I. Field of Invention

The present invention relates to novel substituted urea and related compounds, as well as to the use of such compounds for the modulaton of cell adhesion.

The compounds have application to the study and treatment of disease conditions mediated by cell adhesion. Specifically, the compounds have application to the study, diagnosis, treatment or prevention of diseases and conditions such as, for example, cardiovascular disease, harmful platelet aggregation, neoplastic disease including metastasis of neoplastic growth, wound healing, inflammation and autoimmune disease or other diseases or conditions involving cell adhesion.

II. Background of the Invention

The extracellular matrix is that material which surrounds the muscle and is the major component of connective tissue of all mammals. The extracellular matrix provides for structural integrity, and promotes cell migration and cellular differentiation. As part of these functions, the extracellular matrix has been shown to support adhesion for various types of cells in vitro. Molecules such as the collagens, fibronectin, vitronectin, laminin, von Willebrand factor, thrombospondin, bone sialoprotein, fibrinogen, and tenascin have been found to possess this property of mediating cell adhesion.

The above cell-adhesive molecules have been found to exhibit a structural similarity in their respective binding sites, each of which contains the amino acid sequence arginine-glycine-aspartic acid, or RGD using conventional single letter

nomenclature. The cell-binding site in fibronectin has been reproduced synthetically. In turn, the cellular receptor site for fibronectin has been identified for various cells, in addition, cellular receptors that recognize RGD-containing sequences in other extracellular matrix proteins (e.g., the vitronectin receptor) have been identified.

Such cellular receptors, responsive to RGD-containing proteinaceous compounds, have been characterized. The complete, primary structure of the fibronectin receptor has been deduced from cDNA, and physical properties have been determined. Argraves, ef al., J.Biol. Chem., 1986, 261 , 12922; Argraves et at., J.Cell Biol., 1987, 105, 1183. The protein exists at the cell surface as a heterodimeric complex (although the larger polypeptide is enzymatically processed) having both polypeptide chains inserted into the membrane. Each chain extends 30-40 residues into the cytoplasmic space, and at least one of the cytoplasmic peptides appears to interact with the cytoskeleton. Horwitz et al., Nature, 1986, 320, 531. The larger of the two polypeptides, the subunit, contains a number of regions that are structurally similar to caimodulin and that apparently mediate the binding of calcium to the receptor. The presence of such divalent cations is required for the receptor to bind ligand. The β subunit is somewhat smaller and conformationally compact due to numerous intrachain disulfide bonds. The cytoplasmic domain of the β subunit contains a potentially phosphorylated tyrosine. Hirst ef al., PNAS-USA, 1986, 83, 6470; Tamkun et a!., Cell, 1986, 46, 271-282.

Other RGD-directed receptors, as well as other "orphan" receptors the ligand for which is unknown, have also been characterized. This putative RGD commonality of the ligand matrix proteins has revealed a superfamily of cell surface receptor proteins that share a high degree of structural similarity and probably also functional similarity. The members of this superfamily of cell

surface proteins collectively are known as the integrins. The integrins can be grouped on the basis of the identity of their β subunit. The β subunit, as disclosed above for the fibronectin receptor, is corhpact due to a high degree of cross-linking. The first group of integrins includes the very late activation antigen (VLA) proteins, which themselves include the fibronectin receptor

(VLA-5), the collagen receptor (VLA-2), and the laminin receptor. The second group includes the lymphocyte associated antigen-1 (LFA-1), macrophage antigen-1 (MAC-1), and p150,95. The third group includes the vitronectin receptor, and platelet glycoprotein gpllb/llla. Hynes, Cell, 1987, 48, 549; Hemler, Immunol. Today, 1988, 9, 109; Springer ef al., Annu. Rev. Immunol., 1987, 5,

223; Kishimoto ef al., In Leukocyte Adhesion Molecules, T.A. Springer, D.C. Anderson, A.S. Rosenthal, and R. Rothlein, Eds., Springer-Verlag, New York, 1989, pp. 7-43.

The RGD-directed receptor present on platelets that binds fibronectin, vitronectin, fibrinogen, and von Willebrand factor has also been purified. This receptor is the gpllb/llla protein complex. This receptor is thus not specific to one extracellular matrix protein, as are the above fibronectin and vitronectin receptors. It has been proposed that this lack of specificity is correlated to the lack of conformational specificity in the ligands. Other work has suggested that specificity can be achieved with relatively short, conformationally restricted synthetic peptides containing the RGD sequence. For a literature summary, see: Pierschbacher ef al., Nature, 1984, 309, 30; Pierschbacher et al., PNAS-USA, 1984, 81 , 5985; Ruoslahti ef al., Cell, 1986, 44, 517; Pierschbacher ef al., Science, 1987, 238, 491 ; Pierschbacher ef al., J.Biol.Chem., 1987, 262, 17294; Hynes, Cell, 1987, 48, 549; Ruoslahti, Ann. Rev. Biochem., 1988, 57,

375. It has also been proposed that the receptor affinity for its peptide ligand may be altered as the stereoconformation, or three-dimensional shape, of the

peptide is restricted, typically by cyclization. Pierschbacher and Ruoslahti, PCT International Publication WO 89/05150 (1989).

