-butyl, R-=2-OH-ethylene. and R'= pyrimidin-1-yl ; or for linking an indane ligand of formula (III) (e. g SKB- 209670, where Rs is H, Rb is 2-propanate, R7 is 2-(1-oxy-2-acetic acid)-4-methoxn- benzyl, and R8 is piperonal) are indicated by arrows in the structures (1) and (2) shown below.

Based on known SAR data, presently preferred positions for linking compounds of Formula II, IIa or III are shown by way of illustration in the Reaction Schemes and Examples below.

A preferred embodiment of multi-binding compounds are bivalent compounds having the Formula I which can be represented as L-X-L or L-L, where L is a ligand of Formulas (II), (IIa), (III) or (IV) that is the same or different at each occurrence, and X is the linker. Accordingly, examples of bivalent compounds of Formula I may be prepared as described below, with reference to Figures 7A-7B, 8A-8D and 9-11. It should be noted, however, that the same techniques can be used to generate higher order multi-binding compounds, i. e., the compounds of the invention where p is 3-10. The substituent groups and linker components illustrated in Figures 7A-7B, 8A-8D and 9-11 have the same meanings as described above, unless otherwise specified.

The starting materials and procedure for preparing the ligands for this invention are well known in the art. The preparation of analogs Synthon A and Synthon B for use in the multi-binding compounds is described and illustrated in Example 1, figure 7A and Example 2, figure 7B, respectively. Intermediates 11-15 are created during the synthesis of Synthon A. Synthon B is synthesized from Bosentan by modifying the R2 group. In fact, the product of the synthesis of Synthon B from Bosentan in Example 2 advantageously includes a multi-binding compound of Formula (A) in accordance with one embodiment of the present invention. The compound of Formula (A) is further discussed below.

Figures 8A-8D illustrate representative homodimers and heterodimers in accordance with the preferred embodiment of the invention. Figure 8A exemplifies the multi-binding compound comprising a dimer of Formula (A), wherein L is Bosentan linked at R2 by a 2,6-diaminoacyl-pyridine. As mentioned above, the method of preparing this dimer of Formula (A) is described in Example 2 and illustrated in Figure 7B. Uniquely, this dimer (A) is formed during the preparation of Synthon B from Bosentan. Moreover, dimer (A) may be considered a homodimer of Bosentan or a heterodimer of Bosentan and Synthon B, linked via a covalent bond between the amino group of Synthon B and the hydroxy group of Bosentan both on respective R 2groups.

Figure 8B illustrates a multi-binding compound in accordance with another preferred embodiment of the invention comprising a homodimer of Formula (B), wherein each L is Synthon B covalently linked by a linker X of the formula (V).

The method of preparing this homodimer of Formula (B) is described in Example ß and is illustrated in Figure 9.

Figure 8C illustrates a multi-binding compound in accordance with still another preferred embodiment of the invention comprising a homodimer of Formula (C), wherein each L is known ligand SB-209670 covalently linked by a linker X of the formula (V) via R6. The method of preparing this homodimer of Formula (C) is described in Example 4 and is illustrated in Figure 10.

Figure 8D illustrates a multi-binding compound in accordance with still another preferred embodiment of the invention comprising a heterodimer of Formula (D), wherein each L is independently selected from Synthon A and Synthon B covalently linked by a linker X of the formula (V) via R2 on Synthon B

and via R6 on Synthon A. The method of preparing this heterodimer of Formula (D) is described in Example 5 and is illustrated in Figure 11.

The reaction schemes described below illustrate preferred linking strategies for linking several classes of ligands to linkers according to this invention. The specific ligands employed in the reaction schemes are for illustrative purposes and should not be construed to limit the scope of the invention. These strategies are intended to apply to any endothelin receptor ligand that includes, or can be functionalized with groups compatible with the chosen linker.

In some cases, it is preferred to link ligands directly. using the functionality already present in the monovalent ligand. In other cases, it is preferred to accomplish linking indirectly by first preparing an intermediate that is further reacted to form the multi-binding compounds of the invention. In some cases, it may be necessary to protect portions of the ligand that are not involved in linking reactions. Protecting groups for this purpose are well known to those skilled in the art. Various coupling reactions can be used, some of which are exemplified in the reaction schemes that follow. One skilled in the art will appreciate that synthetically equivalent coupling reactions can be substituted for those illustrated herein.

The linker to which the ligands or ligand precursors are attached comprises a"core"molecule having two or more functional groups with reactivity that is complementary to that of the functional groups on the ligand. The linkers described and illustrated in the Examples and those illustrated in Figures 6A-6B exemplify the diversity of"cores"that are useful for varying the linker size, shape. length, orientation, rigidity, acidity/basicity, hydrophobicityihydrophilicity, hydrogen bonding characteristics and number of ligands connected. This pictorial representation is intended only to illustrate the invention, and not to limit its scope to the structures shown. In the Figures and Examples that follow, a solid circle is used to generically represent a core molecule.

Reactions performed under standard amide coupling conditions are carried out in an inert polar solvent (e. g., DMF, DMA) in the presence of a hindered base (e. g., TEA, DIPEA) and standard amide coupling reagents (e. g., DPPA, PyBOP, HATU, DCC).

The present invention provides pharmaceutical compositions comprising a pharmaceutically acceptable excipient and a therapeutically effective amounts of one or more multi-binding compounds (or pharmaceutically acceptable salts thereof) comprising 2 to 10 ligands which may be the same or different and which are covalently attached to a linker or linkers, which may be the same or different.

Each of said ligands comprises a ligand domain capable of binding to an endothelin receptor of a cell mediating mammalian diseases or conditions, thereby modulating the diseases or conditions.

The pharmaceutical compositions comprise multi-binding compounds that are represented by Formula I defined above. Each ligand L comprises a ligand domain capable of binding to a endothelin receptor ; thereby inhibiting the action of endothelin agonists, such as ET-1, ET-2 and ET-3 at the endothelin receptors.

Such pharmaceutical compositions are useful for modulating diseases of the cardiovascular, renal and endocrine and nervous systems in mammals, which are modulated by endothelin receptors.

Preferably, the pharmaceutical compositions of this invention comprise ligands L independently having the structures of Formulas (II), (IIa), (III) or (IV), as defined above, and linkers X independently having the structures of Formulas (V) or (VI), where q is less than p or the linker is a covalent bond between respective ligands. More preferably, the ligands of the multi-binding compounds are selected from the group consisting of known endothelin ligand antagonists listed in Table 1 and analogs thereof. Most preferably, the ligands are selected from SB 20967032, SB-217242, Bosentan", Ro-48-5695, TBC- 11251, ZD-1611, and LU-135252, analogs thereof, and Synthon A and Synthon

B. Each of these ligands L have a ligand domain capable of selectively binding to the endothelin receptor.

The invention also provides a method of modulating the activity of an endothelin receptor in a biologic tissue, which method comprises contacting a tissue having an endothelin receptor with a multi-binding compound (or pharmaceutically acceptable salts thereof) under conditions sufficient to produce a change in the activity of the receptor in said tissue. The multi-binding compound used in this method comprises 2 to 10 ligands which may be the same or different and which are covalently attached to a linker or linkers, which may be the same or different, each of said ligands comprises a ligand domain capable of binding to an endothelin receptor.

The multi-binding compounds used in the method of modulating the activity of endothelin receptors in biologic tissue are represented by Formula I as defined above, wherein each ligand is covalently attached to the linker and each ligand comprises a ligand domain capable of binding to a endothelin receptor.

When the multi-binding compound binds to the endothelin receptor, it inhibits the action of endothelin agonists, such as ET-1, ET-2 and ET-3 at the endothelin receptors. Preventing or inhibiting the action of endothelin agonists at the endothelin receptors modulates the diseases and conditions mediated by such receptors. In particular, the method is useful for modulating diseases, such as congestive heart failure, pulmonary hypertension, cerebral vasospasm following subarachnoid hemorrhage, essential hypertension, myocardial infarction. myocardial ischemia, unstable angina, restenosis, renal failure ofischemic origin. portal hypertension, cardiac hypertrophy, atherosclerosis, eclampsia, cerebrovascular disease, vascular disease, migraines, and auto-immune diseases, such as Morbus Wegener and Morbus Raynaud, to name a few in mammals, which are mediated by endothelin receptors.

Preferably, the multi-binding compounds used in the method of modulating comprise ligands L having the structure of Formulas (II), (IIa), (III) or (IV) and linkers X having the structure of Formula (V) or (VI), as defined above. More preferably, the ligands L of the multi-binding compounds are selected from the group consisting of known ligand antagonists, such as those listed in Table 1, and analogs and the ligand precursors thereof. Most preferably, the ligands are selected from SB 209670, SB-217242, Bosentan, Ro- 48-5695, TBC-11251, ZD-1611, and LU-135252, analogs thereof, and Synthon A and Synthon B. Each of these ligands L have a ligand domain capable of selectively binding to the endothelin receptor.

This invention also provides a method of preparing the multi-binding compounds represented by Formula I, as defined above. The method of preparing in accordance with the invention comprises the steps of : (a) providing at leastp equivalents of a ligand L or precursors thereof and at least q equivalents of linker or linkers X; and (b) covalently attaching said ligands to said linkers to produce a multi- binding compound; or (b') covalently attaching said ligand precursors to said linkers and completing the synthesis of said ligands thereupon, thereby to produce a multi- binding compound. Preferably, the ligands L have the structure of Formula (II), (IIa), (III) or (IV) and the linkers X have the structure of Formula (V) or (VI), where q is less than p, or the linker is a covalent bond between respective ligands. Several ligands and multi-binding compounds have been prepared and the chemical structures of and reaction schemes for making them are described below in the Examples taken in conjunction with Figures 7A, 7B, 8A-8D, and 9-11.

The present invention also provides a method for treating a disease or condition in a mammal resulting from an activity of an endothelin receptor. The method of treating in accordance with the invention comprises administering to a mammal a therapeutically effective amount of a pharmaceutical composition

comprising a pharmaceutically acceptable excipient and one or more multi-binding compounds (or pharmaceutically acceptable salts thereof) comprising 2 to 10 ligands which may be the same or different and which are covalently attached to a linker or linkers, which may be the same or different, each of said ligands comprising a ligand domain capable of binding to an endothelin receptor of a cell mediating mammalian diseases or conditions.

Accordingly, the multi-binding compounds, or pharmaceutically acceptable salts thereof, for the method of treating are represented by Formula I, as defined above. The action of the multi-binding compound effectively inhibits the action of endothelin agonists, such as ET-1, ET-2, and ET-3 at the endothelin receptors and modulates the diseases and conditions resulting therefrom.

A preferred embodiment of the method of treating is the use of pharmaceutical compositions comprising ligands L having the structure of Formula (II), (IIa), (III) or (IV) and linkers X having the structure of Formula (V) or (VI), where q is less than p, or the linker is a covalent bond between respective ligands. Most preferably, the multi-binding compounds comprise ligands with ligand binding domains capable of selectively binding to the endothelin receptors in mammals.

The multi-binding compounds, pharmaceutical compositions and methods of treating and modulating in accordance with the invention target endothelin receptors, which mediate diseases or conditions associated with the cardiovascular, renal, endocrine and nervous systems in mammals. Conditions, such as congestive heart failure, pulmonary hypertension, cerebral vasospasm following subarachnoid hemorrhage, essential hypertension, myocardial infarction, myocardial ischemia, unstable angina, restenosis, renal failure of ischemic origin, portal hypertension, cardiac hypertrophy, atherosclerosis, eclampsia, cerebrovascular disease, vascular disease, migraines, and auto-immune diseases,

such as Morbus Wegener and Morbus Raynaud, to name a few, may be treatable with the novel multi-binding compounds of this invention.

