AU2002320734B2 - Substituted 2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindolines and their use to reduce TNFalpha levels - Google Patents

Description

-1- 1 SUBSTITUTED 2-(2,6-DIOXO-3-FLUOROPIPERIDIN-3-YL)-ISOINDOLINES

AND

2 THEIR USE TO REDUCE TNFa LEVELS 3 4 Background of the Invention 6 7 Tumor necrosis factor a, or TNFa, is a cytokine which is released primarily by 8 mononuclear phagocytes in response to a number immunostimulators. It is a key 9 proinflammatory cytokine in the inflamation cascade causing the production and/or release of other cytokines and agents. When administered to animals or humans, it 11 causes inflammation, fever, cardiovascular effects, hemorrhage, coagulation, and acute 12 phase responses similar to those seen during acute infections and shock states. Excessive 13 or unregulated TNFa production thus has been implicated in a number of disease 14 conditions. These include endotoxemia and/or toxic shock syndrome {Tracey et a., Nature 330,662-664 (1987) and Hinshaw et al., Circ. Shock 30,279-292 (1990)}; 16 cachexia {Dezube et al., Lancet, 335(8690), 662 (1990)} and Adult Respiratory Distress 17 Syndrome where TNFa concentration in excess of 12,000 pg/mL have been detected in 18 pulmonary aspirates from ARDS patients {Millar et al., Lancet 2 (8665), 712-714 19 (1989)}. Systemic infusion of recombinant TNFa also resulted in changes typically seen in ARDS {Ferrai-Baliviera et al., Arch. Surg. 124(12), 14001405 (1989)}.

21 22 TNFa appears to be involved in bone resorption diseases, including arthritis.

23 When activated, leukocytes will produce bone-resorption, an activity to which the data 24 suggest TNFa contributes. {Bertolini et al., Nature 319,516-518 (1986) and Johnson et al., Endocrinology 124(3), 1424-1427 (1989).} TNFa also has been shown to stimulate 26 bone resorption and inhibit bone formation in vitro and in vivo through stimulation of 27 osteoclast formation and activation combined with inhibition of osteoblast function.

28 Although TNFu may be involved in many bone resorption diseases, including arthritis, 29 the most compelling link with disease is the association between production of TNFa by tumor or host tissues and malignancy associated hypercalcemia {Calci. Tissue Int. (US) 31 46(Suppl.), S3-10 (1990)}. In Graft versus Host Reaction, increased serum TNFa levels 32 have been associated with major -2- 33 complication following acute allogenic bone marrow transplants {Holler et at, Blood, 34 75(4), 1011-1016 (1990)}.

Cerebral malaria is a lethal hyperacute neurological syndrome associated with high 36 blood levels of TNFat and the most severe complication occurring in malaria patients.

37 Levels of serum TNFa correlated directly with the severity of disease and the 38 prognosis in patients with acute malaria attacks {Grau et al., N. Engl. J. Med 320(24), 39 1586-1591 (1989)}.

Macrophage-induced angiogenesis is known to be mediated by TNFa. Leibovich et 41 al. {Nature, 329, 630-632 (1987)} showed TNFa induces in vivo capillary blood 42 vessel formation in the rat cornea and the developing chick chorioallantoic membranes 43 at very low doses and suggest TNFa is a candidate for inducing angiogenesis in 44 inflammation, wound repair, and tumor growth. TNFa production also has been associated with cancerous conditions, particularly induced tumors {Ching et al., Brit.

46 J Cancer, (1955) 72, 339-343, and Koch, Progress in Medicinal Chemistry, 22, 166- 47 242(1985)).

48 TNFa also plays a role in the area of chronic pulmonary inflammatory diseases.

49 The deposition of silica particles leads to silicosis, a disease of progressive respiratory failure caused by a fibrotic reaction. Antibody to TNFa completely blocked the silica- 51 induced lung fibrosis in mice {Pignet et al., Nature, 344, 245-247 (1990)}. High 52 levels of TNFa production (in the serum and in isolated macrophages) have been 53 demonstrated in animal models of silica and asbestos induced fibrosis (Bissonnette et 54 at, Inflammation 13(3), 329-339 (1989)). Alveolar macrophages from pulmonary sarcoidosis patients have also been found to spontaneously release massive quantities 56 of TNFa as compared with macrophages from normal donors (Baughman et al, J 57 Lab. Clin. Med. 115(1), 36-42 (1990)}.

58 TNFa is also implicated in the inflammatory response which follows reperfusion, 59 called reperfusion injury, and is a major cause of tissue damage after loss of blood flow {Vedder et al, PNAS 87, 2643-2646 (1990)). TNFa also alters the properties of 61 endothelial cells and has various pro-coagulant activities, such as producing an increase 62 in tissue factor pro-coagulant activity and suppression of the anticoagulant protein C 63 pathway as well as down-regulating the expression of thrombomodulin {Sherry et at, 64 J. Cell BioL 107, 1269-1277 (1988)}. TNFa has pro-inflammatory activities which 66 67 68 69 71 72 73 74 76 77 78 79 81 82 83 84 86 87 88 89 91 92 93 94 96 97 together with its early production (dining the initial stage of an inflammatory event) make it a likely mediator of tissue injury in several important disorders including but not limited to, myocardial infarction, stroke and circulatory shock. Of specific importance may be TNFc-induced expression of adhesion molecules, such as intercellular adhesion molecule (ICAM) or endothelial leukocyte adhesion molecule (ELAM) on endothelial cells (Munro et al., Am. J. Path. 135(1), 121-132 (1989)).

TNFc blockage with monoclonal anti-TNFx. antibodies has been shown to be beneficial in rheumatoid arthritis (Elliot et al., Int. J Pharmac. 1995 17(2), 141-145).

High levels of TNFc are associated with Crohn's disease (von Dullemen el al., Gastroenterology, 1995 109(1), 129-135) and clinical benefit has been acheived with TNFa antibody treatment.

Moreover, it now is known that TNFat is a potent activator of retrovirus replication including activation of HIV-1. {Duh et al., Proc. Nat. Acad. Sci. 86, 5974-5978 (1989); Poll el al., Proc. Nat. Acad Sci. 87, 782-785 (1990); Monto et al., Blood 79, 2670 (1990); Clouse et al., Immunol. 142, 431438 (1989); Poll et al., AIDS Res.

Hum. Retrovirus, 191-197 (1992)). AIDS results from the infection of T lymphocytes with Human Immunodeficiency Virus (HTV). At least three types or strains of HIV have been identified, Le., HIV-1, HV-2 and HIV-3. As a consequence of MV infection, T-cell mediated immunity is impaired and infected individuals manifest severe opportunistic infections and/or unusual neoplasms. HIV entry into the T lymphocyte requires T lymphocyte activation. Other viruses, such as HIV-1, IRV-2 infect T lymphocytes after T cell activation and such virus protein expression and/or replication is mediated or maintained by such T cell activation. Once an activated T lymphocyte is infected with HIV, the T lymphocyte must continue to be maintained in an activated state to permit IV gene expression and/or HIV replication. Cytokines, specifically TNFaL, are implicated in activated T-cell mediated HIV protein expression and/or virus replication by playing a role in maintaining T lymphocyte activation. Therefore, interference with cytokine activity such as by prevention or inhibition of cytokine production, notably TNFcx, in an B/V-infected individual assists in limiting the maintenance of T lymphocyte caused by IIV infection.

Monocytes, macrophages, and related cells, such as kupffer and glial cells, also have been implicated in maintenance of the [MV infection. These cells, like T cells, are targets for viral replication and the level of viral replication is dependent upon the

I

98 activation state of the cells. {Rosenberg et al., The Immunopathogenesis of HIV 99 Infection, Advances in Immunology, 57 (1989)}. Cytokines, such as TNFoL, have been 100 shown to activate HIV replication in monocytes and/or macrophages {Poli et al., Proc.

101 Nafl. Acad Sci., 87, 782-784 (1990)}, therefore, prevention or inhibition of cytokine 102 production or activity aids in limiting HIV progression for T cells. Additional studies 103 have identified TNFct as a common factor in the activation of HIV in vitro and has 104 provided a clear mechanism of action via a nuclear regulatory protein found in the 105 cytoplasm of cells (Osbom, et al., PNAS 86 2336-2340). This evidence suggests that a 106 reduction of TNFct synthesis may have an antiviral effect in HIV infections, by 107 reducing the transcription and thus virus production.