A limited number of compounds containing sequences of natural amino acids or derivatives other than RGD may also possess the capability for affecting cell adhesion. These non-RGD-containing peptides are not well characterized. See,

Graf, J. ef al., Cell, 1987, 48, 989; Kloezewiak, M. et al., Biochemistry, 1984, 23, 1767-1774; Wayner, E.A., ef al., J. Cell. Biol., 1989, 109, 1321.

U.S. Patent No. 4,879,313 to Tjoeng, ef al. reports the utility as platelet aggregation inhibitors of certain peptide mimetic compounds containing, in addition to a guanidinyi group at one terminus and an internal aspartic acid residue, an aromatic structure (phenyl, biphenyl, naphthyl, pyridyl or thienyl groups, and certain methoxy-substituted forms thereof) at another defined position in the compound. Related structures containing an internal glycine residue are reported in U.S. Patent No. 4,857,508 (Adams, ef al.).

All publications, patents and other reference materials to which reference is made in the present specification are incorporated herein by reference.

III. Summary of the Invention

The present invention relates to compounds having activity as cell adhesion modulators. The compounds are characterized by the structure Y

I' ₃

Z N C N R ³

wherein Z is preferably an aromatic or electron-withdrawing group such as a sulfonyl, substituted sulfonyl, nitro, or haloalkyi group, or a substituted carbonyl, oxy, carboxyl, oxycarbonyl, amino or thio-containing group; Y is a suitable double-bonded atom or substituent group, preferably including sulfur, nitrogen or oxygen; and each R-group is hydrogen or a suitable hydrocarbon-containing or heteroatomic substituent, including amidinyl.

Where Y is a double-bonded oxygen atom, the resulting compound is a substituted urea compound. Where Y is a double-bonded sulfur atom, a substituted thiourea compound results; and where Y includes a double-bonded nitrogen atom, a substituted guanidino (i.e., an iminoanalog of a urea compound) or a substituted biguanidino results.

The compounds, in one aspect, sufficiently mimic extracellular matrix ligands or other cell adhesion ligands so as to bind to cell surface receptors. Such receptors include integrin receptors in general, including the fibronectin, collagen, laminin, LFA-1 , MAC-1 , p150,95, vitronectin and gpllb/llla receptors.

The present compounds have been found to modulate cell adhesion by competing, for example, with RGD-containing ligands and by binding to RGD-directed receptors on ceil surfaces. Such cell adhesion ligands, including (but not limited to) fibronectin, are sufficiently inhibited from binding to the cell's receptor as to prevent or reduce cell adhesion. Other uses include enhancing cell adhesion by using the compounds to attach cells to a surface, or by other promotion of cell adhesion. The useful compounds herein described function as cell-adhesion modulators.

One object of the present invention is to provide novel compounds which act to modulate cell adhesion.

Another object of the present invention is to provide substituted urea and related thiourea, guanidino and biguanidino compounds which are capable of binding with a cellular receptor.

Another object of the present invention is to provide a novel method for modulating cell adhesion using the present compounds.

Another object of the present invention is to provide novel compounds, formulations, and methods which may be used in the study, diagnosis, treatment or prevention of diseases and conditions which relate to cell adhesion, including but not limited to rheumatoid arthritis, asthma, allergies, adult respiratory distress syndrome (ARDS), cardiovascular disease, thrombosis or harmful platelet aggregation, reocclusion following thrombolysis, neoplastic disease including metastasis of neoplastic growth, wound healing, Type I diabetes, inflammatory conditions including ophthalmic inflammatory conditions and inflammatory bowel disease (e.g, ulcerative colitis and regional enteritis), autoimmune diseases, and acquired immunodeficiency syndrome (AIDS).

Another object is to provide derivative compounds, such as, but not limited to, antibodies and anti-idiotype antibodies to the compounds disclosed and claimed in order to study, diagnose, treat or prevent diseases and conditions which relate to cell adhesion, including but not limited to rheumatoid arthritis, asthma, allergies, adult respiratory distress syndrome (ARDS), cardiovascular disease, thrombosis or harmful platelet aggregation, reocclusion following thrombolysis, neoplastic disease including metastasis of neoplastic growth, wound healing, Type I diabetes, inflammatory conditions, autoimmune diseases, and acquired immunodeficiency syndrome (AIDS).

IV. Detailed Description

The compounds of the present invention are those having the property of modulating cell adhesion.

While cell adhesion is required for certain normal physiological functions, situations exist in which cell adhesion is undesirable, or in which modulated cell adhesion is desirable.

Altered leukocyte-endothelial interactions are implicated in adult respiratory distress syndrome (ARDS). Here, the attachment of inappropriate cells to the lung lining induces an inflammatory response. This results in lung injury, ARDS and in some cases, asthma. Preliminary in vitro results show that such detrimental attachment, in which the leukocyte adheres to endothelial cells or the lung extracellular matrix, is mediated by RGD-containing protein and RGD-recognizing receptors on the leukocytes. In this situation, compounds with a binding affinity to RGD receptors are desirable as competitive antagonists and should be useful in treating ARDS and asthma. Such compounds are disclosed herein.