Representative pharmaceutical formations are provided below, along with representative Assays for evaluating the effectiveness of the multi-binding compounds in treating the various pathological conditions mentioned above.

These Formulations, Assays or Examples are illustrative of the many other compounds, reaction schemes, formulations and tests that can be used in accordance with the invention. Therefore, it is not the intent of the inventors to be limited in the scope of this invention to these Formulations, Assays or Examples.

Combinatorial Libraries The methods described above lend themselves to combinatorial approaches for identifying multimeric compounds which possess multibinding properties.

Specifically, factors such as the proper juxtaposition of the individual ligands of a multibinding compound with respect to the relevant array of binding sites on a target or targets is important in optimizing the interaction of the multibinding compound with its target (s) and to maximize the biological advantage through multivalency. One approach is to identify a library of candidate multibinding compounds with properties spanning the multibinding parameters that are relevant for a particular target. These parameters include: (1) the identity of ligand (s), (2) the orientation of ligands, (3) the valency of the construct, (4) linker length, (5) linker geometry, (6) linker physical properties, and (7) linker chemical functional groups.

Libraries of multimeric compounds potentially possessing multibinding properties (i. e., candidate multibinding compounds) and comprising a multiplicity of such variables are prepared and these libraries are then evaluated via conventional assays corresponding to the ligand selected and the multibinding parameters desired. Considerations relevant to each of these variables are set forth

below : Selection of ligand (s) : A single ligand or set of ligands is (are) selected for incorporation into the libraries of candidate multibinding compounds which library is directed against a particular biological target or targets. The only requirement for the ligands chosen is that they are capable of interacting with the selected target (s). Thus, ligands may be known drugs, modified forms of known drugs, substructures of known drugs or substrates of modified forms of known drugs (which are competent to interact with the target), or other compounds. Ligands are preferably chosen based on known favorable properties that may be projected to be carried over to or amplified in multibinding forms. Favorable properties include demonstrated safety and efficacy in human patients, appropriate PK/ADME profiles, synthetic accessibility, and desirable physical properties such as solubility, log P, etc. However, it is crucial to note that ligands which display an unfavorable property from among the previous list may obtain a more favorable property through the process of multibinding compound formation ; i. e., ligands should not necessarily be excluded on such a basis. For example, a ligand that is not sufficiently potent at a particular target so as to be efficacious in a human patient may become highly potent and efficacious when presented in multibinding form. A ligand that is potent and efficacious but not of utility because of a non-mechanism-related toxic side effect may have increased therapeutic index (increased potency relative to toxicity) as a multibinding compound. Compounds that exhibit short in vivo half-lives may have extended half-lives as multibinding compounds. Physical properties of ligands that limit their usefulness (e. g. poor bioavailability due to low solubility, hydrophobicity, hydrophilicity) may be rationally modulated in multibinding forms, providing compounds with physical properties consistent with the desired utility.

Orientation: selection of ligand attachment points and linking chemistry Several points are chosen on each ligand at which to attach the ligand to the linker. The selected points on the ligand/linker for attachment are functionalized to contain complementary reactive functional groups. This permits probing the effects of presenting the ligands to their receptor (s) in multiple relative

orientations, an important multibinding design parameter. The only requirement for choosing attachment points is that attaching to at least one of these points does not abrogate activity of the ligand. Such points for attachment can be identified by structural information when available. For example, inspection of a co-crystal structure of a protease inhibitor bound to its target allows one to identify one or more sites where linker attachment will not preclude the enzyme: inhibitor interaction. Alternatively, evaluation of ligand/target binding by nuclear magnetic resonance will permit the identification of sites non-essential for ligand/target binding. See, for example, Fesik, et al., U. S. Patent No. 5,891,643. When such structural information is not available, utilization of structure-activity relationships (SAR) for ligands will suggest positions where substantial structural variations are and are not allowed. In the absence of both structural and SAR information, a library is merely selected with multiple points of attachment to allow presentation of the ligand in multiple distinct orientations. Subsequent evaluation of this library will indicate what positions are suitable for attachment.

It is important to emphasize that positions of attachment that do abrogate the activity of the monomeric ligand may also be advantageously included in candidate multibinding compounds in the library provided that such compounds bear at least one ligand attached in a manner which does not abrogate intrinsic activity. This selection derives from, for example, heterobivalent interactions within the context of a single target molecule. For example, consider a receptor antagonist ligand bound to its target receptor, and then consider modifving this ligand by attaching to it a second copy of the same ligand with a linker which allows the second ligand to interact with the same receptor molecule at sites proximal to the antagonist binding site, which include elements of the receptor that are not part of the formal antagonist binding site and/or elements of the matrix surrounding the receptor such as the membrane. Here, the most favorable orientation for interaction of the second ligand molecule with the receptor/matrix may be achieved by attaching it to the linker at a position which abrogates activity of the ligand at the formal antagonist binding site. Another way to consider this is that the SAR of individual ligands within the context of a multibinding structure is often different from the SAR of those same ligands in momomeric form.

The foregoing discussion focused on bivalent interactions of dimeric compounds bearing two copies of the same ligand joined to a single linker through different attachment points, one of which may abrogate the binding/activity of the monomeric ligand. It should also be understood that bivalent advantage may also be attained with heterodimeric constructs bearing two different ligands that bind to common or different targets. For example, a 5HT4 receptor antagonist and a bladder-selective muscarinic M3 antagonist may be joined to a linker through attachment points which do not abrogate the binding affinity of the monomeric ligands for their respective receptor sites. The dimeric compound may achieve enhanced affinity for both receptors due to favorable interactions between the 5HT, ligand and elements of the M3 receptor proximal to the formal M, antagonist binding site and between the M, ligand and elements of the 5HT, receptor proximal to the formal 5HT4 antagonist binding site. Thus, the dimeric compound may be more potent and selective antagonist of overactive bladder and a superior therapy for urinary urge incontinence.

Once the ligand attachment points have been chosen, one identifies the types of chemical linkages that are possible at those points. The most preferred types of chemical linkages are those that are compatible with the overall structure of the ligand (or protected forms of the ligand) readily and generally formed, stable and intrinsically inocuous under typical chemical and physiological conditions, and compatible with a large number of available linkers. Amide bonds, ethers, amines. carbamates, ureas, and sulfonamides are but a few examples of preferred linkages.

Linkers: spanning relevant multibinding parameters through selection of valency, linker length, linker geometry, rigidity, physical properties, and chemical functional groups In the library of linkers employed to generate the library of candidate multibinding compounds, the selection of linkers employed in this library of linkers takes into consideration the following factors: Valency : In most instances the library of linkers is initiated with divalent linkers.

The choice of ligands and proper juxtaposition of two ligands relative to their binding sites permits such molecules to exhibit target binding affinities and

specificities more than sufficient to confer biological advantage. Furthermore, divalent linkers or constructs are also typically of modest size such that they retain the desirable biodistribution properties of small molecules.

Linker length : Linkers are chosen in a range of lengths to allow the spanning of a range of inter-ligand distances that encompass the distance preferable for a given divalent interaction. In some instances the preferred distance can be estimated rather precisely from high-resolution structural information of targets, typically enzymes and soluble receptor targets. In other instances where high-resolution structural information is not available (such as 7TM G-protein coupled receptors), one can make use of simple models to estimate the maximum distance between binding sites either on adjacent receptors or at different locations on the same receptor. In situations where two binding sites are present on the same target (or target subunit for multisubunit targets), preferred linker distances are 2-20, with more preferred linker distances of 3-12. In situations where two binding sites reside on separate (e. g., protein) target sites, preferred linker distances are 20-100, with more preferred distances of 30-70.

Linker geometry and rigidity : The combination of ligand attachment site, linker length, linker geometry, and linker rigidity determine the possible ways in which the ligands of candidate multibinding compounds may be displayed in three dimensions and thereby presented to their binding sites. Linker geometry and rigidity are nominally determined by chemical composition and bonding pattern, which may be controlled and are systematically varied as another spanning function in a multibinding array. For example, linker geometry is varied by attaching two ligands to the ortho, meta, and para positions of a benzene ring, or in cis-or trans- arrangements at the 1, 1- vs. 1,2- vs. 1,3- vs. 1,4- positions around a cyclohexane core or in cis-or trans-arrangements aL a point of ethylene unsaturation. Linker rigidity is varied by controlling the number and relative energies of different conformational states possible for the linker. For example, a divalent compound bearing two ligands joined by 1,8-octyl linker has many more degrees of freedom, and is therefore less rigid than a compound in which the two ligands are attached to

the 4,4' positions of a biphenyl linker.

Linker physical properties : The physical properties of linkers are nominally determined by the chemical constitution and bonding patterns of the linker, and linker physical properties impact the overall physical properties of the candidate multibinding compounds in which they are included. A range of linker compositions is typically selected to provide a range of physical properties (hydrophobicity, hydrophilicity, amphiphilicity, polarization, acidity, and basicity) in the candidate multibinding compounds. The particular choice of linker physical properties is made within the context of the physical properties of the ligands they join and preferably the goal is to generate molecules with favorable PK/ADME properties. For example, linkers can be selected to avoid those that are too hydrophilic or too hydrophobic to be readily absorbed and/or distributed in vivo.

Linker chemical functional groups : Linker chemical functional groups are selected to be compatible with the chemistry chosen to connect linkers to the ligands and to impart the range of physical properties sufficient to span initial examination of this parameter.

Combinatorial svnthesis : Having chosen a set of ii ligands (n being determined by the sum of the number of different attachment points for each ligand chosen) and jn linkers by the process outlined above, a library of (n !) m candidate divalent multibinding compounds is prepared which spans the relevant multibinding design parameters for a particular target. For example, an array generated from two ligands, one which has two attachment points (Al, A2) and one which has three attachment points (Bl, B2, B3) joined in all possible combinations provide for at least 15 r possible combinations of multibinding compounds: Al-Al A1-A2 Al-Bl Al-B2 A1-B3 A2-A2 A2-B1 A2-B2 A2-B3 Bl-B1 B1-B2 B1-B3 B2-B2 B2-B3 B3-B3 When each of these combinations is joined by 10 different linkers, a library

of 150 candidate multibinding compounds results.

Given the combinatorial nature of the library, common chemistries are preferably used to join the reactive functionalies on the ligands with complementary reactive functionalities on the linkers. The library therefore lends itself to efficient parallel synthetic methods. The combinatorial library can employ solid phase chemistries well known in the art wherein the ligand and/or linker is attached to a solid support. Alternatively and preferably, the combinatorial libary is prepared in the solution phase. After synthesis, candidate multibinding compounds are optionally purified before assaying for activity by, for example, chromatographic methods (e. g., HPLC).

