WO1999020618A1

WO1999020618A1 - Thiadiazoles amides useful as antiinflammatory agents

Info

Publication number: WO1999020618A1
Application number: PCT/US1998/021629
Authority: WO
Inventors: Ronald B. Gammill; Susan Vander Velde; Richard Allen Nugent
Original assignee: Active Biotech Ab
Priority date: 1997-10-21
Filing date: 1998-10-20
Publication date: 1999-04-29
Also published as: AU1083499A

Abstract

The present invention provides a compound of formula (I) wherein R1, R2 and R3 are as defined herein. The compounds of the present invention are therapeutically useful in the treatment of a broad range of inflammatory disease such as, for example, hypersensitivity reactions, asthma, rheumatoid arthritis, bacterial meningitis, aspiration lung injury, inflammatory bowel disorder and related complications.

Description

THIADIAZOLES AMIDES USEFUL AS ANTIINFLAMMATORY AGENTS

FIELD OF THE INVENTION This invention relates to novel thiadiazole amides, to pharmaceutical compositions containing them, and to methods of using them. The compounds of the invention are pharmaceutically active in the treatment of inflammatory diseases.

BACKGROUND OF THE INVENTION Inflammation is an integral part of a wide array of human diseases, ranging from bacterial pneumonia, in which the response is life-saving, to adult respiratory distress syndrome, in which it is life-threatening. Inflammation may result in substantial tissue damage or initiate processes leading to excessive fibrous repair, and therefore, it is desirable to interrupt its progression. Today, many investigators are attempting to identify new therapeutic agents designed to directly block adhesive events involved in an array of disease processes.

LFA-1 and Mac-1, members of the β2 integrin family of adhesion molecules, are thought to play a critical role in several types of inflammatory disease processes by interacting with intercellular adhesion molecule (ICAM), which promotes the migration of the leukocyte rapidly into surrounding tissue. Support for the importance of β2 integrin in mediating inflammatory responses has been demonstrated by the evidence that transendothelial migration in vitro is markedly inhibited by monoclonal antibodies against β2 integrins or ICAM-1. C. W. Smith, Can. J. Physiol. Pharmacol, Vol. 71, pp 76-87 (1993). Furthermore, blockade of the LFA-1 complex has been shown to inhibit neutrophil influx in almost every system, including skin, peritoneum, synovium, lung, kidney, and heart. As one of the primary ligands for the β2 integrins, it would also be expected that blockade of ICAM-1 would inhibit the inflammatory response. S. M. Albelda et al., The FASEB J., Vol. 8, pp 504-512 (1994).

We now have discovered that certain novel thiadiazole amides are LFA-1 and Mac-1 inhibitors. Molecules that inhibit LFA-1 and Mac-1 binding with ICAM-1 down regulate inappropriate leukocyte wreaking havoc on healthy tissues seen in acute and chronic inflammatory diseases. As such, these compounds are therapeutically useful in the treatment of a broad range of inflammatory disease such as, for example, hypersensitivity reactions, asthma, rheumatoid arthritis, bacterial meningitis, aspiration lung injury, inflammatory bowel disorder and related complications. INFORMATION DISCLOSURE The following references disclose thiadiazole derivatives. International Publication No. WO 96/30370 discloses thiazole and thiadiazole derivatives useful in the treatment of thrombocytopenia. U. S. Patent 4,775,408 discloses pyridine substituted thiadiazole ureas which have herbicidal and plant growth regulatory properties.

U. S. Patent 4,576,629 discloses herbicidal thiadiazole ureas wherein the 5- position of the thiadiazole ring is hetero substituted and which exhibit enhanced selective herbicidal activity. Abstract of Japanese Patent 1160-976-A discloses 1,3,4-thiadiazole derivatives useful as antiulcer agents.

SUMMARY OF THE INVENTION The present invention presents novel compounds of formula I

I or pharmaceutically acceptable salts thereof wherein:

a) -aryl, b) -aryl wherein aryl is substituted with one to three R₄,

0 -Q, d) -Q wherein Q is substituted with one to three R₄, e) -Het, f) -Het wherein Het is substituted with one to three R₄,

h)

, optionally substituted with C_M alkyl or C₃.₆ cycloalkyl, i) C_j.g carboalkoxy, j) -C(=0)-CH₂C0₂(C₁.₄ alkyl), or k) -C(=0)NH(CH₂)_ήR₅,

1) C_{1 0} alkyl, m) C^o alkyl substituted with one to three R₆,

n) C^o alkenyl, or o) C_{1 0} alkenyl substituted with one to three R₆; R₂ is a) -(C=0),.(CH₂)/CR₇R₈V;

R₃ is a) -(CR₉R₁₀)_r(CH₂)_raryl, b) -(CR₉R₁₀)_r(CH₂)_raryl wherein aryl is substituted with one to three R_π, c) -(CR₉R₁₀)_r(CH₂)_rQ, d) -(CR₉R₁₀).-(CH₂)_rQ wherein Q is substituted with one to three R_n, e) -(CR₉R₁₀)_;-(CH₂)_rHet, f) -(CR₉R₁₀)_r(CH₂)_rHet wherein Het is substituted with one to three R_n, or g) -(CR₉R₁₀)_r(CH₂)_rpentafluorophenyl;

R₄ is a) halo, b) C_1-4 alkyl, c) C₃.₆ cycloalkyl, d) C_j.₄ alkoxy, e) aryl, f) Q, g) Het, h) C₁ carboalkoxy, i) C_l monoalkylamino, j) C_1A dialkylamino, k) amido,

