WO1999063988A1 - Inhibitors of nitric oxide synthase - Google Patents

Abstract

Disclosed are multibinding compounds which inhibit nitric oxide synthases (NOSs), enzymes which form nitric oxide and L-citrulline from L-arginine. The multibinding compounds of this invention contain from 2 to 10 ligands covalently attached to one or more linkers. Each ligand binds to a nitric oxide synthase. The multibinding compounds of this invention are useful in the treatment of chronic inflammation, arthritis, sepsis and the like.

Description

INHIBITORS OF NITRIC OXIDE SYNTHASE

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No. 60/088,448 filed June 8, 1998 and U.S. Provisional Patent Application Serial No. 60/093,072 filed July 16, 1998, both of which are incorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates to novel multibinding compounds (agents) that inhibit the enzyme nitric oxide synthase and to pharmaceutical compositions comprising such compounds. Accordingly, the multibinding compounds and pharmaceutical compositions of this invention are useful in the treatment and prevention of various disorders mediated by nitric oxide synthase, such as inflammation, sepsis and the like.

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

¹ Nakane et al. , FEBS Letters, 316, 175-182, 1993

² Marsden et al. , FEBS Letters, 307, 287-293, 1992

³ Geller et al., PNAS, 90, 3491-5, 1993

⁴ Moncada et al. , Pharmacol. Reviews, 43, 109-142, 1991

⁵ Nathan, FASEB J. , 6, 3051-64, 1992 Wright et al., Card. Res., 26,48-57, 1992

Kilbourn et al. , PNAS, 87, 3629-32, 1990

Beasley and Brenner, Kidney Int. , 42, Suppl. , 38, S96-S100, 1992

Hibbs et al., J. Clin. Invest., 89, 867-77, 1992

Miller et al., J. Pharmacol. Exp. Ther., 264, 11-16, 1990

Ialenti et al., Br. J. Pharmacol., 110, 701-6, 1993

Stevanovic-Racic et al., Arth. & Rheum. , 37, 1062-9, 1994

Huang et al. , Science, 265, 1883-5, 1994

U.S. Patent No. 5,902,810

U.S. Patent No. 5,866,569

U.S. Patent No. 5,723,451 U.S. Patent No. 5,866,612

U.S. Patent No. 5,863,931 U.S. Patent No. 5,830,917 U.S. Patent No. 5,807,886

Wickelgren, I, 1997, "Biologists Catch their First Detailed Look at NO enzyme" Science 278:389

Crane et al., 1998, "Structure of Nitric Oxide Synthase Oxygenase Dimer with Pterin and Substrate" Science 279:2121-

2126

Raman et al., 1998, "Crystal Structure of constitutive endothelial Nitric oxide Synthase: A paradigm for pterin function involving a novel metal center" Cell 95:939-950

Crane et al., 1997, "The Structure of Nitric Oxide synthase Oxygenase domain and Inhibitor Complexes" Science 278:425-430 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

The emergence of nitric oxide (NO), a reactive inorganic radical gas, as a molecule contributing to important physiological and pathological processes is one of the major biological revelations of recent times. This molecule is produced under a variety of physiological and pathological conditions by cells mediating vital biological functions. Examples include endothelial cells lining the blood vessels. Nitric oxide derived from these cells relaxes smooth muscle and regulates blood pressure and has significant effects on the function of circulating blood cells such as platelets and neutrophils as well as on smooth muscle, both of the blood vessels and also of other organs such as the airways. In the brain and elsewhere nitric oxide serves as a neurotransmitter in non-adrenergic, non-cholinergic neurons. In these instances nitric oxide appears to be produced in small amounts on an intermittent basis in response to various endogenous molecular signals. In the immune system, nitric oxide can be synthesized in much larger amounts on a protracted basis. Its production is induced by exogenous or endogenous inflammatory stimuli, notably endotoxin and cytokines elaborated by cells of the host defense system in response to infectious and inflammatory stimuli. This induced production results in prolonged nitric oxide release which contributes both to host defense processes such as the killing of bacteria and viruses, as well as pathology associated with acute and chronic inflammation in a wide variety of diseases. The discovery that nitric oxide production is mediated by a unique series of three closely related enzymes, named nitric oxide synthases, which utilize the amino acid arginine and molecular oxygen as co-substrates, has provided an understanding of the biochemistry of this molecule and provides distinct pharmacological targets for the inhibition of the synthesis of this mediator, which should provide significant beneficial effects in a wide variety of diseases.

Nitric Oxide Synthases Nitric oxide and L-citrulline are formed from L-arginine via the dioxygenase activity of specific nitric oxide synthases (NOSs) in mammalian cells. In this reaction, L-arginine, O₂ and NADPH are co-substrates while FMN, FAD and tetrahydrobiopterin are co-factors. NOSs fall into two distinct classes, constitutive NOS (cNOS) and inducible NOS (iNOS). Two constitutive NOSs have been identified.

They are: (i) a constitutive, Ca^{+ +} /calmodulin dependent enzyme, located in the endothelium (eNOS or NOS 3), that releases NO in response to receptor or physical stimulation;

(ii) a constitutive, Ca. ^{+ +}/calmodulin dependent enzyme, located in the brain (nNOS or NOS 1) and elsewhere, that releases NO in response to receptor or physical stimulation.

The third isoform identified is inducible NOS (iNOS or NOS 2):

(iii) a Ca. ^{+ +} independent enzyme which is induced after activation of vascular smooth muscle, macrophages, endothelial cells, and a large number of other cells by endotoxin and cytokines.

Once expressed, this inducible NO synthase produces NO in relatively large amounts for long periods of time. Spectral studies of both the mouse macrophage iNOS and rat brain nNOS have shown that these enzymes (which have been classified as P-450-like enzymes from their CO-difference spectra) contain a heme moiety. See Fig. 1 for the binding sites for calmodulin (caM), FMN, FAD (including diphosphate (PPi) and 7,8 dimethylisoalloxazine (ISO) sites) and NADPH (including the riboside (ribo) and adenine (ade) sites) in the various NOS monomers and cytochrome P450 reductase . The structural similarity between NOS and the P-450-flavoprotein complex suggests that the NOS reaction mechanism may be similar to P-450 hydroxylation and/or peroxidation. This indicates that NOS belongs to a class of flavohemeproteins which contain both heme and flavin binding regions within a single protein in contrast to the multiprotein NADPH oxidase or Cytochrome P-450/NADPH Cyt C reductase complexes. See Fig. 2 for the assembly of monomers to dimers.

Distinct Functions of NO Produced by Different Nitric Oxide Synthases

The NO released by the constitutive enzymes (NOS 1 and NOS 3) acts as an autocoid mediating a number of physiological responses. Two distinct cDNAs accounting for the activity of NOS 1 and NOS 3 in man have been cloned, one for NOS 1 (Nakane et al., FEBS Letters, 316, 175-182, 1993¹) which is present in the brain and a number of peripheral tissues, the other for an enzyme present in endothelium (NOS 3) (Marsden et al., FEBS Letters, 307, 287-293, 1992²). This latter enzyme is critical for production of NO to maintain vasorelaxation. A second class of enzyme, iNOS or NOS 2, has been cloned from human liver (Geller et al., PNAS, 90, 3491-5, 1993³), and identified in more than a dozen other cells and tissues, including smooth muscle cells, chondrocytes, the kidney and airways. As with its counterpart from the murine macrophage, this enzyme is induced upon exposure to cytokines such as gamma interferon (IFN-γ), interleukin-lβ (IL-lβ), tumor necrosis factor (TNF-α) and LPS (lipopolysaccharide). Once induced, iNOS expression continues over a prolonged period of time.

Endothelium derived relaxation factor (EDRF) has been shown to be produced by NOS 3 (Moncada et al. , Pharmacol. Reviews, 43, 109-142, 1991⁴). Studies with substrate analog inhibitors of NOS have shown a role for NO in regulating blood pressure in animals and blood flow in man, a function attributed to NOS 3. NO has also been shown to be an effector of the cytotoxic effects of activated macrophages (Nathan, FASEB J., 6, 3051-64, 1992⁵) for fighting tumour cells and invading microorganisms (Wright et al., Card. Res., 26,48-57, 1992⁶ and Moncada et al., Pharmacological Review, 43, 109-142, 1991⁴). It also appears that the adverse effects of excess NO production, in particular pathological vasodilation and tissue damage, may result largely from the effects of NO synthesized by the NOS 2.

NO generated by NOS 2 has been implicated in the pathogenesis of inflammatory diseases. In experimental animals, hypotension induced by LPS or TNF-α can be reversed by NOS inhibitors and reinitiated by L-arginine (Kilbourn et al., PNAS, 87, 3629-32, 1990⁷). Conditions which lead to cytokine-induced hypotension include septic shock, hemodialysis (Beasley and Brenner, Kidney Int. , 42, Suppl. , 38, S96-S100, 1992⁸) and IL-2 therapy in cancer patients (Hibbs et al., J. Clin. Invest., 89, 867-77, 1992°). NOS 2 is implicated in these responses, and thus the possibility exists that a NOS inhibitor would be effective in ameliorating cytokine-induced hypotension. Recent studies in animal models have suggested a role for NO in the pathogenesis of inflammation and pain, and NOS inhibitors have been shown to have beneficial effects on some aspects of the inflammation and tissue changes seen in models of inflammatory bowel disease, (Miller et al., J. Pharmacol. Exp. Ther., 264, 11-16, 1990¹⁰), cerebral ischemia, and arthritis (Ialenti et al., Br. J. Pharmacol. , 110, 701-6, 1993¹¹; Stevanovic-Racic et al., Arth. & Rheum., 37, 1062-9, 1994¹²). Moreover, transgenic mice deficient in NOS 1 show diminished cerebral ischemia (Huang et al., Science, 265, 1883-5, 1994¹³).

Further conditions where there is an advantage in inhibiting NO production from L-arginine include therapy with cytokines such as TNF, IL-1 and IL-2 or therapy with cytokine-inducing agents, for example, 5,6-dimethylxanthenone acetic acid, and as an adjuvant to short term immunosuppression in transplant therapy. In addition, compounds which inhibit NO synthesis may be of use in reducing the NO concentration in patients suffering from inflammatory conditions in which an excess of NO contributes to the pathophysiology of the condition, for example, adult respiratory distress syndrome (ARDS) and myocarditis.

There is also evidence that an NO synthase enzyme may be involved in the degeneration of cartilage which takes place in autoimmune and/or inflammatory conditions such as arthritis, rheumatoid arthritis, chronic bowel disease and systemic lupus erythematosis (SLE). It is also thought that an NO synthase enzyme may be involved in insulin-dependent diabetes mellitus. Therefore, a yet further aspect of the present invention provides compounds for use in cytokine or cytokine-inducing therapy, as an adjuvant to short term immunosuppression in transplant therapy, and for the treatment of patients suffering from inflammatory conditions in which an excess of NO contributes to the pathophysiology of the condition.

A number of compounds have been identified which act as inhibitors of NOS, these include pteridine derivatives (U.S. Patent No. 5,902,81c)¹⁴) naphthalenedione compounds (U.S. Patent Nos. 5,866,569¹⁵ and 5,723,451¹⁶) , acetamidine derivatives ( U.S. Patent No. 5,866,612¹⁷) amidino derivatives (U.S. Patent No. 5,863,931¹⁸ and U.S. Patent No. 5,830,917¹⁹) and bicyclic amidine derivatives (U.S. Patent No. 5,807,886²⁰).

It has now been discovered that selective nitric oxide synthase inhibitors having surprising and unexpected properties can be prepared by linking from 2 to 10 nitric oxide synthase inhibitors to one or more linkers. Such multibinding compounds provide improved biological and/or therapeutic effects compared to the aggregate of the unlinked ligands due to their multibinding properties. SUMMARY OF THE INVENTION

This invention is directed to novel multibinding compounds (agents) that inhibit nitric oxide synthase (NOS). Preferably, the multibinding compounds selectively inhibit either only i-NOS or only n-NOS. The multibinding compounds of this invention are useful in the treatment and prevention of disorders mediated by NOS, such as inflammation, sepsis and the like.

Accordingly, in one of its composition aspects, this invention provides a multibinding compound comprising from 2 to 10 ligands covalently attached to one or more linkers wherein each of said ligands independently comprises a moiety capable of binding to nitric oxide synthase; and pharmaceutically- acceptable salts thereof; with the proviso that the multibinding compound is not formula III or IV:

wherein R is hydrogen or C,_₈ hydrocarbyl;

T is a C_j.g hydrocarbyl group optionally containing a 5- or 6-membered heterocyclic ring ot T is a ^ hydrocarbyl group containing a phenylene ring.

In another of its composition aspects, this invention provides a multibinding compound of formula I: (L)_p(X)_q ^]

wherein each L is independently a ligand comprising a moiety capable of binding to nitric oxide synthase; each X is independently a linker; p is an integer of from 2 to 10; and q is an integer of from 1 to 20; and pharmaceutically- acceptable salts thereof; with the proviso that the multibinding compound is not formula III or IV:

wherein R³⁵ is hydrogen or C,._g hydrocarbyl;

T is a C,_.8 hydrocarbyl group optionally containing a 5- or 6-membered heterocyclic ring or T is a ^ hydrocarbyl group containing a phenylene ring.

Preferably, q is less than/? in the multibinding compounds of this invention.

Preferably, each ligand, L, in the multibinding compound of formula I is independently selected from the group consisting of:

(a) a compound selected from the group consisting of formula IA', IA", IA'", IB', IB", IB'":

IA'"

IA' IA"

IB' IB" IB'"

wherein

X is selected from the group consisting of O, S;

Y is selected from the group consisting of OR¹ , SR¹, NR'R²;

R¹ and R² are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, heteroaryl, aryl, heterocyclic, acyl, substituted acyl;

R³ , R³ , R^{3 '"}, R⁴ , R⁴ and R⁴ are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, acyl, substituted acyl, aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, heterocyclic and wherein R³ and R⁴ together form a heterocyclic or a heteroaryl;

R⁵ , R⁵ , R^{5 '"}, R⁶ , R⁶ and R⁶ are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, alkoxy, substituted alkoxy, alkylthioalkoxy, acylamino, cycloalkyl, substituted cycloalkyl,; and

R⁷, R⁸ and R⁸ are selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl; with the proviso that one of R¹, R², R³ , R³ , R ^", R⁴ , R⁴ , R⁴ ,R⁵ , R^5", R^{5 "}, R^6', R⁶ , R⁶ , R⁷, R⁸ or R^8" is a covalent linkage to a linker;

(b) a compound of formula IC or IC"

IC IC" wherein

R¹⁰ and R¹¹ are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkaryl, alkoxy, substituted alkoxy, alkyalkoxy, acyl, acylamino, amino, substituted amino, aminoacyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, heterocyclic; and R¹⁰ and R¹¹ may optionally together form a cycloalkyl, subsituted cycloalkyl or heterocyclic; and

R^12', R^12", R^13', R^13", R^14', R^14" are selected from the group consisting of hydrogen, alkyl, substituted alkyl, acyl, substituted acyl, aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, heterocyclic and wherein R³ and^~R⁴ together form a heterocyclic or a heteroaryl; with the proviso that one of R^{11 '} R^{11 "}, R^12', R^12", R¹ , R¹³ , R¹⁴ , R^14' is a covalent linkage to a linker; and (c) a compound of formula ID

R23

wherein

R²⁰ is selected from the group consisting of NH, O, S, NOH, NR²⁴

R²¹, R²², and R²⁴ are independently selected from the group consisting of hydrogen, an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, a substituted alkynyl, an aryl, a heterocyclic, a cycloalkyl and a substituted cycloalkyl;

R^{23 1S} independently selected from the group consisting of an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, a substituted alkynyl, an aryl, a heterocyclic, a cycloalkyl and a substituted cycloalkyl; and

Z is an alkylene, a substituted alkylene, an amino, a substituted amino, -S-; with the proviso that one of R²¹, R²², R²³, or R²⁴ is a covalent linkage to a linker, and their tautomeric forms and also their pharmaceutically acceptable salts.

In still another of its composition aspects, this invention provides a multibinding compound of formula II:

L'- X'- L' II

wherein each L' is independently a ligand comprising a moiety capable of binding nitric oxide synthase and X' is a linker; and pharmaceutically-acceptable salts thereof; with the proviso that the multibinding compound is not formula III or IV:

wherein R is hydrogen or C,_₈ hydrocarbyl; T is a C,_₈ hydrocarbyl group optionally containing a 5- or 6-membered heterocyclic ring or T is a C^ hydrocarbyl group containing a phenylene ring..