Analysis of array by biochemical anatytical. pharmacofogicai, and computational methods: Various methods are used to characterize the properties and activities of the candidate multibinding compounds in the library to determine which compounds possess multibinding properties. Physical constants such as solubility under various solvent conditions and logD/clogD values can be determined. A combination of NMR spectroscopy and computational methods is used to determine low-energy conformations of the candidate multibinding compounds in fluid media. The ability of the members of the library to bind to the desired target and other targets is determined by various standard methods, which include radioligand displacement assays for receptor and ion channel targets, and kinetic inhibition analysis for many enzyme targets. In vitro efficacy, such as for receptor agonists and antagonists, ion channel blockers, and antimicrobial activity, can also be determined. Pharmacological data, including oral absorption, everted gut penetration, other pharmacokinetic parameters and efficacy data can be determined in appropriate models. In this way, key structure-activity relationships are obtained for multibinding design parameters which are then used to direct future work.

The members of the library which exhibit multibinding properties, as defined herein, can be readily determined by conventional methods. First those members which exhibit multibinding properties are identified by conventional methods as described above including conventional assays (both in vitro and in

vivo).

Second, ascertaining the structure of those compounds which exhibit multibinding properties can be accomplished via art recognized procedures. For example, each member of the library can be encrypted or tagged with appropriate information allowing determination of the structure of relevant members at a later time. See, for example, Dower, et al., International Patent Application Publication No. WO 93/06121 ; Brenner, et al., Proc. Natl. Acad. Sci., USA, 89 : 5181 (1992) ; Gallop, et al., U. S. Patent No. 5,846,839; each of which are incorporated herein by reference in its entirety. Alternatively, the structure of relevant multivalent compounds can also be determined from soluble and untagged libaries of candidate multivalent compounds by methods known in the art such as those described by Hindsgaul, et al., Canadian Patent Application No. 2,240,325 which was published on July 11,1998. Such methods couple frontal affinity chromatography with mass spectroscopy to determine both the structure and relative binding affinities of candidate multibinding compounds to receptors.

The process set forth above for dimeric candidate multibinding compounds can, of course, be extended to trimeric candidate compounds and higher analogs thereof.

Follow-up synthesis and analysis of additional arrav (s) : Based on the information obtained through analysis of the initial library, an optional component of the process is to ascertain one or more promising multibinding"lead"compounds as defined by particular relative ligand orientations, linker lengths, linker geometries, etc. Additional libraries can then be generated around these leads to provide for further information regarding structure to activity relationships. These arrays typically bear more focused variations in linker structure in an effort to further optimize target affinity ancblor actin, ity at the target (antagonism, partial agonism, etc.), and/or alter physical properties. By iterative redesign/analysis using the novel principles of multibinding design along with classical medicinal chemistry, biochemistry, and pharmacology approaches, one is able to prepare and identify optimal multibinding compounds that exhibit biological advantage towards their targets and as therapeutic agents.

To further elaborate upon this procedure, suitable divalent linkers include, by way of example only, those derived from dicarboxylic acids, disulfonylhalides, dialdehydes, diketones, dihalides, diisocyanates, diamines, diols, mixtures of carboxylic acids, sulfonylhalides, aldehydes, ketones, halides, isocyanates, amines and diols. In each case, the carboxylic acid, sulfonylhalide, aldehyde, ketone, halide, isocyanate, amine and diol functional group is reacted with a complementary functionality on the ligand to form a covalent linkage. Such complementary functionality is well known in the art as illustrated in the following table : COMPLEMENTARY BINDING CHEMISTRIES First Reactive Group Second Reactive Group Linkage hydroxyl isocyanate urethane amine epoxide-amine hydroxyamine sulfonyl halide sulfonamide carboxyl acid amine amide hydroxyl alkyl/aryl halide ether aldehyde amine/NaCNBH4 amine ketone aminelNaCNBH4 amine amine isocyanate urea Exemplary linkers include the following linkers identified as X-I through X-418 as set forth below:

Diacide HOO OH HO OH SH HO 0--S S-/--o 0 S S X-20 X-21 HO OU X-22 0 HO oh 0 0 OH OU X-24 o 0 HO X-25 OH OH p p- X25OH OH O 04 /\ S 0 °°) ChN OH X-27 0 0 HO0 0 HO 0 ou <9 0 0 OH HO OH 0 CH -l-2 ° X29 OH X-28X-30 OH 0 i HOSx~ fa OH v X-31 H p HO HOOH f O ; g, N OH , 0 thiol X-32 \ OU HO HO N HO 0 N OH OH H3 C 0 CH3 0 X-34 X-35 0 Chiral Chiral HO HO X-33 0 OH 0 p HO 0 HO F OH X-38 X-37 X-36OH 0 ou 0 0 0 0 S HO CH3 H3 0 zu 0 Ch irol H, 3 OH X-39 0 b X-40 X-41 H3 C CH3 0 0 OH 0 OH HO 0 0 HO o 0 CHj 0 zu ou X-44 HO0 HO 0 = CHj 0 OH CH3 OH HO l-p 0 0 s 0 OH X-45 X-46 Ch iral H3 C OH X-47X-48 oo HO 0 0CH3 OHX-49 F F HO 0 OU OH zon Choral X-51 0Ch irol F F X-52 X-50 H2NHN 0 0 HO s OU C' HOChiral HO Chiral X-53X-54 0 OH OH ou 0 04, OHCH3 H H : H C 0 0 0 3 OH X-57 Chiral X-58 X-56 00 \"-N OH HO Ho- HO 00 Chiral X-60 X-590 OH OH HOy : A I HO OH 0 Thiol X-61 X-62 C C C 0 H3C CH H3C ° ON ° 0 OH 0'y OH I CL HO S S 0 HO ( 0 X-63 Chiral if ! X-64 0 0 OH zozo HO HOHO X-66 X-67 NO 0 0 0 HOOH HO OH 0 \ l X-68 L S 0 N HO Ch irol 0 X-69 X-70 OH 0 0 HO HO OH 0 FFFFFFFF 0 / -7/-z ? -/j X-72 X-73 HO HO <O A ° OH HO} = O °OH HO 0 0 , N 0 -7 0 0 X-74 00 OHOH 0 0 0 H3 C HO HO X-77X-78 0 HOX CH3 OHC 0X-79 onto H CC Nv N I N''N 3 CH3 0 Y ClAo xChl8aÓ ° OH Chira O OH0H O 0 o ou zou II Hp 0 HO NOH H H Hp p 0 0 Chiral X-83 X-84 X-82 0 0 HO < +O X-85OH CH3 p CH3, y OH H : , 0 N,,,, N OH O 0 0 ß Chiral « OH OH HOirol HO O OH H X-87 X-88 0 II I 0 N 0 OH 0 H, 2z 1110 j, N, >' OH, H \ J D ( X-91 X-89 X-90 0 0 oh oh II H2 C N,,.. OH f.. p S S 'OH X-94 Cl3 oh k. ck. s" c Chiral X-92 0 O FF FF FF FF OU 0 0 OH HO HO OH i i i FF FF FF FF FF o 7 X-96 --lulu-- 0 OH ON OH OH Cul OH 0 HO chu CI 3 Ch irol B,- X_ Ch irol X-110X-112 oh oh 0 0 HO pH 0 H D ; 0 N,,, ° OH OH OH 0 X1 14 HO OH H OH X-113Chiral X-115 0r 0 -</v 0 0 N N, N oh Chiral 0 OH OH Ch ir l OH X-1 6 0 OH HO 0 sOH OH X-118X-120 X-119 0 0 OH 0 II N HO>~S4S40 HOXN f OH X-121o o X-122 ho 0 0 S-SOH OH HO 0 0 0 ? o 6 H2N X125 X/26 Ch irol X-12J OH0 OH Ho 0X-128 cN OH OH 0 S _, o Chiral 0 X-129 X-127 HO 0 o 0 H,,. I OH i I OH <H HO O HO HO --o X131 OH X-130X-132 Disulfon yl Halides 0== : : S S-C/ cri 0 X-134 p 0 p i S_F p i, Cl S S X-135 0 0 X-135 -7J _SO 0 S 0 C/J ' 0 F 110-51 0 0 0 X-136 0N So Q5 0 S. Cl X-136 -7J zunez x-lj9 X-140 o'c o X-141 F o=s =o C, C 0 c/ Hj CHj 0/C/ c f YT) CI N s c/x 0r-s s C/ v ir CI p pS 0 S, chu X-142 p F. i Cl o, F, S. p S IV F lV w zuy 0x-4s X-145 0 fT---f1 0 ! ! ! I//o 0 F C/ SCI S ,, 5 S S 0 -S 0 H C I CH I 0 C F I, , S 3 3 C/ CHj$ (90 X X-147X-149 X-150 0 I I ci. s S ci C/IS"0X-152 11 00 0//'u X-151 Dialdehydes, o \/ 0w 0 0 w 0 X-154 0 X-156 CH X-157 3 X-158 X-156 p X-155 0 0 CH3, o 0 1 0 0 X-157 CH3 X-158 0 o'' I o I c" r< 0. = 0 "o 0 J 0ó X-160 o CHj X-159 0 1 N 0 0 0 0 0 0 nu 1 0 0 X-1610X-162 X-164 o H3Coo 0 10 10 X-7bb OH o w X-166 X-165 0 X-167 -0 H30 l S < HO A X-168 s I X-169X-170 OH X-171 00 X-172 HO X-174 X-173 Dihalides Cl _/sJS ci-N, ClsX-176 X-175 BrBr Br OH OH X-178 X-179 X-180 Diisocyanates / N N X-215 0 X-216 0 0 111 N 0 \ o 0 N N //II /7jC-0 0-CH3 X218 0 X-217 FF 0--NF F N H3C l N 0 (i/0 0 N N X-219 X-220 0 X-221 0 0 zozo \\ Br CH3 0 N l l N CH3 X-222 H3C CH3 X-224 o x223, ° N N N N 0 N I i I/X-227 o X-226 CHHj i 0 N N / N p I I D X-225 X-229 X-228 Cl Cl3 N N/N/ N N-t- Cl) : X zu N CI Cl NI X-2Jl X-2J2 N 0 X-231 X-232 X-230 0 o 0 N 0 0 l /j V H C N ll ,. p/X-246 3 I I X-247 X-248 Diamines w NO pN k -2 X-249 N \ H2N NH2 N' N X-251 CH, 3 cl N H2N NzO X-252 X-250 CH3 CH3 CH3 N l l2/l NH2 H2N w X-25J H2N ur7j N Nu X-2542 X-256 NH H2N,-, I---N X-255 X-257 H2N NH 2 H2N NH2 » Z58 X259 H3CN NCH H2NONH2 3 X-261X-262 NU nu H2N NH X-265 X-263H2N X-264 0 H2N,,...., NH I I H2NNH2 X-304 X-303 CH3 OH Huns o X ! 0s H2N NH2 2 2 X-305 ., N CH3 X-307 Chircl X-306 NH2 NH2 H2N NH cl chu -C 3- X-308X-309 HAN NU2 X-ill H2NNH H2N NH2 X311 X-310 NH2 CH3 H2N H3/w Chu HjC 4-DNH ChirolX313 NH2 X-312X-314 H2N NH2 CH3 X-315H3C J\N/\N X-316 C/C/ C/C/ CL X-Jl7 \/C/\ (i X-317 NU Chiral \ CH3 JkJ8 w I _NH2 H2N H2N X-39 H ! H H2 3 3 X-320 NH 2 X-321 X-322 H3C oN v NsCH H3C sNz Nz CH3 H2N X O zNH2 X-323 X-324 X-325 -J27 - Diols H HO-- 3 HO Br X327 < HO-- 0 0 OH X-J27 Br Br Oh X-326 Sr' X-J26-, _,,-OH zOH 0 HON'AN OH X-328 X-329 N OH w Dithiols

Representative ligands for use in this invention include, by way of example, L-1 through L-_ as identified above.