Cell adhesion also contributes to metastasis of cancerous tumors. Metastasis has been called "the major underlying cause of death from cancer." Welch, ef al., Intern. J. Cancer, 1989, 43, 449. A compound which would prevent cell adhesion to basement membrane components may be useful to prevent or eliminate metastasis. See, Humphries, M.J. ef al., Science, 1986, 233, 467; Liotta, L , Cancer Res., 1986, 46, 1 ; Roose, E., Biochem. Biophys. Acta., 1986,

I

738, 263. A compound with suitable affinity for RGD receptors, such as disclosed herein, should likewise have anti-metastasis utility.

Harmful blood clotting is also caused by inappropriate cell adhesion, particularly ceil adhesion to the extracellular matrix. The attachment, spreading and aggregation of platelets on extracellular matrices are central events in thrombus formation. These events can be regulated by the family of platelet adhesive glycoproteins, fibrinogen, fibronectin, and von Willebrand factor. Fibrinogen functions as a cofactor for platelet aggregation, while fibronectin supports platelet attachment and spreading reactions. Von Willebrand factor is important in platelet attachment to and spreading on subendothelial matrices. Plow ef al., PNAS-USA, 1985, 82, 8057. A compound, such as these herein, which would function as an antagonist and bind to ceil receptors which recognize the matrix glycoprotein RGD site would be beneficial as a thrombolytic.

Human immunodeficiency virus (HIV) has a gene for transactivating protein, termed faf, which contains an RGD sequence. It is reported that this faf protein functions in the ceil adhesion of the virus to target cells, and that, by inhibiting such cell adhesion function, transcriptional activation by the faf protein can be inhibited. It has further been found that such inhibition of faf mediated transcriptional activation inhibits, i.e., prevents or slows, progression, including initiation, of disease states resulting from abnormal gene expression. Such disease states include immune dysfunction resulting in immunodeficiency as well as other disease states associated with Immunodeficiency Virus infestion such as Kaposi's Sarcoma. See, e.g., Vogel ef al., Nature, 1988, 335, 606.

International Patent Application WO91/15,224 describes RGD-containing proteins and mimics thereof being an inhibitor of such cell adhesion. The RGD proteins or mimics bind to the RGD cell adhesion receptor, or otherwise inhibit the binding of faf to cells. The present compounds would also inhibit the faf protein of HIV from adhering to target cell's receptors, as well as would inhibit the consequent transcriptional activation by the virus. Furthermore, the present

compounds, being non-proteinaceons, would offer advantages as pharmaceuticals over the RGD-containing proteins in terms of the ease of formulation or oral activity.

Other physiological conditions may be treated by stimulatory modulation of cell adhesion. Wound healing, for example, is undesirably prolonged when insufficient cell adhesion occurs. A compound with suitable affinity for RGD receptors, attached for example to a suitably positioned matrix or surface, may be able to promote beneficial cell adhesion and resultant wound healing by binding cells with the appropriate RGD-recognizing receptor. Also, in prosthetic implantation, such compounds coating the prosthesis would provide a means for covering the prosthesis with a surface of cells. This cell surface would provide a surface compatible with the organism, and thus minimize rejection that might otherwise occur due to stimulation of the immune system by the prosthesis itself. The compounds of the present invention are believed to be useful in this cell adhesion modulation application as well. The compounds of this invention are those of the formula:

Y

-N C N R ³ (I)

I I

R ¹ R ²

and pharmaceutically acceptable salts thereof, wherein

Z is a pharmaceutically suitable substituent group or salt thereof, preferably including an aromatic or electron-withdrawing group, and most preferably one selected from R ⁴ -

O O O II It II

R ⁴ -S- R' -O-S- II \ ,3C-S- II O O

O O O II il II R ⁴ -C- R ⁴ -C-O- R ⁴ -O-C-

O X ₃ C-C- , R ⁴ O- , R ⁴ NH- ,

R ⁴ ₂ N- , R ⁴ S- , and NO ₂ - ;

wherein each X- is individually a halogen atom; Y is a double-bonded atom or substituent group, preferably one selected from

S NR ⁵ and O

II II II

R ³ is a pharmaceutically suitable substituent group, preferably one selected from hydrogen, amino from linear and branched, unsubstituted and substituted C C _e lower alkyl, C ₂ -C ₈ alkenyls, C ₂ -C ₈ alkynyls, C ₃ -C ₁₄ cycloalkyls, from groups of the form Ar — , from a group of the form

NH II

— C NH ₂

and from a group of the form NH NH

II II C NH (CH ₂ ) _n NH C NH Z

wherein n is an integer of from 1 to 4 and Ar is an unsubstituted or substituted aryl, aralkyl group, preferably one having from 5 to about 14

ring atoms, and optionally containing one or more of 0, N or 5 as a ring heteroatom; and each of R ¹ , R ² , R ⁴ and R ⁵ is individually a pharmaceutically suitable substituent group, preferably one selected from hydrogen, amino, from linear and branched, unsubstituted and substituted C^C _g lower alkyls, C ₂ -C _β alkenyls, C ₂ -C ₈ alkynyls, C ₃ -C ₁₄ cycloalkyls, from groups of the form Ar — ,and, in the case of -NR ² R ³ and R ⁴ ₂ N-, from cyclized groups forming (in attachment with the nitrogen atom) a 5-8 membered heterocyclic ring optionally containing one or more of 0, N or S as a further ring heteroatom.