1) C_j.₄ alkylthio, m) trihalomethyl, n) -(CH₂)_rO-(C₁.₄ alkyl), o) nitro, P) mercapto, q) ni trine, r) cyano, s) hydroxy. t) -NHC(=0)(C₁.₄ alkyl), or u) -NHS0₂(C₁.₄ alkyl);

R₅ is a) C _s alkyl, b) aryl, c) Q, or d) Het;

R₆ is a) halo, b) hydroxy, c) C_j_₄ alkoxy, d) 0^₄ carboalkoxy, e) amido, f) nitro, g) trihalomethyl, h) cyano, i) mercapto, j) C_j.₄ alkylthio, or k) C_j.₈ alkyl;

R₇ and R₈ are the same and different a) H, b) C_j.₆ alkyl, c) C_3.6 cycloalkyl, d) -(CH^-O-C^ alkyl, e) -(CH₂)_rQ, or ) -(CH₂)_rHet;

R₉ and R₁₀ are the same and different a) H, b) C_1-4 alkyl, c) 0^₄ alkoxy, d) C_3.6 cycloalkyl, or e) C_x.4 carboalkoxy; R_π is a) C₁.₄ alkyl, b) Cj.₄ alkoxy, c) trihalomethyl, d) halo, e) nitro, f) cyano, g) nitrine, h) C_j.₄ acyl, i) C₁ carboalkoxy, or j) carboxyl; aryl is monocarbocyclic, or bicarbocyclic aromatic moiety;

Q is 5- to 10-membered saturated heterocyclic moiety having one to three atoms selected from the group consisting of oxygen, nitrogen, and sulfur; Het is 5- to 10-membered unsaturated heterocyclic moiety having one to three atoms selected from the group consisting of oxygen, nitrogen, and sulfur; h is 0, 1, 2 or 3; i is 0 or 1; j is 0, 1, 2, 3, 4 or 5; k is 0, 1, 2 or 3;

I is 0, 1, 2, 3, 4 or 5; n is 0, 1 or 2; and with the following provisos: a) where both R₇ and R₈ are hydrogen, j + k is other than 1; b) where R₃ is phenyl substituted with fluoro, R_x is other than unsubstituted phenyl.

These compounds are therapeutically useful in the treatment of a broad range of inflammatory disease such as, for example, hypersensitivity reactions, asthma, rheumatoid arthritis, bacterial meningitis, aspiration lung injury, inflammatory bowel disorder and related complications.

DETAILED DESCRIPTION OF THE INVENTION For the purpose of the present invention, the carbon content of various hydrocarbon containing moieties is indicated by a prefix designating the minimum and maximum number of carbon atoms in the moiety, i.e., the prefix C^ defines the number of carbon atoms present from the integer "i" to the integer "j", inclusive.

Thus, for example, C^ alkyl refers to alkyl of one to four carbon atoms, inclusive, or methyl, ethyl, propyl, butyl and isomeric forms thereof.

The terms "C₁₄ alkyl", "C_w alkyl", "C_H alkyl", and "C_{1 0} alkyl" refer to an alkyl group having one to four, one to six, one to eight, or one to ten carbon atoms respectively such as, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl and their isomeric forms thereof.

The term "C₂.₁₀ alkenyl" refers to at least one double bond alkenyl group having two to ten carbon atoms respectively such as, for example, ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, heptdienyl, octenyl, octadienyl, octatrienyl, nonenyl, undecenyl, dodecenyl, and their isomeric forms thereof. The term "C₃.₆ cycloalkyl" refers to a cycloalkyl having three to six carbon atoms such as, for example, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl and their isomeric forms thereof.

The terms "C_M alkoxy" refers to an alkyl group having one to four carbon atoms attached to an oxygen atom of hydroxyl group such as, for example, methoxy, ethoxy, propyloxy, butyloxy and their isomeric forms thereof.

The term "C alkylthio" refers to an alkyl group having one to four carbon atoms attached to an thiohydroxy moiety, for example, methythio, ethylthio, propylthio, butylthio and isomeric forms thereof.

The terms "C,_.4 acyl" and "C^ acyl" refer to a carbonyl group having an alkyl group of one to four or one to six carbon atoms respectively.

The terms "C_H carboalkoxy" and

carboalkoxy" refer to an ester group having an alkyl group of one to four or one to six carbon atoms respectively.

The term "C₁₄ monoalkylamino" refers to an alkyl group having one to four carbon atoms attached to an amino moiety, for example, methylamine, ethylamine, n-propylamine, n-butylamine, and isomeric forms thereof.

The term "C,_.4 dialkylamino" refers to two alkyl groups having one to four carbon atoms attached to an amino moiety, for example, dimethylamine, methylethylamine, diethylamine, dipropylamine, methypropylamine, ethylpropylamine, dibutylamine, and isomeric forms thereof. The term "halo" refers to fluoro, chloro, bromo, or iodo.

The term trihalomethyl refers to trifluoromethyl, trichloromethyl or tribr omomethyl .