7 108 AIDS viral replication of latent HIV in T cell and macrophage lines can be induced 109 by TNFac {Folks et al., PNAS 86, 2365-2368 (1989)}. A molecular mechanism for the 110 virus inducing activity is suggested by TNFTo's ability to activate a gene regulatory 111 protein (NFicB) found in the cytoplasm of cells, which promotes HIV replication 112 through binding to a viral regulatory gene sequence (LTR) {Osborn et al., PNAS 86, 113 2336-2340 (1989)}. TNFac in AIDS associated cachexia is suggested by elevated 114 serum TNFa and high levels of spontaneous TNFa production in peripheral blood 115 monocytes from patients {Wright et al., J. Immunol. 141(1), 99-104 (1988)}. TNFa 116 has been implicated in various roles with other viral infections, such as the cytomegalia 117 virus (CMV), influenza virus, adenovirus, and the herpes family of viruses for similar 118 reasons as those noted.

119 The nuclear factor .B (NFiB) is a pleiotropic transcriptional activator (Lenardo, et 120 al., Cell 1989, 58, 227-29). NFicB has been implicated as a transcriptional activator in 121 a variety of disease and inflammatory states and is thought to regulate cytokine levels 122 including but not limited to TNFac and also to be an activator of HIV transcription 123 (Dbaibo, et al., J Biol. Chem. 1993, 17762-66; Duh et aL, Proc. Natl. Acad Sci.

124 1989, 86, 5974-78; Bachelerie et al., Nature 1991, 350, 709-12; Boswas et al., J.

125 Acquired Immune Deficiency Syndrome 1993, 6, 778-786; Suzuki et al., Biochem.

126 And Biophys. Res. Comm. 1993, 193, 277-83; Suzuki et al., Biochem. And Biophys.

127 Res Comm. 1992, 189, 1709-15; Suzuki et al., Biochem. Mol. Bio. Int. 1993, 31(4), 128 693-700; Shakhov et al., Proc. NatL Acad. Sci. USA 1990, 171, 35-47; and Staal et 129 al., Proc. Natl. Acad Sci. USA 1990, 87, 9943-47). Thus, inhibition of NFicB binding 130 can regulate transcription of cytokine gene(s) and through this modulation and other 131 mechanisms be useful in the inhibition of a multitude of disease states. The compounds 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 described herein can inhibit the action of NFicB in the nucleus and thus are useful in the treatment of a variety of diseases including but not limited to rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, other arthritic conditions, septic shock, septis, endotoxic shock, graft versus host disease, wasting, Crohn's disease, ulcerative colitis, multiple sclerosis, systemic lupus erythrematosis, ENL in leprosy, HIV, AIDS, and opportunistic infections in AIDS. TNFa and NFicB levels are influenced by a reciprocal feedback loop. As noted above, the compounds of the present invention affect the levels of both TNFa and NFicB.

Many cellular functions are mediated by levels of adenosine 3',5'-cyclic monophosphate (cAMP). Such cellular functions can contribute to inflammatory conditions and diseases including asthma, inflammation, and other conditions (Lowe and Cheng, Drugs of the Future, 17(9), 799-807, 1992). It has been shown that the elevation of cAMP in inflammatory leukocytes inhibits their activation and the subsequent release of inflammatory mediators, including TNFct and NFicB. Increased levels of cAMP also leads to the relaxation of airway smooth muscle. Phosphodiesterases control the level of cAMP through hydrolysis and inhibitors of phosphodiesterases have been shown to increase cAMP levels.

Decreasing TNFa levels and/or increasing cAMP levels thus constitutes a valuable therapeutic strategy for the treatment of many inflammatory, infectious, immunological or malignant diseases. These include but are not restricted to septic shock, sepsis, endotoxic shock, hemodynamic shock and sepsis syndrome, post ischemic reperfusion injury, malaria, mycobacterial infection, meningitis, psoriasis, congestive heart failure, fibrotic disease, cachexia, graft rejection, cancer, autoimmune disease, opportunistic infections in AIDS, rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, other arthritic conditions, Crohn's disease, ulcerative colitis, multiple sclerosis, systemic lupus erythrematosis, ENL in leprosy, radiation damage, and hyperoxic alveolar injury.

Prior efforts directed to the suppression of the effects of TNFa have ranged from the utilization of steroids such as dexamethasone and prednisolone to the use of both polyclonal and monoclonal antibodies (Beutler et al., Science 234, 470-474 (1985); WO 92/11383}.

-6- 162 Detailed Description 163 The present invention is based on the discovery that certain classes of non- 164 polypeptide compounds more fully described herein decrease the levels of 165 TNFa, increase cAMP levels, and inhibit phosphodiesterase. The present invention thus 166 relates to 2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindolines, the method of reducing 167 levels of tumor necrosis factor ao and other inflammatory cytokines in a mammal 168 through the administration of such derivatives, and pharmaceutical compositions 169 containing such derivatives.

170 In particular, the invention pertains to 171 a 2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline of the formula: 172 RI 0 R2 C F

H

0

R

4

Y

173 174

I.

175 in which 176 Y is oxygen or H 2 and 177 each of R, R 2

R

3 and R 4 independently of the others, is hydrogen, halo, alkyl of 1 to 178 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, or amino, and 179 the acid addition salts of said 2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindolines 180 which contain a nitrogen atom capable of being protonated.

181 Unless otherwise defined, the term alkyl denotes a univalent saturated branched or 182 straight hydrocarbon chain containing from 1 to 8 carbon atoms. Representative of 183 such alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, and 184 tert-butyl. Alkoxy refers to an alkyl group bound to the remainder of the molecule 185 through an ethereal oxygen atom. Representative of such alkoxy groups are methoxy, 186 ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, and tert-butoxy.

-7- 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 S 207 208 The compounds of Formula I are used, under the supervision of qualified professionals, to inhibit the undesirable effects of TNFa and other inflammatory cytokines including IL-1, IL-6, and IL-12. The compounds can be administered orally, rectally, or parenterally, alone or in combination with other therapeutic agents including antibiotics, steroids, etc., to a mammal in need of treatment.

The compounds of the present invention also can be used topically in the treatment or prophylaxis of topical disease states mediated or exacerbated by excessive TNFca production, respectively, such as viral infections, such as those caused by the herpes viruses, or viral conjunctivitis, psoriasis, atopic dermatitis, etc.

The compounds also can be used in the veterinary treatment of mammals other than humans in need of prevention or inhibition of TNFa production. TNFat mediated diseases for treatment, therapeutically or prophylactically, in animals include disease states such as those noted above, but in particular viral infections. Examples include feline immunodeficiency virus, equine infectious anaemia virus, caprine arthritis virus, visna virus, and maedi virus, as well as other lentiviruses.

The compounds of Formula I are readily prepared through a number of routes. In a first embodiment, the ring nitrogen of a 2-(2,6-dioxopiperidin-3-yl)-isoindoline of Formula II is protected with a conventional amino protecting group to yield a protected 2-(2,6-dioxopiperidin-3-yl)-isoindoline of Formula I. This is then fluorinated to yield a protected 2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline of Formula IV, following which removal of the protecting group yields the compounds of Formula V: -7 209 V

U

210 In the foregoing reactions, each ofR R, R 3

R

4 and Y are as defined above and X 211 is amino protecting group. When any of R 1

R

2

R

3 and R 4 is amino, it too should be 212 protected prior to the fluorination step.

213 The intermediates of Formula IV can be isolated and purified prior to removal of 214 the protecting group X or can be converted directly to the final compounds of Formula 215 V in situ.

216 Some of the compounds of Formula II which are here utilized as intermediates are 217 known, as for example 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline and 218 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-5-aminoisoindoline. See, J6nsson, Acta 219 Pharma. Succica, 9, 521-542 (1972). Others are described in copending application 220 Serial No. 08/701,494, the disclosure of which is incorporated herein by reference, or 221 can be prepared by methods analogous thereto.

222 The fluorination can be effected with a variety of reagents, as for example, N- 223 fluorobenzenesulfonimide, perchloryl fluoride, N-fluorobenzenedisulfonimide, and the 224 like, in the presence of a strong base such as n-butyl lithium, sodium hydride, lithium 225 diisopropylamide, lithium bis(trimethylsilyl)amide, and the like.