The term "substituted" used throughout this specification means a group having one or more substituents selected from C _j -C ₈ lower alkyls, C^C _g lower alkoxy, C^C _g carboxyalkyl, C^C _g alkoxy carboxyl, C ₃ -C ₁₄ cycloalkyl optionally containing one or more heteroatoms, amino, carboxyl, halo, hydroxyl, nitro, trihalomethyl and uryl. The term "halo" means fluoro, bromo, chloro and iodo.

The compounds of the invention further include pharmaceutically acceptable base- or acid-addition salts of the compositions of Formula I. The pharmaceutical compositions of the invention include such compounds (including salts thereof) formulated with a pharmaceutically acceptable excipient.

With respect generally to substituents R ¹ through R ⁵ , hydrogen, as well as unsubstituted and substituted lower alkyl, lower (C ₃ -C _β ) cycloalkyl and single ring aryl, aralkyl and alkaryl moieties, are most preferred. Methyl, ethyl, isopropyl, 2-hydroxymethyl and cyclohexyl are examples of generally preferred hydrocarbon substituents in positions R ¹ through R ⁵ .

With respect particularly to substituent R ¹ , hydrogen is especially preferred, and relatively non-bulky substituents such as lower alkyls are also preferred.

Substituents R ² and R ³ are most preferably hydrogen or unsubstituted or substituted lower alkyl or cycloalkyl groups. Methyl, ethyl, isopropyl, 2-hydroxyethyl and cyclohexyl are preferred hydrocarbon substituents in one

(preferably) or both of positions R ² and R ³ (with any nonhydrocarbon substituent being most preferably hydrogen). R ² or R ³ may be amino so as to form a hydrazino structure. Where R ² and R ³ together form a nitrogen-attached cyclized structure, the piperidine structure is especially preferred, and the heterocyclic morpholine structure is also preferred.

With respect to Z, the preferred substituents are aromatic groups of the form Ar as well as aromatic sulfonyl groups of the form Ar(S0 ₂ )-where Ar is an aromatic group as defined above such as a C ₆ -C ₁₄ aryl, a C ₇ -C ₁₄ alkaryl or a C ₇ -C ₁₄ aralkyl. The most highly preferred substituents are unsubstituted or substituted single-ring aryl, alkaryl, arylsulfonyl and alkarylsulfonyl groups, especially p- toluenesulfonyl and 3'- and/or 4'-substituted (o- and/or p-substituted) phenyl substituents. Where one or more substituents occur on the aromatic portion of such a Z-group, these are most preferably lower alkyl (e.g., methyl), lower alkoxy (e.g., methoxy) or electron-withdrawing groups (e.g., nitro, chloro, fiuoro or trifluoromethyl).

Where the substituent Z comprises a portion of the X ₃ C- moiety, each X may be a halogen atom, most preferably fiuoro. Trifluoromethyl is especially preferred for such a Z-group.

In the Y-position, the preferred substituent is sulfur (i.e., of the form S=) such that a thiourea compound is formed. Where Y is of the form R ⁵ N=, the most

highly preferred R ⁵ substituent is hydrogen, and relatively non-bulky substituents such as lower alkyl groups are also preferred.

Where one or more of R ¹ through R ⁵ is chosen to be a heterocyclic group, the cyclic structure may contain one or more heteroatoms selected from N, O and S, and may be mono- or polycyclic. Single-ring structures are preferred. The cyclic structure can be saturated, as in morphoiinyi, thiamorpholinyi, piperidyl, piperazinyl, pyrrolidinyl, pyrazoiidinyl, quinuclidinyl, imidazolidinyi, and other structures, or unsaturated or aromatic, as in imidazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyrazolyl, pyrrolyl, pyrrolinyl, pyridazinyl, pyrrodiazolyl, isothiazolyl, thiophenyl, thiazinyi, isoxazolyl, furazanyl and other structures. Polycyclic structures such as indolyl, quinoiyl, quinazoiinyl, phenoxazinyl, phenazinyl, phenothiazinyl, benzo[to]thienyl, phenanthrolinyl or others may be employed. Attachment of such cyclic substituents with the remainder of the compound may occur through a carbon or (provided that a point of bonding is present on a heteroatom) through a heteroatom within the heterocyclic group, or attachment may be achieved through, for example, an intermediate alkyiene moiety which links the cyclic group with the remainder of the compound. A preferred aralkyl group having such a structure is the benzyl group. Such cyclic substituents may also be substituted with pharmaceutically suitable substituents as is now discussed.

Where R ₃ is amidinyl, the R ₃ group may be further substituted and of the form:

NR ⁶ -C N R ⁸

wherein R ₆ , R ₇ and R ₈ are each a pharmaceutically suitable substituent group, preferably one selected from hydrogen, amino, from linear and branched, unsubstituted and substituted C^C _Q lower alkyls, C ₂ -C ₈ alkenyls; C ₂ -C ₈ alkynyls, C ₃ -C ₁₄ cycloalkyls, from gropus of the form Ar — , and, in the case of -NR ⁷ R ⁸ , from cyciized groups forming (in attachment with the nitrogen atom) a 5-8 membered heterocyclic ring optionally containing one or more of O, N or S as a further ring heteroatom, wherein Ar — is an unsubstituted or substituted aryl or aralkyl ("aromatic") group, preferably one having from 5 to about 14 ring atoms, and optionally containing one or more of O, N or S as a ring heteroatom.

