AU2005239663A1 - Substituted 2-(2,6-dioxo-3-fluoropiperidin-3-YL)-isoindolines and their use to reduce TNFalpha levels - Google Patents

Description

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

AND

2 THEIR USE TO REDUCE TNFa LEVELS 3 S 4 Background of the Invention Cc 6 s 7 Tumor necrosis factor a, or TNFa, is a cytokine which is released primarily by m 8 mononuclear phagocytes in response to a number immunostimulators. It is a key in 9 proinflammatory cytokine in the inflamation cascade causing the production and/or O 10 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 al., 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 TNFa may be involved in many bone resorption diseases, including arthritis, 29 the most compelling link with disease is the association between production of TNF 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 33 complication following acute allogenic bone marrow transplants {Holler et al., Blood, S34 75(4), 1011-1016(1990)}.

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

'O 37 Levels of serum TNFa correlated directly with the severity of disease and the S38 prognosis in patients with acute malaria attacks {Grau et al., N. Engl. J. Med. 320(24), S39 1586-1591 (1989)}.

O 40 Macrophage-induced angiogenesis is known to be mediated by TNFao. 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 al., Inflammation 13(3), 329-339 (1989)}. Alveolar macrophages from pulmonary sarcoidosis patients have also been found to spontaneously release massive quantities 56 of TNFc 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 al., 64 J. Cell Biol. 107, 1269-1277 (1988)}. TNFac has pro-inflammatory activities which 0 65 together with its early production (during the initial stage of an inflammatory event) Z 66 make it a likely mediator of tissue injury in several important disorders including but C 67 not limited to, myocardial infarction, stroke and circulatory shock. Of specific 68 importance may be TNFc-induced expression of adhesion molecules, such as rcr 69 intercellular adhesion molecule (ICAM) or endothelial leukocyte adhesion molecule (ELAM) on endothelial cells (Munro et al., Am. J. Path. 135(1), 121-132 (1989) S 71 TNFo blockage with monoclonal anti-TNFa antibodies has been shown to be 0 72 beneficial in rheumatoid arthritis (Elliot e al., Int. J. Pharmac. 1995 17(2), 141-145).

O 73 High levels of TNFa are associated with Crohn's disease {von Dullemen et at.

74 Gastroenterology, 1995 109(1), 129-135) and clinical benefit has been acheived with TNFoc antibody treatment.

76 Moreover, it now is known that TNFct is a potent activator of retrovirus replication 77 including activation of HIV-1. {Duh et al., Proc. Nat. Acad. Sci. 86, 5974-5978 78 (1989); Poll et al., Proc. Nat. Acad Sci. 87, 782-785 (1990); Monto et al., Blood 79, 79 2670 (1990); Clouse et al., J. Immunol. 142, 431-438 (1989); Poll et al., AIDS Res.

Hum. Retrovirus, 191-197 (1992)). AIDS results from the infection of T lymphocytes 81 with Human Immunodeficiency Virus (HIV). At least three types or strains of HIV 82 have been identified, HIV-1, HIV-2 and HIV-3. As a consequence of HIV 83 infection, T-cell mediated immunity is impaired and infected individuals manifest severe 84 opportunistic infections and/or unusual neoplasms. HIV entry into the T lymphocyte requires T lymphocyte activation. Other viruses, such as HIV-1, HIV-2 infect T 86 lymphocytes after T cell activation and such virus protein expression and/or replication 87 is mediated or maintained by such T cell activation. Once an activated T lymphocyte is 88 infected with HIV, the T lymphocyte must continue to be maintained in an activated 89 state to permit HIV gene expression and/or HIV replication. Cytokines, specifically TNFa, are implicated in activated T-cell mediated HIV protein expression and/or virus 91 replication by playing a role in maintaining T lymphocyte activation. Therefore, inter- 92 ference with cytokine activity such as by prevention or inhibition of cytokine 93 production, notably TNFca, in an HIV-infected individual assists in limiting the 94 maintenance of T lymphocyte caused by HIV infection.

Monocytes, macrophages, and related cells, such as kupffer and glial cells, also have 96 been implicated in maintenance of the HIV infection. These cells, like T cells, are 97 targets for viral replication and the level of viral replication is dependent upon the -4- 98 activation state of the cells. (Rosenberg et al., The Immunopathogenesis of HIV Z 99 Infection, Advances in Immunology, 57 (1989)}. Cytokines, such as TNFa, have been 0 100 shown to activate HIV replication in monocytes and/or macrophages {Poli et al., Proc.