-9- 226 In a second method, an appropriately substituted glutanic acid diester of Formula 227 VI is fluorinated to yield the corresponding fluoroglutainic acid diester of Formula VI.

228 This is then convented to the fluorinated glutarnic acid anhydride of Formula VII 229 which, in turn, is amidated to yield the compounds of Formula V:

R

1 0 RI 0 2 12

CF

ON O

R

4 y

I

cI 0. C Z IV F 1 0

O

230R 231 In the foregoing reactions, each of BR', R(2, 1(4, and Y are as defined above and Z 232 and Z' are lower alkyl. Again when any of R1, R(2, R(3, and 1(4 is amino, it too should 233 be protected prior to the fluorination step.

234 Protecting groups utilized herein denote groups which generally are not found in the 235 final therapeutic compounds but which are intentionally introduced at some stage of 236 the synthesis in order to protect groups which otherwise might be altered in the course 237 of chemical manipulations. Such protecting groups are removed at a later stage of the 238 synthesis and compounds bearing such protecting groups thus are of importance 239 primarily as chemical intermediates (although some derivatives also exhibit biological 240 activity). Accordingly the precise structure of the protecting group is not critical.

241 Numerous reactions for the formation and removal of such protecting groups are 242 described in a number of standard works including, for example, "Protective Groups in 243 Organic Chemistry", Plenum Press, London and New York, 1973; Greene, Th. W.

244 "Protective Groups in Organic Synthesis", Wiley, New York, 1981; "The Peptides", 245 Vol. I, Schrbder and Lubke, Academic Press, London and New York, 1965; 246 "Methoden der organischen Chemie", Houben-Weyl, 4th Edition, Vol. 15/I, Georg 247 Thieme Verlag, Stuttgart 1974, the disclosures of which are incorporated herein by 248 reference.

249 In any of the foregoing reactions, a nitro compound can be employed with the nitro 250 group being convenrted to an amino group by catalytic hydrogenation. Alternatively, a 251 protected amino group can be cleaved to yield the corresponding amino compound.

252 An amino group can be protected as an amide utilizing an acyl group which is selec- 253 tively removable under mild conditions, especially benrzyloxycarbonyl, formyl, or a 254 lower alkanoyl group which is branched in 1- or a position to the carbonyl group, 255 particularly tertiary alkanoyl such as pivaloyl, a lower alkanoyl group which is substi- 256 tuted in the position ac to the carbonyl group, as for example trifluoroacetyl.

257 The carbon atom to which the depicted fluorine atom is bound in the compounds of 258 Formula I constitutes a center of chirality, thereby giving rise to optical isomers: Kl-&: R' 0

R

2

Y

INml

IA

0

R

4

Y

IA

R

2 C F /Ne 259 260 Both the racemates of these isomers and the individual isomers themselves, as well 261 as diastereomers when a second chiral center is present, are within the scope of the 262 present invention. The racemates can be used as such or can be separated into their 263 individual isomers mechanically as by chromatography using a chiral absorbant. Alter- 264 natively, the individual isomers can be prepared in chiral form or separated chemically 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 from a mixture by forming salts with a chiral acid or base, such as the individual enantiomers. of lo-camnphorsulfonic acid, camphoric acid, a-bromocanphoric acid, inethoxyacetic acid, tartaric acid, diacetyltartaric acid, malic acid, carboxylic acid, and the like, and then freeing one or both of the resolved bases, optionally repeating the process, so as obtain either or both substantially free of the other;, in a form having an optical purity of The present invention also pertains to the physiologically acceptable non-toxic acid addition salts of' the compound of Formula I which contain a group capable of being protonated; amino. Such salts include those derived from organic and inorganic acids such as, without limitation, hydrochloric acid, hydrobromnic acid, phosphoric acid, sulfuric acid, methanesuiphonic acid, acetic acid, tartaric acid, lactic acid, succinic acid, citric acid, malic. acid, maleic. acid, sorbic acid, aconitic acid, salicylic acid, phthalic acid, embonic; acid, enanthic acid, and the like.

Particularly preferred compounds include 1 l,3-dioxo-2-(2,6-dioxo-3-fluoropipcridin-3-yl)-isoindoline, I ,3-dioxo-2-(2,6-dioxo-3 -f luoropiperidin-3 -yl)-4aminoisoindoline, 1,-ix--26doo3furppeii--l--mnionoie 1 ,3-dioxo-2-(2,6-dioxo-3 -fluoropiperidin-3-yl)-4-methylisoindOlilC, I ,3-dioxo-2-(2,6dioxo-3-fluoropiperidin-3-yl)-5-nethylisoindolile, 1 -oxo-2-(2,6-dioxo-3-fluorOpiperidin-3-yl)-5-methylisoindollne, I -oxo-2-(2,6-dioxo-3-fluoropipefldin4 -yl)-4methylisoinidoline, 1 ,3-dioxo-2-(2,6-dioxo-3 -fluoropiperidin-3-yl)-4, 5,6, 7-tetrafluoroisoindoline, 1 ,3-dioxo-2-(2,6-dioxo-3 -fluoropiperidin-3-yl)-4,5, 6 ,ltetrachloroisoindoline, 1 ,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3 i'i)- 4 5 6 7 tetramnethylisoindoline, 1 ,3-dioxo-2-(2,6-dioxo-3-fluoropiperidifl-3-yl)-4,5, 6 7 tetramethoxyisoindoline, I -oxo-2-(2,6-dioxo-3-fluoropiperidifl-3-Yfr-5 aminoisoindoline, 1 x--26doo--looieii-3y)ionoie I -oxo-2- (2,6-dioxo-3-fluoropiperidin-3-yl)-4-aminoisoindoline, I -oxo-2-(2,6-dioxo-3fluoropipcridin-3-yl)-4,5,6,7-tetrafluoroisoindoine, 1 -oxo-2-(2,6-dioxo-3 fluoropiperidin-3-yl)- 4,5,6,7-tetrachloroisoindoline, 1 -oxo-2-(2,6-dioxo-3 fluoropiperidin-3-yl)-4, 5,6,7-tetraniethylisoindoline, and 1 -oxo-2-(2,6-dioxo-3 fluoropiperidin-3-yl)-4,5,6,7-tetramethoxyisoindolile. Of these, 1, 3-dioxo-2-(2,6dioxo-3-fluoropiperidin-3-yl)-isoindoline and 1-oxo-2-(2,6-dioxo-3-fluoopiPeidifl-3yl)-isoindoline are particularly preferred.

297 Oral dosage forms include tablets, capsules, dragees, and similar shaped, corn- 298 pressed pharmaceutical forms containing from I to 100 mg of dwug per unit dosage.

-12- 299 Isotonic saline solutions containing from 20 to 100 mg/mL can be used for parenteral 300 administration which includes intramuscular, intrathecal, intravenous and intra-arterial 301 routes of administration- Rectal administration can be effected through the use of 302 suppositories formulated from conventional carriers such as cocoa butter.

303 Pharmaceutical compositions thus comprise one or more compounds of Formulas I 304 associated with at least one pharmaceutically acceptable carrier, diluent or excipient.

305 In preparing such compositions, the active ingredients are usually mixed with or 306 diluted by an excipient or enclosed within such a carrier which can be in the form of a 307 capsule or sachet. When the excipient serves as a diluent, it may be a solid, semi-solid, 308 or liquid material which acts as a vehicle, carrier, or medium for the active ingredient.

309 Thus, the compositions can be in the form of tablets, pills, powders, elixirs, 310 suspensions, emulsions, solutions, syrups, soft and hard gelatin capsules, suppositories, 311 sterile injectable solutions and sterile packaged powders. Examples of suitable 312 excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, 313 calcium silicate, microcrystalline cellulose, polyvinylpyrrolidinone, cellulose, water, 314 syrup, and methyl cellulose, the formulations can additionally include lubricating agents 315 such as talc, magnesium stearate and mineral oil, wetting agents, emulsifying and 316 suspending agents, preserving agents such as methyl- and propylhydroxybenzoates, 317 sweetening agents or flavoring agents.