Where one or more of the groups R ¹ through R ⁵ in the compound is itself additionally substituted, preferred substituents include hydroxyl, amino, lower (C _t -C ₈ ) alkoxy I, and, in the case particularly of aromatic groups, the foregoing substituents as well as nitro and halogen (especially chloro and bromo) moieties. Such substituents to one or more of R ¹ through R ⁵ may be used, for example, to alter bioactivity, solubility and/or biodistribution characteristics of the subject compounds. As noted above with respect to R ² and R ³ , a hydroxyl substituent on a lower alkyl R-group (as in 2-hydroxyethyl) is particularly preferred. Also, where the R-group includes an aromatic portion (as in certain preferred Z groups), substituents occurring on the meta and/or para positions (i.e., 3'- and/or 4'-positions) are most preferred. Preferred forms of such aromatic substituents thus include 3'- and/or 4'[di/mono]methylphenyl,

I

[di/mono]methoxyphenyl, [di/mono]nitrophenyl and [di/mono]chlorophenyl groups, optionally bonded through a sulfonyl residue as in the 3',4'- dichlorophenylsulfonyl Z-group.

EXAMPLES

The compounds of this invention may be prepared using synthetic methods such as those described hereinafter, or, In view of the present disclosure, by other synthetic methods known in the art. The following examples are intended to be illustrative only, and should not be construed as limiting the scope of the present invention.

Sulfonyl thioureas, such as p-toluenesulfonyl thioureas, that are substituted with hydrogen at each of positions R ² and R ³ may be prepared by reacting an appropriate sulfonyl compound (such as p-toluenesulfonyl chloride, 1) with a reactive cyano-containing compound (such as calcium cyanamide) to yield a reactive sulfonylcyano intermediate (such as a p-toluenesulfonyl-cyanamide salt, 2), and thereafter reacting with an appropriate reducing agent (such as sodium thiosulfate) to yield the sulfonyl thiourea product (3). This may be exemplified by the following scheme:

M «

Sulfonylthioureas that are singly or multiply substituted with non-hydrogen moieties at one or more of positions R ² and R ³ may be prepared by reacting an appropriate sulfonamide (such as p-toluenesulfonamide, 4) with carbon disulfide to form a sulfoniminodithiocarbonate (such as p-toluenesulfσniminodithio- carbonate salt, 5), then reacting to form a sulfonylisothiocyanate compound (e.g., 6), followed by reaction with one or more amine compounds to form the amino-substituted sulfonylthiourea product (e.g., 7). Such a synthetic scheme is exemplified below:

Example 1 Synthesis of p-Toluenesulfonylthiourea

The procedure of Bodesinsky, et al., Czechoslov. Farm., 1960, 9, 440, cited in Chem. Abstr., 55:104351 (1961), was followed to prepare the intermediate sodium salt of p-toluenesulfonyl-cyanamide (2). A suspension of 35 g (0.43 mole) of calcium cyanamide in 140 mL of water was stirred overnight and filtered. The clear filtrate was heated to 40° C and 33.3 g (0.17 mole) of p-toluenesulfonyl chloride was added over 3 hours. Both the pH and the temperature were monitored during the course of the addition; the temperature was kept below 42 "C and pH was maintained near 11 by the occasional addition of solid sodium hydroxide. The next day, the solution was heated to 80° C, filtered, and 35 g of NaCI added to the filtrate. Cooling and filtration gave 39.4 g (36.9 g theory) of a white solid (mp>310°C, with softening at 292 °C). The melting point is cited as 292° C.

The p-toluene-sulfonylthiourea product (3) was prepared according to the procedure of Bodesinsky, ef al., Czechoslov. Farm., 1959, 8, 129, cited in Chem. Abstr., 54:3197a (1960). The crude material from above and 70.8 g (0.28 mmoie) of sodium thiosulfate pentahydrate was dissolved in 150 mL of water. The solution was heated at reflux for 10 hours, cooled, and acidified to a pH of 3 with concentrated HCl. Filtration and drying gave 23.8 g of crude material.

Recrystallization from methanol/water gave a first crop of 14.6 g (mp 118-122°) and a second crop of 3.81 g (mp 117-126°C). The total yield for the two steps was 47%. Recrystallization of a portion of the first crop from isopropanol gave a reference sample (mp 123-126° (lit 129-130°); IR(CHCI ₃ ) 1598 (ar), 1456 (CS), 1404 (S0 ₂ ), 1356 (CS) Cm ^'1 ).

Example 2

Synthesis of 1 - (p-Toluenesulfonyl)

-3-cyclohexy|-thiourea Salt

The dipotassium salt of p-toluenesulfoniminodithio-carbonate (5) was first prepared by mixing 15.27 g (0.2 mole) of carbon disulfide and 34.28 g (0.2 mole) of p-toluenesulfonamide (4) in 200 mL of dimethylformamide and stirring mechanically as 11.35 g (86.4%, 0.0.175 mole) of potassium hydroxide was added under N ₂ over 1-hour. After an additional hour, an additional 14.66 g (0.225 mole) of KOH was added. In one additional hour 200 mL of ethyl

I acetate was added and the resulting golden slurry cooled in an ice bath.