Combinations of ligands (L) and linkers (X) per this invention include, by way example only, homo-and hetero-dimers wherein a first ligand is selected from L-1 through L-_ above and the second ligand and linker is selected from the following: L-I/X-1-L-l/X-2-L-I/X-3-L-l/X-4-L-l/X-5-L-l/X-6- L-I/X-7-L-I/X-8-L-I/X-9-L-I/X-10-L-I/X-11-L-l/X-12-<BR> ; L-l/X-13-L-l/X-14-L-1/X-15-L-l/X-16-L-l/X-17-L-l/X-18- L-1/X-19-L-1/X-20-L-1/X-21-L-1/X-22-L-1/X-23-L-1,X-24- L-I/X-25-L-I/X-26-L-I/X-27-L-l/X-28-L-l/X-29-L-I/X-30-<BR > L-1/X-31-L-l/X-32-L-I/X-33-L-l/X-34-L-I/X-35-L-1/X-36-<BR > L-l/X-37-L-l/X-38-L-l/X-39-L-l/X-40-L-l/X-41-L-l/X-42- L-1/X-43-L-1/X-44-L-1/X-45-L-1/X-46-L-1/X-47-L-1/X-48- L-1/X-49-L-1/X-50-L-1/X-51-L-1/X-52-L-1/X-53-L-1/X-54- L-l/X-55-L-l/X-56-L-l/X-57-L-l/X-58-L-l/X-59-L-l'X-60-<BR > L-l/X-61-L-l/X-62-L-l/X-63-L-l/X-64-L-l/X-65-L-liX-66-<BR > L-l/X-67-L-l/X-68-L-l/X-69-L-l/X-70-L-l/X-71-L-l/X-7'-<BR > L-l/X-73-L-l/X-74-L-l/X-75-L-l/X-76-L-l/X-77-L-ISX-7S-<BR > L-l/X-79-L-l/X-80-L-l/X-81-L-l/X-82-L-l/X-83-L-1'X-84-<BR > L-l/X-85-L-l/X-86-L-l/X-87-L-l/X-88-L-l/X-89-L-l : X-90- L-l/X-91-L-l/X-92-L-1/X-93-L-l/X-94-L-1/X-95-L-l/X-96- <BR> <BR> L-l/X-97-L-l/X-98-L-l/X-99-L-l/X-100-L-l/X-101-L-1 ! X-102-<BR> L-l/X-103-L-l/X-104-L-l/X-105-L-l/X-106-L-l/X-107-L-1 :'X-108-<BR> L-l/X-109-L-l/X-110-L-l/X-111-L-l/X-112-L-l/X-113-L-I. X-H4-<BR> L-l/X-115-L-I/X-116-L-I/X-117-L-I/X-118-L-1/X-119-L-1'C-120- L-1/X-121-L-1/X-122-L-1/X-123-L-I/X-124-L-1/X-125-L-1/X-126- <BR> <BR> L-l/X-127-L-l/X-128-L-l/X-129-L-l/X-130-L-l/X-131-L-l/X-132- <BR> L-l/X-133-L-l/X-134-L-l/X-135-L-l/X-136-L-l/X-137-L-l/X-138- L-l/X-139-L-l/X-140-L-l/X-141-L-l/X-142-L-l/X-143-L-l/X-144- <BR> <BR> L-l/X-145-L-l/X-146-L-l/X-147-L-l/X-148-L-l/X-149-L-I/X-150- <BR> L-l/X-151-L-l/X-152-L-l/X-153-L-l/X-154-L-l/X-155-L-l/X-156- <BR> L-l/X-157-L-l/X-158-L-l/X-159-L-l/X-160-L-l/X-161-L-l/X-162-

L-1/X-163-L-l/X-164-L-l/X-165-L-l/X-166-L-l/X-167-L-l/X-168- <BR> L-1n (-169-L-1SX-170-L-l/X-171-L-l/X-172-<BR> L-l/X-173-L-l/X-174-L-l/X-175-L-l/X-176-L-1/X-177-L-I/X-178- <BR> L-l/X-179-L-1/X-180-L-I/X-181-L-l/X-182-L-I/X-183-L-l/X-184- <BR> L-l/X-185-L-l/X-186-L-l/X-187-L-l/X-188-L-l/X-189-L-l/X-190- <BR> L-l/X-191-L-I/X-192-L-l/X-193-L-I/X-194-L-I/X-195-L-I/X-196- <BR> L-I/X-197-L-l/X-198-L-l/X-199-L-1/X-200-L-1/X-201-L-l/X-202- <BR> L-I/X-203-L-l/X-204-L-l/X-205-L-I/X-206-L-I/X-207-L-l/X-208- L-1/X-209-L-1/X-210-L-1/X-211-L-1/X-212-L-1/X-213-L-1/X-214- <BR> <BR> L-l/X-215-L-l/X-216-L-l/X-217-L-l/X-218-L-l/X-219-L-l/X-220- <BR> L-l/X-221-L-l/X-222-L-1/X-223-L-l/X-224-L-1/X-225-L-liX-226- <BR> L-l/X-227-L-l/X-228-L-I/X-229-L-l/X-230-L-l/X-231-L-l'X-232- <BR> L-l/X-233-L-I/X-234-L-I/X-235-L-I/X-236-L-I/X-237-L-1/X-238- <BR> L-l/X-239-L-l/X-240-L-l/X-241-L-l/X-242-L-l/X-243-L-liX-244- <BR> L-l/X-245-L-l/X-246-L-l/X-247-L-l/X-248-L-l/X-249-L-liX-950- <BR> L-1/X-251-L-I/X-252-L-l/X-253-L-l/X-254-L-l/X-255-L-l/X-256- L-1/X-257-L-1/X-258-L-1/X-259-L-1/X-260-L-1/X-261-L-1 X-262- L-1/X-263-L-1/X-264-L-1/X-265-L-1/X-266-L-1/X-267-L-1/X-268- L-1/X-269-L-1/X-270-L-1/X-271-L-1/X-272-L-1/X-273-L-1/X-274- L-l/X-275-L-1/X-276-L-l/X-277-L-I/X-278-L-I/X-279-L-I X-280-<BR> L-l/X-281-L-l/X-282-L-l/X-283-L-l/X-284-L-l/X-285-L-l/X-286- <BR> L-l/X-287-L-l/X-288-L-l/X-289-L-l/X-290-L-l/X-291-L-l/X-292- <BR> L-l/X-293-L-l/X-294-L-l/X-295-L-l/X-296-L-l/X-297-L-lX-298-& lt;BR> L-l/X-299-L-l/X-300-L-1/X-301-L-l/X-302-L-l/X-303-L-1 ! X-304-<BR> L-l/X-305-L-l/X-306-L-l/X-307-L-l/X-308-L-l/X-309-L-l ! X-310-<BR> L-l/X-311-L-l/X-312-L-l/X-313-L-l/X-314-L-I/X-315-L-I/X-316- L-1/X-317-L-1/X-318-L-1/X-319-L-1/X-320-L-1/X-321-L-1/X-322- L-l/X-323-L-l/X-324-L-l/X-325-L-l/X-326-L-l/X-327-L-l/X-328- <BR> L-l/X-329-L-l/X-330-L-l/X-331-L-l/X-332-L-l/X-333-L-l/X-334- L-1/X-335-L-1/X-336-L-1/X-337-L-1/X-338-L-1/X-339-L-1/X-340- L-l/X-341-L-l/X-342-L-I/X-343-L-l/X-344-L-I/X-345-L-I X-346-<BR> L-l/X-347-L-l/X-348-L-l/X-349-L-l/X-350-L-l/X-351-L-liX-35'- L-1/X-353-L-1/X-354-L1/X-355-L-1/X-356-L-1/X-357-L-1/X-358- L-l/X-359-L-l/X-360-L-l/X-361-L-l/X-362-L-l/X-363-L-l/X-364- <BR> L-1/X-365-L-1/X-366-L-1/X-367-L-l/X-368-L-l/X-369-L-l/X-370- <BR> L-1/X-371-L-l/X-372-L-l/X-373-L-l/X-374-L-l/X-375-L-l/X-376-

L-l/X-377-L-l/X-378-L-l/X-379-L-l/X-380-L-l/X-381-L-l/X-382- <BR> L-l/X-383-L-l/X-384-L-l/X-385-L-I/X-386-L-l/X-387-L-l/X-388- <BR> L-l/X-389-L-1/X-390-L-l/X-391-L-l/X-392-L-I/X-393-L-I/X-394- <BR> L-l/X-395-L-I/X-396-L-l/X-397-L-l/X-398-L-l/X-399-L-I/X-400- <BR> L-l/X-401-L-l/X-402-L-I/X-403-L-I/X-404-L-l/X-405-L-I/X-406- <BR> L-l/X-407-L-l/X-408-L-l/X-409-L-l/X-410-L-l/X-411-L-l/X-412- L-1/X-413-L-1/X-414-L-1/X-415-L-1/X-416-L-1/X-417-L-1/X-418- L-2/X-I-L-2/X-2-L-2/X-3-L-2/X-4-L-2/X-5-L-2 X-6-<BR> L-2/X-7-L-2/X-8-L-2/X-9-L-2/X-IO-L-2/X-H-L-2/X-I2-<BR> L-2/X-13-L-2/X-14-L-2/X-15-L-2/X-16-L-2/X-17-L-2?'X-I8-<B R> L-2/X-19-L-2/X-20-L-2/X-21-L-2/X-22-L-2/X-23-L-2?-X-24-<B R> L-2/X-25-L-2/X-26-L-2/X-27-L-2/X-28-L-2/X-29-L-2/X-30-<BR > L-2/X-31-L-2/X-32-L-2/X-33-L-2/X-34-L-2/X-35-L-21X-36-<BR > L-2/X-37-L-2/X-38-L-2/X-39-L-2/X-40-L-2/X-41-L-2/X-42-<BR > L-2/X-43-L-2/X-44-L-2/X-45-L-2/X-46-L-2/X-47-L-2/X-48-<BR > L-2/X-49-L-2/X-50-L-2/X-51-L-2/X-52-L-2/X-53-L-2/X-54-<BR > L-2/X-55-L-2/X-56-L-2/X-57-L-2/X-58-L-2/X-59-L-2/X-60- L-2/X-61-L-2/X-62-L-2/X-63-L-2/X-64-L-2/X-65-L-2/X-66- L-2/X-67-L-2/X-68-L-2/X-69-L-2/X-70-L-2/X-71-L-'X-7'-<BR& gt; L-2/X-73-L-2/X-74-L-2/X-75-L-2/X-76-L-2, X-77-L-'Y-7S-<BR> L-2/X-79-L-2/X-80-L-2/X-81-L-2/X-82-L-2/X-83-L-2X-84-<BR& gt; L-2/X-85-L-2/X-86-L-2/X-87-L-2/X-88-L-2/X-89-L-2 X-90-<BR> L-2/X-91-L-2/X-92-L-2/X-93-L-2/X-94-L-2/X-95-L-2/X-96-<BR > L-2/X-97-L-2/X-98-L-2/X-99-L-2/X-100-L-2/X-101-L-'X-102-< BR> L-2/X-103-L-2/X-104-L-2/X-105-L-2/X-106-L-2/X-107-L-2/X-108- L-2/X-109-L-2/X-110-L-2/X-111-L-2/X-112-L-2/X-113-L-2/X-114- L-2/X-115-L-2/X-116-L-2/X-117-L-2 X-118-L-2/X-119-L-2/X-120-<BR> L-2/X-121-L-2/X-122-L-2/X-123-L-2/X-124-L-2/X-125-L-2-X-1 ? 6- L-2/X-127-L-2/X-128-L-2/X-129-L-2/X-130-L-2/X-131-L-2/X-132- L-2/X-133-L-2/X-134-L-2/X-135-L-2/X-136-L-2/X-137-L- ? ; X-138-<BR> L-2/X-139-L-2/X-140-L-2/X-141-L-2/X-142-L-2/X-143-L-2/X-144- <BR> L-2/X-145-L-2/X-146-L-2/X-147-L-2/X-148-L-2/X-149-L-2/X-150- <BR> L-2/X-151-L-2/X-152-L-2/X-153-L-2/X-154-L-2/X-155-L-2 X-156-<BR> L-2/X-157-L-2/X-158-L-2/X-159-L-2/X-160-L-2/X-161-L-2/X-162-