318 The compositions preferably are formulated in unit dosage form, meaning physically 319 discrete units suitable as a unitary dosage, or a predetermined fraction of a unitary dose 320 to be administered in a single or multiple dosage regimen to human subjects and other 321 mammals, each unit containing a predetermined quantity of active material calculated 322 to produce the desired therapeutic effect in association with a suitable pharmaceutical 323 excipient. The compositions can be formulated so as to provide an immediate, sustain- 324 ed or delayed release of active ingredient after administration to the patient by 325 employing procedures well known in the art.

326 Enzyme-linked immunosorbent assays for TNFat can be performed in a conventional 327 manner. PBMC is isolated from normal donors by Ficoll-Hypaque density centrifugation.

328 Cells are cultured in RPM[ supplemented with 10% AB+ serum, 2mM L-glutamine, 100 329 U/mL penicillin, and 100 mg/mL streptomycin. Drugs are dissolved in dimethylsulfoxide 330 (Sigma Chemical) and further dilutions are done in supplemented RPMI. The final 331 dimethylsulfoxide concentration in the presence or absence of drug in the PBMC 13- 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 suspensions is 0.25 wt Drugs are assayed at half-log dilutions starting at 50 mg/mL.

Drugs are added to PBMC (106 cells/mL) in 96 wells plates one hour before the addition of LPS. PBMC (106 cells/mL) in the presence or absence of drug are stimulated by treatment with 1 mg/mL of LPS from Salmonella mirmesota R595 (List Biological Labs, Campbell, CA). Cells are then incubated at 370 C for 18-20 hours. Supematants are harvested and assayed immediately for TNFa levels or kept frozen at -70 0 C (for not more than 4 days) until assayed. The concentration of TNFa in the supernatant is determined by human TNFa ELISA kits (ENDOGEN, Boston, MA) according to the manufacturers directions.

The following examples will serve to further typify the nature of this invention but should not be construed as a limitation in the scope thereof, which scope is defined solely by the appended claims.

EXAMPLE

To a stirred suspension of 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)isoindoline (2.0 g, 7.75 mmol) and di-tert.-butyl dicarbonate (1.86 g, 8.52 mmol) in 1,4-dioxane (3.0 mL) is added dimethylaminopyridine (1.00 mg) at room temperature. The solution is stirred at room temperature for 18 hours and the solvent then removed in vacua. The residue is stirred with ether (30 mL) for 30 minutes, filtered, and washed with ether to give 1,3-dioxo-2-(l-tert.-butoxycarbonyl-2,6-dioxopiperidin- 3 -yl)isoindoline.

A typical run produced 2.5 g (90% yield) of 1,3-dioxo-2-(l-tert.-butoxycarbonyl- 2,6-dioxopiperidin-3-yl)isoindoline, mp, 274.0-275.0 'H NMR (DMSO-d6); 6 1.47 9H, CH3), 2.08-2.15 1H, CHH), 2.50-2.70 1H, CHH), 2.69-2.82 1H, CHH), 3.02-3.17 1H, CHH), 5.42 (dd, J 5.4, 12.9 Hz, 1H, NCH), 7.90-7.94 (m, 4H, Ar); 13C NMR (DMSO-dc) 5 21.11, 27.03, 30.69, 48.77, 86.01, 123.52, 131.16, 134.99, 148.28, 166.99, 167.55, 169.99; Anal. Calc'd for C 1 sHisN 2 0 6 C, 60.33; H, 5.06; N, 7.82. Found: C, 60.01; H, 5.21; N, 7.47.

EXAMPLE 2 Similarly to the procedure of Example 1, there are respectively obtained from 1,3dioxo-2-(2,6-dioxopiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoline, 1,3-dioxo-2-(2,6dioxopiperidin-3-yl)- 4,5,6,7-tetrachloroisoindoline, 1,3-dioxo-2-(2,6-dioxopiperidin- 3-yl)-4,5,6,7-tetramethylisoindoline, 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4,5,6,7tetramethoxyisoindoline, 1-oxo-2-(2,6-dioxopiperidin-3-yl)-isoindoline, 1-oxo-2-(2, 6 dioxopiperidin-3-yl)- 4 ,5,6,7-tetrafluoroisoindoline, 1-oxo-2-(2,6-dioxopiperidin-3-yl)- -14- 364 4, 5,6,7-tetrachloroisoildOlifle, I -oxo-2-(2,6-dioxopiperidifl-3-yl)- 4,5,6,7- 365 tetramethylisoinckiline, 1,-iox--26dooieii-3y)4mtyionoie 1,3 366 dioxo-2-(2,6-dioxopiperidifl-3 -yl)-5-methylisoindoline, I -oxo-2-(2,6-dioxopiperidifl-J- 367 yl)-5-methylisoindoline, 1 x--26dixpprdn--i--ehyionoie and 1- 368 oxo-2-(2,6-dioxopiperidil- 3 -yl)-4,5,6,7-tetramethoxyisolfldohle, the compounds 1,3 369 cioxo-2-( 1-terd. butoxycarbony-2,6-dioxOPiPefldil- 3 5,6,7-tetrafluoroisoildO- 370 line, 1,3-dioxo-2-(1 -tert-uoyabnl26dooieidn3y)4567ttahoo 371 isoindoline, I ,3-dioxo I -MrL -butoxycarbonyl-2,6-dioxOpiPefdif 3 l)- 4

,SGJ

372 tetramethylisoindoline, I ,3-dioxo-2-(l-tert. -butoxycarbonyL-2,6-diOXOoiPefldifl 3 -yl)- 373 4,5,6,7-tetranethoxyisOindOlifle, 1 -oxo-2-(1 -tert.-butoxycarbony-2,6-dioxopiPefldrn- 374 3-yl)-isoindoline, 1-oxo-2-(1 -tert -butoxycarbony1-2,6-diOXOPiPefdif- 3 1tyi-5 6 375 tetrafluoroisoindoline, I -oxo-2-( I-ten. butoxycarbonyl-2,6-dioXOPiPefldifl- 3 -yl>- 376 4,5,6,7-tetrachloroisoindolifle, 1 -oxo-2-( 1-tent-butoxycarbonyl-2,6-diOXOpiPefldifl 3 377 yE)- 4,5,6,7-tetramethylisoildolifle, 1 ,3-dioxo-2-( 1-teM butoxycarbony-2,6-diOXO- 378 piperidin-3-yl)-4-metbylisoindolile, 1,3 -dioxo-2-(1 -lent -butoxycarbonyl-2,6-diOXO- 379 piperidi n-3-yl)-5-methylisoildolifle, 1 -oxo-2-(1 -tent-butoxycarbony-2, 6 -diOXO- 380 piperidin-3-yy-5-methylisoiidOtife, 1 -oxo-2-(1-tert -butoxycarbonyl-2,6-diOXOPiPefl- 381 din-3-yl)-4-methylisoindolifle, and 1 -oxo-2-(1 -tent-butoxycartonyl-2,6-diOXOPiPefldifl' 382 3-yl)-4, 5,6,7-tetranethoxyisoildolifle 383 Similarly utilizing equivalent amounts of 1,-ix--26doxpprdn3y)4 384 axinoisoindoline, 1 ,3-dioxo-2-(2,6-dioxOpipefldil-3 -yI)-5-aminoisoindOlifle, 1-oxo-2- 385 (2,&-dioxopiperidin-3-yl)-5-aminoisoildOhfe, and 1 -oxo-2-(2,6-dioxopiperidifl3-Yl)A4 386 aminoisoindoline, but utilizing 3.72 g of di-rerr.-butyl dicarbonate in the procedure of 387 Example 1, there are respectively obtained 1 ,3-dioxo-2-(1-tent.-butoxycarbonyl-2, 6 388 dioxopiperidin-3-yl)-4-(l -rert.-butoxycarbonylamino)-iSOildolifle, 1,3 -dioxo-2-{1 -tent- 389 butoxycarbonyl-2,6-dioxopiperidifl- 3 tert.-butoxycarbonyalnflOYisoifline, 390 1 -oxo-2-(WI-ent -butoxycarbonyl-2,6-dioxOpipCridifl- 3 -Yl)hS( I -tent.- 391 butoxycarbonylamino)-isoildOlifle, and I -oxo-2-(1 -tert.-butoxycarboiYl-2,6-dioKopiP- 392 eridin-3-yl)-4-(l 1 tert.-butoxycarbonylaniinO)-iSOifline.