Filtration and drying yielded 55.9 g (mp 230-239 °C). Dilution of the mother liquor with 200 mL more of ethyl acetate yielded another 7.7 g.

The p-toluenesulfonyiisothiocyanate intermediate (6) was prepared by mixing the first crop from the previous reaction with 3 g of potassium chloride and grinding to a fine powder with a mortar and pestle. The powder was suspended in 200 mL of methylene chloride and the slurry cooled to 0°F. A 90 mL portion of 1.93 M solution of phosgene in toluene was added dropwise over 45 minutes. Stirring was continued as the reaction mixture warmed to room temperature overnight. Filtration and evaporation gave a yellow solid which was distilled on the Kugelrohr apparatus at 80 to 100°C/0.05 mmHg. The yield was 13 g (35%) of a yellow oil which solidified upon standing (IR (CHCl ₃ ) 1 00 (NC), 1593 (Ar), and 1568 cm ^"1 ).

The 1 -(p-toluenesulfonyl)-3-cyc!ohexylthiourea salt (Z) was prepared by dissolving a 0.49 g (2.3) mmole) portion of the isothiocyanate from above in a mixture of 10 mL of isopropyl ether and 5 mL of ethyl acetate. To this solution

0.27 g (2.72 mmole) of cyclohexylamine was added and the resulting mixture

allowed to stand overnight. Filtration and drying gave 0.57 g of a yellow solid (mp 162-168°). Microanalysis showed the presence of 2/3 of an equivalent of cyclohexylamine. Based on this stoichiometry, the yield was 92%. NMR (CDCI ₃ ): 8 7.90 (d, J=7.8, 1 H, NH); 7.82 (d, J=8.0, 0.5H, O ₂ SNHCS); 7.75 (d, J=8.2, 2H, ar); 7.30 (d, J=8.1 , 2H, ar); 4.12 (b, 0.57H, ⁺ NCH); 299 (b, 1H,

NCH); 2.42 (S, 3H, CH ₃ ); 1.96 (b, 2.7H, CH ₂ CN ⁺ ); 1.71 (b, 4H, CH ₂ CN); 1.24 (b, 10H, CCH ₂ C); IR (CHCI ₃ ): 3018, 2936, 1590 (w), 1533, 1595 (CS) 1130 (CS) cm ^"1 . Anal, calcd. for C ₁₄ H ₂ OS ₂ N ₂ O ₂ 2/3 C ₆ H ₁₃ N: C, 57.12; H, 7.64; N, 9.87; S, 16.92; found: C, 57.11 ; H, 7.64; N, 9.85; S, 16.67.

Example 3

Synthesis of 1 -(3.4-Dichlorophenyl) biguanide Nitrate

The procedure of Furukawa, ef al., Chem. Pharm. Bull. 9, 914 (1961), was followed with some modification.

3,4-dichlorophenylguanidine carbonate (Parish Chemical Co.) (ca. 2.0g) was partitioned between 2N KOH (50 mL) and CH ₂ CI ₂ (50 mL) and the organic layer was dried (Na ₂ SO ₄ ) and concentrated to provide the 3,4- dichlorophenylguanidine free base. The free base (240 mg, 1.2 mmol) was dissolved in MeOH (5 mL) and 3,5-dimethylpyrazolecarboxamidine nitrate (213 mg, 1.1 mmol) was added. The solution was heated at 110°C with evaporation of MeOH and the resulting residue was heated at 110° C for 2 hr. The mixture was then dissolved in a minimum amount of hot MeOH (ca. 4 mL) and poured into cold Et (ca. 40 mL). The resulting white solid was collected by filtration and washed with E^O (3x30 mL). The product was recrystallized from hot MeOH-Et ₂ O to provide (123 mg, 0.40 mmol) (33%) of 1 -(3,4- dichlorophenyl)biguanide nitrate as a white powder: mp 195-200° C (dec).

NMR (DMSO d ₆ ) 9.80 (br s, 2H, NH ₂ ), 7.85 (br s, 4H, 4xNH), 7.80-7.65 (m, 2H,

2xArH), 7.42-7.30 (m, 1 H, ArH).

* * * *

Purification of the compounds of the invention may be achieved using methods known in the art. When any of the synthetic procedures involve the use of fluorine or fluoride ion, a potentially important aspect in final purification is the removal of fluoride, which if present in even small amounts may alter the biological profile of the compound.

Salts of acidic groups of the product compounds may be prepared in the usual manner by contacting the compound with one or more equivalents of a desired base such as, for example, a metallic hydroxide base such as, for example, sodium hydroxide; a metal carbonate base such as, for example, sodium carbonate; or an amine base such as, for example, triethylamine, triethanolamine, and the like.

Acid salts of the compounds may be prepared by contacting a basic group in the compound with one or more equivalents of the desired inorganic or organic acid, such as, for example, hydrochloric, acetic, citric, oxalic, maionic, saiicyciic, malic, gluconic, fumaric, succinic, ascorbic, maleic, sulfuric, phosphoric, methanesuifonic or other acid.