101 Natl. 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 ,O 103 have identified TNFa as a common factor in the activation of HIV in vitro and has a* 104 provided a clear mechanism of action via a nuclear regulatory protein found in the r n 105 cytoplasm of cells (Osbom, et al., PNAS 86 2336-2340). This evidence suggests that a 106 reduction of TNFo synthesis may have an antiviral effect in HIV infections, by 0 107 reducing the transcription and thus virus production.

108 AIDS viral replication of latent HIV in T cell and macrophage lines can be induced 109 by TNFa (Folks et al., PNAS 86, 2365-2368 (1989)}. A molecular mechanism for the 110 virus inducing activity is suggested by TNFa's ability to activate a gene regulatory 111 protein (NFriB) 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)}. TNFa 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)}. TNFoa 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 KB (NFicB) 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 TNFa 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 ofNFKB 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 o 132 described herein can inhibit the action of NFKB in the nucleus and thus are useful in the Z 133 treatment of a variety of diseases including but not limited to rheumatoid arthritis, O 134 rheumatoid spondylitis, osteoarthritis, other arthritic conditions, septic shock, septis, c 135 endotoxic shock, graft versus host disease, wasting, Crohn's disease, ulcerative colitis, 136 multiple sclerosis, systemic lupus erythrematosis, ENL in leprosy, HIV, AIDS, and C0 137 opportunistic infections in AIDS. TNFa and NFiB levels are influenced by a O 138 reciprocal feedback loop. As noted above, the compounds of the present invention 139 affect the levels of both TNFac and NFiB.

8 140 Many cellular functions are mediated by levels of adenosine 3',5'-cyclic monophosphate 141 (cAMP). Such cellular functions can contribute to inflammatory conditions and diseases 142 including asthma, inflammation, and other conditions (Lowe and Cheng, Drugs of the 143 Future, 17(9), 799-807, 1992). It has been shown that the elevation of cAMP in 144 inflammatory leukocytes inhibits their activation and the subsequent release of inflammatory 145 mediators, including TNFa and NFKB. Increased levels of cAMP also leads to the 146 relaxation of airway smooth muscle. Phosphodiesterases control the level of cAMP 147 through hydrolysis and inhibitors of phosphodiesterases have been shown to increase 148 cAMP levels.

149 Decreasing TNFa levels and/or increasing cAMP levels thus constitutes a valuable 150 therapeutic strategy for the treatment of many inflammatory, infectious, immunological 151 or malignant diseases. These include but are not restricted to septic shock, sepsis, 152 endotoxic shock, hemodynamic shock and sepsis syndrome, post ischemic reperfusion 153 injury, malaria, mycobacterial infection, meningitis, psoriasis, congestive heart failure, 154 fibrotic disease, cachexia, graft rejection, cancer, autoimmune disease, opportunistic 155 infections in AIDS, rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, other 156 arthritic conditions, Crohn's disease, ulcerative colitis, multiple sclerosis, systemic 157 lupus erythrematosis, ENL in leprosy, radiation damage, and hyperoxic alveolar injury.

158 Prior efforts directed to the suppression of the effects of TNFa have ranged from the 159 utilization of steroids such as dexamethasone and prednisolone to the use of both 160 polyclonal and monoclonal antibodies {Beutler et al., Science 234, 470-474 (1985); 161 WO 92/11383).

-6- 2 Detailed Description 0 162 163 The present invention is based on the discovery that certain classes of non- Cc, 164 polypeptide compounds more fully described herein decrease the levels of 165 TNFc, increase cAMP levels, and inhibit phosphodiesterase. The present invention thus S166 relates to 2-(2,6-dioxo3-fuoropiperidin-3-yl)-isoindoines the method of reducing S167 levels of tumor necrosis factor c and other inflammatory cytokines in a mammal M0 168 through the administration of such derivatives, and pharmaceutical compositions tr 169 containing such derivatives.