Preferably, in the above embodiments, each linker (i.e. , X, X' or X" independently has the formula:

-X^a-Z-(Y^a-Z)_m-Y^b-Z-X^a- wherein m is an integer of from 0 to 20;

X^a at each separate occurrence is selected from the group consisting of -O-, -S-, -NR-, -C(O)-, -C(O)O-, -C(O)NR-, -C(S), -C(S)O-, -C(S)NR- or a covalent bond where R is as defined below;

Z is at each separate occurrence is selected from the group consisting of alkylene, substituted alkylene, cycloalkylene, substituted cylcoalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, cycloalkenylene, substituted cycloalkenylene, arylene, heteroarylene, heterocyclene, or a covalent bond;

Y^a and Y^b at each separate occurrence are selected from the group consisting of -C(O)NR'-, -N'C(O)-, -NR'C(O)NR'-, -C(=NR')-NR'-, -NR'-C(=NR')-, -NR'-C(O)-O-, -N=C(X^a)-NR'-, -P(O)(OR')-O-, -S(O)_nCR'R", -S(O)_n-NR'-, -S-S- and a covalent bond; where n is 0, 1 or 2; and R, R' and R' ' at each separate occurrence are 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.

In yet another of its composition aspects, this invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a multibinding compound comprising from 2 to 10 ligands covalently attached to one or more linkers wherein each of said ligands independently comprises a moiety capable of binding nitric oxide synthase; with the proviso that the multibinding compound is not formula III or IV:

wherein R³⁵ is hydrogen or C,.₈ hydrocarbyl;

T is a C_j.g hydrocarbyl group optionally containing a 5- or 6-membered heterocyclic ring or T is a .₄ hydrocarbyl group containing a phenylene ring, and pharmaceutically-acceptable salts thereof.

This invention is also directed to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an effective amount of a multibinding compound of formula I or II.

Certain of the multibinding compounds of this invention are effective inhibitors of the enzyme i-NOS, an enzyme involved in the biosynthesis of nitric oxide involved in sepsis. Accordingly, in one of its method aspects, this invention provides a method for treating sepsis in a patient, the method comprising administering to a patient having sepsis a pharmaceutical composition comprising a pharmaceutically-acceptable carrier and a therapeutically-effective amount of a multibinding compound comprising from 2 to 10 ligands covalently attached to one or more linkers wherein each of said ligands independently comprises a moiety capable of binding i-NOS; with the proviso that the multibinding compound is not formula III or IV:

wherein R³⁵ is hydrogen or C,__g hydrocarbyl;

T is a C,„₈ hydrocarbyl group optionally containing a 5- or 6-membered heterocyclic ring or T is a .₄ hydrocarbyl group containing a phenylene ring; and pharmaceutically-acceptable salts thereof.

Certain of the multibinding compounds of this invention are effective inhibitors of the enzyme i-NOS, an enzyme involved in the biosynthesis of nitric oxide involved in chronic inflammation. Accordingly, in one of its method aspects, this invention provides a method for treating chronic inflammation in a patient, the method comprising administering to a patient having inflammation or an inflammation-related disorder a pharmaceutical composition comprising a pharmaceutically-acceptable carrier and a therapeutically-effective amount of a multibinding compound comprising from 2 to 10 ligands covalently attached to one or more linkers wherein each of said ligands independently comprises a moiety capable of binding i-NOS; with the proviso that the multibinding compound is not formula III or IV:

wherein R³⁵ is hydrogen or C_j_₈ hydrocarbyl;

T is a C,.₈ hydrocarbyl group optionally containing a 5- or 6-membered heterocyclic ring or T is a ^ hydrocarbyl group containing a phenylene ring; and pharmaceutically-acceptable salts thereof.

The multibinding compounds of this invention are effective inhibitors of the enzyme nitric oxide synthase, an enzyme involved in the biosynthesis of nitric oxide involved in arthritis. Accordingly, in one of its method aspects, this invention provides a method for treating arthritis in a patient, the method comprising administering to a patient having arthritis a pharmaceutical composition comprising a pharmaceutically-acceptable carrier and a therapeutically-effective amount of a multibinding compound comprising from 2 to 10 ligands covalently attached to one or more linkers wherein each of said ligands independently comprises a moiety capable of binding nitric oxide synthase; with the proviso that the multibinding compound is not formula III or IV:

wherein R³⁵ is hydrogen or _₈ hydrocarbyl;

T is a C,_₈ hydrocarbyl group optionally containing a 5- or 6-membered heterocyclic ring or T is a C^ hydrocarbyl group containing a phenylene ring, and pharmaceutically-acceptable salts thereof.

This invention is also directed to general synthetic methods for generating large libraries of diverse multimeric compounds which multimeric compounds bind nitric oxide synthases and 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 bind nitric oxide synthases and 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 bind nitric oxide synthases which method comprises:

(a) identifying a ligand or a mixture of ligands wherein each ligand binds a nitric oxide synthase and 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 which bind nitric oxide synthases possessing multibinding properties which method comprises: (a) identifying a library of ligands wherein each ligand binds a nitric oxide synthase and 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 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.

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 compounds prepared are homomultimeric compounds.

The assay protocols recited in (d) can be conducted on the multimeric ligand compound library or portions thereof 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 bind a nitric oxide synthase and may possess multivalent properties which library is prepared by the method comprising:

(a) identifying a ligand or a mixture of ligands which bind a nitric oxide synthase 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 bind a nitric oxide synthase and may possess multivalent properties which library is prepared by the method comprising: (a) identifying a library of ligands wherein each ligand binds a nitric oxide synthase and 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/or polarizability 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 100A.

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, pseudohalides, isocyanates, vinyl unsaturation, ketones, aldehydes, thiols, alcohols, anhydrides, boronates 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 heteromeric (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 iterative 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 bind to a nitric oxide synthase 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 preferably at least 5-50 times.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, illustrates the sequence relationship of NOS monomers. The unlabeled black areas are the putative binding sites of arginine and tetrahydrobiopterin .

FIG. 2 illustrates the pathway for synthesis of active NOS.

FIG. 3 illustrates examples of multibinding compounds comprising 2 ligands attached in different formats to a linker. FIG. 4 illustrates examples of multibinding compounds comprising 3 ligands attached in different formats to a linker.

FIG. 5 illustrates examples of multibinding compounds comprising 4 ligands attached in different formats to a linker.

FIG. 6 illustrates examples of multibinding compounds comprising >4 ligands attached in different formats to a linker.

FIG. 7 illustrates the starting materials used in a representative synthesis of a ligand precursor.

FIG. 8 illustrates synthesis of Synthon C.

FIG. 9 illustrates the synthesis of Synthon G.

FIG. 10 illustrates the synthesis of Compound I.

FIG. 11 illustrates the synthesis of Compound II.

FIG. 12 illustrates the synthesis of Compound III.

FIG. 13 illustrates the synthesis of Compound IV.

FIG. 14 illustrates the synthesis of Compound V.

FIG. 15 illustrates the synthesis of Compound VI.

FIG. 16 illustrates the synthesis of Compound VII. FIG. 17 illustrates the synthesis of Compound VIII.

FIG. 18 illustrates the synthesis of Compound IX.

FIG. 19 illustrates the synthesis of Compound X.

FIG. 20 illustrates the synthesis of Compound XL

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to multibinding compounds which inhibit the enzyme nitric oxide synthase, pharmaceutical compositions containing such compounds and methods for treating sepsis. When discussing such compounds, compositions or methods, the following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings.

The term "eNOS" or NOS3 " means a nitric oxide synthase which is a constitutive Ca⁺⁺/calmodulin dependent enzyme, located in the endothelium that releases NO is response to receptor or physical stimulation.

The term "nNOS" or "NOS1 " means a nitric oxide synthase which is a constitutive Ca⁺⁺/calmodulin dependent enzyme, located in the brain and elsewhere in the body that releases NO is response to receptor or physical stimulation.

The term "iNOS" or "NOS2" means a nitric oxide synthase which is an inducible Ca^{+ +} independent enzyme which is induced after activation of vascular smooth muscle, macrophages, endothelial cells, and a large number of other cells by endotoxin and cytokines. 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, rø-propyl, iso-propyl, n-butyl, wø-butyl, n-hexyl, «-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, -SO₂-alkyl, -SO₂-substituted alkyl, -SO₂-aryl and -SO₂-heteroaryl.

The term "alkylene" refers to a diradical of a 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 methylene (-CH₂-), ethylene (-CH₂CH₂-), the propylene isomers (e.g. , -CH₂CH₂CH₂- and -CH(CH₃)CH₂-) 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, -SO-heteroaryl, -SO₂-alkyl, -SO₂-substituted alkyl, -SO₂-aryl and -SO₂-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, /z-propoxy, iso- propoxy, n-butoxy, tert-butoxy, .sec-butoxy, n-pentoxy, «-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 alkylene are as defined herein. Preferred alkylalkoxy groups are alkylene-O-alkyl and include, by way of example, methylenemethoxy (-CH₂OCH₃), ethylenemethoxy (-CH₂CH₂OCH₃), rc-propylene-/.rø-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 (-CH₂SCH₃), ethylenethiomethoxy (-CH₂CH₂SCH₃), n-propylene-wo-thiopropoxy (-CH₂CH₂CH₂SCH(CH₃)₂), methylene-t-thiobutoxy (-CH₂SC(CH₃)₃) 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 1 and preferably from 1-6 sites of vinyl unsaturation. Preferred alkenyl groups include ethenyl (-CH = CH₂), /z-propenyl (-CH₂CH=CH₂), isø-propenyl (-C(CH₃)=CH₂), and the like.

The term "substituted alkenyl" refers to an alkenyl 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, -SO₂-alkyl, -SO₂-substituted alkyl, -SO₂-aryl and -SO₂-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., -CH₂CH=CH- and -C(CH₃) = 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, -SO₂-alkyl, -SO₂-substituted alkyl, -SO₂-aryl and -SO₂-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, -SO₂-alkyl, -SO₂-substituted alkyl, -SO₂-aryl and -SO₂-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 (-CH₂C≡C-) and the like.

The term "substituted alkynylene" refers to an alkynylene 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, -SO₂-alkyl, -SO₂-substituted alkyl, -SO₂-aryl and -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(O)O-, substituted alkyl- C(O)O-, cycloalkyl-C(O)O-, substituted cycloalkyl-C(O)O-, aryl-C(O)O-, heteroaryl-C(O)O-, and heterocyclic-C(O)O- 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, oxy acylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, - SO₂-alkyl, -SO₂-substituted alkyl, -SO₂-aryl, -SO₂-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 -NH₂.

The term "substituted amino 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 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, -SO₂-substituted alkyl, -SO₂-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 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, -SO₂-substituted alkyl, -SO₂-aryl and -SO₂ -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 1 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, -SO₂-substituted alkyl, -SO₂-aryl, -SO₂- 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 unsaturated group having a single ring or multiple condensed rings, from 1 to 40 carbon atoms and from 1 to 10 hetero atoms, preferably 1 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 alkyl, -SO-aryl, -SO-heteroaryl, -SO₂-alkyl, -SO₂- substituted alkyl, -SO₂-aryl and -SO₂-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, benzimidazole, 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.

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-morpholino, 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 which 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.

The term "pharmaceutically-acceptable salt" refers to salts which retain the biological effectiveness and properties of the multibinding compounds of this invention and which are not biologically or otherwise undesirable. ^~Tn many cases, the multibinding 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, dialkenyl 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, diary 1 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(wø-propyl) amine, tτi(n- propyl) amine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaihe, 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, sulfur ic 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, /?-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 ally 1 , 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, acyl groups, benzyl groups, and the like. Preferred removable amino blocking groups include conventional substituents such as t-butyoxycarbonyl (t-BOC), benzyloxycarbonyl (CBZ), fluorenylmethoxy carbonyl (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 "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 "ligand" as used herein denotes a compound that binds to the enzyme nitric oxide synthase. The specific region or regions of the ligand that is (are) recognized by the enzyme is designated as the "ligand domain" . A ligand may be either capable of binding to an enzyme by itself, or may require the presence of one or more non-ligand components for binding (e.g., Ca⁺², Mg⁺² or a water molecule is required for the binding of a ligand to various ligand binding sites).

Examples of ligands useful in this invention are described herein. Those skilled in the art will appreciate that portions of the ligand structure that are not essential for specific molecular recognition and binding activity may be varied substantially, replaced or substituted with unrelated structures (for example, with ancillary groups as defined below) and, in some cases, omitted entirely without affecting the binding interaction. The primary requirement for a ligand is that it has a ligand domain as defined above. It is understood that the term ligand is not intended to be limited to compounds known to be useful in binding to nitric oxide synthase (e.g. , known drugs). Those skilled in the art will understand that the term ligand can equally apply to a molecule that is not normally associated with enzyme 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 multivalency.

The ligands and linkers which comprise the multibinding agents of the invention may have various stereoisomeric forms, including enantiomers and diastereomer s..

The term "multibinding compound or agent" refers to a compound that is capable of multivalency, as defined below, and which has 2-10 ligands covalently bound to one or more linkers which may be the same or different. In all cases, each ligand and linker in the multibinding compound is independently selected such that the multibinding compound includes both symmetric compounds (i.e. where each ligand as well as each linker is identical) and asymmetric compounds (i.e. , where at least one of the ligands is different from the other ligand(s) and/or at least one linker is different from the other linker(s). Additionally, the term is intended to include the racemic forms of the multibinding compound as well as individual enantiomers and diastereomers and non-racemic mixtures thereof. It is to be understood that the invention contemplates all possible stereoisomeric forms of mutibinding compounds and mixtures thereof. Multibinding compounds provide a biological and/or therapeutic effect greater than the aggregate of unlinked ligands equivalent thereto which are made available for binding. That is to say that the biological and/or therapeutic effect of the ligands attached to the multibinding compound is greater than that achieved by the same amount of unlinked ligands made available for binding to the ligand binding sites (receptors). The phrase "increased biological or therapeutic effect" includes, for example: increased affinity, increased selectivity for target, increased specificity for target, increased potency, increased efficacy, decreased toxicity, improved duration of activity or action, decreased side effects, increased therapeutic index, improved bioavailability, improved pharmacokinetics, improved activity spectrum, and the like. The multibinding compounds of this invention will exhibit at least one and preferably more than one of the above-mentioned affects.

The term "potency" 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-linear ly correlated with its affinity. In comparing the potency of two drugs, e.g. , a multibinding agent and the aggregate of its unlinked ligand, the dose-response curve of each is determined under identical test conditions (e.g. , in an in vitro or in vivo assay, in an appropriate animal model such a human patient). The finding that the multbinding agent produces an equivalent biological or therapeutic effect at a lower concentration than the aggregate unlinked ligand is indicative of enhanced potency.

The term "univalency" 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 compound having multiple copies of a ligand (or ligands) exhibit univalency when only one ligand is interacting with a ligand binding site. Examples of univalent interactions are depicted below.

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 enzymes which may be the same or different.

For example, two ligands connected through a linker that bind concurrently to two ligand binding sites would be considered as bivalency; three ligands thus connected would be an example of trivalency. An example of trivalent binding, illustrating a multibinding compound 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 or to linkers do not necessarily exhibit the phenomena of multivalency, i.e., that the biological and/or therapeutic effect of the multibinding agent is greater than the sum of the aggregate of unlinked ligands made available for binding to the ligand binding site (receptor). For multivalency to occur, the ligands that are connected by a linker or linkers have to be presented to their ligand binding sites by the linker(s) in a specific manner in order to bring about the desired ligand-orienting result, and thus produce a multibinding event.

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 K_d (i.e., the dissociation constants for each ligand-receptor complex) or, in cases where a biological effect is observed below the Kj , the ratio of the respective EC₅₀'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 term "ligand binding site" denotes the site on the nitric oxide synthase enzyme that recognizes a ligand domain and provides a binding partner for the 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, modulatory effects, may maintain an ongoing biological event, and the like.

The terms "agonism" and "antagonism" are well known in the art. The term "modulatory 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 should be recognized that the ligand binding sites of the enzyme that participate in biological multivalent binding interactions are constrained to varying degrees by their intra- and inter-molecular associations (e.g. , such macromolecular structures may be covalently joined to a single structure, noncovalently associated in a multimeric structure, embedded in a membrane or polymeric matrix, and so on) and therefore have less translational and rotational freedom than if the same structures were present as monomers in solution.

The term "inert organic solvent" or "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 "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 disease condition;

(ii) inhibiting the pathologic condition, i.e. , arresting its development; (iii) relieving the pathologic condition, i.e. , causing regression of the pathologic condition; or (iv) relieving the conditions mediated by the pathologic condition.

The term "pathologic condition which is modulated by treatment with a ligand" covers all disease states (i.e., pathologic conditions) which are generally acknowledged in the art to be usefully treated with a ligand for the enzyme nitric oxide synthase in general, and those disease states which have been found to be usefully treated by a specific multibinding compound of our invention. Such disease states include, by way of example only, the treatment of a mammal afflicted with inflammation, pain, fever and the like.

The term "therapeutically effective amount" refers to that amount of multibinding compound which 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 "linker", identified where appropriate by the symbol X, X' or X", refers to a group or groups that covalently links from 2 to 10 ligands (as identified above) in a manner that provides for a compound capable of multivalency. 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 itself 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 understood that the multibinding compounds of this invention can be attached to a solid support if desired. For example, such attachment to solid supports can be made for use in separation and purification processes and similar applications.