Therapeutic Utility

In the practice of the therapeutic methods of the present invention, an effective amount of the active compound, including derivatives or salts thereof, or a pharmaceutical composition containing the same, as described below, is administered via any of the usual and acceptable methods known in the art, either singly or in combination with another compound or compounds of the

present invention or other pharmaceutical agents such as immunosuppressants, antihistamines, corticosteroids, and the like. These compounds or compositions can thus be administered orally, sublingually, topically (e.g., on the skin or in the eyes), parenterally (e.g., intramuscularly, intravenously, subcutaneously or intradermally), or by inhalation, and in the form of either solid, liquid or gaseous dosage including tablets, suspensions, soluates, hydrates and aerosols, as is discussed in more detail below. The administration can be conducted in single unit dosage form with continuous therapy or in single dose therapy ad libitum.

It is expected in particular that the present compounds may have suitably high cell adhesion modulation activity when administered via the oral route in view of the relative nonlabiiity of the chemical bonds in the compounds.

Useful pharmaceutical carriers for the preparation of the pharmaceutical compositions hereof can be solids, liquids or gases; thus, the compositions can take the form of tablets, pills, capsules, powders, entericaliy coated or other protected formulations (such as by binding on ion exchange resins or other carriers, or packaging in lipid protein vesicles or adding additional terminal amino acids), sustained release formulations, solutions (e.g., ophthalmic drops), suspensions, elixirs, aerosols, and the like. Water, saline, aqueous dextrose, and glycols are preferred liquid carriers, particularly (when isotonic) for injectable solutions. The carrier can be selected from various oils including those of petroleum, animal, vegetable or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Suitable pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like. The compositions may be subjected to

conventional pharmaceutical expedients such as sterilization and may contain conventional pharmaceutical additives such as preservatives, stabilizing agents, wetting or emulsifying agents, salts for adjusting osmotic pressure, buffers, and the like. Suitable pharmaceutical carriers and their formulations are described in Martin, Remington's Pharmaceutical Sciences, 15th Ed. (Mack Publishing Co.,

Easton 1975) (see, e.g., pp. 1405-1412, 1461-1487). Such compositions will, in general, contain an effective amount of the active compound together with a suitable amount of carrier so as to prepare the proper dosage form for proper administration to the host.

In one preferred embodiment, the therapeutic methods of the present invention are practiced when the relief of symptoms is specifically required or perhaps imminent; in another preferred embodiment, the method hereof is effectively practiced as continuous or prophylactic treatment.

In the practice of the therapeutic methods of the invention, the particular dosage of pharmaceutical composition to be administered to the subject will depend on a variety of considerations including the nature of the disease, the severity thereof, the schedule of administration, the age and physical characteristics of the subject, and so forth. Proper dosages may be established using clinical approaches familiar to the medicinal arts. It is presently believed that dosages in the range of 0.1 to 100 mg of compound per kilogram of subject body weight will be useful, and a range of 1 to 100 mg per kg generally preferred, where administration is by injection or ingestion. Topical dosages may utilize formulations containing generally as low as 0.1 mg of compound per ml of liquid carrier or excipient, with multiple daily applications being appropriate.

The compounds and therapeutic or pharmaceutical compositions of the invention are useful in the study or treatment of diseases or other conditions which are mediated by the binding of integrin receptors to ligands, including conditions involving inappropriate (i.e., excessive or insufficient) binding of cells to natural or other ligands. Such diseases and conditions include inflammatory diseases such as rheumatoid arthritis, asthma, allergy conditions, adult respiratory distress syndrome, inflammatory bowel diseases (e.g., ulcerative colitis and regional enteritis) and ophthalmic inflammatory diseases; autoimmune diseases; thrombosis or inappropriate platelet aggregation; reocculusion following thrombolysis conditions, cardiovascular disease; neoplastic disease including metastasis conditions, and acquired immunodeficiency syndrome (AIDS); as well as conditions wherein increased cell binding is desired, as in wound healing or prosthetic implantation situations as discussed in more detail above. Examples of "excessive" binding of cells to ligands include conditions, such as metastasis, where particular cell binding is inappropriate to the health of the subject and is sought to be eliminated to the maximum extent possible.

In addition, derivatives of the present compounds may be useful in the generation of antigens which, in turn, may be useful to generate antibodies. These antibodies will, in some cases, themselves be effective in inhibiting cell adhesion or modulating immune activity by acting as receptors for matrix proteins or other cell adhesion ligands, or, if anti-idiotypic, by acting to block cellular receptors.

EXAMPLE 3 Cell Adhesion Inhibition Assay

The following assay established the activity of the present compounds in inhibiting cell adhesion in a representative in vitro system. The assay was a competition assay in which both fibronectin and a test compound were present.

Microtiter plates were first precoated with fibronectin. The test compound was then added in increasing concentrations with cells known to contain the fibronectin receptor. The plates were then washed and stained for quantitation

I of attached cells. The present assay directly demonstrates the anti-cell adhesion activity and modulatory activity of the present compounds. For example, by immobilizing the compound on a surface, one could adhere appropriate ceils to that surface. Other ceil adhesion modulation activity, and utilities pertinent thereto, will be apparent to those skilled in the art.

The cell line U937 was purchased from American Type Tissue Culture Collection. The cells were cultured in RPMI media (J.R. Scientific Company,

Woodland Hiiis, California 95695) containing 10% fetal calf serum. Fibronectin was purified from human plasma according to the procedure of Engvall, E. and Ruoslahti, E., Int. J. Cancer 1977, 20, 1-4.