CN 170 In particular, the invention pertains to 171 a 2 2 6 -dioxo-3-fluoropiperidin-3-yl)-isoindoline of the formula: 172

R

1

O

R2 C F O N

R

3

O

R4 y 173 174 175 in which 176 Y is oxygen or H2 and 177 each ofR 1

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- 0 187 The compounds of Formula I are used, under the supervision of qualified profes- Z 188 sionals, to inhibit the undesirable effects of TNFa and other inflammatory cytokines 189 including IL-1, IL-6, and L-12. The compounds can be administered orally, rectally, or 190 parenterally, alone or in combination with other therapeutic agents including antim 191 biotics, steroids, etc., to a mammal in need of treatment.

rr 192 The compounds of the present invention also can be used topically in the treatment S193 or prophylaxis of topical disease states mediated or exacerbated by excessive TNFcc S194 production, respectively, such as viral infections, such as those caused by the herpes C' 195 viruses, or viral conjunctivitis, psoriasis, atopic dermatitis, etc.

196 The compounds also can be used in the veterinary treatment of mammals other than 197 humans in need of prevention or inhibition of TNFa production. TNFo mediated 198 diseases for treatment, therapeutically or prophylactically, in animals include disease 199 states such as those noted above, but in particular viral infections. Examples include 200 feline immunodeficiency virus, equine infectious anaemia virus, caprine arthritis virus, 201 visna virus, and maedi virus, as well as other lentiviruses.

202 The compounds of Formula I are readily prepared through a number of routes. In a 203 first embodiment, the ring nitrogen of a 2 2 6 .dioxopiperidin-3-yl)-isoindoline of 204 Formula II is protected with a conventional amino protecting group to yield a 205 protected 2-(2,6dioxopipeidin-3-yl)-isoindoline of Formula I. This is then 206 fluorinated to yield a protected 2 2 ,6dioxo-3-fluoropiperidin-3-yl)-isoindoline of 207 Formula IV, following which removal of the protecting group yields the compounds of 208 Formula

V:

-8- O RI O RI O SR2 C R2 C

IND

4 Y 14 Y R' O C R2 O H

X

2 Y R Y 215 V Yn R4 Y

IV

V n 209 210 Inthe foregoing reactions, each of R, R 2 R3u R 4 and Y are as defined above and X 211 is amino protecting group. When any of Rl) R 3 and R is amino, it too should be 212 protected prior to the fluorination step.

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

216 Some of the compounds of Formula I1 which are here utilized as intermediates are 217 known, as for example l,3-dioxo-2-(2.6-dioxopiperidin-3-ylH-ainoisoindolne and 218 1 ,3-dioxo-2-(2,6-dioxopiperidin- 3 -yl)-5-aminoisoindolhne. See, Jonsson, Acta 219 Pharnna. 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- 226 227 228 229 In a second method, an appropriately substituted glutamic acid diester of Formula VI is fluorinated to yield the corresponding fluoroglutamic acid diester of Formula

VI.

This is then converted to the fluorinated glutamic acid anhydride of Formula

VIII

which, in turn, is amidated to yield the compounds of Formula

V:

RI 0 RI 0 RC R2 C F 0> O

OZ

R

4 y R Y VI Z

I

R'I 0 R

CFF

NO

3/

R

4

Y

VIfI 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 in the foregoing reactions, each of R 2

W

4 and Y are as defined above and Z and Z' are lower alkyl. Again when any of R 1 RBand R? is amino, it too should be protected prior to the fluorination step.

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

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

"Protective Groups in Organic Synthesis", Wiley, New York, 1981; "The Peptides", O 245 Vol. I, Schr6der and Lubke, Academic Press, London and New York, 1965; Z 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 converted to an amino group by catalytic hydrogenation. Alternatively, a C~ 251 protected amino group can be cleaved to yield the corresponding amino compound.