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

The linkers used in this invention are selected to allow multivalent binding of ligands to the ligand binding sites of nitric oxide synthase, whether such sites are located interiorly, both interiorly and on the periphery of the enzyme structure, or at any intermediate position thereof. Crystal structures of nitric oxide synthase with bound inhibitors are known. See for example, Wickelgren²¹, I, 1997, "Biologists Catch their First Detailed Look at NO enzyme" Science 278:389: Crane et al.²², 1998, "Structure of Nitric Oxide Synthase Oxygenase Dimer with Pterin and Substrate" Science 279:2121-2126; Raman et al.²³, 1998, "Crystal Structure of constitutive endothelial Nitric oxide Synthase: A paradigm for pterin function involving a novel metal center" Cell 95:939-950; and Crane et al.²⁴, 1997, "The Structure of Nitric Oxide synthase Oxygenase domain and Inhibitor Complexes" Science 278:425-430 all of which are incorporated herein. FIG. 2 illustrates a nitric oxide synthase dimer. There is a single biochemically and structurally distinct active site in each monomer comprising a heme and a site for binding arginine and a site for binding biopterin. The active sites in the dimer are spaced roughly 40 angstroms apart. In the case where the multibinding compound binds to two ligand binding sites on the same monomer ( for example, the heme and the arginine binding site) the preferred linker distances are 2 - 20A, preferably 3 - 12A. In the case where the multibinding compound binds to ligand binding sites on different monomers, the distance between the nearest neighboring ligand domains is preferably greater than about 20A, more preferably in the range of about 30A to about lOOA.

The ligands are covalently attached to the linker or linkers using conventional chemical techniques providing for covalent linkage of the ligand to the linker or linkers. Reaction chemistries resulting in such linkages are well known in the art and involve the use of complementary functional groups on the linker and ligand. Preferably, the complementary functional groups on the linker are selected relative to the functional groups available on the ligand for bonding or which can be introduced onto the ligand for bonding. Again, such complementary functional groups are well known in the art. For example, 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 I below illustrates numerous complementary reactive groups and the resulting bonds formed by reaction there between.

Table I

Representative Complementary Binding Chemistries First Reactive Group Second Reactive Group Linkage hydroxyl isocyanate urethane amine epoxide β-hydroxyamine sulfonyl 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-ligand binding site interaction and specifically which 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.

The relative orientation in which the ligand domains are displayed derives from the particular point or points of attachment of the ligands 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 the synthetic methods for covalent attachment are well known in the art. Following attachment to the selected linker (or attachment to a significant portion of the linker, for example 2-10 atoms of the linker), the univalent linker-ligand conjugate may be tested for retention of activity in the relevant assay.

Suitable linkers are discussed more fully below.

At present, it is preferred that the multibinding agent is a bivalent compound, e.g., two ligands which are covalently linked to linker X.

Methodology

The linker, when covalently attached to multiple copies of the ligands, provides a biocompatible, substantially non- immunogenic multibinding compound. The biological activity of the multibinding compound is highly sensitive to the valency, geometry, composition, size, flexibility or rigidity, etc. of the linker and, in turn, on the overall structure of the multibinding compound, 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 multibinding compound. The linker 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 their ligand binding sites to permit multivalency. 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 multibinding compound.

For example, different orientations can be achieved by including in the framework groups containing mono- or polycyclic groups, including aryl and/or heteroaryl groups, or structures incorporating one or more carbon-carbon multiple bonds (alkenyl, alkenylene, alkynyl or alkynylene groups). Other groups can also include oligomers and polymers which are branched- or straight- chain species. In preferred embodiments, rigidity is imparted by the presence of cyclic groups (e.g., aryl, heteroaryl, cycloalkyl, heterocyclic, etc.). In other preferred embodiments, the ring is a six or ten member ring. In still further preferred embodiments, the ring is an aromatic ring such as, for example, phenyl or naphthyl.

Different hydrophobic/hydrophilic characteristics of the linker as well as the presence or absence of charged moieties can readily be controlled by the skilled artisan. For example, the hydrophobic nature of a linker derived from hexamethylene diamine (H₂N(CH₂)₆NH₂) 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" .

Different frameworks can be designed to provide preferred orientations of the ligands. Such frameworks may be represented by using an array of dots (as shown below) wherein each dot may potentially be an atom, such as C, O, N, S, P, H, F, CI, Br, and F or the dot may alternatively indicate the absence of an atom at that position. To facilitate the understanding of the framework structure, the framework is illustrated as a two dimensional array in the following diagram, although clearly the framework is a three dimensional array in practice:

» ■ ■ ■ I

0 1 2 3 4 5 6 7 8

Each dot is either an atom, chosen from carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, or halogen, or the dot represents a point in space (i.e., an absence of an atom). As is apparent to the skilled artisan, only certain atoms on the grid have the ability to act as an attachment point for the ligands, namely, C, O, N, S and P.

Atoms can be connected to each other via bonds (single, double or triple bonds with acceptable resonance and tautomeric forms), with regard to the usual constraints of chemical bonding. Ligands may be attached to the framework via single, double or triple bonds (with chemically acceptable tautomeric and resonance forms). Multiple ligand groups (2 to 10) can be attached to the framework such that the minimal, shortest path distance between adjacent ligand groups does not exceed 100 atoms. Preferably, the linker connections to the ligand is selected such that the maximum spatial distance between two adjacent ligands is no more than 100A. An example of a linker as presented by the grid is shown below for a biphenyl construct.

Nodes (1,2), (2,0), (4,4), (5,2), (4,0), (6,2), (7,4), (9,4), (10,2), (9,0), (7,0) all represent carbon atoms. Node (10,0) represents a chlorine atom. All other nodes (or dots) are points in space (i.e., represent an absence of atoms).

Nodes (1,2) and (9,4) are attachment points. Hydrogen atoms are affixed to nodes (2,4), (4,4), (4,0), (2,0), (7,4), (10,2) and (7,0). Nodes (5,2) and (6,2) are connected by a single bond.

The carbon atoms present are connected by either a single or double bonds, taking into consideration the principle of resonance and/or tautomerism.

The intersection of the framework (linker) and the ligand group, and indeed, the framework (linker) itself can have many different bonding patterns. Examples of acceptable patterns of three contiguous atom arrangements are shown in the following diagram:

CCC NCC OCC SCC PCC

CCN NCN OCN SCN PCN CCO NCO OCO SCO PCO

CCS NCS OCS SCS PCS

CCP NCP OCP SCP PCP

CNC NNC ONC SNC PNC

CNN NNN ONN SNN PNN

CNO NNO ONO SNO PNO

CNS NNS ONS SNS PNS

CNP NNP ONP SNP PNP

COC NOC OOC SOC POC

CON NON OON SON PON

COO NOO OOO SOO POO

COS NOS OOS SOS POS

COP NOP OOP SOP POP

CSC NSC OSC SSC PSC

CSN NSN OSN SSN PSN

CSO NSO OSO SSO PSO

CSS NSS OSS SSS PSS

CSP NSP OSP SSP PSP

CPC NPC OPC SPC PPC

CPN NPN OPN SPN PPN

CPO NPO OPO SPO PPO

CPS NPS OPS SPS PPS

CPP NPP OPP SPP PPP

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, "Advanced Organic Chemistry", 4th Edition, Wiley-Interscience, New York, New York (1992). These arrangements are described in the grid of dots shown in the scheme above. All of the possible arrangements for the five most preferred atoms are shown. Each atom has a variety of acceptable oxidation states. The bonding arrangements underlined are less acceptable and are not preferred.

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

The identification of an appropriate framework geometry and size for ligand domain presentation are important steps in the construction of a multibinding compound with enhanced activity. Systematic spatial searching strategies can be used to aid in the identification of preferred frameworks through an iterative process. FIG. 3 illustrates a useful strategy for determining an optimal framework display orientation for ligand domains. Various other strategies are known to those skilled in the art of molecular design and can be used for preparing compounds of this invention.

As shown in FIG. 3, display vectors around similar central core structures such as a phenyl structure and a cyclohexane structure can be varied, as can the spacing of the ligand domain from the core structure (i.e., the length of the attaching moiety). 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 process may require the use of multiple copies of the same central core structure or combinations of different types of display cores.

The above-described process can be extended to trimers (FIG. 4) and compounds of higher valency.

Assays of each of the individual compounds of a collection generated as described above will lead to a subset of compounds with the desired enhanced activities (e.g., potency, selectivity, etc.). The analysis of this subset using a technique such as Ensemble Molecular Dynamics will provide a framework orientation that favors the properties desired. A wide diversity of linkers is commercially available (see, e.g., Available Chemical Directory (ACD)). Many of the linkers that are suitable for use in this invention fall into this category. Other can be readily synthesized by methods well known in the art and/or are described below.

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

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 or onto the linker, for example, to change the solubility of the multibinding 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 or into the linker enhances the hydrophilicity and water solubility of the multibinding 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 may decrease antigenicity and potentially enhances the overall rigidity of the linker.

Ancillary groups which enhance the water solubility /hydrophilicity of the linker and, accordingly, the resulting multibinding 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., tetraethylenepentamine), and the like) to enhance the water solubility and/ or hydrophilicity of the multibinding compounds of this invention. In preferred embodiments, the ancillary group used to improve water solubility/hydrophilicity will be a polyether . The incorporation of lipophilic ancillary groups within the structure of the linker to enhance the lipophilicity and/or hydrophobicity of the multibinding compounds described herein is also within the scope of this invention. Lipophilic groups useful with the linkers of this invention include, by way of example only, aryl and heteroaryl groups which, as above, may be either unsubstituted or substituted with other groups, but are at least substituted with a group which allows their covalent attachment to the linker. Other lipophilic groups useful with the linkers of this invention include fatty acid derivatives which do not form bilayers in aqueous medium until higher concentrations are reached.

Also within the scope of this invention is the use of ancillary groups which result in the multibinding compound being incorporated or anchored into a vesicle or other membranous structure such as a liposome or a micelle. The term "lipid" refers to any fatty acid derivative that is capable of forming a bilayer or a 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, sulfato, 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 phosphglycerides and sphingolipids, representative examples of which include phosphatidylcholine , phosphatidy lethanolamine , phosphatidy lserine , phosphatidylinositol, phosphatidic acid, palmitoyleoyl phosphatidylcholine, ly sophosphatidylcholine , lysophosphatidyl-ethanolamine , dipalmitoylphosphatidylcholine , dioleoylphosphatidylcholine , distearoyl- phosphatidylcholine or dilinoleoylphosphatidylcholine could be used. 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 which 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 lability is restrained by the presence of rings and/or multiple bonds within the group, 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 which 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.

In preferred embodiments, rigidity is imparted by the presence of cyclic groups (e.g., aryl, heteroaryl, cycloalkyl, heterocyclic, etc.). 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. 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 presenter 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 which allows bonding between the oppositely charged groups. The addition of ancillary groups which are charged, or alternatively, bear a latent charge when deprotected, following addition to the linker, include deprotectation of a carboxyl, hydroxyl, thiol or amino group by a change in pH, oxidation, reduction or other mechanisms known to those skilled in the art which result in removal of the protecting group, is within the scope of this invention.

In view of the above, it is apparent that the appropriate selection of a linker group providing suitable orientation, restricted/unrestricted rotation, the desired degree of hydrophobicity /hydrophilicity, etc. is well within the skill of the art. Eliminating or reducing antigenicity of the multibinding compounds described herein is also within the scope of this invention. In certain cases, the antigenicity of a multibinding compound may be eliminated or reduced by use of groups such as, for example, poly (ethylene glycol).

As explained above, the multibinding compounds describedTierein comprise 2-10 ligands attached to a linker that links the ligands in such a manner that they are presented to the enzyme 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 of the multibinding compound as compared to the same number of ligands made available in monobinding form.

The 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 multivalency, and a more detailed explanation is described 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 multibinding 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 each X is independently the linker. Examples of such bivalent compounds are provided in FIG. 3 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 can X. However, a trimer can also be a radial multibinding compound comprising three ligands attached to a central core, and thus represented as (Lpc, where the linker X could include, for example, an aryl or cycloalkyl group. Illustrations of trivalent and tetravalent compounds of this invention are found in FIG.s 4 and 5 respectively where, again, the shaded circles represent ligands. Tetravalent compounds can be represented in a linear array, e.g. ,

L-X-L-X-L-X-L in a branched array, e.g. ,

L-X-L-X-L I

L

(a branched construct analogous to the isomers of butane — «-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 alkyl, aryl or cycloalkyl derivative as above with four (4) ligands attached to the core linker.

The same considerations apply to higher multibinding compounds of this invention containing 5-10 ligands as illustrated in FIG. 6 where, as before, the shaded circles represent ligands. However, for multibinding agents attached to a central linker such as aryl or cycloalkyl, 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: X ^L

X X

X^

and variants thereof.

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 the following formula:

-X^a-Z-(Y^a-Z)_m-Y^b-Z-X

in which: m is an integer of from 0 to 20;

X^a at each separate occurrence is selected from the group consisting of -O-, -S-, -NR-, -C(O)-, -C(O)O-, -C(O)NR-, -C(S), -C(S)O-, -C(S)NR- or a covalent bond where R is as defined below;

Z is at each separate occurrence is selected from the group consisting of alkylene, substituted alkylene, cycloalkylene, substituted cylcoalkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, cycloalkenylene, substituted cycloalkenylene, arylene, heteroarylene, heterocyclene, or a covalent bond;

Y and Y^b at each separate occurrence are selected from the group consisting of:

-S-S- or a covalent bond; in which: n is 0, 1 or 2; and

R, R' and R" at each separate occurrence are 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.

Additionally, the linker moiety can be optionally substituted at any atom therein by one or more alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl and heterocyclic group.

In one embodiment of this invention, the linker (i.e. , X, X' or X") is selected those shown in Table II: Table II Representative Linkers

Linker

-HN-(CH₂) ₂-NH-C(O)-(CH₂)-C(O)-NH-(CH₂) ₂-NH-

-HN-(CH₂) ₂-NH-C(O)-(CH₂) ₂-C(O)-NH-(CH₂) ₂-NH-

-HN-(CH₂)₂-NH-C(O)-(CH₂)₃-C(O)-NH-(CH₂)₂-NH-

-HN-(CH₂) ₂-NH-C(O)-(CH₂)₄-C(O)-NH-(CH₂) ₂-NH-

-HN-(CH₂) ₂-NH-C(O)-(CH₂)₅-C(O)-NH-(CH₂) ₂-NH-

-HN-(CH₂) ₂-NH-C(O)-(CH₂)₆-C(O)-NH-(CH₂) ₂-NH-

-HN-(CH₂) ₂-NH-C(O)-(CH₂)₇-C(O)-NH-(CH₂) ₂-NH-

-HN-(CH₂) ₂-NH-C(O)-(CH₂)₈-C(O)-NH-(CH₂) ₂-NH-

-HN-(CH₂) ₂-NH-C(O)-(CH₂)₉-C(O)-NH-(CH₂) ₂-NH-

-HN-(CH₂) ₂-NH-C(O)-(CH₂)₁₀-C(O)-NH-(CH₂) ₂-NH-

-HN-(CH₂) ₂-NH-C(O)-(CH₂), ,-C(O)-NH-(CH₂) ₂-NH-

-HN-(CH₂) ₂-NH-C(O)-(CH₂)₁₂-C(O)-NH-(CH₂) ₂-NH-

-HN-(CH₂)₂-NH-C(O)-Z-C(O)-NH-(CH₂) ₂-NH- where Z is 1,2-phenyl

-HN-(CH₂)₂-NH-C(O)-Z-C(O)-NH-(CH₂)₂-NH- where Z is 1,3-phenyl

-HN-(CH₂)₂-NH-C(O)-Z-C(O)-NH-(CH₂)₂-NH- where Z is 1,4-phenyl

-HN-(CH₂)₂-NH-C(O)-Z-O-Z-C(O)-NH-(CH₂)₂-NH- where Z is 1,4-phenyl

-HN-(CH₂)₂-NH-C(O)-(CH₂)₂-CH(NH-C(O)-(CH₂)₈-CH₃)-C(O)-NH-(CH₂)₂- NH-

-HN-(CH₂)₂-NH-C(O)-(CH₂)-O-(CH₂)-C(O)-NH-(CH₂)₂-NH-

-HN-(CH₂) ₂-NH-C(O)-Z-C(O)-NH-(CH₂) ₂-NH- where Z is 5-(«-octadecyloxy)-l,3-phenyl

-HN-(CH₂) ₂-NH-C(O)-(CH₂) ₂-CH(NH-C(O)-Z)-C(O)-NH-(CH₂) ₂-NH- where Z is 4-biphenyl

-HN-(CH₂)₂-NH-C(O)-Z-C(O)-NH-(CH₂)₂-NH- where Z is 5-(n-butyloxy)- 1,3-phenyl

-HN-(CH₂) ₂-NH-C(O)-(CH₂)₈-trαra-(CH-=CH)-C(O)-NH-(CH₂) ₂-NH- Linker

-HN-(CH₂)₂-NH-C(O)-(CH₂)₂-CH(NH-C(O)-(CH₂)_!2-CH₃)-C(O)-NH-(CH₂)₂-

NH-

-HN-(CH₂)₂-NH-C(O)-(CH₂) ₂-CH(NH-C(O)-Z)-C(O)-NH-(CH₂) ₂-NH- where Z is 4-(n-octyl)-phenyl

-HN-(CH₂)-Z-O-(CH₂)₆-O-Z-(CH₂)-NH- where Z is 1,4-phenyl

-HN-(CH₂)₂-NH-C(O)-(CH₂)₂-NH-C(O)-(CH₂)₃-C(O)-NH-(CH₂)₂-C(O)-NH- (CH₂)₂-NH-

-HN-(CH₂) ₂-NH-C(O)-(CH₂) ₂-CH(NH-C(O)-Ph)-C(O)-NH-(CH₂) ₂-NH-

-HN-(CH₂)₂-NH-C(O)-(CH₂)-N+((CH₂)₉-CH₃)(CH,-C(O)-NH-(CH₂)₂-NH₂)- (CH₂)-C(O)-NH-(CH₂) ₂-NH-

-HN-(CH₂)₂-NH-C(O)-(CH₂)-N((CH₂)₉-CH₃)-(CH₂)-C(O)-NH-(CH₂)₂-NH-

-HN-(CH₂) ,-NH-C(O)-(CH₂) ₂-NH-C(O)-(CH₂) ₂-NH-C(O)-(CH₂) ₃-C(O)-NH- (CH₂) ₂-C(O)-NH-(CH₂) ₂-C(O)-NH-(CH₂)₂-NH-

-HN-(CH₂) ₂-NH-C(O)-Z-C(O)-NH-(CH₂) ₂-NH- where Z is 5-hydroxy-l,3-phenyl

In another embodiment of this invention, the linker (i.e. , X, X' or X") has the formula:

wherein each R^a is independently selected from the group consisting of a covalent bond, alkylene, substituted alkylene and arylene; each R^b is independently selected from the group consisting of hydrogen, alkyl and substituted alkyl; and n' is an integer ranging from 1 to about 20. In view of the above description of the linker, it is understood that the term "linker" when used in combination with the term "multibinding compound" includes both a covalently contiguous single linker (e.g. , L-X-L) and multiple covalently non-contiguous linkers (L-X-L-X-L) within the multibinding compound.