Microtiter plates (96-well, Falcon) were coated overnight at 4°C with 5 μg/ml fibronectin (FN) (for a total volume of 0.1 ml) or, as a control, 5 μg/ml bovine serum albumin (BSA) diluted in phosphate buffered saline (PBS, 0.01 M NaP0 ₄ in 0.9% NaCI at pH 7.2 to 7.4). Unbound proteins were removed from plates by washing with PBS. The plates were then coated with 100 μl of PBS containing 2.5 mg/ml BSA for one hour at 37 °C. This procedure is a modification of a previously published method, Cardareili, P.M. and M.D. Pierschbacher,

PNAS-USA 1986, 83, 2647-2651. The containment in the wells of functional

amounts of immobilized protein has been confirmed by independent assay of fibroblast attachment and ELISA (Engvall, E., Methods Enzymol. 1980, 70, 419- 43), although the actual amount of protein bound to the plate in these assays was not determined.

A U937 culture was collected and washed two times with Hanks Balanced Salt

Solution. The cells were counted and adjusted to 1.5 x 10 ⁶ cells per ml in Dulbecco's Modified Eagles Medium (DMEM) plus BSA (2.5 mg/ml) for cell attachment assay. Subject compounds were then dissolved in DMEM and BSA, and the pH was adjusted to 7.4 with 7.5% sodium bicarbonate. The compounds (100 μl) were added to FN-coated wells, at 1.5, 0.75, 0.375, 0.188,

0.094, 0.047, 0.023, 0.012, 0.006 and 0.003 mg/ml final concentration and U937 cells (100 μl) were added per well. The plates were then incubated at 37° C for 60 minutes. Following this incubation, the plates were washed once with PBS. Attached ceils were fixed with 3% paraformaldehyde in PBS and stained with 0.5% toluidine blue in 3.7% formaldehyde. The cells were stained overnight at room temperature and the optical density at 590 nm of toluidine blue-stained cells was determined using a vertical pathway spectrophotometer to quantitate attachment (VMAX Kinetic Microplate Reader, Molecular Devices, Menlo Park, California 94025).

Results. Table 1 below shows the results of the cell adhesion inhibition assay for representative compounds of the invention. Potency is expressed in μM units.

TABLE 1

Activity of Subject Compounds in the

U937 Fibronectin Adhesion Assay

Y

II

-N- -C- -N- (i) L

E! B I≤so ϋM

^» - ^Q H H H 310

(p-toluenesulfonyl)

(p-toluenesulfonyl) H H H 670 (ammonium salt)

(p-toluenesulfonyl) H H 914 (cyclohexylamine salt)

(p-toluenesulfonyl) H H -CH,CH,OH S 815 (ethanolamine salt)

(p-toluenesulfonyl) H -CH(CH ₃ ) ₂ -CH(CH ₃ ) ₂ 480 (diisopropylamine salt)

(p-toluenesulfonyl) H H H NH 42

H H H 1100

H H H NH 180

H0

// W . _c u — H H H NH 890

B Y JCgoJiM

H H NH 61

ln addition, the compounds of Table 2 were also synthesized and tested in the cell adhesion inhibition assay described above. Specific activity levels were not established inasmuch as the IC _gg of the compounds was determined to be in excess of 1.5 mg/ml. Thus, although such compounds are believed to be active as cell adhesion modulators at higher dosage levels, they are presently not as highly preferred as the compounds exemplified above.

TABLE 2 Additional Compounds

El B

(p-toluenesulfonyl )) HH HH --CCHH, ₂ (benzylamine salt)

(p-toluenesulfonyl) H H (aniline salt)

(p-toluenesulfonyl) H -CH ₂ CH ₃ -CH ₂ CH ₃ (diethylamine salt)

(p-toluenesulfonyl) H (piperidine salt)

(p-toluenesulfonyl) H H -CH ₂ CH ₂ CH ₂ CH ₃

(butylamine salt)

(p-toluenesulfonyl) H

(dicyclohexylamine salt)

(p-toluenesulfonyl) H H —CH _J CH,-//

(phenethylamine salt) \—/

(p-toluenesulfonyl) H H S

(p-methoxyaniline salt)

(p-toluenesulfonyl) H H -ft ^CH ₂ C0 _j H S (p-amiπophenylacetic \=J || acid salt)

(p-toluenesulfonyl) H H -C H. CH.-M ) S

V_/ 1

(p-toluenesulfonyl) H H

(p-toluenesulfonyl) H H -NH

(p-toluenesulfonyl) H H

(p-toluenesulfonyl) H H -CH,CH.

H H H

NH ₂ C H- /_ - H H H

H _j

0

II H H H NH

H-C-

H0 ₂ CCH ₂ CH ₂ CH ₂ — H H H NH

H0 ₂ C(CH ₂ ) ₆ - H H H NH

H H H H NH

H0 ₂ CCHCH ₂ CH ₂ CH ₂ — H H H NH

NH,

The foregoing examples are given to enable those skilled in the art to more fully understand and practice the present invention. They should not be construed as a limitation upon the scope of the invention, which is set forth in the appended claims, but merely as being illustrative and representative thereof.