L-2/X-163-L-2/X-164-L-2/X-165-L-2/X-166-L-2/X-167-L-2/X-168- L-2/X-169-L-2/X-170-L-2/X-171-L-2/X-172- <BR> <BR> L-2/X-173-L-2/X-174-L-2/X-175-L-2/X-176-L-2/X-177-L-2/X-178- <BR> L-2/X-179-L-2/X-180-L-2/X-181-L-2/X-182-L-2/X-183-L-2/X-184- <BR> L-2/X-185-L-2/X-186-L-2/X-187-L-2/X-188-L-2/X-189-L-2/X-190- L-2/X-191-L-2/X-192-L-2/X-193-L-2/X-194-L-2/X-195-L-2/X-196- <BR> <BR> L-2/X-197-L-2/X-198-L-2/X-199-L-2/X-200-L-2/X-201-L-2/X-202- L-2/X-203-L-2/X-204-L-2/X-205-L-2/X-206-L-2/X-207-L-2/X-208- <BR> <BR> L-2/X-209-L-2/X-210-L-2/X-211-L-2/X-212-L-2/X-213-L-2/X-214- <BR> L-2/X-215-L-2/X-216-L-2/X-217-L-2/X-218-L-2/X-219-L-2'X-2'0- <BR> L-2/X-221-L-2/X-222-L-2/X-223-L-2/X-224-L-2/X-225-L-2/X-226- <BR> L-2/X-227-L-2/X-228-L-2/X-229-L-2/X-230-L-2/X-231-L-'X-'3'-& lt;BR> L-2/X-233-L-2/X-234-L-2/X-235-L-2/X-236-L-2/X-237-L-2 X-238-<BR> L-2/X-239-L-2/X-240-L-2/X-241-L-2/X-242-L-2/X-243-L-'X-244-& lt;BR> L-2/X-245-L-2/X-246-L-2/X-247-L-2/X-248-L-2/X-249-L-2/X-250- <BR> L-2/X-251-L-2/X-252-L-2/X-253-L-2/X-254-L-2/X-255-L-2/X-256- <BR> L-2/X-257-L-2/X-258-L-2/X-259-L-2/X-260-L-2/X-26I-L-2/X-262- <BR> L-21X-263-L-21X-264-L-2/X-265-L-2/X-266-L-2/X-267-L-'X-'68-& lt;BR> L-2/X-269-L-2/X-270-L-2/X-271-L-2/X-272-L-2/X-273-L-2'X-274- <BR> L-2/X-275-L-2/X-276-L-2/X-277-L-2/X-278-L-2/X-279-L-2/X-280- <BR> L-2EX-281-L-2/X-282-L-21X-283-L-21X-284-L-2/X-285-L-2/X-286- <BR> L-2/X-287-L-2/X-288-L-2/X-289-L-2/X-290-L-2/X-291-L-2'X-'92- <BR> L-2/X-293-L-2/X-294-L-2/X-295-L-2/X-296-L-2/X-297-L-2X-298-& lt;BR> L-2/X-299-L-2/X-300-L-2/X-301-L-2/X-302-L-2/X-303-L-2,X-304- L-2/X-305-L-2/X-306-L-2/X-307-L-2/X-308-L-2/X-309-L-2/X-310- <BR> <BR> L-2/X-311-L-2/X-312-L-2/X-313-L-2/X-314-L-2/X-315-L-'X-316- L-2/X-317-L-2/X-318-L-2/X-319-L-2/X-320-L-2/X-321-L-2/X-322- <BR> <BR> L-2/X-323-L-2/X-324-L-2/X-325-L-2/X-326-L-2/X-327-L-2/X-328- <BR> L-2/X-329-L-2/X-330-L-2/X-331-L-2/X-332-L-2/X-333-L-2/X-334- <BR> L-2/X-335-L-2/X-336-L-2/X-337-L-2/X-338-L-2/X-339-L-2 : C-340-<BR> L-2/X-341-L-2/X-342-L-2/X-343-L-2/X-344-L-2/X-345-L-'/X-346- <BR> L-2/X-347-L-2/X-348-L-2/X-349-L-2/X-350-L-2/X-351-L-2% X-352-<BR> L-2/X-353-L-2/X-354-L-2/X-355-L-2/X-356-L-2/X-357-L-2/X-358- <BR> L-2/X-359-L-2/X-360-L-2/X-361-L-2/X-362-L-2/X-363-L-2/X-364- L-2/X-365-L-2/X-366-L-2/X-367-L-2/X-368-L-2/X-369-L-2/X-370- <BR> <BR> L-2/X-371-L-2/X-372-L-2/X-373-L-2/X-374-L-2/X-375-L-2 X-376-

L-2/X-377-L-2/X-378-L-2/X-379-L-2/X-380-L-2/X-381-L-2/X-382- <BR> L-2/X-383-L-2/X-384-L-2/X-385-L-2/X-386-L-2/X-387-L-2/X-388- L-2/X-389-L-2/X-390-L-2/X-391-L-2/X-392-L-2/X-393-L-2/X-394- <BR> <BR> L-2/X-395-L-2/X-396-L-2/X-397-L-2/X-398-L-2/X-399-L-2/X-400- <BR> L-2/X-401-L-2/X-402-L-2/X-403-L-2/X-404-L-2/X-405-L-2/X-406- <BR> L-2/X-407-L-2/X-408-L-2/X-409-L-2/X-4 10-L-2/X-41 I-L-2/X-412-<BR> L-2/X-413-L-2/X-414-L-2/X-415-L-2/X-416-L-2/X-417-L-2/X-418- and so on.

Pharmaceutical Formulations When employed as pharmaceuticals, the compounds of formula I are usually administered in the form of pharmaceutical compositions. This invention therefore provides pharmaceutical compositions which contain, as the active ingredient, one or more of the compounds of Formula I above or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients, carriers, diluents, permeation enhancers, solubilizers and adjuvants. The compounds may be administered alone or in combination with other therapeutic agents (e. g., vasoconstrictors, anti-inflammatory agents, antibiotics, other monobinding endothelin ligand antagonists, counter-irritants), carriers, adjuvants, permeation enhancers, and the like. Such compositions arc prepared in a manner well known in the pharmaceutical art (see, e. g., Remiugron s Pharmaceutical Sciences, Mace Publishing Co., Philadelphia, PA 17"'Ed. (1985) and"Modern Pharmaceutics", Marcel Dekker, Inc. 3rd Ed. (G. S. Banker & C. T.

Rhodes, Eds.).

The compounds of Formula I may be administered by any of the accepted modes of administration of agents having similar utilities, for example, by oral, parenteral, rectal, buccal, intranasal or transdermal routes. The most suitable route will depend on the nature and severity of the condition being treated. Oral administration is a preferred route for the compounds of this invention. In making the compositions of this invention, the active ingredient is usually diluted by an excipient or enclosed within such a carrier which can be in the form of a capsule.

sachet, paper or other container. When the excipient serves as a diluent, in can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.

Pharmaceutically acceptable salts of the active agents may be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry and described, e. g., by J. March, Advanced Organic Chemist.

Reactions, Mechanisms and Structure, 4"'Ed. (New York: Wiley-Interscience, 1992).

Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl-and propylhydroxy-benzoates; sweetening agents; and flavoring agents.

The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art. Controlled release drug delivery systems for oral administration include osmotic pump systems and dissolutional systems containing polymer-coated reservoirs or drug-polymer matrix formulations. Examples of controlled release systems are given in U. S. Patent Nos. 3,845,770; 4,326,525; 4,902514; and 5,616,345. Another preferred formulation for use in the methods of the present invention employs transdermal delivery devices ("patches"). Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in

controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, e. g., U. S. Patent Nos. 5, 023,252,4,992,445 and 5,001,139. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.

The compositions are preferably formulated in a unit dosage form. The term"unit dosage forms"refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient (e. g., a tablet, capsule, ampoule). The active compound is effective over a wide dosage range and is generally administered in a pharmaceutically effective amount.

Preferably, for oral administration, each dosage unit contains from 1-250 mg of a compound of Formula I, and for parenteral administration, preferably from 0.1 to 60 mg of a compound of Formula I or a pharmaceutically acceptable salt thereof.

It will be understood, however, that the amount of the compound actuallv administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered and its relative activity, the age. weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.

The tablets or pills of the present invention may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged

action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.

The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

The liquid forms in which the novel compositions of the present invention may be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as corn oil, cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra. Preferably the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions in preferably pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device or the nebulizing device may be attached to a face mask tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.

The following formulation examples illustrate representative pharmaceutical compositions of the present invention.

Formulation Example 1 Hard gelatin capsules containing the following ingredients are prepared:

Quantity Ingredient (mg/capsule) Active Ingredient 30.0 Starch 305. 0 Magnesium stearate 5.0 The above ingredients are mixed and filled into hard gelatin capsules in 340 mg quantities.

Formulation Example 2 A tablet formula is prepared using the ingredients below: Quantity Ingredient tablet Active Ingredient 25.0 Cellulose, microcrystalline 200.0 Colloidal silicon dioxide 10.0 Stearic acid 5. 0 The components are blended and compressed to form tablets, each weighing 240 mg.

Formulation Example 3 A dry powder inhaler formulation is prepared containing the following components: Ingredient Weight % Active Ingredient 5 Lactose 95 The active ingredient is mixed with the lactose and the mixture is added to a dry powder inhaling appliance.

Formulation Example 4 Tablets, each containing 30 mg of active ingredient, are prepared as follows: Quantity Ingredient (mg/tablet Active Ingredient 30.0 mg Starch 45.0 mg Microcrystalline cellulose 35.0 mg Polyvinylpyrrolidone (as 10% solution in sterile water) 4.0 mg Sodium carboxymethyl starch 4.5 mg Magnesium stearate 0.5 mg Talc _ 1. 0 mg Total 120 mg The active ingredient, starch and cellulose are passed through a No. 20 mesh U. S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone is mixed with the resultant powders, which are then passed through a 16 mesh U. S. sieve. The granules so produced are dried at 50° to 60°C and passed through a 16 mesh U. S. sieve. The sodium carboxymethyl starch, magnesium stearate, and talc, previously passed through a No. 30 mesh U. S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets each weighing 120 mg.