Preparation of Multibinding Compounds

The multibinding compounds of this invention can be prepared from readily available starting materials using the following general methods and procedures. It will be 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. 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. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Second Edition, Wiley, New York, 1991, and references cited therein.

Any compound which binds to nitric oxide synthase can be used as a ligand in this invention. As discussed in further detail below, numerous such nitric oxide synthase ligands are known in the art and any of these known compounds or derivatives thereof may be employed as ligands in this invention. Typically, a compound selected for use as a ligand will have at lease one functional group, such as an amino, hydroxyl, thiol or carboxyl group and the like, which allows the compound to be readily coupled to the 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 patents and publications set forth below provide numerous examples of suitably functionalized nitric oxide synthase ligands and intermediates thereof which may be used as ligands in this invention.

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 bind to nitric oxide synthase. Certain sites of attachment of the linker to the ligand are preferred based on known structure- activity relationships. Preferably, the linker is attached to a site on the ligand where structure-activity studies show that a wide variety of substituents are tolerated without loss of enzyme inhibition activity.

A first group of preferred ligands for use in this invention are those ligands having formula IA', IA", IA'", IB', IB", IB'", IC, IC" and ID as follows:

IA'" IA' IA"

wherein

X is selected from the group consisting of O, S; Y is selected from the group consisting of OR¹ , SR¹, NR'R²; R¹ and R² are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, heteroaryl, aryl, heterocyclic, acyl, substituted acyl; R³ , R³ , R^{3 '"}, R⁴ , R⁴ and R⁴ are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, acyl, substituted acyl, aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, heterocyclic and wherein R³ and R⁴ together form a heterocyclic or a heteroaryl;

R⁵ , R^{5 "}, R^{5 '"}, R⁶ , R⁶ and R⁶ are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, alkoxy, substituted alkoxy, alkylthioalkoxy, acylamino, cycloalkyl, substituted cycloalkyl,; and R⁷, R⁸ and R⁸ are selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl; with the proviso that one of R¹, R², R³ , R^3" , R^{3 "}, R⁴ , R^4", R⁴ ,R⁵ , R⁵ , R^5" , R^6', R^6", R^6" , R⁷, R⁸ or R^8" is a covalent linkage to a linker;

IC IC" wherein

R¹⁰ and R¹¹ are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkaryl, alkoxy, substituted alkoxy, alkyalkoxy, acyl, acylamino, amino, substituted amino, aminoacyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, heterocyclic; and R^10" and R^!1 may optionally together form a cycloalkyl, subsituted cycloalkyl or heterocyclic; and R^12', R^12", R¹³ , R^13", R^14', R^14" are selected from the group consisting of hydrogen, alkyl, substituted alkyl, acyl, substituted acyl, aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, heterocyclic and wherein R³ and R⁴ together form a heterocyclic or a heteroaryl; with the proviso that one of Rⁿ R^n", R¹² , R¹² , R¹³ , R^13", R¹⁴ , R^14" is a covalent linkage to a linker; and

wherein

R²⁰ is selected from the group consisting of NH, O, S, NOH, NR²⁴

R²¹, R²², and R²⁴ are independently selected from the group consisting of hydrogen, an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, a substituted alkynyl, an aryl, a heterocyclic, a cycloalkyl and a substituted cycloalkyl;

R²³ is and independently selected from the group consisting of an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, a substituted alkynyl, an aryl, a heterocyclic, a cycloalkyl and a substituted cycloalkyl Z is an alkylene, a substituted alkylene, an amino, a substituted amino, -S-; with the proviso that one of R²¹, R²², R²³, and R²⁴ is a covalent linkage to a linker, and their tautomeric forms and also their pharmaceutically acceptable salts.

Ligands of formula IA', IA", IA'", IB', IB", IB'", IC, IC" and ID (and the precursors thereof) are well-known in the art and can be readily prepared using art-recognized starting materials, reagents and reaction conditions. By way of illustration, the following patents and publications disclose compounds, intermediates and procedures useful in the preparation of ligands of formula IA', IA", IA'", IB', IB", IB'", IC, IC" and ID or related compounds suitable for use in this invention: U.S. Patent No. 5,866,612; U.S. Patent No. 5,723,451; U.S. Patent No. 5,866,569; U.S. Patent No. 5,807,886; U.S. Patent No. 5,854,234; U.S. Patent No. 5,863,931; U.S. Patent No. 5,902,810; U.S. Patent No. 5,821,261; U.S. Patent No. 5,830,917; International Patent Application No. WO'95/09619; Schmidt et al., 1999, Ear. J. Biochem 259:25-31; Bommel et al. , 1998, J. of Biol. Chem. 273:33142-33149; and Hamley and Tinker, 1995, Bioorganis & Medicinal Chemistry Letters 5(15): 1573-1576. Each of these patents and publications is incorporated herein by reference in its entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference in its entirety.

Examples of ligands suitable in this invention are the following:

Pterin scaffold and substituents

Type R₃ R< X

I 1 H H CHj-O-CO (cyclic ^■ R3 R4) 0

II 1 H H 0

H CSNHPh

II 1 COCH(CH₃)₂ Ph Ph n^OT 0

and

and

* In L-NAME the acid function (-CO ) becomes (-COOCH₃)

A representative synthesis of certain ligand precursors is illustrated in the Examples. It will be understood by those skilled in the art that the following methods set forth in the examples may be used to prepare other multibinding compounds of this invention. FIG. 10 - 20 illustrates the synthesis of multibinding compounds of the present invention.

As will be readily apparent to those of ordinary skill in the art, the synthetic procedures described herein or those known in the art may be readily modified to afford a wide variety of compounds within the scope of this invention.

Isolation and Purification of the Compounds

Isolation and purification of the compounds and intermediates described herein can be effected, if desired, by any suitable separation or purification such as, for example, filtration, extraction, crystallization, column chromatography, thin-layer chromatography, thick-layer chromatography, preparative low or high- pressure liquid chromatography or a combination of these procedures. Specific illustrations of suitable separation and isolation procedures can be had by reference to the Examples herein below. However, other equivalent separation or isolation procedures could, of course, also be used. Combinatorial Libraries

The methods described above lend themselves to combinatorial approaches for identifying multimeric compounds which bind nitric oxide synthase and 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 nitric oxide synthase. The only requirement for the ligands chosen is that they are capable of binding to nitric oxide synthase. 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, logP, 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 the nitric oxide synthase 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 nitric oxide synthase ligand bound to its target allows one to identify one or more sites where linker attachment will not preclude the enzyme: ligand interaction. Alternatively, evaluation of ligand/nitric oxide synthase 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 modifying 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 5HT₄ ligand and elements of the M₃ receptor proximal to the formal M₃ antagonist binding site and between the M₃ ligand and elements of the 5HT₄ receptor proximal to the formal 5HT₄ antagonist binding site. Thus, the dimeric compound may be a 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 innocuous 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, such as enzymes. In other instances where high- resolution structural information is not available, 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 A, with more preferred linker distances of 3-12 A. In situations where two binding sites reside on separate (e.g., protein) target sites, preferred linker distances are 20^100 A, with more preferred distances of 30-70 A.

Linker- eometry 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 at 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, polarizability, 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 synthesis Having chosen a set of n ligands (n being determined by the sum of the number of different attachment points for each ligand chosen) and m 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 possible combinations of multibinding compounds:

Al-Al A1-A2 Al-Bl A1-B2 A1-B3 A2-A2 A2-B1 A2-B2 A2-B3 Bl-Bl 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 library 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, analytical, pharmacological, 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 kinetic inhibition analysis for many enzyme targets. In vitro efficacy 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 array(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 and/or activity 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 β-aminohydroxy sulfonyl halide amine sulfonamide carboxyl acid amine amide hydroxyl alkyl/aryl halide ether aldehyde amine/NaCNBH₃ amine ketone amine/NaCNBH₃ amine amine isocyanate urea

Exemplary linkers include the following linkers identified as X-1 through

X-418 as set forth below:

Representative ligands for use in this invention include, by way of example, L-1 which corresponds to any ligand falling within the scope of IA' , IA" , IA'" , L-2 which corresponds to any ligand falling within the scope of IB' , IB", IB'", L-3 which corresponds to any ligand falling within the scope of IC and IC" , and L-4 which corresponds to any ligand falling within the scope of ID.

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 above and the second ligand and linker is selected from the following:

L-l/X-1- L-l/X-2- L-l/X-3- L-l/X-4- L-l/X-5- L-l/X-6-

L-l/X-7- L-l/X-8- L-l/X-9- L-l/X-10- L-l/X-11- L-l/X-12-

L-l/X-13- L-l/X-14- L-l/X-15- L-l/X-16- L-l/X-17- L-l/X-18-

L-l/X-19- L-l/X-20- L-l/X-21- L-l/X-22- L-l/X-23- L-l/X-24- L-l/X-25- L-l/X-26- L-l/X-27- L-l/X-28- L-l/X-29- L-l/X-30-

L-l/X-31- L-l/X-32- L-l/X-33- L-l/X-34- L-l/X-35- L-l/X-36-

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-l/X-43- L-l/X-44- L-l/X-45- L-l/X-46- L-l/X-47- L-l/X-48-

L-l/X-49- L-l/X-50- L-l/X-51- L-l/X-52- L-l/X-53- L-l/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-

L-l/X-61- L-l/X-62- L-l/X-63- L-l/X-64- L-l/X-65- L-l/X-66-

L-l/X-67- L-l/X-68- L-l/X-69- L-l/X-70- L-l/X-71- L-l/X-72-

L-l/X-73- L-l/X-74- L-l/X-75- L-l/X-76- L-l/X-77- L-l/X-78-

L-l/X-79- L-l/X-80- L-l/X-81- L-l/X-82- L-l/X-83- L-l/X-84- 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-l/X-93- L-l/X-94- L-l/X-95- L-l/X-96-

L-l/X-97- L-l/X-98- L-l/X-99- L-l/X-100- L-l/X-10 - L-l/X-102-

L-l/X-103- L-l/X-104- L-l/X-105- L-l/X-106- L-l/X-107- L-l/X-108-

L-l/X-109- L-l/X-110- L-l/X-111- L-l/X-112- L-l/X-113- L-l/X-114- L-l/X-115- L-l/X-116- L-l/X-117- L-l/X-118- L-l/X-119- L-l/X-120-

L-l/X-121- L-l/X-122- L-l/X-123- L-l/X-124- L-l/X-125- L-l/X-126- L-l/X-127- L-l/X-128- L-l/X-129- L-l/X-130- L-l/X-131- L-l/X-132-

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-

L-l/X-145- L-l/X-146- L-l/X-147- L-l/X-148- L-l/X-149- L-l/X-150- L-l/X-151- L-l/X-152- L-l/X-153- L-l/X-154- L-l/X-155- L-l/X-156-

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-l/X-163- L-l/X-164- L-l/X-165- L-l/X-166- L-l/X-167- L-l/X-168-

L-l/X-169- L-l/X-170- L-l/X-171- L-l/X-172- L-l/X-173- L-l/X-174-

L-l/X-175- L-l/X-176- L-l/X-177- L-l/X-178- L-l/X-179- L-l/X-180- L-l/X-181- L-l/X-182- L-l/X-183- L-l/X-184- L-l/X-185- L-l/X-186-

L-l/X-187- L-l/X-188- L-l/X-189- L-l/X-190- L-l/X-191- L-l/X-192-

L-l/X-193- L-l/X-194- L-l/X-195- L-l/X-196- L-l/X-197- L-l/X-198-

L-l/X-199- L-l/X-200- L-l/X-201- L-l/X-202- L-l/X-203- L-l/X-204-

L-l/X-205- L-l/X-206- L-l/X-207- L-l/X-208- L-l/X-209- L-l/X-210- L-l/X-211- L-l/X-212- L-l/X-213- L-l/X-214- L-l/X-215- L-l/X-216-

L-l/X-217- L-l/X-218- L-l/X-219- L-l/X-220- L-l/X-221- L-l/X-222-

L-l/X-223- L-l/X-224- L-l/X-225- L-l/X-226- L-l/X-227- L-l/X-228-

L-l/X-229- L-l/X-230- L-l/X-231- L-l/X-232- L-l/X-233- L-l/X-234-

L-l/X-235- L-l/X-236- L-l/X-237- L-l/X-238- L-l/X-239- L-l/X-240- L-l/X-241- L-l/X-242- L-l/X-243- L-l/X-244- L-l/X-245- L-l/X-246-

L-l/X-247- L-l/X-248- L-l/X-249- L-l/X-250- L-l/X-251- L-l/X-252-

L-l/X-253- L-l/X-254- L-l/X-255- L-l/X-256- L-l/X-257- L-l/X-258-

L-l/X-259- L-l/X-260- L-l/X-261- L-l/X-262- L-l/X-263- L-l/X-264-

L-l/X-265- L-l/X-266- L-l/X-267- L-l/X-268- L-l/X-269- L-l/X-270- L-l/X-271- L-l/X-272- L-l/X-273- L-l/X-274- L-l/X-275- L-l/X-276-

L-l/X-277- L-l/X-278- L-l/X-279- L-l/X-280- L-l/X-281- L-l/X-282-

L-l/X-283- L-l/X-284- L-l/X-285- L-l/X-286- L-l/X-287- L-l/X-288-

L-l/X-289- L-l/X-290- L-l/X-291- L-l/X-292- L-l/X-293- L-l/X-294-

L-l/X-295- L-l/X-296- L-l/X-297- L-l/X-298- L-l/X-299- L-l/X-300- L-l/X-301- L-l/X-302- L-l/X-303- L-l/X-304- L-l/X-305- L-l/X-306-

L-l/X-307- L-l/X-308- L-l/X-309- L-l/X-310- L-l/X-311- L-l/X-312-

L-l/X-313- L-l/X-314- L-l/X-315- L-l/X-316- L-l/X-317- L-l/X-318- L-l/X-319- L-l/X-320- L-l/X-321- L-l/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- 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-l/X-335- L-l/X-336-

L-l/X-337- L-l/X-338- L-l/X-339- L-l/X-340- L-l/X-341- L-l/X-342- L-l/X-343- L-l/X-344- L-l/X-345- L-l/X-346- L-l/X-347- L-l/X-348-

L-l/X-349- L-l/X-350- L-l/X-351- L-l/X-352- L-l/X-353- L-l/X-354-

L-l/X-355- L-l/X-356- L-l/X-357- L-l/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- L-l/X-365- L-l/X-366-

L-l/X-367- L-l/X-368- L-l/X-369- L-l/X-370- L-l/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- L-l/X-383- L-l/X-384-

L-l/X-385- L-l/X-386- L-l/X-387- L-l/X-388- L-l/X-389- L-l/X-390-

L-l/X-391- L-l/X-392- L-l/X-393- L-l/X-394- L-l/X-395- L-l/X-396-

L-l/X-397- L-l/X-398- L-l/X-399- L-l/X-400- L-l/X-401- L-l/X-402- L-l/X-403- L-l/X-404- L-l/X-405- L-l/X-406- 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-l/X-413- L-l/X-414-