The term "aryl" refers to monocarbocyclic or bicarbocyclic aromatic moiety such as, for example phenyl, naphthyl or biphenyl. Each of these moieties may be substituted as appropriate. Aryl is preferably substituted and unsubstituted phenyl. The term "Het" refers to a 5- to 10-membered unsaturated heterocyclic moiety having one or more atoms selected from the group consisting of oxygen, nitrogen, and sulfur such as; for example, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4- pyrimidinyl, 5-pyrimidinyl, 3-pyridazinyl, 4-pyridazinyl, 3-pyrazinyl, 2-quinolyl, 3- quinolyl, 1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 2-quinazolinyl, 4-quinazolinyl, 2- quinoxalinyl, 1-phthalazinyl, 2-imidazolyl, 4-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5- isoxazolyl, 3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 2- thiazolyl, 4-thiazolyl, 5-thiazolyl, 3-isothiazole, 4-isothiazole, 5-isothiazole, 2-indolyl, 3-indolyl, 3-indazolyl, 2-benzoxazolyl, 2-benzothiazolyl, 2-benzimidazolyl, 2- benzofuranyl, 3-benzofuranyl, benzoisothiazole, benzoisoxazole, 2-furanyl, 3-furanyl, 2-thienyl, 3-thienyl, 2-pyrrolyl, 3-pyrrolyl, 3-isopyrrolyl, 4-isopyrrolyl, 5-isopyrrolyl, 1-indolyl, 1-indazolyl, 2-isoindolyl, 1-purinyl, 3-isothiazolyl, 4-isothiazolyl and 5- isothiazolyl, preferably pyridyl, quionlinyl, pyrrolyl, thienyl, thiazolyl, or indolyl. The term "Q" refers to a 5- to 10-membered saturated heterocyclic moiety having one to two atoms selected from the group consisting of oxygen, nitrogen, and sulfur such as, for example, piperidinyl, 2-, 3-, or 4-piperidinyl, [l,4]piperazinyl, morpholinyl, 2- or 3-morpholinyl, thiomorpholinyl, dioxolanyl, imidazolidinyl, [l,3]oxathiolanyl, [l,3]oxazolidinyl, pyrrolidinyl, butyrolactonyl, butyrolactamyl, succinimidyl, glutarimidyl, valerolactamyl, 2,5-dioxo-[l,4]-piperazinyl, pyrazolidinyl, 3-oxopyrazolidinyl, 2-oxo-imidazolidinyl, 2,4-dioxo-imidazolidinyl, 2-oxo-[l,3]- oxazolidinyl, 2,5-dioxo-[l,3]-oxazolidinyl, isoxazolidinyl, 3-oxo-isoxazolidinyl, [1,3]- thiazolidinyl, 2- or 4-oxo-[l,3]-thiazolidinyl, butyrolactamyl, succinimidyl, glutarimidyl, valerolactamyl, 2,5-dioxo-[l,4]-piperazinyl, 3-oxopyrazolidinyl, 2-oxo- imidazolidinyl, 2,4-dioxo-imidazolidinyl, 2-oxo-[l,3]-oxazolidinyl, 2,5-dioxo-[l,3]- oxazolidinyl, 3-oxo-isoxazolidinyl, 2- or 4-oxo-[l,3]-thiazolidinyl. Within the definition of the terms "Het" and "Q", the nitrogen atom forming the hetero rings may have a protective group such as an acetyl or hydroxyacetyl group.

Certain reagents are abbreviated herein. THF refers to tetrahydrofuran, DMF refers to dimethyl formamide. The compounds of the present invention can be converted to their salts, where appropriate, according to conventional methods.

The term "pharmaceutically acceptable salts" refers to addition salts useful for administering the compounds of this invention and include hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, acetate, propionate, lactate, mesylate, maleate, malate, succinate, tartrate, citric acid, 2-hydroxyethyl sulfonate, fumarate and the like. These salts may be in hydrated form. Some of the compounds of this invention may form metal salts such as sodium, potassium, calcium and magnesium salts and these are embraced by the term "pharmaceutically acceptable salts."

Depending on substituents, the compounds of formula I of this invention may contain a chiral center and other isomeric forms and this invention embraces all possible stereoisomers and geometric forms.

Typical antiinflammatory thiadiazoles amides of this invention are a. 3-Fluoro-N-[5-[(l-phenylpropyl)thio]-l,3,4-thiadiazol-2-yl]benzeneacetamide, b. (E)-3-Nitro-N-[5-[(3J-dimethyl-2,6-octadienyl)thio]-l,3,4-thiadiazol-2- yl]benzamide, c. (E)-3-Trifluoromethyl-N-[5-[(3J-dimethyl-2,6-octadienyl)thio]-l,3,4-thiadiazol- 2-yl]benzamide, d. N-[5-[[6-(l,3-Dihydro-l,3-dioxo-2H-isoindol-2-yl)hexyl]thio]- l,3,4-thiadiazol-2- yl]-3-nitrobenzamide, e. N-[5-[[6-(l,3-Dihydro-l,3-dioxo-2H-isoindol-2-yl)hexyl]thio]-l,3,4-thiadiazol-2- yl] -3-trifluoromethylbenzamide, f . N- [5- [ [6-( 1 ,3-Dihydro- 1 ,3-dioxo-2H-isoindol-2-yl)hexyl] thio] - 1 ,3 ,4-thiadiazol-2- yl] -3-cyanobenzamide, g. N-[5-[[6-(l,3-Dihydro-l,3-dioxo-2H-isoindol-2-yl)hexyl]thio]-l,3,4-thiadiazol-2- yl] -2,3,4,5,6-pentafluorobenzamide, h. (E)-N-[5-[(3J-Dimethyl-2,6-octadienyl)thio]-l,3,4-thiadiazol-2-yl]-2,3,4,5,6- pentafluorobenzamide, i. (E)-N-[5-[(3J-Dimethyl-2,6-octadienyl)thio]-l,3,4-thiadiazol-2-yl]-2,3,4,5,6- pentafluorobenzeneacetamide, j. (E)-N-[5-[(3J-Dimethyl-2,6-octadienyl)thio]-l,3,4-thiadiazol-2-yl]-2(3,4,5,6- pentafluorobenzene)propyl, k. N-[5-[[2-Oxo-2-(4-pyridinyl)ethyl]thio]-l,3,4-thiadiazol-2-yl]-3-