O 252 An amino group can be protected as an amide utilizing an acyl group which is selec- 253 tively removable under mild conditions, especially benzyloxycarbonyl, 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 a 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:

R

1 0 R

N

/H

R

4

Y

R

4

Y

IB

259IB 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 I1I >0 25 fo amitre by forming salts with a chiral acid or base, such as the individual Z266 enantiomerS of 10-camphorsulfoniic acid, camphoric acid, c*.-bromocamlphoric acid, r 267 methoxyacetic acid, tartaric acid, diacetyltartaric acid, malic acid, pyrrolidone-S- 268 carboxylic acid, and the like, and then freeing one or both of the resolved bases, 269 optionally repeating the process, so as obtain either or both substantially free of the 270 other; in a form having an optical purity of S271 The present invention also pertains to the physiologically acceptable non-toxic acid C) 272 addition salts of the compound of Formula I which contain a group capable of being N- 273 protonated; amino. Such salts include those derived from organic and inorganic 274 acids such as, without limitation, hydrochloric acid, hydrobromnic acid, phosphoric 275 acid, sulfuric acid, methanesulphornc acid, acetic acid, tartaric acid, lactic acid, succinic 276 acid, citric acid, malic acid, maleic acid, sorbic acid, aconitic acid, salicylic acid, 277 plithalic acid, embonic acid, enanthic acid, and the like.

278 Particularly preferred compounds include ,3-dioxo-2(2,6dioxo3-fuoro- 279 piperidin-3-yl)-isoifldoline, 1,-ix--26doo3furpprdn3y)4 280 aminloisoindolile, I ,3-dioxo-2-(2,6-dioxo- 3 -fiuorpiperidi-3-yl>-54mliioisoindoline, 281 1 ,3-dioxo-2-(2,6-dioxo--oo pieii--3--mtyionoie 1 ,3-dioxo-2-(2,6- 282 dix--looieii--l--ehlsidhe I-oxo-2(2,-dioxo3-fuoro- 283 piperidin-3-yl)-5-methylisoindouine, I-x--26doo3furpprdn3y)4 284 methylisoindoine, 1,-ix--26doo3furpprdn3y)4567ttaloo 285 isoindoline, 1,-ix--26doo3furpprdn3y)4567 286 tetrachioroisoindoine, 1,-ix--26doo-- ooieii--yl)- 4 ,5, 6 7 287 tetramethylisoiuidolifle, 1.-ix--26doo3flooieii- 288 tetramethoxyisoindolifle, I-x--26doo3fuooiei~n3y)5 289 aminoisoindoline, 1 x--26doo--looieii-3y)ionoie I -oxo-2- 290 126doo3furpprdn--l--rfnionoie I oxo-2-(2,6-dioxo- 3 29 looieii--l-,,67ttaloosidhe I-oxo-2-(2,6-diOoo3- 292 fluoropiperidin-3-yl)- 4,5,6,7-tetrachloroisoildolifle, I -oxo-2-(2,6dioxo- 3 293 flooieii--l-,,,-ermtyionoie and 1 ~oxo-2-(2,6-dioxo- 3 294 fluoropiperidif-3-y)4,5,6,7-tetramethoxyisoifldoline. Of these, 1 ,3-dioxo-2-(2, 6 295 dioxo-3-fluoropiperidifl3-yl)-isoindoline and l-oxo- 2 2 6 -dioxo3-fuoropiperidin- 3 296 yl)-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 drug per unit dosage.

-12- 299 Isotonic saline solutions containing from 20 to 100 mg/mL can be used for parenteral 0 Z 300 administration which includes intramuscular, intrathecal, intravenous and intra-arterial 0 301 routes of administration. Rectal administration can be effected through the use of 302 suppositories formulated from conventional carriers such as cocoa butter.

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

c 305 In preparing such compositions, the active ingredients are usually mixed with or n) 306 diluted by an excipient or enclosed within such a carrier which can be in the form of a 0 307 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 TNFa can be performed in a conventional 327 manner. PBMC is isolated from normal donors by Ficoll-Hypaque density centrifugation.

328 Cells are cultured in RPMI 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- 0 332 suspensions is 0.25 wt Drugs are assayed at half-log dilutions starting at 50 mng/mL.

S333 Drugs are added to PBMC (106 cells/mL) in 96 wells plates one hour before the addition of S334 LPS. PBMC (106 cells/mL) in the presence or absence of drug are stimulated by treatment 335 with 1 mg/mL of LPS from Salmonella minnesota R595 (List Biological Labs, Campbell, r 336 CA). Cells are then incubated at 370 C for 18-20 hours. Supernatants are harvested and S337 assayed immediately for TNFa levels or kept frozen at -70 0 C (for not more than 4 days) 338 until assayed. The concentration of TNFL in the supernatant is determined by human S339 TNFa ELISA kits (ENDOGEN, Boston, MA) according to the manufacturer's directions.