393 EXAMPLE 3 394 To a stirred solution of 1,-ix--Itr.btxcroy-,-ixpprdn3 395 yI)isoindoline (1.0 g, 2.8 nunol) in tetrahydrofuran (10 mL) is added n-butyl lithium 396 (1.2 mL, 3.0 mmol, 2.5 M) at -781C2. After 20 minutes, N-fluorobeIZefesUflfnimde 397 (0-8 g, 3.2 nunol) is added to the mixture. The mixture is allowed to reach room 398 temperature and the solvent then removed in vacuo- The residue is stirred with ethyl 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 acetate (10 mL) and IN hydrochloric acid (10 mL) for one hour. The organic layer is separated and the solvent removed in vacuo to yield 1,3-dioxo-2-(2,6-dioxo-3fluoropiperidin-3-yl)isoindoline which can be further purified through chromatography.

In a similar fashion, lithium bis(trimethylsilyl)amide (24 mL, 24 mmol, 1.0 M) is added to a stirred solution of 1,3-dioxo-2-(I-tert-butoxycarbonyl-2,6-dioxopiperidin- 3-yl)isoindoline(7.16 g, 20 mmol) in tetrahydrofuran (250 mL) at -40 OC. After 1 hour, N-fluorobenzenesulfonimide (7.6 g, 24 mmol) is added to the mixture. The mixture is allowed to reach room temperature and kept overnight. The solution is stirred with ethyl acetate (200 mL), aqueous ammonium chloride (100 ml, sat), and water (100 mL). The aqueous layer is separated and extracted with ethyl acetate (200 mL). The combined organic layers are washed with water (100 mL), brine (100 mL), and dried over sodium sulfate. The solvent is removed in vacuo. The residue is purified by chromatography (silica gel) to give the intermediate 1,3-dioxo-2-(l-tertbutoxycarbonyl-2,6-dioxo-3-fluoropiperidin-3-yl)isoindoline.

A typical run produced 700 mg (10 yield) of give 1,3-dioxo-2-(1-tert-butoxycarbonyl-2,6-dioxo-3-fluoropiperidin-3-yl)isoindoline, mp, 156.0-157.0 'H NMR (CDC13); 8 1.62 9H, CH3), 2.41-2.72 2H, CH 2 2.87-3.03 1H, CHH), 3.52-3.65 1H, CHH), 7.81-7.97 4H, Ar); 3 C NMR (CDC13) 6 26.80 2 JC-F 27 Hz), 27.39, 28.98 7.5 Hz), 67.15, 93.50 (Jc-F 218 Hz), 124.31, 130.84, 135.29, 147.17, 161.80 2 Jc.- 28 Hz), 165.93, 167.57; Anal Calcd for CisH 7

N

2 0F: C, 57.45; H, 4.55; N, 7.44; F, 5.05. Found: C, 57.78; H, 4.62; N, 7.23; F, 4.94.

The intermediate 1,3-dioxo-2-(1-tert-butoxycarbonyl-2,6-dioxo-3-fluoropiperidin- 3-yl)isoindoline can be converted to 1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3yl)isoindoline by stirring a solution of 1,3-dioxo-2-(1-tert-butoxycarbonyl-2,6-dioxo- 3-fluoropiperidin-3-yl)isoindoline (620 mg, 1.64 mmol) and hydrogen chloride in 1,4dioxane (15 mL, 4 M, 60 mmol) at room temperature for 3 days. The solvent is removed in vacuo and the residue stirred with ether (10 mL) for 1 hour and filtered to give 1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)isoindoline. A typical run produced 350 mg (77 yield) of to 1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3yl)isoindoline, mp, 228.0-230.0 'H NMR (DMSO-d 6 5 2.44-2.61 2H, CH 2 2.84-2.99 1H, CHH), 3.24-3.31 1H, CHH), 7.93 (brs, 4H, Ar), 11.49 1H, NH); 3C NMR (DMSO-d) 6 26.91 2 JC.F 27 Hz), 28.41 3 JC.F 8 Hz), 93.57 (Jc-F 211 Hz), 123.75, 130.91, 135.29, 164.29 (3c-F 6 Hz), 164.70 (3C-F 6 Hz), 166.21 -16- 432 1 Hz), 171.58 (Jc.F 6 Hz); Anal Calcd for C 13

HN

2 0 4 F +0.2 H20: C, 55.80; 433 H, 3.39;N, 10.01; F, 6.79. Found: C, 55.74; H, 3.30; N, 9.86; F, 7.18.

434 EXAMPLE 4 435 The procedure of Example 3 is followed, substituting, however, 0.76 g of N-fluoro- 436 benzenedisulfonimide for the 0.8 g of N-fluorobenzenesulfonimide. There is thereby 437 obtained 1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)isoindoline which can be 438 further purified through column chromatography.

439 EXAMPLE 440 To a stirred solution of 1,3-dioxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3- 441 yl)isoindoline (1.0 g. 2.8 mmol) in tetrahydrofuran (10 mL) is added lithium 442 diisopropylamide (1.5 mL, 3.0 mmol, 2 M) at. After 30 minutes, perchloryl fluoride 443 (5 mmol) is bubbled into the mixture. The mixture is allowed to reach room 444 temperature and the solvent then removed in vacuo. The residue is stirred with ethyl 445 acetate (10 mL) and IN hydrochloric acid (10 mL) for one hour. The organic layer is 446 separated and the solvent removed in vacuo to yield 1,3-dioxo-2-(2,6-dioxo-3- 447 fluoropiperidin-3-yl)isoindoline which can be further purified through chromatography.

448 EXAMPLE 6 449 To a stirred solution of 1,3-dioxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3- 450 yl)isoindoline (1.0 g. 2.8 mmol) in dimethylformamide (10 mL) is added sodium 451 hydride (112 mg, 2.8 mmol, 60%) at room temperature. After about 30 minutes, 452 perchloryl fluoride (5 mmol) is bubbled into the mixture The mixture is stirred with 453 methylene chloride (10 mL) and IN hydrochloric acid (10 mL) for one hour. The 454 organic layer is separated and the solvent removed in vacuo to yield 1,3-dioxo-2-(2,6- 455 dioxo-3-fluoropiperidin-3-yl)isoindoline which can be further purified through 456 chromatography.

457 EXAMPLE 7 458 To a stirred solution of 1,3-dioxo-2-(l-tert.-butoxycarbonyl-2,6-dioxopiperidin-3- 459 yl)isoindoline (1.0 g, 2.8 mmol) and tetramethylethylene diamine (0.5 g, 4.3 mmol) in 460 tetrahydrofuran (10 mL) is added n-butyl lithium (1.2 mL, 3.0 mmol, 2.5 M) at -78 0

C.

461 After 30 minutes, N-fluorobenzenesulfonimide (0-8 g, 3.2 mmol) is added to the 462 mixture. The mixture is allowed to reach room temperature and the solvent then -17- 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 removed in vacuo. The residue is stirred with ethyl acetate (10 niL) and IN hydrochloric acid (10 mL) for one hour. The organic layer is separated and the solvent removed in vacvo to yield 1 ,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-y1)isoindorne which can be flurther purified through column chromatography.