(trifluoromethyl)benzamide, 1. N-[5-[[2-Oxo-2-(3-pyridinyl)ethyl]thio]-l,3,4-thiadiazol-2-yl]-3- (trifluoromethyl)benzamide, m. 3,4-Dichloro-N-[5-[[l-(3-pyridinyl)propyl]thio]-l,3,4-thiadiazol-2-yl]benzamide, n. 3,5-Difluoro-N-[5-[[l-(3-pyridinyl)propyl]thio]-l,3,4-thiadiazol-2-yl]benzamide, o. 3,5-Dimethoxy-N-[5-[[l-(3-pyridinyl)propyl]thio]-l,3,4-thiadiazol-2- yl]benzamide, p. -Methyl-N-[5-[[l-(3-pyridinyl)propyl]thio]-l,3,4-thiadiazol- 2- yljbenzeneacetamide, q. -Cyclopropyl-N- [5- [( l-phenylpropyl)thio] - 1 , 3 ,4-thiadiazol- 2- yl]benzeneacetamide, or r. α-Methoxy-N-[5-t[l-(3-pyridinyl)propyl]thio]-l,3,4-thiadiazol-2- yl]benzeneacetamide. The compounds of formula I are generally prepared by coupling an alkylating agent A

R' -halo (A) with commercially available 5-amino-l,2,5-thiadiazole-2-thiol in the presence of appropriate base such as, for example, triethylamine or sodium hydride. R' is Rj-R₂-radical as defined previously and halo is fluoro, chloro, bromo or iodo. The alkylating agents A are either commercially available or can be prepared from the corresponding alcohols with an activating agents such as methanesulfonyl chloride or thionyl chloride. The coupling results in the formation of the intermediate B

N-N

^R " ^R2^s^s^^NH; (B)

in the presence of an appropriate solvent such as, for example, THF, EtOAc, DMF, CH₃C1 or CHgCN at room or slightly elevated temperature. Particularly useful starting compounds in the preparation of compounds of formula I of the present invention is a compound of formula D

wherein R₄ is as defined previously, R" is R₇ or R₈ are as defined previously, the ring E is aryl, Q or Het as defined previously. All these starting compounds are either commercially available or can be easily prepared according to the methods well known in the art and are illustrated in examples as described hereinafter. To provide compounds of formula I of the present invention, the intermediate

B is converted to the corresponding thiadiazoles amides. Reaction of the intermediate B with acid chlorides, R₃COCl, in the presence of appropriate base such as triethylamine generates thiadiazole amides. The methods of these reactions are well known to those skilled in the art. When desirable, the sulfur atom of the side chain can be oxidized by an appropriate oxidizer using the methods well known to those skilled in the art in an early synthetic step or at the end of the synthetic sequence to the corresponding sulfones and sulfoxides, respectively.

The pharmaceutical compositions of this invention may be prepared by combining the compounds of formula I of this invention with a solid or liquid pharmaceutically acceptable carrier, and optionally, with pharmaceutically acceptable adjuvants and excipients employing standard and conventional techniques. Solid form compositions include powders, tablets, dispersible granules, capsules and suppositories. A solid carrier can be at least one substance which may also function as a diluent, flavoring agent, solubilizer, lubricant, suspending agent, binder, tablet disintegrating agent, and encapsulating agent. Inert solid carriers include magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, cellulosic materials, low melting wax, cocoa butter, and the like. Liquid form compositions include solutions, suspensions and emulsions. For example, there may be provided solutions of the compounds of this invention dissolved in water, water-propylene glycol, and water-polyethylene glycol systems, optionally containing conventional coloring agents, flavoring agents, stabilizers and thickening agents.

The pharmaceutical composition is provided by employing conventional techniques. Preferably the composition is in unit dosage form containing an effective amount of the active component, that is, the compounds of formula I according to this invention.

The quantity of active component, that is, the compounds of formula I according to this invention, in the pharmaceutical composition and unit dosage form thereof may be varied or adjusted widely depending upon the particular application method, the potency of the particular compound and the desired concentration.

Generally, the quantity of the active component will range between 0.5% to 90% by weight of the composition.

In therapeutic use for treating inflammatory complications in humans and other animals that have been diagnosed with inflammatory disease, the compounds or pharmaceutical compositions thereof will be administered orally, parenterally, aerosol, and/or topically at a dosage to obtain and maintain a concentration, that is, an amount, or blood-level of active component in the animal undergoing treatment which will be antiflammatory effective. Generally, such antiinflammatory effective amount of dosage of the active component will be in the range of about 0.1 to about 200 mg kg, more preferably about 3.0 to about 50 mg/kg of body weight/day. It is to be understood that the dosages may vary depending upon the requirements of the patient, the severity of the inflammatory complication being treated, and the particular compounds being used. Also, it is to be understood that the initial dosage administered may be increased beyond the above upper level in order to rapidly achieve the desired blood-level or the initial dosage may be smaller than the optimum and the daily dosage may be progressively increased during the course of treatment depending on the particular situation. If desired, the daily dose may also be divided into multiple doses for administration, e.g., two to four times per day.