340 The following examples will serve to fuirther typify the nature of this invention but 341 should not be construed as a limitation in the scope thereof, which scope is defined 342 solely by the appended claims.

343 EXAMPLE 1 344 To a stirred suspension of 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)isoindoline (2.0 g, 345 7.75 mmol) and di-lert.-butyl dicarbonate (1.86 g, 8.52 mmol) in 1,4-dioxane (3.0 mL) 346 is added dimethylaminopyridine (1.00 mg) at room temperature. The solution is stirred 347 at room temperature for 18 hours and the solvent then removed in vacuo. The residue 348 is stirred with ether (30 nimL) for 30 minutes, filtered, and washed with ether to give 349 1;3-dioxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)isoindoline.

350 A typical run produced 2.5 g (90% yield) of 1,3-dioxo-2-(1-tert.-butoxycarbonyl- 351 2,6-dioxopiperidin-3-yl)isoindoline, mp, 274.0-275.0 'H NMR (DMSO-d6); 8 1.47 352 9H, CH 3 2.08-2.15 1, CHH), 2.50-2.70 1H, CHH), 2.69-2.82 1H, 353 CHH), 3.02-3.17 1, CHH), 5.42 (dd, J= 5.4, 12.9 Hz, 1H, NCH), 7.90-7.94 (m, 354 4, Ar); 3 C NMR (DMSO-d6) 8 21.11, 27.03, 30.69, 48.77, 86.01, 123.52, 131.16, 355 134.99, 148.28, 166.99, 167.55, 169.99; Anal. Calc'd for C 1 jHsN 2 06: C, 60.33; H, 356 5.06; N, 7.82. Found: C, 60.01; H, 5.21; N, 7.47.

357 EXAMPLE 2 358 Similarly to the procedure of Example 1, there are respectively obtained from 1,3- 359 dioxo-2-(2,6-dioxopiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoine, 1,3-dioxo-2-(2,6- 360 dioxopiperidin-3-yl)- 4,5,6,7-tetrachloroisoindoline, 1, 3 -dioxo-2-(2,6-dioxopiperidin- 361 3-yl)-4,5,6,7-tetramethylisoindoline, 1,3-dioxo-2-(2,6-dioxopiperidin-3-yl)-4,5,6,7- 362 tetramethoxyisoindoline, 1-oxo-2-(2,6-dioxopiperidin- 3 -yl)-isoindoline, 1-oxo-2-( 2 6 363 dioxopiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoline, 1 -oxo-2-(2,6-dioxopiperidin- 3 -yl)- 14 o 364 4, 5,6,7-tetrachloroisoildolifle, I -oxo-2-(2,6-dioxopiperidin-3-yl)- 4,5,6,7- Z 365 tetramethylisoindoline, 1 3 -dioxo-2(2,6dioxopipeidi-flY1$methylisoindohne, 1,3- S366 dioxo-2-(2,6-dioxopiperidin3-yi)5methylisondoline, 1-oxo-2-(2,6-dioxopiperldifl-3- 367 yl)-5-methylisoindoife, I-x--26dooieii-3y)4mtyionoie and 1- 368 oxo-2-(2,6-dioxopiperidil-3-y1)- 4 5,6,7-tetramethoxyisoindoline, the compounds 1,3- .C 369 dioxo-2-( 1 -tert. -butoxycarbonyl-2,6-dioxopiperidifl 3 -yl)- 4 5,6,7-tetrafluoroisoindo- C* 370 line, 1 ,3-dioxo-2-( I-tert.-btxcroy ,-dooieii--l-4567ttahoo N 371 isoindoline, 1,3 -dioxo-2-( I-tert.-btxcroy 26dooieiin3y)4567 '~372 tetramethylisoindoline, I ,3-dioxo-2-(1 -tert. -butoxycarbony1-2,6-dioxooiperidin- 3 -yl)- S373 4,5,6,7-tetramethoxyisoildolifle, I1-oxo-2-(1 -tert. -butoxycarbony-2,6-dioxopiperidin- 374 3-yl)-isoindoline, 1 -oxo-2-(1 -tert. -butoxycarbonyl-2,6-dioxopiperidin- 3 -yl)- 4 5,6,7- 375 tetrafluoroisoindoline, 1 -oxo-2-(1 -ter. butoxycarbonyl-2,6-dioxopiperidi- 3 -yl)- 376 4,5,6,7-tetrachloroisoindohle, I -oxo-2-(1 -tert. -butoxycarbonyl-2,6-dioxopiperldifl- 3 377 yl)- 4,5,6,7-tetramethylisoindoline, 1 ,3-dioxo-2-( 1-tert. -butoxycarbonyl-2,6-diOoo 378 piperidin-3 -yl)-4-methylisoindoline, I ,3-dioxo-2-(1 -ten. butoxycarbonyl-2,6-dioxo- 379 piperidin-3-yl)-5-methylisoildolifle, l-oxo-2-(1 -tert.-butoxycarboflyl-2,6-dioxo' 380 piperidin-3-yl)-5-methyisoildohfe, 1 -oxo-2-(1 -lert. -butoxycarbonyl-2,6-dioxopiperi- 381 din-3-yl)-4-methylisoindoline, and 1-oxo-2-(1 -tert. -butoxycarboflyl-2,6-dioxopiperidin- 382 3-y)4,5,6,7-tetramethoxyisoifldolife 383 Similarly utilizing equivalent amounts of 1,3-dioxo-2-(2,6-diOopipeidin-3-yl)- 384 aminoisoindoline, 1,3doo2(,-ixpprdn3y)5annionoe I 1-oxo-2- 385 (2,6-dioxopiperidin3-yl)-5-a~ioisoinfdohfl, and 1 -oxo-2-(2,6dioxopipeidin3yl)- 4 386 amninoisoindoline, but utilizing 3.72 g of di-tert.-butyl dicarbonate in the procedure of 387 Example 1, there are respectively obtained 1,3-dioxo-2-(l-tert.-butoxycarbonyl- 2 6 388 dioxopiperidin-3-yl)-4-(1 -,ert.-butoxycarbonylamino)-isoindolifle, 1,3 -dioxo-2-(1 -tert. 389 butoxycarbonyl-2,6-dioxopiperidin-3 -tert.-butoxycarbonylarino)-isoindolifle, 390 1-oxo-2-(1-tert. -butoxycarbonyl-2,6-dioxopiperidin-3-y)-5-l -tent.- 391 butoxycarbonylamnino)-isoindoline, and I -oxo-2-(1-tert. -butoxycarbonyl-2,6-dioxopip- 392 eridin-3-yl)-4-(1 -lert.-butoxycarbonylamino)-isoindoline.