FAMPL E 8 Starting with each of 1 ,3-dioxo-2-( 1-1cr. -butoxycarbonyl-2,6-dioxopiperidin-3-yI)- 4-fl -ten.-butoxycarbonylamino)-isoindoline, 1 ,3-dioxo-2-(1 -sen. -butoxycarhonyl-2,6dioxopiperidin-3-yl)-5-( 1-tert. -butoxycarbonylamino)-isoindoline, 1 ,3-dioxo-2-(1I-terf.butoxycarbonyl-2,6-dioxopiperidin-3-yD-4, 5,6,7-tetrafluoroisoindoline, 1,3 -dioxo-2- (1 -ScM -butoxycarbonyl-2,6-dioxopiperidin-3-yI)- 4,5,6, 7-tetrachloroisoindoline, 1,3dioxo-2-( 1-tei. -butoxycarbonyl-2,6-dioxopiperidin-3-yD)- 4 ,5, 6 ,7tetramethylisoindoline, I ,3-dioxo-2-( 1-lert. -butoxycarbonyl-2,6-dioxopiperidin-3-YI)- 4,5,6,7-tetraniethoxyisoindoline, 1-oxo-2-( 1-furL -butoxycarbonyl-2,6-dioxopiperidin- 3-yl)-5-(l -ten. -butoxycarbonylanino)-isoindoline, 1 -oxo-2-( I -tefl. -butoxycarbonyl- 2,6-dioxopiperidin-3-yl)-isoindoline, I -oxo-2-( 1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4-( i-tert.-butoxycarbonylanino)-isoindoine, 1 -oxo-2-(1 -tedf. butoxycarbonyl-2,6-dioxopiperidin-3-y)4,5,6,7-tetrafluoroisoildolile, 1 -oxo-2-( 1terf. -butoxycarbonyl-2,6-dioxopiperidin-3-yl)- 4,5,6,7-tetrachloroisoindoline, 1 -oxo-2- (1-tent.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)- 4,5,6,7-tetramethylisoindolirle, 1,3 dioxo-2-( 1-tern. butoxycarbony-2,6-dioxopiperidin-3-y)-4-methylisoild0iC, 1 ,3-dioxo-2-( I-sert-butoxycarbony1-2,6-dioxopiperidin-3-yl-5-nethylisoidOlilC, I -oxo-2- (1 -ten. -butoxycarbonyl-2,6-dioxopiperidin-3-yl)-5-methylisoindoline, I -oxo-2-(1 -turd. butoxycarbonyl-2,6-dioxopipcridin-3-yl)-4-methylisoindoline, and I -oxo-2-(WI-ent.

butoxycarbony-2,6-dioxopiperidin-3-y)-4,5,6,7-ttramethOxyiSOidolile there are respectively obtained by following the procedures of Examples 3, 4, 5, 6, or 7, 1,3dioxo-2-(2,6-dioxo-3-fiuoropiperidin-3-yl)-4-aninoisOildolile, I ,3-dioxo-2-(2, 6 dioxo-3-fluoropiperidin-3-y)-5-atninoisoindoline, I ,3-dioxo-2-(2,6-dioxo-3fluoropiperidin-3 -yl)-4,5,6,7-tetrafiuoroisoindoline, 1,3 -dioxo-2-(2,6-dioxo-3-fluoropiperidin-3 4,5,6,7-tetrachloroisoindoline, 1 ,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetramethylisoindoline, 1 ,3-dioxo-2-(2,6-di0x-3fiuoropiperidin-3 -yl)-4,5,6,7-tetramethoxyisoindoline, 1 -oxo-2-(2,6-dioxo-3% fluoropiperidin-3-y)-5--aminoisoindoline, I -oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)isoindoline, 1-oxo-2-(2,6-dioxo-3 -fluoropiperidin-3-yl)-4-aminoisoindolifle, I-oxo-2- (2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetrafiuoroisoindoline, 1 -oxo-2-(2,6-dioxo- 3-fluoropiperidin-3-yl)- 4,5,6,7-tetrachloroisoindoline, 1-oxo-2-(2,6-dioxo-3 18- 498 fluoropiperidin-3-yl)- 4,5,6,7-tetramethylisoindoline, 1,3-dioxo-2-(2,6-dioxo-3-fluoro- 499 piperidin-3-yl)-4-methylisoindoline, 1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-5- 500 methylisoindoline, 1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-5-methylisoindoline, 1- 501 oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4-methylisoindoline, and I-oxo-2-(2,6-dioxo- 502 3-fluoropiperidin-3-yl)-4,5,6,7-tetramethoxyisoindoline.

503 EXAMPLE 9 504 Part A. A solution of L-glutamic acid dimethyl ester (2.6 g, 14.8 mmol), isoamyl 505 nitrite (2.13 mL, 15.9 mmol) and acetic acid (0.22 mL) in benzene (150 mL) is heated 506 to reflux for one hour. The solution is washed with IN aqueous sulfuric acid, water, 507 saturated sodium hydrogen carbonate solution, water and brine (50 mL each). The 508 solvent is removed in vacuo to yield dimethyl 2-diazopentane-l,5-dioate which can be 509 further purified by column chromatography.

510 Part B. To a cold solution of 5 mL of 70% hydrogen fluoride in pyridine and 1.2 g 511 (6.7 mmol) N-bromosuccinimide in 10 mL of ether is added a solution of dimethyl 2- 512 diazopentane-1,5-dioate (1.1 g, 5.9 mmol) in ether (10 mL) at 0°C. The mixture is 513 stirred at G0C for 30 minutes The solution is washed with water (20 mL), brine 514 mL) and dried over sodium sulfate. The solvent is removed in vacuo to yield dimethyl 515 2-bromo-2-fluoropentane-1,5-dioate which can be further purified by column 516 chromatography.

517 Part C. A mixture of dimethyl 2-bromo-2-fluoropentane-l,5-dioate (1.0 g, 3.8 518 mmol) and potassium phthalimide (0.79 g, 4.3 mmol) in dimethylformamide (10 mL) is 519 heated at 80 0 C for 3 hours. The solvent is removed in vacuo and the residue is stirred 520 with ethyl acetate (50 mL) for 10 minutes. The organic layer is washed with water and 521 brine (20 mL each), and dried over sodium sulfate. The solvent is removed in vacuo to 522 yield dimethyl 2-(1,3-dioxoisoindolin-2-yl)-2-fluoropenta-1,5-dioate which can be 523 further purified by column chromatography.

524 Part D. A mixture of dimethyl 2-(1,3-dioxoisoindolin-2-yl)-2-fluoropenta-l,5- 525 dioate (1.3 g, 4.0 mmol), methanol (10 mL) and 4N hydrochloric acid (10 mL) is 526 heated at 80°C for one hour. The solvent is removed in vacuo to yield 2-fluoro-2- 527 (1,3-dioxoisoindolin-2-yl)-propane-l,3-dicarboxylic acid. This is dissolved in acetic 528 anhydride (20 mL) and the solution heated at reflux for 30 minutes. The solvent is 529 remove in vacuo to yield 2-fluoro-2-(1,3-dioxoisoindolin-2-yl)-propane-l,3- 530 dicarboxylic acid anhydride which is mixed with ammonia in methanol (35 mL, 2 M) -19- 531 and stirred at room temperature for 18 hours. The solvent is then removed in vacuo 532 and the residue is stirred with methylene chloride (50 mL) for 10 minutes. The organic 533 layer is washed with water and brine (40 mL each), and dried over sodium sulfate. The 534 solvent is removed in vacuo and the residue is heated at reflux with carbonyl 535 diimidazole (0.65 g. 4 mmol) and dimethylaminopyridine (50 mg) in tetrahydrofuran 536 (30 mL) for 18 hours. 1,3-Dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)isoindoline is 537 isolated extraction with ethyl acetate and further purified by chromatography.

538 EXAMPLE 539 A stirred mixture of L-glutamic acid dimethyl ester (2.0 g, 11.4 mmol) and phthalic S 540 anhydride (1.7 g, 11.4 mmol) in acetic acid (30 mL) is heated to refluxed for one hour.

541 The solvent is removed in vacuo to yield dimethyl 2-(1,3-dioxoisoindolin-2-yl)- 542 pentane-1,5-dioate which is further purified through chromatography.

543 To a stirred solution of dimethyl 2-(1,3-dioxoisoindolin-2-yl)-pentane-l,5-dioate 544 (1.0 g, 3.3 mmol) and tetramethylethylene diamine (0.5 g, 4.3 mmol) in 545 tetrahydrofuran (10 mL) is added 2.5 M n-butyl lithium (1.6 mL, 4 mmol,) at -79 0

C.

546 After 30 minutes, N-fluorobenzenesulfonimide (1 g, 3.2 mmol) is added to the mixture 547 which then is allowed to reach room temperature. The solvent is removed in vacuo 548 and the residue is stirred with methylene chloride (100 mL) for 10 minutes. The 549 organic layer is washed with water and brine (30 mL each), and dried over sodium 550 sulfate. The solvent is removed in vacuo to yield dimethyl 2-(1,3-dioxoisoindolin-2- 551 yl)-2-fluoropentane-l,5-dioate which is further purified by chromatography and 552 converted to 1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)isoindoline as described 553 above in Part D of Example 9.