These compounds are useful for the treatment of inflammatory complications in humans and other warm blooded animals by either parenteral, oral, aerosol or topical administration. In general, the preferred form of administration is orally. Pharmaceutical compositions for parenteral administration will generally contain a pharmaceutically acceptable amount of the compounds according to formula I as a soluble salt (acid addition salt or base salt) dissolved in a pharmaceutically acceptable liquid carrier such as, for example, water-for-injection and a suitably buffered isotonic solution having a pH of about 3.5 - 6.0 Suitable buffering agents include, for example, trisodium orthophosphate, sodium bicarbonate, sodium citrate, N-methylglucamine, L(+)-lysine and L(+)-arginine, to name a few. The compounds according to formula I generally will be dissolved in the carrier in an amount sufficient to provide a pharmaceutically acceptable injectable concentration in the range of about 1 mg/ml to about 400 mg/ml. The resulting liquid pharmaceutical composition will be administered so as to obtain the above mentioned antiflammatory effective amount of dosage. The compounds of formula I according to this invention are advantageously administered orally in solid and liquid dosage forms. The compounds of this invention are useful antiinflammatory agents, effective against a broad range of inflammatory disease states in which neutrophils wreak havoc on healthy tissues. Therefore, they are therapeutically useful in the treatment of chronic or acute inflammatory disease such as, for example, hypersensitivity reactions, asthma, rheumatoid arthritis, bacterial meningitis, aspiration lung injury, inflammatory bowel disorder and related complications. Humans or animals suffered with such complications are readily diagnosed by a physician or veterinarian of ordinary skill.

The compounds and their preparations of the present invention will be better understood in connection with the following examples, which are intended as an illustration of and not a limitation upon the scope of the invention. I. Preparation of intermediate Compound B. Method A:

5-Amino-l,3,4-thiadiazole-2-thiol (1 equiv.) is partially dissolved in CH₃CN. Triethylamine (2-3 equiv.) is added, followed by the alkyl chloride. The chloride is either commercially available, or generated from the alcohol with thionyl chloride (2 equiv.) in chloroform. The excess thionyl chloride is removed under reduced pressure, and the neat alkyl chloride was then added to the thiadiazole in CH₃CN. The reaction is stirred at 25-65°C overnight. The CH₃CN is removed in vacuo, and the residual oil is partitioned between CHC1₃ and H₂0. After the layers are separated, the aqueous phase is extracted with CHC1₃. The combined organics are washed with brine, dried over MgS0₄, and concentrated to crude material. Product is purified by either recrystallization or flash chromatography. Method B:

The mesylate of the appropriate alcohol is prepared in situ. The alcohol (1 equiv.) is dissolved in THF, and triethylamine (2 equiv.) is added. The reaction is cooled to 0°C, and methanesulfonyl chloride (1.1 equiv.) is added. The reaction is allowed to warm to room temperature. After 1 hour, 5-amino-l,3,4-thiadiazole-2- thiol (1 equiv.) is added. The reaction is stirred overnight. The reaction is diluted with EtOAc and H₂0. After separation, the aqueous phase is extracted with EtOAc. The combined organics are washed with brine, dried over MgS0₄, and concentrated to crude material. The product is purified by flash chromatography or recrystallization. Method C:

5-Amino-l,3,4-thiadiazole-2-thiol (1 equiv.) is dissolved in DMF and cooled to 0°C. Sodium hydride (1.1 equiv) is added, and the reaction is stirred at 0°C until all the solids are dissolved (1-2 hours). The alkyl chloride is generated from the alcohol (1 equivi) with thionyl chloride (2 equivi) in chloroform. The excess thionyl chloride is removed in vacuo. The alkyl chloride is added to the sodium anion of the thiadiazole. The reaction is allowed to warm to room temperature and stirred for 5- 12 hours. The reaction is quenched and then diluted with H₂0. The aqueous solution is extracted with EtOAc, and the combined organics are washed with brine. After drying over MgS0₄, the solvent is removed in vacuo to yield crude material. The product is purified by flash chromatography or recrystallization. Method D:

The appropriate alcohol (1 equiv.) and triethylamine (1.1 equiv.) is dissolved in THF and cooled to 0°C. Methanesulfonyl chloride (1.1 equiv.) is then added, and the reaction is stirred at room temperature for 1 hour. The reaction is diluted with EtOAc and H₂0, and the layers are separated. The organic phase is washed with brine and dried over MgS0₄. The solvent is removed in vacuo, yielding pale yellow oil. The mesylate is added neat to the sodium anion of the thiadiazole. The thiadiazole is deprotonated by added sodium hydride (1.1 equiv.) to a 0°C solution of 5-amino-l,3,4-thiadiazole-2-thiol (1 equiv.) and dissolved in DMF. The reaction is allowed to warm to room temperature and stirred overnight. The reaction is quenched and diluted with H₂0. The aqueous phase is extracted with EtOAc, and the combined organics are washed with brine. After drying over MgS0₄, the solvent is removed in vacuo yielding crude material. The product is isolated by flash chromatography or recrystallization.

II. Preparation Thiadiazoles Amides. Method E:

To a solution (or slurry) of alkylated thiadiazole (1 equiv.) in THF is added triethylamine or sodium hydride (2 equiv.). Next, acid chloride (1.1 equiv.) is added, and the reaction is stirred at room temperature for 5-12 hours. The reaction is diluted with CH₂C1₂ and H₂0, and the layers are separated. The aqueous phase is extracted with CH₂C1₂. The combined organics is washed with brine and dried over MgS0₄. Solvent is removed in vacuo, and the product is then purified by recrystallization or flash chromatography.

EXAMPLE 1 Preparation of α-Methoxy-N-[5-[[l-(3-pyridinyl)propyl]thio]-

1 , 3 ,4-thiadiazol-2-yl] benzeneacetamide .

Step 1 Preparation of 5-[(l-pyridinylpropyl)thio]-l,3,4-thiadiazol-2-amine. Following the general procedure outlined in Method B and making non- critical variations but starting with 1-phenylpropyl alcohol and 2-amino-5-mercapto- 1,3,4-thiadiazole, the title compound is obtained as a solid. The crude product is purified by flash chromatography (5% CH₃0H/CH₂C1₂). mp 113-114°C. Η NMR (CDClg) δ 0.94, 1.97-2.15, 4.40, 5.30, 7.25-7.31. ¹³C NMR (DMSO) δ 11.8, 28.5, 54.9, 127.5, 127J, 128.4, 140.5, 148.0, 170.4.