393 EXAMPLE 3 394 To a stirred solution of 1,-ix--Itr.btxcroy-,-ixpprdn3 395 yl)isoindoline (1.0 g, 2.8 mmol) in tetrahydrofuran (10 mL) is added n-butyl lithium 396 (1.2 mL, 3.0 mmol, 2.5 NM) at -78 0 C. After 20 minutes, N-fluorobenzelesulfornimde 397 (0-8 g, 3.2 mmol) 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 o -99 acetate (10 mL) and 1N hydrochloric acid (10 mL) for one hour. The organic layer is S400 separated and the solvent removed in vacuo to yield 1,3-dioxo-2-(2,6-dioxo- 3 O 401 fluoropiperidin-3-yl)isoindoline which can be further purified through chromatography.

402 In a similar fashion, lithium bis(trimethylsilyl)amide (24 mL, 24 mmol, 1.0 M) is 403 added to a stirred solution of 1,3-dioxo-2-(-tert-butoxycarbonyl-2,6-dioxopiperid- 0 404 3-yl)isoindoline(7.16 g, 20 mmol) in tetrahydrofuran (250 mL) at -40 C. After 1 405 hour, N-fluorobenzenesulfonimide (7.6 24 mmol) is added to the mixture. The 406 mixture is allowed to reach room temperature and kept overnight. The solution is S407 stirred with ethyl acetate (200 mL), aqueous ammonium chloride (100 ml, sat), and 408 water (100 mL). The aqueous layer is separated and extracted with ethyl acetate (200 409 mL). The combined organic layers are washed with water (100 mL), brine (100 mL), 410 and dried over sodium sulfate. The solvent is removed in vacuo. The residue is 411 purified by chromatography (silica gel) to give the intermediate 1,3-dioxo-2-(1-tert- 412 butoxycarbonyl-2,6-dioxo-3-fluoropiperidin-3-yl)isoindoline.