L-l/X-415- L-l/X-416- L-l/X-417- L-l/X-418-

L-2/X-1- L-2/X-2- L-2/X-3- L-2/X-4- L-2/X-5- L-2/X-6-

L-2/X-7- L-2/X-8- L-2/X-9- L-2/X-10- L-2/X-11- L-2/X-12- L-2/X-13- L-2/X-14- L-2/X-15- L-2/X-16- L-2/X-17- L-2/X-18-

L-2/X-19- L-2/X-20- L-2/X-21- L-2/X-22- L-2/X-23- L-2/X-24-

L-2/X-25- L-2/X-26- L-2/X-27- L-2/X-28- L-2/X-29- L-2/X-30-

L-2/X-31- L-2/X-32- L-2/X-33- L-2/X-34- L-2/X-35- L-2/X-36-

L-2/X-37- L-2/X-38- L-2/X-39- L-2/X-40- L-2/X-41- L-2/X-42- L-2/X-43- L-2/X-44- L-2/X-45- L-2/X-46- L-2/X-47- L-2/X-48-

L-2/X-49- L-2/X-50- L-2/X-51- L-2/X-52- L-2/X-53- L-2/X-54-

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-2/X-72- L-2/X-73- L-2/X-74- L-2/X-75- L-2/X-76- L-2/X-77- L-2/X-78-

L-2/X-79- L-2/X-80- L-2/X-81- L-2/X-82- L-2/X-83- L-2/X-84-

L-2/X-85- L-2/X-86- L-2/X-87- L-2/X-88- L-2/X-89- L-2/X-90- L-2/X-91- L-2/X-92- L-2/X-93- L-2/X-94- L-2/X-95- L-2/X-96-

L-2/X-97- L-2/X-98- L-2/X-99- L-2/X-100- L-2/X-101- L-2/X-102-

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-

L-2/X-121- L-2/X-122- L-2/X-123- L-2/X-124- L-2/X-125- L-2/X-126-

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-2/X-138-

L-2/X-139- L-2/X-140- L-2/X-141- L-2/X-142- L-2/X-143- L-2/X-144- L-2/X-145- L-2/X-146- L-2/X-147- L-2/X-148- L-2/X-149- L-2/X-150-

L-2/X-151- L-2/X-152- L-2/X-153- L-2/X-154- L-2/X-155- L-2/X-156-

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- L-2/X-173- L-2/X-174- L-2/X-175- L-2/X-176- L-2/X-177- L-2/X-178- L-2/X-179- L-2/X-180-

L-2/X-181- L-2/X-182- L-2/X-183- L-2/X-184- 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- 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- L-2/X-209- L-2/X-210-

L-2/X-211- L-2/X-212- L-2/X-213- L-2/X-214- L-2/X-215- L-2/X-216-

L-2/X-217- L-2/X-218- L-2/X-219- L-2/X-220- L-2/X-221- L-2/X-222-

L-2/X-223- L-2/X-224- L-2/X-225- L-2/X-226- L-2/X-227- L-2/X-228-

L-2/X-229- L-2/X-230- L-2/X-231- L-2/X-232- L-2/X-233- L-2/X-234- L-2/X-235- L-2/X-236- L-2/X-237- L-2/X-238- L-2/X-239- L-2/X-240-

L-2/X-241- L-2/X-242- L-2/X-243- L-2/X-244- L-2/X-245- L-2/X-246-

L-2/X-247- L-2/X-248- L-2/X-249- L-2/X-250- L-2/X-251- L-2/X-252-

L-2/X-253- L-2/X-254- L-2/X-255- L-2/X-256- L-2/X-257- L-2/X-258-

L-2/X-259- L-2/X-260- L-2/X-261- L-2/X-262- L-2/X-263- L-2/X-264- L-2/X-265- L-2/X-266- L-2/X-267- L-2/X-268- L-2/X-269- L-2/X-270-

L-2/X-271- L-2/X-272- L-2/X-273- L-2/X-274- L-2/X-275- L-2/X-276-

L-2/X-277- L-2/X-278- L-2/X-279- L-2/X-280- L-2/X-281- L-2/X-282- L-2/X-283- L-2/X-284- L-2/X-285- L-2/X-286- L-2/X-287- L-2/X-288-

L-2/X-289- L-2/X-290- L-2/X-291- L-2/X-292- L-2/X-293- L-2/X-294-

L-2/X-295- L-2/X-296- L-2/X-297- L-2/X-298- 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- L-2/X-311- L-2/X-312-

L-2/X-313- L-2/X-314- L-2/X-315- L-2/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- L-2/X-323- L-2/X-324-

L-2/X-325- L-2/X-326- L-2/X-327- L-2/X-328- L-2/X-329- L-2/X-330-

L-2/X-331- L-2/X-332- L-2/X-333- L-2/X-334- L-2/X-335- L-2/X-336- L-2/X-337- L-2/X-338- L-2/X-339- L-2/X-340- L-2/X-341- L-2/X-342-

L-2/X-343- L-2/X-344- L-2/X-345- L-2/X-346- L-2/X-347- L-2/X-348-

L-2/X-349- L-2/X-350- L-2/X-351- L-2/X-352- L-2/X-353- L-2/X-354-

L-2/X-355- L-2/X-356- L-2/X-357- L-2/X-358- 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- 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- 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- L-2/X-395- L-2/X-396- L-2/X-397- L-2/X-398- L-2/X-399- L-2/X-400- L-2/X-401- L-2/X-402-

L-2/X-403- L-2/X-404- L-2/X-405- L-2/X-406- L-2/X-407- L-2/X-408-

L-2/X-409- L-2/X-410- L-2/X-411- L-2/X-412- L-2/X-413- L-2/X-414-

L-2/X-415- L-2/X-416- L-2/X-417- L-2/X-418-

L-3/X-1- L-3/X-2- L-3/X-3- L-3/X-4- L-3/X-5- L-3/X-6- L-3/X-7- L-3/X-8- L-3/X-9- L-3/X-10- L-3/X-11- L-3/X-12-

L-3/X-13- L-3/X-14- L-3/X-15- L-3/X-16- L-3/X-17- L-3/X-18-

L-3/X-19- L-3/X-20- L-3/X-21- L-3/X-22- L-3/X-23- L-3/X-24-

L-3/X-25- L-3/X-26- L-3/X-27- L-3/X-28- L-3/X-29^ L-3/X-30-

L-3/X-31- L-3/X-32- L-3/X-33- L-3/X-34- L-3/X-35- L-3/X-36- L-3/X-37- L-3/X-38- L-3/X-39- L-3/X-40- L-3/X-41- L-3/X-42-

L-3/X-43- L-3/X-44- L-3/X-45- L-3/X-46- L-3/X-47- L-3/X-48-

L-3/X-49- L-3/X-50- L-3/X-51- L-3/X-52- L-3/X-53- L-3/X-54- L-3/X-55- L-3/X-56- L-3/X-57- L-3/X-58- L-3/X-59- L-3/X-60-

L-3/X-61- L-3/X-62- L-3/X-63- L-3/X-64- L-3/X-65- L-3/X-66-

L-3/X-67- L-3/X-68- L-3/X-69- L-3/X-70- L-3/X-71- L-3/X-72-

L-3/X-73- L-3/X-74- L-3/X-75- L-3/X-76- L-3/X-77- L-3/X-78- L-3/X-79- L-3/X-80- L-3/X-81- L-3/X-82- L-3/X-83- L-3/X-84-

L-3/X-85- L-3/X-86- L-3/X-87- L-3/X-88- L-3/X-89- L-3/X-90-

L-3/X-91- L-3/X-92- L-3/X-93- L-3/X-94- L-3/X-95- L-3/X-96-

L-3/X-97- L-3/X-98- L-3/X-99- L-3/X-100- L-3/X-101- L-3/X-102-

L-3/X-103- L-3/X-104- L-3/X-105- L-3/X-106- L-3/X-107- L-3/X-108- L-3/X-109- L-3/X-110- L-3/X-111- L-3/X-112- L-3/X-113- L-3/X-114-

L-3/X-115- L-3/X-116- L-3/X-117- L-3/X-118- L-3/X-119- L-3/X-120-

L-3/X-121- L-3/X-122- L-3/X-123- L-3/X-124- L-3/X-125- L-3/X-126-

L-3/X-127- L-3/X-128- L-3/X-129- L-3/X-130- L-3/X-131- L-3/X-132-

L-3/X-133- L-3/X-134- L-3/X-135- L-3/X-136- L-3/X-137- L-3/X-138- L-3/X-139- L-3/X-140- L-3/X-141- L-3/X-142- L-3/X-143- L-3/X-144-

L-3/X-145- L-3/X-146- L-3/X-147- L-3/X-148- L-3/X-149- L-3/X-150-

L-3/X-151- L-3/X-152- L-3/X-153- L-3/X-154- L-3/X-155- L-3/X-156-

L-3/X-157- L-3/X-158- L-3/X-159- L-3/X-160- L-3/X-161- L-3/X-162-

L-3/X-163- L-3/X-164- L-3/X-165- L-3/X-166- L-3/X-167- L-3/X-168- L-3/X-169- L-3/X-170- L-3/X-171- L-3/X-172- L-3/X-173- L-3/X-174-

L-3/X-175- L-3/X-176- L-3/X-177- L-3/X-178- L-3/X-179- L-3/X-180-

L-3/X-181- L-3/X-182- L-3/X-183- L-3/X-184- L-3/X-185- L-3/X-186-

L-3/X-187- L-3/X-188- L-3/X-189- L-3/X-190- L-3/X-191- L-3/X-192-

L-3/X-193- L-3/X-194- L-3/X-195- L-3/X-196- L-3/X-197- L-3/X-198- L-3/X-199- L-3/X-200- L-3/X-201- L-3/X-202- L-3/X-203- L-3/X-204-

L-3/X-205- L-3/X-206- L-3/X-207- L-3/X-208- L-3/X-209- L-3/X-210-

L-3/X-211- L-3/X-212- L-3/X-213- L-3/X-214- L-3/X-215- L-3/X-216-

L-3/X-217- L-3/X-218- L-3/X-219- L-3/X-220- L-3/X-221- L-3/X-222-

L-3/X-223- L-3/X-224- L-3/X-225- L-3/X-226- L-3/X-227- L-3/X-228- L-3/X-229- L-3/X-230- L-3/X-231- L-3/X-232- L-3/X-233- L-3/X-234-

L-3/X-235- L-3/X-236- L-3/X-237- L-3/X-238- L-3/X-239- L-3/X-240-

L-3/X-241- L-3/X-242- L-3/X-243- L-3/X-244- L-3/X-245- L-3/X-246- L-3/X-247- L-3/X-248- L-3/X-249- L-3/X-250- L-3/X-251- L-3/X-252-

L-3/X-253- L-3/X-254- L-3/X-255- L-3/X-256- L-3/X-257- L-3/X-258-

L-3/X-259- L-3/X-260- L-3/X-261- L-3/X-262- L-3/X-263- L-3/X-264-

L-3/X-265- L-3/X-266- L-3/X-267- L-3/X-268- L-3/X-269- L-3/X-270-

L-3/X-271- L-3/X-272- L-3/X-273- L-3/X-274- L-3/X-275- L-3/X-276-

L-3/X-277- L-3/X-278- L-3/X-279- L-3/X-280- L-3/X-281- L-3/X-282-

L-3/X-283- L-3/X-284- L-3/X-285- L-3/X-286- L-3/X-287- L-3/X-288-

L-3/X-289- L-3/X-290- L-3/X-291- L-3/X-292- L-3/X-293- L-3/X-294-

L-3/X-295- L-3/X-296- L-3/X-297- L-3/X-298- L-3/X-299- L-3/X-300-

L-3/X-301- L-3/X-302- L-3/X-303- L-3/X-304- L-3/X-305- L-3/X-306-

L-3/X-307- L-3/X-308- L-3/X-309- L-3/X-310- L-3/X-311- L-3/X-312-

L-3/X-313- L-3/X-314- L-3/X-315- L-3/X-316- L-3/X-317- L-3/X-318-

L-3/X-319- L-3/X-320- L-3/X-321- L-3/X-322- L-3/X-323- L-3/X-324-

L-3/X-325- L-3/X-326- L-3/X-327- L-3/X-328- L-3/X-329- L-3/X-330-

L-3/X-331- L-3/X-332- L-3/X-333- L-3/X-334- L-3/X-335- L-3/X-336-

L-3/X-337- L-3/X-338- L-3/X-339- L-3/X-340- L-3/X-341- L-3/X-342-

L-3/X-343- L-3/X-344- L-3/X-345- L-3/X-346- L-3/X-347- L-3/X-348-

L-3/X-349- L-3/X-350- L-3/X-351- L-3/X-352- L-3/X-353- L-3/X-354-

L-3/X-355- L-3/X-356- L-3/X-357- L-3/X-358- L-3/X-359- L-3/X-360-

L-3/X-361- L-3/X-362- L-3/X-363- L-3/X-364- L-3/X-365- L-3/X-366-

L-3/X-367- L-3/X-368- L-3/X-369- L-3/X-370- L-3/X-371- L-3/X-372-

L-3/X-373- L-3/X-374- L-3/X-375- L-3/X-376- L-3/X-377- L-3/X-378-

L-3/X-379- L-3/X-380- L-3/X-381- L-3/X-382- L-3/X-383- L-3/X-384-

L-3/X-385- L-3/X-386- L-3/X-387- L-3/X-388- L-3/X-389- L-3/X-390-

L-3/X-391- L-3/X-392- L-3/X-393- L-3/X-394- L-3/X-395- L-3/X-396-

L-3/X-397- L-3/X-398- L-3/X-399- L-3/X-400- L-3/X-401- L-3/X-402-

L-3/X-403- L-3/X-404- L-3/X-405- L-3/X-406- L-3/X-407- L-3/X-408-

L-3/X-409- L-3/X-410- L-3/X-411- L-3/X-412- L-3/X-4I3- L-3/X-414-

L-3/X-415- L-3/X-416- L-3/X-417- L-3/X-418-

L-4/X-1- L-4/X-2- L-4/X-3- L-4/X-4- L-4/X-5- L-4/X-6-

L-4/X-7- L-4/X-8- L-4/X-9- L-4/X-10- L-4/X-11- L-4/X-12-

L-4/X-13- L-4/X-14- L-4/X-15- L-4/X-16- L-4/X-17- L-4/X-18- L-4/X-19- L-4/X-20- L-4/X-21- L-4/X-22- L-4/X-23- L-4/X-24-

L-4/X-25- L-4/X-26- L-4/X-27- L-4/X-28- L-4/X-29- L-4/X-30-

L-4/X-31- L-4/X-32- L-4/X-33- L-4/X-34- L-4/X-35- L-4/X-36-

L-4/X-37- L-4/X-38- L-4/X-39- L-4/X-40- L-4/X-41- L-4/X-42- L-4/X-43- L-4/X-44- L-4/X-45- L-4/X-46- L-4/X-47- L-4/X-48-

L-4/X-49- L-4/X-50- L-4/X-51- L-4/X-52- L-4/X-53- L-4/X-54-

L-4/X-55- L-4/X-56- L-4/X-57- L-4/X-58- L-4/X-59- L-4/X-60-

L-4/X-61- L-4/X-62- L-4/X-63- L-4/X-64- L-4/X-65- L-4/X-66-

L-4/X-67- L-4/X-68- L-4/X-69- L-4/X-70- L-4/X-71- L-4/X-72- L-4/X-73- L-4/X-74- L-4/X-75- L-4/X-76- L-4/X-77- L-4/X-78-

L-4/X-79- L-4/X-80- L-4/X-81- L-4/X-82- L-4/X-83- L-4/X-84-

L-4/X-85- L-4/X-86- L-4/X-87- L-4/X-88- L-4/X-89- L-4/X-90-

L-4/X-91- L-4/X-92- L-4/X-93- L-4/X-94- L-4/X-95- L-4/X-96-

L-4/X-97- L-4/X-98- L-4/X-99- L-4/X-100- L-4/X-101- L-4/X-102- L-4/X-103- L-4/X-104- L-4/X-105- L-4/X-106- L-4/X-107- L-4/X-108-

L-4/X-109- L-4/X-110- L-4/X-111- L-4/X-112- L-4/X-113- L-4/X-114-

L-4/X-115- L-4/X-116- L-4/X-117- L-4/X-118- L-4/X-119- L-4/X-120-

L-4/X-121- L-4/X-122- L-4/X-123- L-4/X-124- L-4/X-125- L-4/X-126-

L-4/X-127- L-4/X-128- L-4/X-129- L-4/X-130- L-4/X-131- L-4/X-132- L-4/X-133- L-4/X-134- L-4/X-135- L-4/X-136- L-4/X-137- L-4/X-138-

L-4/X-139- L-4/X-140- L-4/X-141- L-4/X-142- L-4/X-143- L-4/X-144-

L-4/X-145- L-4/X-146- L-4/X-147- L-4/X-148- L-4/X-149- L-4/X-150-

L-4/X-151- L-4/X-152- L-4/X-153- L-4/X-154- L-4/X-155- L-4/X-156-

L-4/X-157- L-4/X-158- L-4/X-159- L-4/X-160- L-4/X-161- L-4/X-162- L-4/X-163- L-4/X-164- L-4/X-165- L-4/X-166- L-4/X-167- L-4/X-168-