Following the general procedure outlined in Method E and making non- critical variations but starting with the product of Step 1, Example 1 and - methoxyphenyl acetyl chloride, the title compound is obtained as a solid.

*H NMR (MEOH) 0.98, 1.98-2.19, 3.40, 4.63, 7.33-7.46, 7.84-7.88, 8.37, 8.44.

EXAMPLE 2 Preparation of α -Methyl-N-[5-[[l-(3-pyridinyl)propyl]thio]-l,3,4- thiadiazol- 2-yl]benzeneacetamide.

Following the general procedure outlined in Method E and making non- critical variations but starting with the product of Step 1, Example 1 and 2-phenyl proprionyl chloride, the title compound is obtained as a solid, mp 156-158°C.

*H NMR (DMSO) 0.87, 1.40, 1.94-2.07, 3.98, 4.64, 7.23-7.36, 7.77-7.79, 8.42-8.44, 8.50.

EXAMPLE 3 Preparation of 3,4-Dichloro-N-[5-[[l-(3-pyridinyl)propyl]thio]- l,3,4-thiadiazol-2-yl]benzamide.

Following the general procedure outlined in Method E and making non- critical variations but starting with the product of Step 1, Example 1 and 3,4- dichlorobenzoyl chloride, the title compound is obtained as a solid, mp 152-155°C. !H NMR (DMSO) 0.90, 1.97-2.12, 4.72, 7.34-7.38, 7.81-7.83, 7.98-8.02, 8.32, 8.44, 8.55.

EXAMPLE 4 Preparation of 3,5-Difluoro-N-[5-[[l-(3-pyridinyl)propyl]thio]- l,3,4-thiadiazol-2-yl]benzamide.

Following the general procedure outlined in Method E and making non- critical variations but starting with the product of Step 1, Example 1 and 3,5- difluorobenzoyl chloride, the title compound is obtained as a solid, mp 174-177°C. !H NMR (DMSO) 0.90, 2.00-2.12, 4.73, 7.34-7.37, 7.57-7.63, 7.77-7.84, 8.44, 8.55.

EXAMPLE 5 Preparation of 3,5-Dimethoxy-N-[5-[[l-(3-pyridinyl)propyl]thio] l,3,4-thiadiazol-2-yl]benzamide.

Following the general procedure outlined in Method E and making non- critical variations but starting with the product of Step 1, Example 1 and 3,5- dimethoxybenzoyl chloride, the title compound is obtained as a solid, mp 169-171°C.

*H NMR (DMSO) 0.90, 1.99-2.12, 3.79, 4.71, 6.74, 7.24-7.25, 7.34-7.28, 7.80-7.83, 8.44, 8.54.

INHIBITION OF β₂ INTEGRIN LIGAND BINDING ASSAYS The compounds may be tested in one of several biological assays to determine the concentration of compound which is required to have a given pharmacological effect.

To identify inhibitors of β₂ integrin ligand binding function, two primary and two secondary assays are performed. The assays are established to identify compounds which inhibit the interaction of either LFA-1 or Mac-1 with immobilized ICAM-1. The interaction of the β₂ integrins with ICAM-1 plays as important role in a number of adhesive events during normal immune and inflammatory responses including antigen presentation to T cells, T cell mediated cytotoxicity, and the firm attachment and extravasation of circulating leukocytes into the surrounding tissue. Both the primary LFA-1 and Mac-1 adhesion assays are performed using the well- known scintillation proximity assay (SPA) bead technology which is discussed in further in Cook, N.D. et. al. Pharmaceutical Manufacturing International (1992) pp. 49-53, "SPA: A revolutionary new technique for drug screening". Bosworth, N. and Towers, P. Nature (1989) 341:167-168, "Scintillation proximity assay". Undefriend, S., Gerber, L. and Nelson, N. Analytical Biochemistry (1987) 161: 494-500 "Scintillation Proximity Assay, a sensitive and continuous isotopic method for monitoring ligand-receptor and antigen-antibody interactions".

Briefly, the assay relies upon three major components: a radiolabeled CHO cell that has been transfected with the heterodimeric either LFA-1 or Mac-1 molecule and is functionally expressed on the cell surface; a secreted soluble form of intercellular ahesion molecule produced from a transfected CHO cell line and which has subsequently been biotinylated; and streptavidin SPA beads to monitor the interaction of these two components. The SPA technology is utilized because it obviates the need for a wash step(s), allowing low affinity interactions to remain undisturbed.