413 A typical run produced 700 mg (10 yield) of give 1,3-dioxo-2-(1-tert-butoxy- 414 carbonyl-2,6-dioxo-3-fluoropiperidin-3-yl)isoindoline, mp, 156.0-157.0 1 H NMR 415 (CDC 3 8 1.62 9H, CH 3 2.41-2.72 2H, CH2), 2.87-3.03 1H, CHH), 416 3.52-3.65 1H, CHH), 7.81-7.97 4H, Ar); 'C NMR (CDCl 3 5 26.80 2 C-F 417 27 Hz), 27.39, 28.98 (JC-F 7.5 Hz), 67.15, 93.50 (JC-F 218 Hz), 124.31, 130.84, 418 135.29, 147.17, 161.80 2 JC- 28 Hz), 165.93, 167.57; Anal Calcd for CjsH 1 7

N

2 0 6

F.

419 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.

420 The intermediate 1,3-dioxo-2-( -tert-butoxycarbonyl-2,6-dioxo-3-fluoropiperiin- 421 3-yl)isoindoline can be converted to 1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3- 422 yl)isoindoline by stirring a solution of 1,3-dioxo-2-(-tert-butoxycarbonyl-2,6-dioxo- 423 3-fluoropiperidin-3-l)isoindoline (620 mg, 1.64 mmol) and hydrogen chloride in 1,4- 424 dioxane (15 mL, 4 M, 60 mmol) at room temperature for 3 days. The solvent is 425 removed in vacuo and the residue stirred with ether (10 mL) for 1 hour and filtered to 426 give 1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)isoindoline. A typical run 427 produced 350 mg (77 yield) of to 1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3- 428 yl)isoindoline, mp, 228.0-230.0 OC; 'H NMR (DMSO-d6); 8 2.44-2.61 2H, CH 2 429 2.84-2.99 1, CHH), 3.24-3.31 1H, CHH), 7.93 (brs, 4H, Ar), 11.49 1H, 430 NH); 3 C NMR (DMSO-d6) 6 26.91 (JC.F 27 Hz), 28.41 3 Jc.v 8 Hz), 93.57 (Jc.iF 431 211 Hz), 123.75, 130.91, 135.29, 164.29 3 JC.F= 6 Hz), 164.70 (Jc.F= 6 Hz), 166.21 -16- 432 4 Jc-F 1 Hz), 171.58 (Jc-F 6 Hz); Anal Calcd for C 1 3

HN

2 0 4 F +0.2 H20: C, 55.80; Z 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 Cf 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 CI 438 further purified through column chromatography.

439 EXAMPLE 440 To a stirred solution of 1,3-dioxo-2-(l-ert.-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-(-tert.-butoxycarbonyl-2,6-dioxopiperidin3- 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-(1-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- 63 removed in vacuc. The residue is stirred with ethyl acetate (10 m.L) and IN 0 1 64 hydrochloric acid (10 mL) for one hour. The organic layer is separated and the solvent C) 165 removed in vacuo to yield 1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)isoidolifle 466 which can be further purified through column chromatography.