L-4/X-169- L-4/X-170- L-4/X-171- L-4/X-172- L-4/X-173- L-4/X-174-

L-4/X-175- L-4/X-176- L-4/X-177- L-4/X-178- L-4/X-179- L-4/X-180-

L-4/X-181- L-4/X-182- L-4/X-183- L-4/X-184- L-4/X-185- L-4/X-186-

L-4/X-187- L-4/X-188- L-4/X-189- L-4/X-190- L-4/X-191- L-4/X-192- L-4/X-193- L-4/X-194- L-4/X-195- L-4/X-196- L-4/X-197- L-4/X-198-

L-4/X-199- L-4/X-200- L-4/X-201- L-4/X-202- L-4/X-203- L-4/X-204-

L-4/X-205- L-4/X-206- L-4/X-207- L-4/X-208- L-4/X-209- L-4/X-210- L-4/X-211- L-4/X-212- L-4/X-213- L-4/X-214- L-4/X-215- L-4/X-216-

L-4/X-217- L-4/X-218- L-4/X-219- L-4/X-220- L-4/X-221- L-4/X-222-

L-4/X-223- L-4/X-224- L-4/X-225- L-4/X-226- L-4/X-227- L-4/X-228-

L-4/X-229- L-4/X-230- L-4/X-231- L-4/X-232- L-4/X-233- L-4/X-234- L-4/X-235- L-4/X-236- L-4/X-237- L-4/X-238- L-4/X-239- L-4/X-240-

L-4/X-241- L-4/X-242- L-4/X-243- L-4/X-244- L-4/X-245- L-4/X-246-

L-4/X-247- L-4/X-248- L-4/X-249- L-4/X-250- L-4/X-251- L-4/X-252-

L-4/X-253- L-4/X-254- L-4/X-255- L-4/X-256- L-4/X-257- L-4/X-258-

L-4/X-259- L-4/X-260- L-4/X-261- L-4/X-262- L-4/X-263- L-4/X-264- L-4/X-265- L-4/X-266- L-4/X-267- L-4/X-268- L-4/X-269- L-4/X-270-

L-4/X-271- L-4/X-272- L-4/X-273- L-4/X-274- L-4/X-275- L-4/X-276-

L-4/X-277- L-4/X-278- L-4/X-279- L-4/X-280- L-4/X-281- L-4/X-282-

L-4/X-283- L-4/X-284- L-4/X-285- L-4/X-286- L-4/X-287- L-4/X-288-

L-4/X-289- L-4/X-290- L-4/X-291- L-4/X-292- L-4/X-293- L-4/X-294- L-4/X-295- L-4/X-296- L-4/X-297- L-4/X-298- L-4/X-299- L-4/X-300-

L-4/X-301- L-4/X-302- L-4/X-303- L-4/X-304- L-4/X-305- L-4/X-306-

L-4/X-307- L-4/X-308- L-4/X-309- L-4/X-310- L-4/X-311- L-4/X-312-

L-4/X-313- L-4/X-314- L-4/X-315- L-4/X-316- L-4/X-317- L-4/X-318-

L-4/X-319- L-4/X-320- L-4/X-321- L-4/X-322- L-4/X-323- L-4/X-324- L-4/X-325- L-4/X-326- L-4/X-327- L-4/X-328- L-4/X-329- L-4/X-330-

L-4/X-331- L-4/X-332- L-4/X-333- L-4/X-334- L-4/X-335- L-4/X-336-

L-4/X-337- L-4/X-338- L-4/X-339- L-4/X-340- L-4/X-341- L-4/X-342-

L-4/X-343- L-4/X-344- L-4/X-345- L-4/X-346- L-4/X-347- L-4/X-348-

L-4/X-349- L-4/X-350- L-4/X-351- L-4/X-352- L-4/X-353- L-4/X-354- L-4/X-355- L-4/X-356- L-4/X-357- L-4/X-358- L-4/X-359- L-4/X-360-

L-4/X-361- L-4/X-362- L-4/X-363- L-4/X-364- L-4/X-365- L-4/X-366-

L-4/X-367- L-4/X-368- L-4/X-369- L-4/X-370- L-4/X-371- L-4/X-372-

L-4/X-373- L-4/X-374- L-4/X-375- L-4/X-376- L-4/X-377- L-4/X-378-

L-4/X-379- L-4/X-380- L-4/X-381- L-4/X-382- L-4/X-383- L-4/X-384- L-4/X-385- L-4/X-386- L-4/X-387- L-4/X-388- L-4/X-389- L-4/X-390-

L-4/X-391- L-4/X-392- L-4/X-393- L-4/X-394- L-4/X-395- L-4/X-396-

L-4/X-397- L-4/X-398- L-4/X-399- L-4/X-400- L-4/X-401- L-4/X-402- L-4/X-403- L-4/X-404- L-4/X-405- L-4/X-406- L-4/X-407- L-4/X-408- L-4/X-409- L-4/X-410- L-4/X-411- L-4/X-412- L-4/X-413- L-4/X-414- L-4/X-415- L-4/X-416- L-4/X-417- L-4/X-418-

Pharmaceutical Formulations

When employed as pharmaceuticals, the compounds of this invention are usually administered in the form of pharmaceutical compositions. These compounds can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal. These compounds are effective as both injectable and oral compositions. Such compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound.

This invention also includes pharmaceutical compositions which contain, as the active ingredient, one or more of the compounds described herein associated with pharmaceutically acceptable carriers. In making the compositions of this invention, the active ingredient is usually mixed with an excipient, 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, it 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.

In preparing a formulation, it may be necessary to mill the active compound to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it ordinarily is milled to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size is normally adjusted by milling to provide a substantially uniform distribution in the formulation, e.g. about 40 mesh.

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.

The compositions are preferably formulated in a unit dosage form, each dosage containing from about 0.001 to about 1 g, more usually about 1 to about 30 mg, of the active ingredient. 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. Preferably, the compound of formula I above is employed at no more than about 20 weight percent of the pharmaceutical composition, more preferably no more than about 15 weight percent, with the balance being pharmaceutically inert carrier(s).

The active compound is effective over a wide dosage range and is generally administered in a pharmaceutically effective amount. It, will be under stood, however, that the amount of the compound actually 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. This solid preformulation is then subdivided into unit dosage forms of the type described above containing from, for example, 0.1 to about 500 mg of the active ingredient of the present invention.

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 numbef 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 (mg/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 Microcry stalline 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 poly vinylpyrrolidone 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:

Quantity Ingredient (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 7

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

A formulation may be prepared as follows:

Quantity Ingredient (rug/capsule 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 1-10 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 known in the art. See, e.g. , U.S. Patent 5,023,252, issued June 11, 1991, herein incorporated by reference in its entirety. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.

Other suitable formulations for use in the present invention can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, PA, 17th ed. (1985).

Utility The multibinding compounds of this invention inhibit one or more of the nitric oxide synthases, enzymes which synthese nitric oxide and L-citrulline from L-arginine. Accordingly, the multibinding compounds and pharmaceutical compositions of this invention are useful in the treatment and prevention of various disorders mediated by certain of the nitric oxide synthases, such as Alzheimer's disease, cancer, malaria, Parkinsons, diabetes, asthma, alergic rhinitis, sunburn, erectile disfunction, stroke, long term depression, chronic inflammation, arthritis, pain perception, virus induced encephalopathy, morphine withdrawal, muscular dystrophy, neurodegeneration, hypertension, aids dementia, colitis, crohn's disease, toxic megacolon, multiple sclerosis, bacterial meningitis, migraine, hypercholesterolemia, sepsis. Preferably, the multibinding compounds inhibit either only iNOS or n NOS.

When used in treating or ameliorating such conditions, the compounds of this invention are typically delivered to a patient in need of such treatment by a pharmaceutical composition comprising a pharmaceutically acceptable diluent and an effective amount of at least one compound of this invention. The amount of compound administered to the patient will vary depending upon what compound and/or composition is being administered, the purpose of the administration, such as prophylaxis or therapy, the state of the patient, the manner of administration, and the like. In therapeutic applications, compositions are administered to a patient already suffering from, for example, sepsis in an amount sufficient to at least partially reduce the condition. Amounts effective for this use will depend on the judgment of the attending clinician depending upon factors such as the degree or severity of the inflammation in the patient, the age, weight and general condition of the patient, and the like. The pharmaceutical compositions of this invention may contain more than one compound of the present invention.

As noted above, the compounds administered to a patient are in the form of pharmaceutical compositions described above which can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, etc.. These compounds are effective as both injectable and oral deliverable pharmaceutical compositions. Such compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound.

The multibinding compounds of this invention can also be administered in the form of pro-drugs, i.e., as derivatives which are converted into a biologically active compound in vivo. Such pro-drugs will typically include compounds in which, for example, a carboxylic acid group, a hydroxyl group or a thiol group is converted to a biologically liable group, such as an ester, lactone or thioester group which will hydrolyze in vivo to reinstate the respective group.

The following synthetic and biological examples are offered to illustrate this invention and are not to be construed in any way as limiting the scope of this invention. Unless otherwise stated, all temperatures are in degrees Celsius. EXAMPLES

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

DMF N, N-dimethy If ormamide

DMSO dimethylsulfoxide

EDTA ethylenediaminetetraacetic acid g gram mg milligram min minute mL milliliter mm millimeter mmol millimol N normal

THF tetrahydrofuran μL microliters μm microns TLC thin layer chromatography LAH

Example A-l Preparation of Synthon C. (Figure 8)

A solution of 100 mmols of Synthon B (Japanese Patent Application Publication No. 61,189,247, Application No. 85/28,307; Chemical Abstracts 106:49617c) in 100 mL of THF with 200 mmols of triethylamine is treated at room temperature with 200 mmols of trifluoroacetic anhydride. After 1 hr., the resulting mixture is slowly added to 200 mL of THF which has been saturated with anhydrous ammonia. After 1 hr., the reaction mixture is concentrated and the residue partitioned between ethyl acetate and water. After washing with water and saturated sodium carbonate, the organic layer is dried over sodium sulfate and the solvent removed in vacuo. The resulting diamide may be purified by chromatography as necessary.

A solution of 120 mL of 1 N LAH in THF diluted with 100 mL of THF is cooled under N₂ to -20°C and a solution of 50 mmols of the above diamide in 50 mL of THF is slowly added. The cooling is removed and the mixture allowed to warm to room temperature then heated to reflux. The reaction is followed by TLC and when judged complete, it is cooled and 4.5 mL of water followed by 4.5 mL of 15% NaOH followed by 13.5 mL of water is slowly added. The resulting mixture is filtered and the cake washed well with THF. The solvent is removed to afford Synthon C which is purified as necessary by chromatography.

Example A-2 Preparation of Synthon G. (Figure 9)

A mixture of 100 mmols of 4-nitrophenol, 100 mmols of 2- bromoethylamine hydrobromide (or a compound where the amine is protected by a protecting group such as BOC) and 200 mmols of potassium carbonate in 50 mL of DMF is warmed as necessary and the reaction followed byTLC. When judged complete, the reaction is partitioned between water and ethyl acetate. After back extraction of the aqueous layer with ethyl acetate the combined organic phases are washed with water dried over sodium sulfate and filtered. To the filtrate is added 100 mmols of triethylamine and 100 mmols of trifluoroacetic anhydride. After lhr., the reaction is washed with water dried over sodium sulfate and filtered. The filtrate is hydrogenated in the usual manner at one atmos. H₂ using 10% Pd/C. When the reduction is complete, the reaction is filtered and 100 mmols of benzoylisothiocyanate is added. When the reaction is complete, the solvent is removed and replaced with 100 mL of methanol and 40 mL of 5 N_ NaOH added. The reaction is warmed as necessary and followed by TLC. When complete, the reaction is concentrated and the residue partitioned between ethyl acetate and water. The organic phase is washed with water, dried over sodium sulfate, filtered and the solvent removed to afford Synthon G which is purified as necessary by crystallization or chromatography.

Example 1 Preparation of a compound of Compound I. (Figure 10)

A. A solution of 20 mmols of Synthon B (Japanese Patent Application

Publication No. 61,189,247, Application No. 85/28,307; Chemical Abstracts 106:49617c) in 100 mL of THF with 80 mmols of triethylamine is treated at room temperature with 40 mmols of trifluoroacetic anhydride. After 1 hr., a solution of 40 mmols of Synthon A (Koster S. et al., Eur. I. Biochem 231:414 (1995)) is added and the reaction followed by TLC. When it is judged complete, the mixture is concentrated and the residue partitioned between water and ethyl acetate. After washing with water and saturated sodium carbonate^" the organic layer is dried over sodium sulfate and the solvent removed in vacuo. The residue is purified by chromatography to afford material of Compound I. — Ill—

B. Other analogs of Compound I may be prepared in a similar fashion by using alternative diacid linker molecules.

Example 2 Preparation of a compound of Compound II. (Figure 11)

A. A mixture of 20 mmols of Synthon G (Example A-2) and 10 mmols of Synthon C (Example A-1) in 20 mL of DMF is stirred an room temperature for 24 hr. The reaction mixture is partitioned between isopropyl acetate and 40 mL of 1 N NaOH and the organic layer washed well with water then dried over sodium sulfate and the solvent removed. The residue is purified by chromatography to afford material of Compound II.

B. Other analogs of Compound II may be prepared in a similar fashion by using alternative diamine linker molecules.

Example 3 Preparation of a compound of Compound III. (Figure 12)

A. A solution of 10 mmols of Synthon C (prepared in Example A-1) in 25 mL of THF with 40 mmols of triethylamine is treated at room temperature with 20 mmols of 4-nitrophenyl chloroformate. After lhr., 20 mmols of o- phenylenediamine is added and the reaction heated to reflux and followed by TLC. When judged complete, the mixture is concentrated and partitioned between saturated sodium carbonate and isopropyl acetate. After washing with water and saturated sodium carbonate, the organic layer is dried over sodium sulfate and the solvent removed in vacuo. The residue is purified by chromatography to afford material of Compound III.

B. Other analogs of Compound III may be prepared in a similar fashion by using alternative diamine linker molecules or using substituted phenylene diamines.

Example 4 Preparation of a compound of Compound IV. (Figure 13)

A. A mixture of 20 mmols of penta(ethylene glycol), 38 mmols of 5-fluoro-2- nitroaniline and 40 mmols potassium t-butoxide is warmed as necessary and the reaction followed by TLC. When judged complete, the mixture is partitioned between water and isopropyl acetate. After washing with water, the organic layer is dried over sodium sulfate and the solvent removed in vacuo. The residue is purified by chromatography to afford nitroaniline intermediate.

This material is hydrogenated at atmospheric pressure in the usual manner in ethanol with 10% Pd/C and followed by TLC. When judged complete, the mixture is filtered and to the filtrate is added two equivalents of cyanogen bromide and two equivalents of triethylamine and the mixture heated to reflux. When judged complete by TLC, the mixture is concentrated and the residue partitioned between ethyl acetate and water. After washing with water, the organic layer is dried over sodium sulfate and the solvent removed in vacuo. The residue is purified by chromatography to afford material of Compound IV.

B. Other analogs of Compound IV may be prepared in a similar fashion by using alternative diol linker molecules or using substituted 5-fluoro-2-nitro anilines.

Example 5 Preparation of a compound of Compound V. (Figure 14)

A. A mixture of 20 mmols of Synthon C (Example A-1), 10 mmols of 5- fluoro-2-nitroaniline and 10 mmols potassium t-butoxide in 10 mL of dioxane is warmed as necessary and the reaction followed by TLC. When judged complete, the mixture is concentrated and the residue partitioned between ethyl acetate and water. After washing with water, the organic layer is dried over sodium sulfate and the solvent removed in vacuo. The residue is purified by chromatography to afford the mono alkylated intermediate.

A solution of 20 mmols of the above intermediate in 25 mL of THF with

40 mmols of triethylamine is treated at room temperature with 20 mmols of 4- nitrophenyl chloroformate. After lhr., 20 mmols of o-phenylenediamine is added and the reaction heated to reflux and followed by TLC. When judged complete, the mixture is concentrated and partitioned between sat. sodium carbonate and isopropyl acetate. After washing with water and sat. sodium carbonate, the organic layer is dried over sodium sulfate and the solvent removed in vacuo. The residue is purified by chromatography to afford the mono benzimidazole intermediate.

This material is hydrogenated at atmospheric pressure in the usual manner in ethanol with 10% Pd/C and followed by TLC. When judged complete, the mixture is filtered and to the filtrate is added one equivalent of cyanogen bromide and one equivalent of triethylamine and the mixture heated to reflux. When judged complete by TLC, the mixture is concentrated and the residue partitioned between ethyl acetate and water. After washing with water, the organic layer is dried over sodium sulfate and the solvent removed in vacuo. The residue is purified by chromatography to afford material of Compound V.

B. Other analogs of Compound V may be prepared in a similar fashion by using alternative diamine linker molecules or other substituted nitroanilines, or other substituted phenyldiamines.