554 EXAMPLE 11 555 A stirred mixture of ethyl bromofluoroacetate (1.0 g, 5.4 mmol) and potassium 556 phthalide (1.0 g, 5.4 mmol) in dimethylformamide (10 mL) is heated at 80°C for 3 557 hours. The mixture is stirred with ether (50 mL) and water (50 mL) and the organic 558 layer then washed with water and brine (30 mL each), and dried over sodium sulfate.

559 The solvent is removed in vacuo to give ethyl 2-(1,3-dioxoisoindolin-2-yl)-2- 560 fluoroacetate which is further purified through chromatography.

561 To a stirred solution of ethyl 2-(1,3-dioxoisoindolin-2-yl)-2-fluoroacetate (0.80 g, 562 3.2 mmol) in tetrahydrofuran (30 mL) is added lithium diisopropylamide (1.7 mL, 3.4 563 mmol, 2 M) at -78 0 C. After 30 minutes, t-butyl acrylate (0.42 g, 3.2 mmol) is added 564 to the mixture which is allowed to reach room temperature. The solvent is removed in 565 vacuo and the residue stirred with methylene chloride (50 mL) and water (30 mL) for 566 10 min. The organic layer is washed with brine (30 mL), and dried over sodium 567 sulfate. The solvent is removed in vacuo to give tert-butyl 4-(1,3-dioxoisoindolin-2- 568 yl)-4-fluoro-4-ethoxycarbonylbutanoate which is further purified by column 569 chromatography.

570 A solution of tert-butyl 4-(1,3-dioxoisoindolin-2-yl)-4-fluoro-4- 571 ethoxycarbonylbutanoate (1.1 g, 3 mmol) and trifluoroacetic acid (5 mL) in methylene 572 chloride (5 mL) is stirred for 18 hours and then with methylene chloride (50 mL) for 573 10 min. The organic layer is washed with water and brine (30 mL each), and dried 574 over sodium sulfate. The solvent is removed in vacuo to yield 4-(1,3-dioxoisoindolin- 575 2-yl)-4-(ethoxycarbonyl)-4-fluorobutanoic acid which can be purified by 576 chromatography or used in the next step without further purification.

577 A mixture of 4-(1,3-dioxoisoindolin-2-yl)-4-(ethoxycarbonyl)-4-fluorobutanoic acid 578 (0.9 g, 2.8 mmol), carbonyl diimidazole (0.46 g, 2.8 mmol) and 579 dimethylaminopyrimidine (0.68 g, 5.6 mmol) in tetrahydrofuran (30 mL) is heated at 580 reflux for 18 hours. The solvent is removed in vacuo and the residue is stirred with 581 methylene chloride (50 mL) for 10 minutes, The organic layer is washed with water 582 and brine (40 mL each) and dried over sodium sulfate. The solvent is removed in 583 vacuo to give 1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)isoindoline which is 584 further purified by column chromatography.

585 EXAMPLE 12 586 Tablets, each containing 50 mg of l,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-- 587 isoindoline, can be prepared in the following manner: 588 Constituents (for 1000 tablets) 589 590 1,3-dioxo-2-(2,6-dioxo- 591 3-fluoropiperidin-3-yl)- 592 isoindoline 50.0 g 593 lactose 50.7 g 594 wheat 7.5 g 595 polyethylene glycol 5.0 g 596 talc 5.0 g -21- 597 magnesium stearate 1.8 g 598 demineralized q.s.

599 600 The solid ingredients are first forced through a sieve of 0.6 mm mesh width. The 601 active ingredient, lactose, talc, magnesium stearate and half of the starch then are 602 mixed. The other half of the starch is suspended in 40 mL of water and this suspension 603 is added to a boiling solution of the polyethylene glycol in 100 mL of water. The 604 resulting paste is added to the pulverulent substances and the mixture is granulated, if 605 necessary with the addition of water. The granulate is dried overnight at 35°C, forced 606 through a sieve of 1.2 mm mesh width and compressed to form tablets of 607 approximately 6 mm diameter which are concave on both sides.

608 EXAMPLE 13 609 Tablets, each containing 100 nmg of 1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-iso- 610 indoline, can be prepared in the following manner: 611 612 613 614 615 616 617 618 619 S 620 621 622 623 624 625 626 627 628 Constituents (for 1000 tablets) 1 -oxo-2-(2,6-dioxo- 3-fluoropiperidin-3-yl)isoindoline 100.0 g lactose 100.0 g wheat 47.0 g magnesium 3.0 g All the solid ingredients are first forced through a sieve of 0.6 mm mesh width. The active ingredient, lactose, magnesium stearate and half of the starch then are mixed.

The other half of the starch is suspended in 40 mL of water and this suspension is added to 100 mL of boiling water. The resulting paste is added to the pulverulent substances and the mixture is granulated, if necessary with the addition of water. The granulate is dried overnight at 35°C, forced through a sieve of 1.2 mm mesh width and compressed to form tablets of approximately 6 mm diameter which are concave on both sides.

EXAMPLE 14 629 Tablets for chewing, each containing 75 mg of 1-oxo-2-(2,6-dioxo-3-fluoro- 630 piperidin-3-yl)-isoindoline, can be prepared in the following manner: -22- 631 Composition (for 1000 tablets) 632 633 1-oxo-2-(2,6-dioxo- 634 3-fluoropiperidin-3-yl)- 635 isoindoline 75.0 g 636 230.0 g 637 lactose 150.0 g 638 talc 21.0 g 639 glycine 12.5 g 640 stearic 10.0 g 641 1.5 g 642 5% gelatin solution q.s.

643 644 All the solid ingredients are first forced through a sieve of 0.25 mm mesh width.

645 The mannitol and the lactose are mixed, granulated with the addition of gelatin 646 solution, forced through a sieve of 2 mm mesh width, dried at 50°C and again forced 647 through a sieve of 1.7 mm mesh width. 1-Oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)- 648 isoindoline, the glycine and the saccharin. are carefully mixed, the mannitol, the lactose 649 granulate, the stearic acid and the talc are added and the whole is mixed thoroughly 650 and compressed to form tablets of approximately 10 mm diameter which are concave 651 on both sides and have a breaking groove on the upper side.

652 EXAMPLE 653 Tablets, each containing 10 mg 1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)- 654 4,5,6,7-tetrafluoroisoindoline, can be prepared in the following manner: 655 Composition (for 1000 tablets) 656 657 1,3-dioxo-2-(2,6-dioxo- 658 3-fluoropiperidin-3-yl)- 659 4,5,6,7-tetrafluoroisoindoline 10.0 g 660 328.5 g 661 corn 17.5 g 662 polyethylene glycol 5.0 g 663 talc 25.0 g 664 magnesium 4.0 g 665 demineralized water q.s.

666 667 The solid ingredients are first forced through a sieve of 0.6 mm mesh width. Then 668 the active imide ingredient, lactose, talc, magnesium stearate and half of the starch are 669 intimately mixed. The other half of the starch is suspended in 65 mL of water and this 670 suspension is added to a boiling solution of the polyethylene glycol in 260 mL of 671 water. The resulting paste is added to the pulverulent substances, and the whole is -23- 672 mixed and granulated, if necessary with the addition of water. The granulate is dried 673 overnight at 35 0 C, forced through a sieve of 1.2 mm mesh width and compressed to 674 form tablets of approximately 10 mm diameter which are concave on both sides and 675 have a breaking notch on the upper side.

676 EXAMPLE 16 677 Gelatin dry-filled capsules, each containing 100 mg of 1-oxo-2-(2,6-dioxo-3-fluoro- 678 piperidin-3-yl)-5-aminoisoindoline, can be prepared in the following manner: S 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 Composition (for 1000 capsules) 1-oxo-2-(2,6-dioxo- 3-fluoropiperidin-3-yl)- 100.0 g microcrystalline 30.0 g sodium lauryl sulfate 2.0 g magnesium 8.0 g The sodium lauryl sulfate is sieved into the 1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3yl)-5-aminoisoindoline through a sieve of 0.2 mm mesh width and the two components are intimately mixed for 10 minutes. The microcrystalline cellulose is then added through a sieve of 0.9 mm mesh width and the whole is again intimately mixed for minutes. Finally, the magnesium stearate is added through a sieve of 0.8 mm width and, after mixing for a further 3 minutes, the mixture is introduced in portions of 140 mg each into size 0 (elongated) gelatin dry-fill capsules.