Formulation Example 5 Capsules, each containing 40 mg of medicament are made as follows: Ingredient Quantity (mg/capsule) Active Ingredient 40.0 mg Starch 109.0 mg Magnesium stearate 1.0 mg Total 150.0 mg The active ingredient, starch, and magnesium stearate are blended, passed through a No. 20 mesh U. S. sieve, and filled into hard gelatin capsules in 150 mg quantities.

Formulation Example 6 Suppositories, each containing 25 mg of active ingredient are made as follows: Ingredient Amount Active Ingredient 25 mg Saturated fatty acid glycerides to 2,000 mg

The active ingredient is passed through a No. 60 mesh U. S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimum heat necessary. The mixture is then poured into a suppository mold of nominal 2.0 g capacity and allowed to cool.

Formulation Example Suspensions, each containing 50 mg of medicament per 5.0 mL dose are made as follows: Ingredient Amount Active Ingredient 50.0 mg Xanthan gum 4.0 mg Sodium carboxymethyl cellulose (11%) Microcrystalline cellulose (89%) 50.0 mg Sucrose 1.75 g Sodium benzoate 10. 0 mg Flavor and Color q. v.

Purified water to 5. 0 mL The active ingredient, sucrose and xanthan gum are blended, passed through a No. 10 mesh U. S. sieve, and then mixed with a previously made solution of the microcrystalline cellulose and sodium carboxymethyl cellulose in water. The sodium benzoate, flavor, and color are diluted with some of the water and added with stirring. Sufficient water is then added to produce the required volume.

Formulation Example 8 Quantity Ingredient (mgicapsule) Active Ingredient 15.0 mg Starch 407. 0 mg Magnesium stearate 3.0 mg Total 425.0 mg The active ingredient, starch, and magnesium stearate are blended, passed through a No. 20 mesh U. S. sieve, and filled into hard gelatin capsules in 425. 0 mg quantities.

Formulation Example 9 A formulation may be prepared as follows: Ingredient Quantity Active Ingredient 5.0 mg Corn Oil 1. 0 mL Formulation Example 10 A topical formulation may be prepared as follows: Ingredient Quantity Active Ingredient I-10 g Emulsifying Wax 30 g Liquid Paraffin 20 g White Soft Paraffin to 100 g The white soft paraffin is heated until molten. The liquid paraffin and emulsifying wax are incorporated and stirred until dissolved. The active ingredient is added and stirring is continued until dispersed. The mixture is then cooled until solid.

Another preferred formulation employed in the methods of the present invention employs transdermal delivery devices ("patches"). Such transdermal patches may be used to provide continuous or discontinuous infusion of the compounds of the present invention in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well knows in the art. See, e. g., U. S. Patent 5,023,252, issued June 11, 1991, herein incorporated by reference. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.

Frequently, it will be desirable or necessary to introduce the pharmaceutical composition to the brain, either directly or indirectly. Direct techniques usually involve placement of a drug delivery catheter into the host's ventricular system to bypass the blood-brain barrier. One such implantable delivery system used for the transport of biological factors to specific anatomical regions of the body is described in U. S. Patent 5,011,472, which is herein incorporated by reference.

Indirect techniques, which are generally preferred, usually involve formulating the compositions to provide for drug latentiation by the conversion of hydrophilic drugs into lipid-soluble drugs. Latentiation is generally achieved through blocking of the hydroxy, carbonyl, sulfate, and primary amine groups present on the drug to render the drug more lipid soluble and amenable to transportation across the blood-brain barrier. Alternatively, the delivery of hydrophilic drugs may be enhanced by intra-arterial infusion of hypertonic solutions which can transiently open the blood-brain barrier.

Other suitable formulations for use in the present invention can be found in Remington's Pharmaceutrcal Sciences.' Utility and Testing The multi-binding compounds of this invention can be used to modulate endothelin receptors in various tissues including vascular smooth muscle tissue of the heart, kidney, endocrine glands and nervous system. They will typically be used for the treatment of diseases and conditions in mammals that involve or are mediated by endothelin receptors, such as congestive heart failure, pulmonary hypertension, cerebral vasospasm following subarachnoid hemorrhage, essential hypertension, myocardial infarction, myocardial ischemia, unstable angina, restenosis, renal failure of ischemic origin, portal hypertension, cardiac hypertrophy, atherosclerosis, eclampsia, cerebrovascular disease, vascular disease, migraines, and auto-immune diseases, such as Morbus Wegener and Morbus Raynaud, to name a few.

The multi-binding compounds of this invention are tested in well-known and reliable assays and their activities are compared with those of the

corresponding unlinked (i. e., monovalent) ligands.

Representative Assays The following assays are used to evaluate the multi-binding compounds of this invention relative to their monovalent ligand counterparts.

In Vitro Assays : Binding of Multi-binding Compounds to Endothelin Receptors Competitive binding of multi-binding compounds (endothelin receptor antagonists) and their monovalent counterparts to endothelin receptors from microsomal membranes is performed as described by Breu, V., et al.,'. Membranes are placed in 250 p1 50 mM Tris buffer [pH 7.4,25 mM MnCI,, 1 mM EDTA, 0.5% (mass/vol.) bovine serum albumin] in the presence of 10-25 ig protein and either 30,000 cpm (32 pM) 12 endothelin-1 or 100,000 cpm (2.1 nM) ['H] multi-binding compound and varying amounts of unlabelled multi-binding compound (and their monovalent counterparts).

After incubation for 2 hr. at 22°C, bound and free multi-binding compounds (and their monovalent counterparts) are separated by filtration. Non-specific binding is assessed in the presence of 100 nM unlabeled endothelin-1. Specific binding is defined as the difference between total and non-specific binding. KD and Bn, ax values are calculated from competition binding curves by direct fit analysis with a competitive binding assay.

In Vivo Assays : A Model of Congestive Heart Failure (CHF) The effect of multi-binding compounds on experimentally induced CHF is examined in vivo following the method described by Oie, E., et al."and compared to the effects of their monovalent counterparts. The left coronary artery of male Wistar rats (300-350g) is ligated resulting in infarction of the left ventricle (LV) free wall. The rats are then anesthetized with halothane and ventilated with the use of a rodent ventilator with a mixture of 30%-70% NrO and 1% halothane. A left thoracotomy is performed and the heart is exteriorized. The proximal section of the left coronary artery is ligated with a silk suture. The heart is replaced in its normal position and the thoracotomy closed. Sham treated rats undergo identical procedures except ligation. Surgical mortality of animals with infarction is about 30%.

Some of the animals are treated with various concentrations of multi-binding compounds (and some treated with their monovalent counterparts) twenty fours hours after left coronary ligation. Multi-binding compounds (and their monovalent counterparts), which are prepared fresh daily, are administered once daily for 15 days by gavage. Control animals are sham-treated with saline.

The effectiveness of ligation in inducing CHF is determined by infarct size. The infarct size is assessed by measuring the segmental length of the scar tissue relative to the circumference of the left ventricle immediately after excision of the heart and by weighing the scar tissue. All ligated animals had infarcts > 40% of the left ventricle circumference.

Hemodynamic measurements are performed on animals 16 days after the induction of myocardial infarction or sham treatment and 24 hours following the last dose of ligand or saline. The animals are anesthetized and ventilated. A 2-F micromanometer-tipped catheter (e. g., Model SPR-407, Miller Instruments, TX) is inserted into the right carotid artery and advanced into the left ventricle. Left ventricle diastolic pressure, LV systolic pressure, and positive first derivative of the LV pressures is recorded, e. g., on a CardioMed Flowmeter CM 4008, Oslo, Norway. The time constant of the isovolume is calculated according to the logarithmic method.

Endocardiographic examination is determined immediately after the dynamic measurements using, for example, a fully digital Vingmed System FiVe (Ving-Sound, Horten, Normay) with a 7.5 MHz linear array transducer. The septal and posterior wall thickness, as well as LV internal dimension, is measured in both M-mode and two- dimensional tracings. LV internal dimensions are recorded as the largest anteroposterior diameter. In all cases the diameter is recorded outside the infarcted areas.

In Vivo : Effects of Multi-binding Compounds on Experimental Essential Hypertension (changes in systolic arterial pressure, renal hemodynamics, and cardiovascular changes) in Sprague-Dawley rats.

The effect of multi-binding compounds (as compared to their monovalent counterparts) on the prevention of experimental hypertension is evaluated in male Sprague-Dawley rats according to the method described by Herizi, A., et al.'2 The animals are maintained in metabolic cages on a diet with normal sodium levels, for one

week prior to study. Baseline values for experimental parameters are obtained 3-days prior to the investigation.

Hypertension is induced by infusing the animals with either angiotensin II (Sigma) or vehicle subcutaneously via osmotic pumps (e. g., model 2002, Alza Corp) at 200 ng/kg/min for 10 days. Animals, which are divided into 4 groups, are treated as follows : 1) Group 1 consists of animals infused with angiotensin II ; 2) Group 2 consists of animals infused with vehicle alone; 3) Group 3 consists of animals infused with angiotensin and multi-binding compound (or alternatively its monovalent counterpart) ; 4) Group 4 consists of animals infused with multi-binding compounds alone. Multi- binding compounds (or their monovalent counterparts) are administered by gavage at 24-hr. intervals beginning 24 hr. before infusion of angiotensin II.

Body weight, food and water intake, urine volume and excretion of creatinin and electrolytes are measured daily. Urinary retention of albumin is determined before and at the end of the treatment period. Changes in systolic arterial pressure are monitored by the tail cuff method (Jarco, Biosystems), in conscious rats, before and every second day during the experimental period.

At the end of the experiments, eight rats from each group are anesthetized and prepared for cardiac output and renal blood flow determination using microsphere technique. Briefly, two catheters (e. g., 50, Merck-Clevenot) are implanted into the left ventricle via the right carotid artery and into the lower aorta via the left femoral artery.

Both catheters are tunneled subcutaneously and exteriorized to the back of the neck.

After a 3-hour recovery period, catheters are connected to a pressure transducer (e. g., Statham P23D) and arterial pressure and heart rate continuously recorded of 30 min in conscious, freely moving animals. During the intraventricular injection of microspheres, blood is sampled at a rate of 0.5 ml/min and prepared for radioactive counting assays and for sodium, potassium and creatinine concentrations in plasma. Animals are killed by intraventricular injection of pentobarbital sodium, the heart and kidneys removed and weighed. The radioactive counts and sizes of hearts and kidneys are expressed relative to the weights of the animals.

The carotid media thickness and lumen diameter are determined in rats anesthetized with pentobarbital (60 x/kg, IP). The right carotid is catheterized (PE 50) and washed with a phosphate buffer in 0.5-mol/L sucrose. The vessel is fixed by a perfusion of 10% formalin at a constant pressure. Carotid arteries are frozen and stored at-80°C. Measurements of carotid media thickness and lumen diameter are made on hematoxylin-stained 20 um thick slices.

In Vivo Effects: Experimental Portal Hypertension Portal hypertension is induced in rats following the method described by Sogni et al."Sprague-Dawley rats are divided into four groups as follows: 1) Normal control animals; 2) Rats with portal hypertension induced by partial ligation of the portal vein; 3) Animals with secondary biliary cirrhosis induced by bile duct ligation in animals anesthetized with pentobarbital; and 4) Animals with CCI, induced cirrhosis (I mg/kg diluted 1: 1 in corn oil administered by intragastic gavage 3 days/week for 0 weeks).

Animals are allowed free access to food and water until 14-16 h before the study, when food is withdrawn.