Example 6 Preparation of a compound of Compound VI. (Figure 15)

A. A solution of 20 mmols of 1,3-diaminopropane in 25 mL of THF with 80 mmols of triethylamine is treated at room temperature with 40 mmols of 4- nitrophenyl chloroformate. After lhr., 40 mmols of o-phenylenediamine is added and the reaction heated to reflux and followed by TLC. When judged complete, the mixture is concentrated and partitioned between saturated sodium carbonate and isopropyl acetate. After washing with water and saturated sodium carbonate, the organic layer is dried over sodium sulfate and the solvent removed in vacuo. The residue is purified by chromatography to afford material of Compound VI.

B. Other analogs of Compound VI may be prepared in a similar fashion by using alternative diamine linker molecules.

Example 7 Preparation of a compound of Compound VII. (Figure 16)

A. A mixture of 20 mmols of 1 ,4-butanediol 38 mmols of 5-fluoro-2- nitroaniline and 40 mmols potassium t-butoxide is warmed as necessary and the reaction followed by TLC. When judged complete, the mixture is partitioned between water and isopropyl acetate. After washing with water, the organic layer is dried over sodium sulfate and the solvent removed in vacuo. The residue is purified by chromatography to afford nitroaniline intermediate.

This material is hydrogenated at atmospheric pressure in the usual manner in ethanol with 10% Pd/C and followed by TLC. When judged complete, the mixture is filtered and to the filtrate is added two equivalents of cyanogen bromide and two equivalents of triethylamine and the mixture heated to reflux. When judged complete by TLC, the mixture is concentrated and the residue partitioned between ethyl acetate and water. After washing with water, the organic layer is dried over sodium sulfate and the solvent removed in vacuo. The residue is purified by chromatography to afford material of Compound VII.

B. Other analogs of Compound VII may be prepared in a similar fashion by using alternative diol linker molecules.

Example 8 Preparation of a compound of Compound VIII. (Figure 17)

A. A mixture of 20 mmols of ethylenediamine 10 mmols of 5-fluoro-2- nitroaniline and 10 mmols potassium t-butoxide in 10 mL of dioxane is warmed as necessary and the reaction followed by TLC. When judged complete, the mixture is concentrated and the residue partitioned between ethyl acetate and water. After washing with water, the organic layer is dried over sodium sulfate and the solvent removed in vacuo. The residue is purified by chromatography to afford the mono alkylated intermediate.

A solution of 20 mmols of the above intermediate in 25 mL of THF with 40 mmols of triethylamine is treated at room temperature with 20 mmols of 4- nitrophenyl chloroformate. After lhr., 20 mmols of o-phenylenediamine is added and the reaction heated to reflux and followed by TLC. When judged complete, the mixture is concentrated and partitioned between sat. sodium carbonate and isopropyl acetate. After washing with water and sat. sodium carbonate, the orgamc layer is dried over sodium sulfate and the solvent removed in vacuo. The residue is purified by chromatography to afford the mono benzimidazole intermediate.

This material is hydrogenated at atmospheric pressure in the usual manner in ethanol with 10% Pd/C and followed by TLC. When judged complete, the mixture is filtered and to the filtrate is added one equivalent of cyanogen bromide and one equivalent of triethylamine and the mixture heated to reflux. When judged complete by TLC, the mixture is concentrated and the residue partitioned between ethyl acetate and water. After washing with water, the organic layer is dried over sodium sulfate and the solvent removed in vacuo. The residue is purified by chromatography to afford material of Compound VIII.

B. Other analogs of Compound VIII may be prepared in a similar fashion by using alternative diamine linker molecules.

Example 9 Preparation of a compound of Compound IX. (Figure 18)

A. A reaction flask is charged with 20 mmols of Synthon D (Roth, B et al., J. Am. Chem Soc. (1951) Vol. 73, 2869) and 25 mmols of Synthon G (Example A- 2) and 20 mL of dioxane added. The flask is equipped for distillation and heated to afford a slow rate of reflux. The reaction is followed by TLC and fresh dioxane is added as needed. When judged complete, the solvent is removed to afford the thioureido intermediate which is purified as necessary by crystallization or chromatography. A solution of 10 mmols of this intermediate in 5 mL of acetonitrile is treated with 12 mmols of iodoethane. When complete, the mixture is concentrated and partitioned between ethyl acetate and sat. sodium bicarbonate. The organic phase is washed with water, dried over sodium sulfate and the solvent removed in vacuo. The residue is purified by chromatography to afford material of Compound IX.

B. In a similar fashion, other analogs of Compound IX may be prepared by using other amino linker molecules.

Example 10 Preparation of a compound of Compound X. (Figure 19)

A. A mixture of 100 mmols of Synthon E (McFadden, H, et al., Z. Naturforsch, C. Biosci (1990) vol. 45, 196) and 100 mmols of 4-chloro-2,6- diaminopyrimidine in 50 mL of tert-butanol with 100 mmols of potassium tert- butoxide is heated to reflux for two hours. The reaction is then neutralized with acetic acid and concentrated to give crude intermediate 1. This material is partitioned between ether and water and the ether layer washed with water. To the organic layer is added 100 mL of sodium nitrite followed by a small amount of water to dissolve any precipitates. The reaction is then acidified to pH 3 by the addition of acetic acid. The resulting precipitate is isolated and washed with ether and water and purified as necessary to give intermediate 2.

Reduction of intermediate 2 (50 mmols) in THF is carried out in the usual way at atmos. pres. and when complete the reaction mixture is filtered under a nitrogen atmosphere. To the filtrate is added 50 mmols of benzoylisothiocyanate and when reaction is complete, 25 mL of 2 N NaOH is added. When hydrolysis of the benzoyl group is complete, The mixture is concentrated in vacuo and the vacuum broken with nitrogen. Under nitrogen, the residue is partitioned between water and methylene chloride and the organic solution isolated and the solvent removed. The residue is dissolved in 100 mL of DMF and 3.7g of glyoxal- hydrate trimer added and the mixture stirred at room temperature for three days. Any insoluble material is removed by filtration and the filtrate diluted with water and extracted with 3X ethyl acetate. The combined extracts are washed with water, dried over sodium sulfate and the solvent removed to afford intermediate 3 which is purified as necessary by crystallization or chromatography. It is important to exclude air from the above series of reactions as the triamine intermediates are easily oxidized.

A solution of intermediate 3 (20 mmols) in 10 mL of acetonitrile is treated with 20 mmols of iodoethane and the reaction followed by TLC. When complete, the mixture is concentrated and partitioned between ethyl acetate and sat. sodium bicarbonate. The organic phase is washed with water, dried over sodium sulfate and the solvent removed in vacuo. The residue is purified by chromatography or crystallization to afford material of Compound X.

B. In a similar fashion, other analogs of Compound X may be prepared by using other alcohol linker molecules or using other substituted -diketones. Example 11 Preparation of a compound of Compound XL (Figure 20)

A. A mixture of 50 mmols of Synthon F (Montgomery, J. et al., J. Heterocyclic Chem. (1979) Vol. 16, 537) and 50 mmols of 5-fluoro-2- nitroaniline and 50 mmols potassium t-butoxide in 30 mL of dioxane is warmed as necessary and the reaction followed by TLC. When judged complete, the mixture is concentrated and the residue partitioned between ethyl acetate and water. After washing with water, the orgamc layer is dried over sodium sulfate and the solvent removed in vacuo. The residue is purified by chromatography to afford the mono alkylated nitroaniline intermediate.

The above intermediate is hydrogenated in the usual way at atmos. pres. using 10% Pd/C to give the triamine intermediate.

A solution of the triamine ( 30 mmols) in 30 mL of THF is treated with 60 mmols of benzoylisothiocyanate and the reaction followed by TLC. When judged complete, 50 mL of 1 N NaOH is added and the mixture heated to reflux for 20 minutes. After cooling, 55 mL of 1 N HCl is added and the mixture concentrated in vacuo. The residue is partitioned between ethyl acetate and water and the organic phase dried over sodium sulfate and the solvent removed. The residue is purified by crystallization or chromatography to afford the bisthioureido intermediate. A solution of the bisthioureido (10 mmols) in 10 mL of acetonitrile is treated with 20 mmols of iodoethane and the reaction followed by TLC. When complete, the mixture is concentrated and partitioned between ethyl acetate and sat. sodium bicarbonate. The organic phase is washed with water, dried over sodium sulfate and the solvent removed in vacuo. The residue is purified by chromatography to afford material of Compound XL

B. In a similar fashion, other analogs of Compound XI may be prepared by using other nitrophenoxy alcohol linker molecules.

Example B-l Nitric Oxide Synthase Assay

Isolation and Purification of Nitric Oxide Synthases Methods demonstrating the isolation and purification of all three isoforms of NOS have been published and reviewed in U. Forstermann, et al. , in Methods in Enzymology, Vol. 233, L. Packer, ed., Academic Press, NY, 1994, Ch. 26, pp. 258-264. Cloned and expressed NOS has also been demonstrated and reviewed in C. J Lowenstein and S. H. Snyder in Methods in Enzymology, Vol. 233, L. Packer, ed., Academic Press, NY, 1994, Ch. 26, pp. 264-269.

Assay Protocol for NOS activity

Various assays for NOS activity have been reported in the literature and are reviewed in the following: M. E. Murphy and E. Noack in Methods in Enzymology, Vol. 233, L. Packer, ed., Academic Press, NY, 1994, Ch. 26, pp. 240-250 and J. M. Hevel and M. A. Marietta in Methods in Enzymology, Vol. 233, L. Packer, ed., Academic Press, NY, 1994, Ch. 26, pp. 250-258 each of which is incorporated by reference herein. Details for one assay protocol to measure NOS activity is set forth in U.S. Patent No. 5,821,261, which is incorporated by reference herein as follows: NOS activity is measured as the formation of L-[2,3,4,5-³ HjCitrulline from L-[2,3,4,5-³ H] Arginine. The incubation buffer (100 μL) contains; 100 mM TES, pH 7.5, 5 μM FAD, 5 μM FMN, 10 μM BIL, 0.5 mM NADPH, 0.5 mM DTT, 0.5 mg/mL BSA, 2 mM CaCl₂, 10 μg/mL calmodulin (bovine), 1 μM L-Arg, 0.2 μCi L-[2,3,4,5-³ H]Arg, and the inhibitor in aqueous DMSO (max. 5%). The reaction is initiated by addition of enzyme. Incubations are performed at room temperature for 30 minutes and stopped by the addition of an equal volume of quenching buffer consisting of 200 mM sodium citrate, pH 2.2, 0.02% sodium azide. Reaction products are separated by passing through a cation exchange resin and quantitated as cpm by scintillation counting. Percent inhibition is calculated relative to enzyme incubated without inhibitor according to: % inhibition =100X (cpm L-[2,3,4,5-³ H]Cit with inhibitor/cpm L-[2,3,4,5-³ H]Cit without inhibitor).

Example B-2 Sepsis Assay

A protocol for evaluating the effect of treatment with a nitric oxide synthase inhibitor on systemic hypotension during sepsis in described in Pedoto et al. , Crit. Care Med Vol. 26, No. 12, 2021-2028 (1998) as follows:

Adult male Spraque-Dawley rats (Taconic Germantown, NY) weighing 350 to 450 g are anesthetized with an intraperitoneal injection of sodium pentobarbitoal (50 mg/kg). Body temperature is monitored using a rectal probe. A midline incision on the ventral surface of the neck is made and The trachea is cannulated. Animals are mechanically ventilated with room air using a rodent ventilator (Model 683, Harvard Apparatus, South Natick MA) with a tidal volume of 3 mL and a respiratory rate of 45 breaths/min. Airway pressure (Paw) is monitored by a Gould pressure transducer (Gould Inc. Cleveland OH) and recorded on a chart paper recorder (7D polygraph, Grass Instruments, Quincy MA) The experiments are conducted at ambient room temperature and no attempt is made to alter the animal temperature with an exogenous source of heat. The external jugular vein is cannulated and anethesia is maintained with continuous infusion of sodium pentobarbital at a rate of 5 mg/kg/hr (infused volume of 2 mL/hr). The carotid artery is cannulated and connected to a pressure transducer for systemic blood pressure recording. The mean systemic arterial pressure (MAP) is averaged electronically. The animals are heparinized (1000 units/kg) and baseline measurements of blood pressure, arterial blood gases and pH, Paw and exhaled nitric oxide (ENO) are obtained. Exhaled gas is collected for > 3 mins. using a 1-L-poly vinyl bag connected to the expiratory port of the ventilator, after collecting > 0.5 L of exhaled gas, the bag is removed and the concentration of NO in the exhaled gas is measured using a chemiluminexcence NO analyzer (270B, Sievers, Boulder CO). The NO analyzer is calibrated daily using nitrogen for a zero and a mixture of 248 ppb NO in nitrogen. Room air is monitored daily and when the NO concentration in room airs exceeds 5 ppb, the rats are ventilated from bag containing room air with zero NO.

With the exception of animals in the control group, all rats receive a slow

(10 - 15 min.) intravenous injection of Escherichia coli lipopolysaccharide (LPS, 100 mg/kg) (L4130, Sigma Chemical, st. Louis MO) diluted in normal saline. All animals are monitored for a total of 5 hrs after receiving LPS. One hour after LPS injection, the animals are injected with the inhibitor.

MPA, Paw and ENO concentrations are measured every 30 mins. Arterial blood gases and pH are measured every 30 mins. for the first 2 hours and every hour thereafter, using a blood gas analyzer maintained at 37 °C (ABL5 radiometer, Copenhagen, Denmark). Using a method similar to this, the effectiveness of certain compounds of the invention for the treatment of sepsis may be measured.

Claims

WHAT IS CLAIMED IS:

1. A multibinding compound comprising from 2 to 10 ligands covalently attached to one or more linkers wherein each of said ligands independently comprises a moiety capable of binding a nitric oxide synthase; with the proviso that the multibinding compound is not formula III or IV:

wherein R³⁵ is hydrogen or C ₈ hydrocarbyl;

T is a .₈ hydrocarbyl group optionally containing a 5- or 6-membered heterocyclic ring or T is a .₄ hydrocarbyl group containing a phenylene ring; and pharmaceutically-acceptable salts thereof.

2. A multibinding compound of formula I:

( )_p(X)_q

wherein each L is independently a ligand comprising a moiety capable of binding a nitric oxide synthase; each X is independently a linker; p is an integer of from 2 to 10; and q is an integer of from 1 to 20; with the proviso that the multibinding compound is not formula III or IV:

wherein R³⁵ is hydrogen or C _s hydrocarbyl; T is a C,._g hydrocarbyl group optionally containing a 5- or 6-membered heterocyclic ring or T is a .₄ hydrocarbyl group containing a phenylene ring; and pharmaceutically-acceptable salts thereof.

3. The multibinding compound of Claim 2 wherein q is less than p.

4. The multibinding compound of Claim 3 wherein each ligand is independently selected from the group consisting of:

(a) a compound selected from the group consisting of formula IA', IA", IA'", IB', IB", IB'":

IA"

IA' IA"

wherein

X is selected from the group consisting of O, S; Y is selected from the group consisting of OR¹ , SR¹, NR*R²; R¹ and R² are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substimted alkenyl, alkynyl, substimted alkynyl, cycloalkyl, heteroaryl, aryl, heterocyclic, acyl, substituted acyl; R³ , R³ , R^3'", R⁴ , R⁴ and R⁴ are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, acyl, substituted acyl, aryl, cycloalkyl, substimted cycloalkyl, heteroaryl, heterocyclic and wherein R³ and R⁴ together form a heterocyclic or a heteroaryl;

R⁵ , R⁵ , R^5'", R⁶ , R⁶ and R⁶ are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, alkoxy, substimted alkoxy, alkylthioalkoxy, acylamino, cycloalkyl, substimted cycloalkyl,; and R⁷, R⁸ and R⁸ are selected from the group consisting of hydrogen, alkyl, substimted alkyl, aryl; with the proviso that one of R', R², R³ , R^3" , R³ , R^4', R^4", R^4",R^5', R^5", R⁵ , R^6', R^6", R^6", R⁷, R⁸ or R^8" is a covalent linkage to a linker;

(b) a compound of formula IC or IC"

IC IC" wherein

R¹⁰ and R¹¹ are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkaryl, alkoxy, substituted alkoxy, alky alkoxy, acyl, acylamino, amino, substimted amino, aminoacyl, cycloalkyl, substimted cycloalkyl, aryl, heteroaryl, heterocyclic; ^* and R¹⁰ and Rⁿ may optionally together form a cycloalkyl, subsituted cycloalkyl or heterocyclic; and

R^12', R^12", R^13', R^13", R^14', R^14' are selected from the group consisting of hydrogen, alkyl, substituted alkyl, acyl, substimted acyl, aryl, cycloalkyl, substimted cycloalkyl, heteroaryl, heterocyclic and wherein R³ and R⁴ together form a heterocyclic or a heteroaryl; with the proviso that one of R^11' R^11", R^12', R^12", R^13', R^13", R^14', R^14" is a covalent linkage to a linker; and

(c) a compound of formula ID

R23

wherein R²⁰ is selected from the group consisting of NH, O, S, NOH, NR ₂₄ R²¹, R²², and R²⁴ are independently selected from the group consisting of hydrogen, an alkyl, a substimted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, a substituted alkynyl, an aryl, a heterocyclic, a cycloalkyl and a substituted cycloalkyl; R²³ is independently selected from the group consisting of an alkyl, a substimted alkyl, an alkenyl, a substimted alkenyl, an alkynyl, a substimted alkynyl, an aryl, a heterocyclic, a cycloalkyl and a substituted cycloalkyl; and Z is an alkylene, a substituted alkylene, an amino, a substimted amino, -S-; with the proviso that one of R²¹, R²², R²³, and R²⁴ is a covalent linkage to a linker, and their tautomeric forms and also their pharmaceutically acceptable salts.