Stable CHO cells expressing either LFA-1 or Mac-1 were established. Cells were grown in modified Dulbecco's media and labeled overnight in a leucine deficient media in the presence of ³H-leucine (10 mCi/10⁶ cells for LFA-1 and 50 mCi/10⁶ cells for Mac-1). After labeling, cells (1 x 10⁴ LFA-1 and 5 x 10⁴ for Mac-1) were activated with phorbol ester (100 nM for LFA-1 and 500 nM for Mac-1) and allowed to react with streptavidin SPA beads previously coated with biotinylated soluble ICAM-1 dispensed into 96 well plates. To inhibit adhesion to ICAM-1 coated SPA beads, 4X stock of compound, blocking antibodies or buffer control were added to the wells immediately prior to the addition of cells. Following incubation for 8 hours, adhesion was quantitated in the wells using a scintillation counter. For further analysis of compounds that inhibit LFA-1 interactions, a secondary adhesion assay using JY and human soluble ICAM-1 was established. JY cells, a human lymphoblastoid cell line, constitutively expresses LFA-1. Microtiter wells were coated with soluble ICAM-1 diluted in 0.1 1M NaC0₃ buffer (pH 8.0) overnight at 4°C. The remaining binding sites on the plastic were blocked with phosphate buffered saline (PBS) containing 1 mM Ca²⁺/Mg²⁺ and 1% human serum albumin (PBS/HSA) for 1 hour at 37°C. JY cells were harvested by centrifugation and fluorescently labeled with 2'7'-bis-(carboxyethyl)-5(6)-carboxy-fluorescien. JY cells were then washed once, in PBS/HSA, and stimulated with phorbol 12-myristate 13-acetate (PMA; 50 ng/ml) for 5 minutes. The microtiter plates was washed once with PBS containing 1 mM Ca²⁺/Mg²⁺ and 0.5% Tween-20 and then immediately washed with PBS/HSA. A 80 mL aliquot of cells (1 x 10⁵) was plated in triplicate on the microtiter wells. To inhibit adhesion to ICAM-1 coated wells, a 20 ml aliquot of 5X stock of compound, blocking antibodies or buffer control were added to the wells immediately prior to the addition of cells to the wells. Following incubation for 30 minutes at 37°C, the plates were washed with PBS/HSA. Fluorescence was quantitated in the wells using a Pandex fluorescence concentration analyzer. For further analysis of compounds that inhibit Mac-1 interactions, a secondary adhesion assay using human neutrophils and human soluble ICAM-1 was established. Human neutrophils were used because of the limited availability of cultured cell lines expressing Mac-1. Mac-1 expressed on stimulated neutrophils play a major role in the adherence of neutrophils to endothelial cells and transendothelial migration via its interaction with ICAM-1. Microtiter wells were coated with soluble ICAM-1 diluted in 0.1 mM NaC0₃ buffer (pH 8.0) overnight at 4°C. The remaining binding sites on the plastic were blocked with PBS containing 1 mM Ca²⁺/Mg²⁺ and 1% fetal calf serum (PBS/FCS) at room temperature for 30 minutes. Neutrophils were purified from the peripheral blood of healthy adult individuals by dextran sedimentation and centrifugation on a Ficoll-Hypaque solution. Neutrophils were then fluorescently labeled with 2'7'-bis-(carboxyethyl)- 5(6)-carboxy-fluorescien. The cells were then washed in PBS/FCS and subjected to hypotonic lysis. To each well, 30 ml of PBS/FCS, 10 ml 10X stock of compound or blocking antibody, 10 ml f-Met-Leu-Phe (10^"7M), and 50 ml of cells (2 X 10⁶ cells/ml) was plated in triplicate. Following incubation for 30 minutes at 37°C, the plates were washed with PBS. Fluorescence was quantitated in the wells using a Pandex fluorescence concentration analyzer.

The inhibition results are given in Table 1. LFA/SPA and Mac-1/SPA refer to LFA-1 and Mac-1 adhesion assays are performed using the SPA technology; JY/ICAM refers to a secondary adhesion assay, inhibition of LFA-1 interactions, using JY and human soluble ICAM-1. PMN/ICAM refers to a secondary adhesion assay, inhibition of Mac-1 interactions, using human neutrophils and human soluble ICAM-1. TABLE 1

Claims

We claim:

1. A compound of a formula I

O

I or pharmaceutically acceptable salts thereof wherein: R_j is a) -aryl, b) -aryl wherein aryl is substituted with one to three R₄, c) -Q, d) - wherein Q is substituted with one to three R₄, e) -Het, (172441 will be provisoed out) f) -Het wherein Het is substituted with one to three R₄,

h) ^N '^N " ^ ' optionally substituted with C₁ alkyl or C₃.₆ cycloalkyl,

N -N i) C_x.₆ carboalkoxy, j) -C(=0)-CH₂C0₂(C₁.₄ alkyl)( 172509), k) -C(=0)NH(CH₂)_ftR₅,

1) C_{1 0} alkyl, m) C_{1 0} alkyl substituted with one to three R₆, n) C_1╬¢0 alkenyl, or o) C_1╬¢0 alkenyl substituted with one to three R₆;

R₂ is a) -(C=0)₍(CH₂),(CR₇R₈V

R₃ is a) -(CR₉R₁₀)_r(CH₂)_raryl, b) -(CR₉R₁₀)_r(CH₂)_raryl wherein aryl is substituted with one to three R n,╬╣> c) -(CR₉R₁₀)_r(CH₂)_rQ, d) -(CR₉R₁₀)_r(CH₂),-Q wherein Q is substituted with one to three R_n, e) -(CR₉R₁₀)_r(CH₂)_rHet, f) -(CR₉R₁₀)_r(CH₂)_rHet wherein Het is substituted with one to three R_n, or g) -(CR₉R₁₀)_r(CH₂)_rpentafluorophenyl;

R₄ is a) halo, b) Cj.₄ alkyl, c) C₃.₆ cycloalkyl, d) 0^₄ alkoxy, e) aryl, f) Q, g) Het, h) C_j.₄ carboalkoxy, i) C_j.₄ monoalkylamino, j) C₁ dialkylamino, k) amido,

1) C_j.₄ alkylthio, m) trihalomethyl, n) -(CH₂)_rO-(C₁.₄ alkyl), o) nitro,

P) mercapto, q) nitrine, r) cyano, s hydroxy. t) -NHC(=0)(C_1.4 alkyl), or u) -NHS0₂(C₁_₄ alkyl);

R₅ is a) C_LB alkyl, b) aryl, c) Q, or d) Het;