EXAMPLE 17 A 0.2% injection or infusion solution can be prepared, for example, in the following manner: 1,3-dioxo-2-(2,6-dioxo- 3-fluoropiperidin-3-yl)- 5-aminoisoindoline hydrochloride 5.0 g sodium 22.5 g phosphate buffer pH 300.0 g demineralized to 2500.0 mL 1,3-Dioxo-2-(2,6-dioxo-3-fiuoropiperidin-3-yl)-5-aminoisoindoline hydrochloride is dissolved in 1000 mL of water and filtered through a microfilter. The buffer solution is -24- 707 added and the whole is made up to 2500 mL with water. To prepare dosage unit forms, 708 portions of 1.0 or 2.5 mL each are introduced into glass ampoules (each containing 709 respectively 2.0 or 5.0 mg of imide).

710 711 EXAMPLE 18 712 713 The efficacy of the 2-(2,6-dioxo-3-fluorpiperidin-3-yl)-isoindoline compound 714 depicted below, as an inhibitor of TNFa production in lipopolysaccharide (LPS) 715 stimulated human peripheral blood mononuclear cells (PBMCs), was determined as 716 follows.

717 OO H N- 0 718 NH 2 719 720 PBMCs were isolated from normal donors by Ficoll-Hypaque density 721 centrifugation. Cells were then cultured in RPMI supplemented with 10% serum, 2mM 722 L-glutamine, 1OOU/mL penicillin and 1OOU/mL streptomycin. The compound was then 723 assayed at half log dilutions starting at 50mg/ml. The compound was then applied to 724 the PBMCs (106 cells/mL) in 96 well plates and incubated for one hour. Cells were 725 then treated with Img/mL LPS from Salmonella Minnesota R595 (List Biological Labs, 726 Campbell, CA). The cells were then further incubated at 37 0 C for 18-20 hours.

727 728 Following this incubation, supernatants were harvested and assayed for TNFa 729 levels. If samples were not used immediately upon harvesting, they were frozen at 730 (for not more than 4 days) prior to assaying. The concentration of TNFa in the 731 supernatant was then determined by human TNFc ELISA kits (ENDOGEN, Boston, 732 MA) according to the manufacturer's instructions.

733 734 The decrease in TNFa production as a function of the concentration of 735 compound added, allowed the determination of the IC 5 o of the test compound. In this 736 case the IC 50 of the compound is the concentration required to elicit a 50% reduction in 737 the TNFa production by PBMCs compared to cells which were not exposed to the 738 compound. The IC 5 0 for this particular fluorine substituted compound was 230nM.

739 Given this result, it is expected that other substituted 2-(2,6-dioxo-3-fluorpiperidin-3- 740 yl)-isoindoline compounds function would also be effective TNFa reducing agents.

Claims (24)

1. A method of reducing or inhibiting undesirable levels of inflammatory cytokines in a maminal, which comprises administering thereto an effective amount of a compound selected from the group consisting of: an optical isomer of 2-(2,6-dioxo-3fluoropiperidin-3-yl)-isoindoline of the formulas: 751 752 753 754 755 756 757 758 759 760 761 in which Y is oxygen or H 2 and each of R 2 R 3 and R 4 independently of the others, is hydrogen, halo, alkyl of 1 to 4 carbon atoms, alkoxy of I to 4 carbon atoms, or amino, and the acid addition salts of said 2-(2,6-dioxo-3-fluoropiperidin-3-yl)- isoindoline isomer which contain a nitrogen atom capable of being protonated.

2. The method according to claim 1, wherein R 2 R 3 and R 4 are hydrogen. -27- 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794

3. The method according to claim 1, wherein R' R 2 R 3 and R 4 are the same and each is chloro, fluoro, methyl, or methoxy.

4. The method according to claim 1, wherein R 3 is amino, and R 2 and R 4 are hydrogen.

5. The method according to claim 1, wherein R 4 is amino, and R 2 and R 3 are hydrogen.

6. The method according to claim 1, wherein R 3 is methyl, and R 2 and R 4 are hydrogen.

7. The method according to claim 1, wherein R4 is methyl, and R 2 and R are hydrogen.

8. The method according to claim 1, wherein the compound is 1,3-dioxo-2-(2,6- dioxo-3-fluoropiperidin-3-yl)-isoindoline.

9. The method according to claim 1, wherein the compound is dioxo-3-fluoropiperidin-3-yl)-4-aminoisoindoline.

10. The method according to claim 1, wherein the compound is dioxo-3-fluoropiperidin-3-yl)-5-aminoisoindoline.

11. The method according to claim 1, wherein the compound is dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoline. 1,3-dioxo-2-(2,6- 1,3-dioxo-2-(2,6- 1,3-dioxo-2-(2,6-

12. The method according to claim 1, wherein the compound is 1,3-dioxo-2-(2,6- dioxo-3-fluoropiperidin-3-yl)- 4 ,5,6,7-tetrachloroisoindoline.

13. The method according to claim 1, wherein the compound is 1,3-dioxo-2-(2,6- dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetramethylisoindoline. -28- 795 796

14. The method according to claim 1, wherein the compound is 1,3-dioxo-2-(2,6- 797 dioxo-3-fluoropipcridin-3-yl)- 4 ,5,6,7-tetramethoxyisoindoline. 798 799

15. The method according to claim 1, wherein the compound is 1-oxo-2-(2,6-dioxo- 3 800 801 802

16. The method according to claim 1, wherein the compound is 1-oxo-2-(2,6-dioxo-3- 803 fluoropiperidin-3-yl)-isoindoline. 804 805

17. The method according to claim 1, wherein the compound is 1-oxo-2-(2,6-dioxo-3- 806 fluoropiperidin-3-yl)-4-aminoisoindoline. 807 808

18. The method according to claim 1, wherein the compound is 1-oxo-2-(2,6-dioxo-3- 809 fluoropiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoline. 810 811

19. The method according to claim 1, wherein the compound is 1-oxo-2-(2,6-dioxo-3- 812 fluoropiperidin-3-yl)-4,5,6,7-tetrachloroisoindoline. 813 814

20. The method according to claim 1, wherein the compound is 1-oxo-2-(2,6-dioxo-3- 815 fluoropiperidin-3-yl)- 4 ,5,6,7-tetramethylisoindoline. 816 817

21. The method according to claim 1, wherein the compound is 1-oxo-2-(2,6-dioxo-3- 818 fluoropiperidin-3-yl)- 4 ,5,6,7-tetramethoxyisoindoline. 819 820

22. The method according to any one of claims 1 to 21, wherein the inflammatory 821 cytokines are at least one member selected from the group consisting of TNF-c, IL-1, IL- 822 6, and IL-12. 823 824

23. The method according to any one of claims 1 to 22, wherein the compound is part 825 of a pharmaceutical composition. 826 827 828 829 -29

24. The use of the compound selected from the group consisting of an optical isomer of 2-(2,6-dioxo-3fluoropiperidin-3-yl)-isoindoline of the formulas 830 831 832 833 or 834 835 836 837 838 839 840 841 842 843 844 845 846 847 in which Y is oxygen or H 2 and each of R 2 R 3 and R 4 independently of the others, is hydrogen, halo, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, or amino, and the acid addition salts of said 2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline isomer which contain a nitrogen atom capable of being protonated, to prepare a medicament for treating a disease selected from the group consisting of septic shock, sepsis, endotoxic shock, hemodynamic shock and sepsis syndrome, post ischemic reperfusion injury, malaria, mycobacterial infection, meningitis, psoriasis, congestive heart failure, fibrotic disease, cachexia, graft rejection, cancer, autoimmune disease, opportunistic infections in AIDS, rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, other arthritic conditions, Chron's disease, ulcerative colitis, multiple 848 sclerosis, systemic lupus erythrematosis, ENL in leprosy, radiation damage, and 849 hyperoxic alveolar injury. 850 851 852 DATED THIS TWENTIETH DAY OF DECEMBER 2002 853 CELGENE CORPORATION 854 BY 855 PIZZEYS PATENT AND TRADE MARK ATTORNEYS