5. The multibinding compound of Claim 4 wherein each ligand is independently selected from the group consisting of formula IA', IA", IA'", IB', IB", IB'":

A«

IB'"

IB' IB" wherein

X is selected from the group consisting of O, S;

Y is selected from the group consisting of OR¹' SR¹' NR^JR²;

R¹ and R² are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substimted alkynyl, cycloalkyl, heteroaryl, aryl, heterocyclic, acyl, substimted acyl;

R³ , R³ , R^{3 '"}, R⁴ , R⁴ and R^{4 "} are independently selected from the group consisting of hydrogen, alkyl, substimted alkyl, acyl, substimted acyl, aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, heterocyclic and wherein R³ and R⁴ together form a heterocyclic or a heteroaryl;

R^5', R^5", R⁵' ", R⁶ , R^6" and R^{6 "} are independently selected from the group consisting of hydrogen, alkyl, substimted alkyl, aryl, alkoxy, substimted alkoxy, alkylthioalkoxy, acylamino, cycloalkyl, substimted cycloalkyl,; and

R⁷, R⁸ and R⁸ are selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl; with the proviso that one of R\ R², R³ , R^3" , R^{3 "}, R⁴ , R^4", R⁴ ,R⁵ , R⁵ , R^5"',

R⁶ , R^6", R^{6 "}, R⁷, R⁸ or R⁸ is a covalent linkage to a linker.

6. The multibinding compound of Claim 4 wherein each ligand is independently selected from formula IC or IC"

IC IC" wherein

R^10' and R¹¹ are independently selected from the group consisting of hydrogen, alkyl, substimted alkyl, alkenyl, substimted alkenyl, alkynyl, substimted alkynyl, alkaryl, alkoxy, substimted alkoxy, alky alkoxy, acyl, acylamino, amino, substituted amino, aminoacyl, cycloalkyl, substimted cycloalkyl, aryl, heteroaryl, heterocyclic; and R^{10 "} and R^11" may optionally together form a cycloalkyl, subsituted cycloalkyl or heterocyclic; and R¹² , R^12", R¹³ , R^13", R^14', R^14" are selected from the group consisting of hydrogen, alkyl, substituted alkyl, acyl, substituted acyl, aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, heterocyclic and wherein R³ and R⁴ together form a heterocyclic or a heteroaryl; and with the proviso that one of R^11' R^11", R^12', R^12", R¹³ , R^13", R¹⁴ , R¹⁴ is a covalent linkage to a linker.

7. The multibinding compound of Claim 4 wherein each ligand is formula ID

wherein

R²⁰ is selected from the group consisting of NH, O, S, NOH, NR²⁴ R²¹, R²², and R²⁴ are independently selected from the group consisting of hydrogen, an alkyl, a substimted alkyl, an alkenyl, a substimted alkenyl, an alkynyl, a substimted alkynyl, an aryl, a heterocyclic, a cycloalkyl and a substituted cycloalkyl;

R²³ is independently selected from the group consisting of an alkyl, a substimted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, a substimted alkynyl, an aryl, a heterocyclic, a cycloalkyl and a substimted cycloalkyl; and

Z is an alkylene, a substituted alkylene, an amino, a substimted amino, -S-; and with the proviso that one of R²¹, R²², R²³, and R²⁴ is a covalent linkage to a linker.

8. The multibinding compound of Claim 4 wherein each linker independently has the formula:

-X^a-Z-(Y^a-Z)_m-Y^b-Z-X^a- wherein m is an integer of from 0 to 20;

X^a at each separate occurrence is selected from the group consisting of -O-, -S-, -NR-, -C(O)-, -C(O)O-, -C(O)NR-, -C(S), -C(S)O-, -C(S)NR- or a covalent bond where R is as defined below; Z is at each separate occurrence is selected from the group consisting of alkylene, substituted alkylene, cycloalkylene, substimted cylcoalkylene, alkenylene, substimted alkenylene, alkynylene, substituted alkynylene, cycloalkenylene, substituted cycloalkenylene, arylene, heteroarylene, heterocyclene, or a covalent bond; Y^a and Y^b at each separate occurrence are selected from the group consisting of -C(O)NR'-, -NR'C(O)-, -NR'C(O)NR'-, -C(=NR')-NR'-, -NR'-C(=NR')-, -NR'-C(O)-O-, -N=C(X^a)-NR'-, -P(O)(OR')-O-, -S(O)_nCR'R"-, -S(O)_n-NR'-, -S-S- and a covalent bond; where n is 0, 1 or 2; and R, R' and R" at each separate occurrence are selected from the group consisting of hydrogen, alkyl, substimted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substimted alkenyl, cycloalkenyl, substimted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl and heterocyclic.

9. A multibinding compound of formula II:

L'- X'- L' II

wherein each L' is independently a ligand comprising a moiety capable of binding a nitric oxide synthase and X' is a linker; with the proviso that the multibinding compound is not formula III or IV:

wherein R is hydrogen or C ₈ hydrocarbyl; T is a C_j.₈ hydrocarbyl group optionally containing a 5- or 6-membered heterocyclic ring or T is a ^ hydrocarbyl group containing a phenylene ring; and pharmaceutically-acceptable salts thereof.

10. The multibinding compound of Claim 9 wherein X' has the formula:

-X^a-Z-(Y^a-Z)_m-Y^b-Z-X^a- wherein m is an integer of from 0 to 20;

X^a at each separate occurrence is selected from the group consisting of -O-, -S-, -NR-, -C(O)-, -C(O)O-, -C(O)NR-, -C(S), -C(S)O-, -C(S)NR- or a covalent bond where R is as defined below;

Z is at each separate occurrence is selected from the group consisting of alkylene, substituted alkylene, cycloalkylene, substimted cylcoalkylene, alkenylene, substimted alkenylene, alkynylene, substimted alkynylene, cycloalkenylene, substituted cycloalkenylene, arylene, heteroarylene, heterocyclene, or a covalent bond;

Y^a and Y^b at each separate occurrence are selected from the group consisting of -C(O)NR'-, -NR'C(O)-, -NR'C(O)NR'-, -C(=NR')-NR'-, -NR'-C(=NR')-, -NR'-C(O)-O-, -N=C(X^a)-NR'-, -P(O)(OR')-O-, -S(O)_nCR'R"-, -S(O)_n-NR'-, -S-S- and a covalent bond; where n is 0, 1 or 2; and R, R' and R" at each separate occurrence are selected from the group consisting of hydrogen, alkyl, substimted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substimted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substimted alkynyl, aryl, heteroaryl and heterocyclic.

11. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a multibinding compound comprising from 2 to 10 ligands covalently attached to one or more linkers wherein each of said ligands independently comprises a moiety capable of binding a nitric oxide synthase; with the proviso that the multibinding compound is not formula III or IV:

wherein R³⁵ is hydrogen or C,_₈ hydrocarbyl;

T is a C ₈ hydrocarbyl group optionally containing a 5- or 6-membered heterocyclic ring or T is a C^ hydrocarbyl group containing a phenylene ring; and pharmaceutically-acceptable salts thereof.

12. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a multibinding compound of formula I:

(L)_p(X)_fl wherein each L is independently a ligand comprising a moiety capable of binding a nitric oxide synthase; each X is independently a linker; p is an integer of from 2 to 10; and q is an integer of from 1 to 20; with the proviso that the multibinding compound is not formula III or IV:

wherein R³⁵ is hydrogen or C _s hydrocarbyl;

T is a .g hydrocarbyl group optionally containing a 5- or 6-membered heterocyclic ring or T is a ^ hydrocarbyl group containing a phenylene ring; and pharmaceutically-acceptable salts thereof.

13. The pharmaceutical composition of Claim 12 wherein q is less than

14. The pharmaceutical composition of Claim 12 wherein each ligand is independently selected from the group consisting of:

(a) a compound selected from the group consisting of formula IA', IA", IA'", IB', IB", IB'":

wherein

X is selected from the group consisting of O, S; Y is selected from the group consisting of OR¹ , SR¹, NR^²; R¹ and R² are independently selected from the group consisting of hydrogen, alkyl, substimted alkyl, alkenyl, substituted alkenyl, alkynyl, substimted alkynyl, cycloalkyl, heteroaryl, aryl, heterocyclic, acyl, substimted acyl; R³ , R³ , R^{3 '"}, R⁴ , R⁴ and R⁴ are independently selected from the group consisting of hydrogen, alkyl, substimted alkyl, acyl, substimted acyl, aryl, cycloalkyl, substimted cycloalkyl, heteroaryl, heterocyclic and wherein R³ and R⁴ together form a heterocyclic or a heteroaryl;

R⁵ , R⁵ , R^{5 '"}, R⁶ , R⁶ and R^{6 "} are independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl, alkoxy, substimted alkoxy, alkylthioalkoxy, acylamino, cycloalkyl, substimted cycloalkyl,; and R⁷, R⁸ and R⁸ are selected from the group consisting of hydrogen, alkyl, substituted alkyl, aryl; with the proviso that one of R¹, R², R³ , R³ , R³ , R , R⁴ , R⁴ ,R⁵ , R⁵ , R⁵ , R^6', R┬░^", R^6'", R⁷, R⁸ or R^8" is a covalent linkage to a linker;

(b) a compound of formula IC or IC"

IC IC" wherein

R¹⁰ and R¹¹ are independently selected from the group consisting of hydrogen, alkyl, substimted alkyl, alkenyl, substituted alkenyl, alkynyl, substimted alkynyl, alkaryl, alkoxy, substituted alkoxy, alkyalkoxy, acyl, acylamino, amino, substituted amino, aminoacyl, cycloalkyl, substimted cycloalkyl, aryl, heteroaryl, heterocyclic; and R^10" and R^11" may optionally together form a cycloalkyl, subsituted cycloalkyl or heterocyclic; and

R¹² , R^12", R^13', R^13", R^14', R^14" are selected from the group consisting of hydrogen, alkyl, substituted alkyl, acyl, substimted acyl, aryl, cycloalkyl, substimted cycloalkyl, heteroaryl, heterocyclic and wherein R³ and R⁴ together form a heterocyclic or a heteroaryl; with the proviso that one of R^11' R^╧Ç", R^12', R^12", R^13', R^13", R^14', R^14" is a covalent linkage to a linker; and

(c) a compound of formula ID

wherein R²⁰ is selected from the group consisting of NH, O, S, NOH, NR²⁴ R²¹, R²², and R²⁴ are independently selected from the group consisting of, hydrogen, an alkyl, a substituted alkyl, an alkenyl, a substimted alkenyl, an alkynyl, a substimted alkynyl, an aryl, a heterocyclic, a cycloalkyl and a substituted cycloalkyl; R²³ is independently selected from the group consisting of an alkyl, a substituted alkyl, an alkenyl, a substimted alkenyl, an alkynyl, a substimted alkynyl, an aryl, a heterocyclic, a cycloalkyl and a substimted cycloalkyl; and Z is an alkylene, a substimted alkylene, an amino, a substimted amino, -S-; with the proviso that one of R²¹, R²², R²³, and R²⁴ is a covalent linkage to a linker, and their tautomeric forms and also their pharmaceutically acceptable salts.

15. A method for treating sepsis in a patient, the method comprising administering to a patient suffering from sepsis a pharmaceutical composition comprising a pharmaceutically-acceptable carrier and a therapeutically-effective amount of a multibinding compound comprising from 2 to 10 ligands covalently attached to one or more linkers wherein each of said ligands independently binds to i-NOS; with the proviso that the multibinding compound is not formula III or IV:

wherein R³⁵ is hydrogen or C ₈ hydrocarbyl;

T is a C_j.₈ hydrocarbyl group optionally containing a 5- or 6-membered heterocyclic ring or T is a C^ hydrocarbyl group containing a phenylene ring; and pharmaceutically-acceptable salts thereof.

16. A method for treating inflammation in a patient, the method comprising administering to a patient having inflammation or an inflammation- related disorder a pharmaceutical composition comprising a pharmaceutically- acceptable carrier and a therapeutically-effective amount of a multibinding compound comprising from 2 to 10 ligands covalently attached to one or more linkers wherein each of said ligands independently binds to i-NOS; with the proviso that the multibinding compound is not formula III or IV:

wherein R³⁵ is hydrogen or C _s hydrocarbyl;

T is a .g hydrocarbyl group optionally containing a 5- or 6-membered heterocyclic ring or T is a C^, hydrocarbyl group containing a phenylene ring; and pharmaceutically-acceptable salts thereof.

17. A method for identifying multimeric ligand compounds which bind nitric oxide synthases and possess multibinding properties which method comprises:

(a) identifying a ligand or a mixture of ligands wherein each ligand binds a nitric oxide synthase and 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 the library prepared in (c) above to identify multimeric ligand compounds possessing multibinding properties.

18. A method for identifying multimeric ligand compounds which bind nitric oxide synthases and possess multibinding properties which method comprises:

(a) identifying a library of ligands wherein each ligand binds a nitric oxide synthase and 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 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 the library prepared in (c) above to identify multimeric ligand compounds possessing multibinding properties.

19. The method according to Claim 17 or 18 wherein 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).

20. The method according to Claim 19 wherein the multimeric ligand compounds comprising the multimeric ligand compound library are dimeric.

21. The method according to Claim 20 wherein the dimeric ligand compounds comprising the dimeric ligand compound library are heterodimeric.

22. The method according to Claim 21 wherein the heterodimeric ligand compound library is prepared by sequential addition of a first and second ligand.

23. The method according to Claim 17 or 18 wherein, prior to procedure (d), each member of the multimeric ligand compound library is isolated from the library.

24. The method according to Claim 23 wherein each member of the library is isolated by preparative liquid chromatography mass spectrometry

(LCMS).

25. The method according to Claim 17 or Claim 18 wherein the linker or linkers employed are 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/or polarizability and amphiphilic linkers.

26. The method according to Claim 25 wherein the linkers comprise linkers of different chain length and/or having different complementary reactive groups.

27. The method according to Claim 26 wherein the linkers are selected to have different linker lengths ranging from about 2 to lOOA.

28. The method according to Claim 17 or 18 wherein the ligand or mixture of ligands is selected to have reactive functionality at different sites on said ligands.

29. The method according to Claim 28 wherein said reactive functionality is selected from the group consisting of carboxylic acids, carboxylic acid halides, carboxyl esters, amines, halides, pseudohalides, isocyanates, vinyl unsaturation, ketones, aldehydes, thiols, alcohols, anhydrides, boronates, and precursors thereof wherein 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.

30. The method according to Claim 17 or Claim 18 wherein the multimeric ligand compound library comprises homomeric ligand compounds.

31. The method according to Claim 17 or Claim 18 wherein the multimeric ligand compound library comprises heteromeric ligand compounds.

32. A library of multimeric ligand compounds which may bind a nitric oxide synthase and may possess multivalent properties which library is prepared by the method comprising:

(a) identifying a ligand or a mixture of ligands which bind a nitric oxide synthase 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.

33. A library of multimeric ligand compounds which may bind a nitric oxide synthase and may possess multivalent properties which library is prepared by the method comprising:

(a) identifying a library of ligands wherein each ligand binds a nitric oxide synthase and 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.

34. The library according to Claim 32 or Claim 33 wherein the linker or linkers employed are 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/or polarizability and amphiphilic linkers.

35. The library according to Claim 34 wherein the linkers comprise linkers of different chain length and/or having different complementary reactive groups.

36. The library according to Claim 35 wherein the linkers are selected to have different linker lengths ranging from about 2 to 100A.

37. The library according to Claim 32 or 33 wherein the ligand or mixture of ligands is selected to have reactive functionality at different sites on said ligands.

38. The library according to Claim 37 wherein said reactive functionality is selected from the group consisting of carboxylic acids, carboxylic acid halides, carboxyl esters, amines, halides, pseudohalides, isocyanates, vinyl unsaturation, ketones, aldehydes, thiols, alcohols, anhydrides, boronates, and precursors thereof wherein 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.

39. The library according to Claim 32 or Claim 33 wherein the multimeric ligand compound library comprises homomeric ligand compounds.

40. The library according to Claim 32 or Claim 33 wherein the multimeric ligand compound library comprises heteromeric ligand compounds.

41. An iterative method for identifying multimeric ligand compounds capable of binding nitric oxide synthases and 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 nitric oxide synthase 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 or are consistent with imparting 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 or are consistent with imparting 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.

42. The method according to Claim 41 wherein steps (e) and (f) are repeated from 2-50 times.

43. The method according to Claim 42 wherein steps (e) and (f) are repeated from 5-50 times.