Hemodynamic measurements are made after hypertension established in the animals. Thus, studies are performed in Group 2 animals 2 weeks following ligation of the portal vein, 4 weeks following bile duct ligation of the Group 3 animals and 1 () weeks following CC14 induced cirrhosis in Group 4 animals.

Hemodynamic measurements are performed by on conscious rats allowed to run freely in cages. Catheters are inserted through subcutaneous tunnels at the back of the neck of the rats after anesthesia by ether. For intravenous drug administration, a 0.7 mm-diameter polyethylene catheter is placed in the left femoral vein. Mean arterial pressure and heart rate are measured with a catheter (e. g., PE-10, Clay Adams, NJ) inserted in the left femoral artery. Mean arterial pressure and heart rate are monitored using a multichannel recorder (e. g., Philips CM 130, Heidovne, and the Netherlands).

For portal pressure measurements, the abdomen is opened and a polyethylene catheter (0.7-mm diameter) is inserted into a small ileal vein and gently advanced up to the bifurcation of the superior mesenteric and splenic. For cardiac output and or, an blood flow measurements, a 0.7-mm-diameter polyethylene catheter with a silastic medical- grade tube tip (e. g., Dow-Corning Corporation Medical Products, Midland, MI) is advanced into the left ventricle the left ventricle via the right carotid artery. This catheter is used for microsphere injections. Cardiac output and organ blood flows are determined in each rat using the radioactive microsphere method (163 urn in diameter,

specific activity: 10 mCi/g, (e. g., New England Nuclear, Boston MA). Cardiac output (ml/min) is calculated as the ratio of the radioactivity (cpm) injected to radioactivity (cpm) in the reference blood sample times 0.8. The cardiac index is calculated as the cardiac output relative to the weight of the animal [ (ml/min)/g].

Regional blood flows are calculated by the following formula: organ blood flow (ml x min-'x long''= organ activity (cpm)/radioactivity injected (cpm) x cardiac index (ml x min~'x 100g').

Portal tributary blood flow is calculated as the sum of spleen, stomach, small intestine, colon, and mesenteric pancreas blood flows.

Systemic vascular resistance (dyn x sec x cm-'x 100-') is calculated as the mean arterial pressure (mm Hg) 80/cardiac index.

Portal territory vascular resistance (dyn x sec x cm-5 100 g-'x 10-') is calculated as: [mean arterial pressure (mm Hg)-portal pressure (mm Hg) x 80/portal tributary blood flow (ml x min-'x 100g l) Hepatocollateral vascular resistance (dyn x sec x cm-'x 100 g'x 10') is calculated as portal pressure (mm Hg) x 80/portal tributary blood flow.

Each group of rats receives an intravenous bolus of either endothelinw or various doses of multi-binding compounds (or their monovalent counterparts). The hemodynamic measurements are performed before and 10 minutes after drug administration.

The Group 3 rats (with induced secondary biliary cirrhosis) are anesthetized with ether. The portal vein is cannulated with a polyethylene catheter (inside diameter is 2 mm) and the hepatic artery ligated. The liver is perfused immediately with 50 ml of Krebs-albumen solution. The rat is killed and the liver excised and transferred to the perfusion chamber. The liver is then perfused by recirculating a solution containing 100 ml of Krebs-Ringer bicarbonate phosphate buffer containing 1-% bovine serum albumin.

1. 5g/L of glucose and 2.5 mmol/L calcium through the portal vein. The perfusate is oxygenated with a mixture of 95% 0,, ard 5% CO2, and the pH maintained at 7. 4=0. 05 by adjusting CO, flow. Livers are perfused at constant temperature (37oC) and pressure (20 mm Hg). The blood flow from the hepatic vein is assessed by measuring the volume of blood (ml) obtained x sec-'x body weight (lOOg-'). After an equilibration period of 10 minutes, blood flows are measured every 10 minutes for 40 minutes. Hepatic

vascular resistance is calculated as follows: resistance pressure (20cm H, O/ blood flow ml/min). Multi-binding compounds (or their monovalent counterparts) or placebo are added to the recirculation system at 10,20, and 30 minutes to obtain a final concentration of 1 ßmol/L, 10 pmol/L or 100 Hmol/L, respectively.

All experimental values are expressed as means SEM. Values before and after drug-administration are compared with a Students t test for paired data. P values < than . 05 are considered statistically significant.

In Vivo Assays: A Model of Pulmonary Hypertension Pulmonary hypertension is induced in 10 week old male Sprague-Dawley rats weighing 325-400g following the method described by Haleen, S., et a/.''* The rats are placed in a 30-L Plexiglas environmental isolation chamber and exposed to either room air (air control) or room air mixed with nitrogen to reduced oxygen content to 10% (hypoxic). The rats receive ground chow and water ad libitum. After 10 days of exposure to air or hypoxia, some of the rats are removed from their chambers weighed and anesthetized with pentobarbital (35 mg/kg, ip). A pulmonary artery catheter is inserted and the rats returned to their respective air or hypoxic chambers with their pulmonary arterial pressure continuously monitored. Mean pulmonary arterial pressure is recorded and averaged for 60 min following recovery of the animals from the anesthetic.

The remaining rats are maintained in their air or hypoxic chambers for an additional 10 days (20 days total). During this period, multi-binding compounds, their monovalent counterparts, or placebos are administered daily by mixing them with food (in varying concentrations x, depending on the body weight and the rate of food consumption of the rats).

The experimental groups consisted of the following: 1) 20-day air control ¢ placebo (rat chow) ; 2) 20-day air control + multi-binding compounds (or their monovalent counterparts) (x concentration) ; 3) 20-day hypoxia-f placebo, 4) 20-day hypoxic + multi-binding compounds (or their monovalent counterparts) (. r concentration) ; 5) 20-day air control + multi-binding compounds (or their monovalent counterparts) (x concentration) ; 6) 20-day hypoxia + placebo; 20-day hypoxic + multi- binding compounds (or their monovalent counterparts) ( : c concentration) ; 7) 20-day air control + multi-binding compounds (or their monovalent counterparts) (x concentration) ; 20-day hypoxia + placebo; 8) 20-day hypoxic + multi-binding compounds (or their monovalent counterparts) (x concentration).

Approximately 16 h after the tenth treatment, rats are removed from their chambers, anesthetized and pulmonary artery catheters are inserted. Mean pulmonary arterial pressure (MPAP) is determined after recovery from anesthesia. The MPAP is averaged for 60 min. Twenty four hours later MPAP is recorded and averaged for 60 min to determine the effects of the washout of multi-binding compounds and their monovalent counterparts on MPAP.

Right heart hypertrophy is determined after the 10-20 day protocols. The animals are anesthetized and arterial blood collected into iced polyethylene tubes containing 2 mg EDTA for measurement of plasma endothelin. The heart is excised and dissected into free right ventricle and left ventricle plus septum. The heart chambers are dried in a dessicator under vacuum for I week and weighed. The ratio of right ventricle weight to left ventricle weight is used as an index of right ventricle hypertrophy.

The effectiveness of multi-binding compounds in preventing or reversing pulmonary hypertension as compared to their monovalent counterparts is also monitored by determining plasma endothelin levels. Blood is collected in chilled EDTA (2 mgiml) tubes and centrifuged at 3,000 rpm for 15 min at 4°C. Plasma samples are stored at- 20°C until assayed. Plasma ET-1 is extracted from 1 ml of plasma with 1. 5 mi of extraction solvent composed of acetone : lM HCI: water (40: 1.5). The mixture is centrifuged for 20 min at 3,000 rpm and 4°C. The supematant is dried with a centnfuga) evaporator and the pellet is reconstituted in sample diluent and assayed using a solid- phase ELISA kit. Optical density readings of unknown samples are estimated against a standard curve of synthetic endothelin spike rat plasma samples over a concentration range of I-113 pg/ml. The recovery from the extraction procure is 36%--_ ; °,'. The inter- assay variation is 6% and the intra-assay variation is 7°, iO.

In Vivo Assays : Effects of Ligands on Nociception.

The analgesic effect of multi-binding compounds is determined on capsaicin- induced nociception in mice following the method described by Piovezan, A., et 1 ls and compared to their monovalent counterparts. Male Swiss mice (25-30g) are housed

in a room with both controlled temperature (22°C2°C) and light (lights on from 0600 to 1800 h) with free access to laboratory chow and tap water. Experiments are performed between 1000 and 1700h.

Nociception is induced in conscious animals with a 20 p1 intraplantar injection of capsaicin (0-3.2 ug) into the right hindpaw. Control animals are injected with 20 p1 of vehicle (1 % dimethylsulfoxide in saline). Immediately after injection each animal is placed in a separate jar underlayed with a mirror set at an angle of about 60° relative to the table to enable full view of the paws at all times. To evaluate the analgesic effect of multi-binding compounds, animals are pretreated with the multi-binding compounds (or their monovalent counterparts) and compared. Control animals are treated similarly with vehicle alone.

The induction of nociception and the analgesic effect of multi-binding compounds (and their rr Dnovalent counterparts) are quantitated by assessing the amount of time a mouse spent licking the injected hindpaw. Licking is recorded cumulatively over the first 5 min using a stopwatch chronometer. The results are statistically evaluated by analysis of variance followed by either a ferroni's test or an unaired Students t-test.

In Vivo : The Effect of Ligands on Cerebral Vasospasm The prevention of subarachnoid hemorrhage-induced cerebral vasospasm by multi-binding compounds is assessed in male New Zealand white rabbits following the method described by Zuccarello, M., et al.'6 and compared to the effectiveness of their monovalent counterparts.

Vertebrobasilar angiograms are obtained on Day 0 and Day 6 of the studies as follows. Rabbits, weighing 2.5-3. 8 kg, are anesthetized by intramuscular injection of 1 mg/kg acepromazine followed by injections of 0.18-0. 23 ml/kg fentanyl-doperidol and 25mg/kg sodium pentobarbital (im and iv, respectively). The rabbits are intubated and ventilated mechanically. Rabbits are given 400 IU heparin via an auricular vein and paralyzed with 0.1 mg/kg Pavulon given IV and ventilated with room air supplement with 0 (0. 3L/min). A gastric tube for multi-binding compound (or its monovalent counterpart) administration is inserted and the tube position verified under fluoroscopy,

or the multi-binding compound (or its monovalent counterpart) is administered via an esophageal tube. The left or right subclavian artery is catheterized and the tip of a No.

4 French polyethylene catheter, for example, is directed toward the ipsilateral vertebral artery to obtain a selective injection of the vertebobasilar system. Arterial blood is collected for blood gas analysis.

Contrast medium (e. g., Angiovist 282) is injected (5 ml/sec for 5 seconds) and images (4 left anterior oblique projection) of the vertebrobasilar system are obtained at two per second for 14 seconds using a rapid sequential angiographic technique. Digital subtraction analysis is performed with the small focal spot at 60 kV and 0.8 mA.

Immediately after the Day 0 angiogram rabbits are immobilized in a sterotactic frame and the cistema magna punctured percutaneously with a 21-gauge-butterfly needle. Arterial blood (0.75 ml/kg) is withdrawn from the central ear artery and injected into the cistema magna (, ver 3 minutes. The injection is repeated on day 2. Various concentrations of multi-binding compounds (and their monovalent counterparts) are administered twice a day orally beginning within 1 hour after the initial subarachnoid hemorrhage. The day 6 angiogram is obtained 6-12 h after the end of the dosing schedule.