R₆ is a) halo, b) hydroxy, c) C_j.₄ alkoxy, d) C_j.₄ carboalkoxy, e) amido, ) nitro, g) trihalomethyl, h) cyano, i) mercapto, j) C_j.₄ alkylthio, or k) C_1-8 alkyl;

R₇ and R₈ are the same and different and are a) H, b) Cj.₆ alkyl, c) C₃.₆ cycloalkyl, d) -(CH^-O-C^ alkyl, e) -(CH₂)_rQ, or f) -(CH₂)_rHet;

R₉ and R₁₀ are the same and different and are a) H, b) C,.₄ alkyl, c) C_l╬¢ alkoxy, d) C₃_₆ cycloalkyl, or e) C_lA carboalkoxy;

R_╧Ç is a) Cj.₄ alkyl, b) C_j.₄ alkoxy, c) trihalomethyl, d) halo, e) nitro, f) cyano, g) nitrine, h) C_1-4 acyl, i) C_j.₄ carboalkoxy, or j) carboxyl; aryl is monocarbocyclic, or bicarbocyclic aromatic moiety;

Q is 5- to 10-membered saturated heterocyclic moiety having one to three atoms selected from the group consisting of oxygen, nitrogen, and sulfur; Het is 5- to 10-membered unsaturated heterocyclic moiety having one to three atoms selected from the group consisting of oxygen, nitrogen, and sulfur; h is 0, 1, 2, or 3; j is 0 or 1; j is 0, 1, 2, 3, 4 or 5; k is 0, 1, 2 or 3; I is 0, 1, 2, 3, 4 or 5; n is 0, 1 or 2; and with the following provisos: a) where both R₇ and R₈ are hydrogen, j + k is other than 1; b) where R₃ is phenyl substituted with F, R_j is other than unsubstituted phenyl.

2. A compound of claim 1 which is a. 3-Fluoro-N-[5-[(l-phenylpropyl)thio]-l,3,4-thiadiazol-2-yl]benzeneacetamide, b. (E)-3-Nitro-N-[5-[(3J-dimethyl-2,6-octadienyl)thio]-l,3,4-thiadiazol-2- yl]benzamide, c. (E)-3-Trifluoromethyl-N-[5-[(3J-dimethyl-2,6-octadienyl)thio]-l,3,4-thiadiazol- 2-yl]benzamide, d. N-[5-[[6-(l,3-Dihydro-l,3-dioxo-2H-isoindol-2-yl)hexyl]thio]- l,3,4-thiadiazol-2- yl]-3-nitrobenzamide, e. N-[5-[[6-(l,3-Dihydro-l,3-dioxo-2H-isoindol-2-yl)hexyl]thio]- l,3,4-thiadiazol-2- yl] -3-trifluoromethylbenzamide, f . N- [5- [ [6-( 1 ,3-Dihydro- 1 ,3-dioxo-2H-isoindol-2-yl)hexyl] thio] - 1,3 ,4-thiadiazol-2- yl] -3-cyanobenzamide, g. N-[5-[[6-(l,3-Dihydro-l,3-dioxo-2H-isoindol-2-yl)hexyl]thio]- l,3,4-thiadiazol-2- yl]-2,3,4,5,6-pentafluorobenzamide, h. (E)-N-[5-[(3J-Dimethyl-2,6-octadienyl)thio]-l,3,4-thiadiazol-2-yl]-2,3,4,5,6- pentafluorophenylbenzamide , i. (E)-N-[5-[(3,7-Dimethyl-2,6-octadienyl)thio]-l,3,4-thiadiazol-2-yl]- 2,3,4,5,6- pentafluorobenzeneacetamide, j. (E)-N-[5-[(3J-Dimethyl-2,6-octadienyl)thio]-l,3,4-thiadiazol-2-yl]- 2(3,4,5,6- pentafluorobenzene)propylamide, k. N-[5-[[2-Oxo-2-(4-pyridinyl)ethyl]thio]-l,3,4-thiadiazol-2-yl]- 3-

(trifluoromethyl)benzamide, 1. N-[5-[[2-Oxo-2-(3-pyridinyl)ethyl]thio]-l,3,4-thiadiazol-2-yl]- 3-

(trifluoromethyl)benzamide , m. 3,4-Dichloro-N-[5-[[l-C3-pjrridinyl)propyl]thio]-l,3,4-thiadiazol-2-yl]benzamide, n. 3,5-Difluoro-N-[5-[[l-(3-pyridinyl)propyl]thio]-l,3,4-thiadiazol-2-yl]benzamide, o. 3,5-Dimethoxy-N-[5-[[l-(3-pyridinyl)propyl]thio]-l,3,4-thiadiazol-2- yl]benzamide, p. -Methyl-N-[5-[[l-(3-pyridinyl)propyl]thio]-l,3,4-thiadiazol- 2- yl]benzeneacetamide, q. ╬▒-Cyclopropyl-N-[5-[(l-phenylpropyl)thio]-l,3,4-thiadiazol- 2- yl]benzeneacetamide, or r. ╬▒-Methoxy-N-[5-[[l-(3-pyridinyl)propyl]thio]-l,3,4-thiadiazol-2- yl]benzeneacetamide.

3. A method of inhibiting LFA-1 and Mac-1 which comprises administering to a patient in need thereof an effective amount of a compound of claim 1.

4. A method of treating a patient suffering from inflammatory diseases which comprises administering to a patient in need thereof an effective amount of a compound of claim 1.

5. A method of claim 4 wherein the inflammatory diseases are hypersensitivity reactions, asthma, rheumatoid arthritis, bacterial meningitis, aspiration lung injury, inflammatory bowel disorder and related complications.

6. A pharmaceutical composition which comprises an effective amount of the compound of claim 1 and a pharmaceutically acceptable carrier.