Rabbits are ventilated and blood gas levels are controlled by adjusting the respiratory rate and/or tidal volume. Core body temperature is monitored using a rectal thermometer and maintained at 37°C with a heating pad.

Angiograms are placed on a light box below a video camera that is connected to an image analysis computer. The angiographic image is captured by the camera, digitized, and reproduced on the video monitor. An observer blind to the experiment measures basilar artery diameter below the basilar-posterior cerebral artery junction. above the basilar vertebral artery junction, and midway between these locations. The means of these three measurements are averaged. Constriction is expressed as a percent of the basilar artery diameter on day 6 relative to day 0.

In order to illustrate further the present invention and advantages thereof, the following specific examples are given but are not meant to limit the scope of the claims in any way.

EXAMPLES Based on the basic pharmacophore for endothelin receptor antagonists and the current available materials, several analogs of ligand antagonist are synthesized and several classes of bivalent endothelin receptor antagonists are designed, the syntheses are described in the following examples.

In the examples below, all temperatures are in degrees Celsius (unless otherwise indicated) and all percentages are weight percentages (also unless otherwise indicated).

In the examples below, the following abbreviations have the following meanings. If an abbreviation is not defined, it has its generally accepted meaning.

A = Angstroms cm = centimeter DIC = 2-dimethylaminoisopropyl chloride hydrochloride DCC NN-dicyclohexylcarbodiimide<BR> DCM = dichloromethane<BR> DIPEA diisopropy lethy lamine<BR> DMA N, N-dimethylacetamide<BR> DMAP 4-N, N-dimethylaminopyridine<BR> DMF N, N-dimethylformamide<BR> DMSO dimethylsulfoxide DPPA = diphenylphosphoryl azide g = gram<BR> HBTU = 1-hydroxybenzotrizole HPLC = high performance liquid chromatography Hunig's base diisopropylethylamine MFC = minimum fungicidal concentration mg milligram MIC = minimum inhibitory concentration min = minute mL = milliliter mm = millimeter mmol millimol N = normal

PyBOP = pyridine benzotriazol-l-yloxy-tris (dimethyl- amino) phosphonium hexafluorophosphate t-BOC tert-butyloxycarbonyl TBAF = tetrabutyl ammonium fluoride TFA trifluoroacetic acid THF = tetrahydrofuran dz = microliters Example 1 : Preparation of Svnthon A.

To a mixture of 0.94 mmols of oil free sodium halide in 100 mL of dimethyl carbonate under N, is added over 30 min. a solution of 3'- nitroacetophenone (10) in 150 mL of dimethyl carbonate. The mixture is then refluxed for 30 min., cooled and quenched by slow addition of 3 N HCI. The reaction is then partitioned between ethyl acetate and water and the aqueous phase extracted with ethyl acetate. The combined organic phases are washed with water, aqueous sodium bicarbonate and dried over sodium sulfate. After filtering, the solvent is removed in vacuo to afford intermediate (11), which may be purified by crystallization or chromatography as necessary.

A mixture of 0.26 mmols of intermediate (11), 0.29 mmols of piperonal.

3.6 mL of acetic acid and 1.2 mL of piperidine in 70 mL of benzene is refluxed with the azeotropic removal of water. After 4hrs., the mixture is concentrated in vacua and the residue purified as necessary by crystallization or chromatography to afford intermediate (12).

To 150 mL of trifluoroacetic acid at 0°C under N, is added 0.19 mmols of intermediate (12). The mixture is warmed to room temperature and after 30 min. concentrated under reduced pressure. The residue is partitioned between ethyl acetate and water and the organic phase washed successively with water, aqueous sodium bicarbonate and brine. After drying over sodium sulfate and filtering, the solvent is removed in vacuo and the residue purified as necessary by chromatography or crystallization. To a solution of 65.6 mmols of this material in

80 mL of dioxane cooled in an ice bath is added 67.8 mmols of 2, 3-dichloro-5,6- dicyano-1, 4-benzoquinone. The mixture is stirred at room temperature for 2hrs. then for Ihr. at 50 C. The reaction is filtered and the solids washed with dioxane and the combined filtrates concentrated under reduced pressure. The residue is partitioned between ethyl acetate and water and washed successively with water, aqueous sodium bicarbonate and brine. After drying over sodium sulfate and filtering, the solvent is removed in vacuo and the residue purified as necessary by crystallization or chromatography to afford intermediate (13).

A solution of 60 mmols of [4-methoxy-2- (methoxymethoxy) phenyl] magnesium bromide (J. Med. Chem., 1994,37,1553-1557) in ether is added to a solution of 40 mmols of intermediate 3 in 200 mL of ether under N, at 0°C. The reaction is warmed to room temperature and after 10 min. partitioned between 1 N HC1 and ethyl acetate and washed successively with water, aqueous sodium bicarbonate and brine. After drying and filtering, the solvent is removed in zuclzo and the residue purified as necessary by chromatography or crystallization. To a solution of 23 mmols of this material in 200 mL of methylene chloride at 0°C under N, is added 29 mmols of triethylsilane followed by 112 mmols of boron trifluoride etherate. The resulting solution is stirred at 0°C for 10 min. and then partitioned between I N HCl and ethyl acetate. The organic phase is washed successively with water, aqueous sodium bicarbonate and brine. After drying over sodium sulfate and filtering, the solvent is removed in vacua and the residue purified as necessary by chromatography or crystallization to afford intermediate (14).

A solution of 20 mmols of intermediate (14) in 50 mL of DMF is added to a suspension of 25 mmols of oil free sodium hydride in 10 mL of DMF and the mixture stirred at room temperature for 10 min. The reaction is then treated with 24 mmols of ethyl bromoacetate and stirring continued for 20 min. followed by quenching with 3 N HCI and extraction with ethyl acetate. The organic phase is washed successively with water, aqueous sodium bicarbonate and brine. After drying over sodium sulfate and filtering, the solvent is removed in vacuo and the

residue purified as necessary by chromatography or crystallization to afford intermediate (15).

A solution of 10 mmols of intermediate (15) in 20 mL of methanol with 200 mg of 10% palladium on carbon is shaken under an atmosphere of 50 psi H, for 6 hrs. After exchanging for an atmosphere of N2 and filtering, the solvent is removed in vacuo and the residue purified by crystallization or chromatography to afford Synthon A. The synthesis of Synthon A is illustrated in Figure 7A.

It is recognized that material produced by the above route will be racemic. but it is understood that a chiral product may be obtained by any of several methods, three of which are indicated below. a. Racemic material may be separated by classical resolution by forming a pair of diastereomeric salts with a chiral acid, such as dibenzoyltartaric acid, separating the diastereomers and freeing the individual enantiomers. b. A preparative chiral HPLC column such as Chiralpak AD could be used to separate the enantiomers. c. A chiral catalyst could be used in the hydrogenation of intermediate 5 to afford a single isomer directly.

Example 2 : Preparation of Synthon B and A Multi-binding Compound of Formula (A) A solution of 20 mmols of Bosentan (20) in 50 mL of isopropyl acetate with 20 mmols of triethylamine at room temperature is treated with 20 mmols of 4- nitrophenyl chloroformate, yielding (21). After lhr., 20 mmols of 2,6- diaminopyridine is added and the reaction warmed and followed by TLC. When judged complete, water is added, the layers separated and the organic phase extracted sequentially with water, sat. sodium carbonate, and brine. After drying

over sodium sulfate and filtering, the solvent is removed in vacuo to give a mixture containing Synthon B and the dimer (22) of formula (A). The mixture is separated by chromatography. The synthesis of Synthon B and dimer (22) is illustrated in Figure 7B.

Example 3: Preparation of a Multi-binding Compound (31) of Formula (B) wherein X is a linker of Formula (V) and wherein n=4 A solution of 10 mmols of Synthon B (30) in 20 mL of ethyl acetate with 10 mmols of triethylamine is treated at room temperature with 5 mmols of a linker. adipoyl chloride (n=4), at room temperature. After 1 hr., the mixture is washed with water, dried over sodium sulfate, filtered and the solvent removed isz vaco The residue is purified by crystallization or chromatography to afford the homodimer (31) of formula (B). The synthesis of homodimer (31) is illustrated in Figure 9.

In a similar manner, other homodimer compounds of formula (B) may be prepared by using alternative linker molecules of Formula (V) or (VI), as defined above.

Example 4: Preparation of a Multi-binding Compound of Formula (C). using a linker of the Formula (V), wherein n=3 A solution of 10 mmols of Synthon A in 20 mL of ethyl acetate with 10 mmols of triethylamine is treated at room temperature with 5 mmols of linker, glutaryl chloride (n=3), at room temperature. After I hr., the mixture is washed with water, dried over sodium sulfate, filtered and the solvent removed in cl The resulting tetraester (not illustrated in Figure 10) is purified as required and then dissolved in 25 mL of methanol and 15 mL of 2 N sodium hydroxide added.

The reaction is warmed and followed by TLC. When judged complete, it is concentrated and 30 mL of IN HCI is added and the mixture extracted with ethyl acetate which is washed with water, dried over sodium sulfate, filtered and the

solvent removed in vacuo. The residue is purified by crystallization or chromatography to afford the homodimer (41) of formula (C). The synthesis of homodimer (41) is illustrated in Figure 10.

In a similar manner, other multi-binding compounds of formula (C) may be prepared by using alternative linker molecules of Formula (V) or (VI).

Example 5: Preparation of a Multi-binding Compound of Formula (D ! usinu a linker of Formula (V), wherein n=6 A solution of 10 mmols of Synthon B in 20 mL of ethyl acetate with 10 mmols of triethylamine is treated at room temperature with 10 mmols of linker, methyl suberyl chloride (n=6), at room temperature. After Ihr., the mixture is washed with water, dried over sodium sulfate, filtered and the solvent removed in vacua. The resulting ester is purified as required and then dissolved in 25 mL of methanol and 10 mL of 2 N sodium hydroxide added. The reaction is warmed and followed by TLC. When judged complete, it is concentrated and 20 mL of IN HO is added and the mixture extracted with ethyl acetate which is washed with water, dried over sodium sulfate, filtered and the solvent removed, yielding product (51).

The product (51) from the preceding reaction is carefully dried and dissolved in 20 mL of dry DMF and 10 mmols of Synthon A and 14 mmols of 1- hydroxybenzotriazole added under N,. The mixture is cooled in an ice bath and 11 mmols of l-ethoxy-3- [3- (dimethylamino) propyl] carbodimiide hydrochloride is added. The cooling bath is removed and the reaction followed by TLC. When judged complete, the reaction mixture is partitioned between water and isopropyl acetate and the organic phase exhaustively washed with water and the solvent removed in vacuo. The resulting ester is purified as required and then dissolved in 25 mL of methanol and 15 mL of 2 N sodium hydroxide added. The reaction is warmed and followed by TLC. When judged complete, it is concentrated and 30 mL of IN HCl is added and the mixture extracted with ethyl acetate which is washed with water, dried over sodium sulfate, filtered and the solvent removed in vacuo. The residue is purified by crystallization or chromatography to afford the heterodimer (52) of formula (D).

Again, in a similar manner, other multi-binding compounds of formula (D) may be prepared by using alternative linker molecules of Formula (V) or (VI).

While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

All of the publications, patent applications and patents cited in this application are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent application or patent was specifically and individually indicated to be incorporated by reference in its entirety.