467 XAMPLE 8 468 Starting with each of 1,3-dioxo-2-(1-tert.-butoxycarbonyl-2,6-dioxopiperidin-3-ylYc1 469 4-(1 -tert. -butoxycarbonylamino)-isoindoine, 1 ,3-dioxo-2-(1 -ten. -butoxycarbonyl-2,6- 470 dioxopiperidin-3-yl)-5-( 1-tert.-butoxycarbonylamino)-isoindoline, I ,3-dioxo-2-(1 -ter. 471 buoyabnl26dooieii--y),,,-erfurionoie 1,3 -dioxo-2- 472 (1 -tent. -butoxycarbonyl-2,6-dioxopiperidin-3-yl)- 4,5,6,7-tetrachloroisoindolile, 1,3- 473 dioxo-2-(1 -tet. -butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4,5,6,7- 474 tetraniethylisoindoline, 1 ,3-dioxo-2-(1 -tent. -butoxycarbonyl-2,6-dioxopiperidim- 3 -yl)- 475 4,5,6,7-tetraniethoxyisoindoline, l-oxo-2-(1 -tent.-butoxycarbonyl-2,6-dioxopiperidifl- 476 1-tert.-butoxycarbonylamino)-isoindoline, 1 -oxo-2-(1 -It. -butoxycarbonyl- 477 2,6-dioxopiperidin-3-yl)-isoindoline, 1 -oxo-2-(1 -tert. -butoxycarbonyl-2,6-dioxopip- 478 eridin-3-yl)-4-(1-tert.-butoxycarbonylamiino)-isoindoline, I -oxo-2-(1 -ten. 479 buoyabnl26dooieii--l4567ttfurionoie I -oxo-2-( 1- 480 tert.-butoxycarbonyl-2,6-dioxopiperidin-3-yl)- 4,5,6,7-tetrachloroisoindoline, I-oxo-2- 481 (1 -tent. -butoxycarbonyl-2,6-dioxopiperidin-3-yl)- 4,5,6,7-tetramethylisoindolifle, 1,3 482 dioxo-2-( 1-tent.-uoyabnl26dooieidn3y)4mtyionoie 1 ,3-di- 483 oxo-2-( 1-tert.-btxcroy-,-ixpprdn3y)5mtyionoie I -oxo-2- 484 (1-tent. -butoxycarbonyl-2,6-dioxopiperidin-3-yl)-5-methyisoifldoline, 1-oxo-2-(1 -tent. 485 butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4-methylisoindoline, and 1 -oxo-2-(1-tet 486 butoxycarbonyl-2,6-dioxopiperidin-3-yl)-4,5,6,7-tetramethoxyisoildohle, there are 487 respectively obtained by following the procedures of Examples 3, 4, 5, 6, or 7, 1,3- 488 dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4-aminoisoindoine, 1 ,3.-dioxo-2-(2,6- 489 dioxo-3 -fluoropiperidin-3-yI)-5-aminoisoindoine, I ,3-dioxo-2-(2,6-dioxo-3- 490 fluoropiperidin-3-yl)-4,5,6,7-tetrafluoroisoindoline, 1,3 -dioxo-2-(2,6-dioxo-3 -fluoro- 491 piperidin-3-yl)- 4,5,6,7-tetrachloroisoindoline, 1,3 -dioxo-2-(2,6-dioxo-3 -fluoro- 492 piperidin-3 -yl)-4,5,6,7-tetramethylisoindoline, I ,3-dioxo-2-(2,6-di0x-3- 493 fluoropiperidin-3-yi)-4,5,6,7-tetramethoxyisoindoine, I -oxo-2-(2,6-dioxo-3 494 fluoropiperidin-3-yl)-5-aminoisoindoline, 1 -oxo-2-(2,6-dioxo-3-fluoropiperidil-3-yl)- 495 isoindoline, 1 -oxo-2-(2,6-dioxo-3-fluoropiperidin-3-y1)-4-aminoisoindolifle, l-oxo-2- 496 (2,6-dioxo-3-fluoropiperidin-3 -yl)-4,5,6,7-tetrafluoroisoindoline, 1 -oxo-2-(2,6-dioxo- 497 3-fluoropipefidin-3-yl)- 4,5,6,7-tetrachloroisaindoline, l-oxo-2-(2,6-dioxo-3 -18- 0 498 fluoropiperidin 3 4 ,5,6,7-tetramethylisoindoline, 1,3-dioxo-2-(2,6-dioxo-3-fluoro- S499 piperidin 3 yl)-4-ymethylisoindoline, 1,3-dioxo2-(2,6-dioxo- 3 500 methylisoindoline, 1-oxo-2-( 2 6 -dioxo-3-fluoropiperidin- 3 -y1)-5-methylisoindo 1- 501 oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4-methyisoindoline, and 1-oxo-2-(2,6dioxo- 502 3-fluoropiperidin- 3 -yl)- 4 ,5,6,7-tetramethoxyisoindoline.

S503 EXAMPLE 9 N 504 Part A. A solution of L-glutamic acid dimethyl ester (2.6 g, 14.8 mmol), isoamyl 0 505 nitrite (2.13 mL, 15.9 mmol) and acetic acid (0.22 mL) in benzene (150 mL) is heated N 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 L each). Th 508 solvent is removed in vacuo to yield dimethyl 2-diazopentane-1,5-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 OC. The mixture is 513 stirred at aC for 30 minutes The solution is washed with water (20 mL), brine 514 nimL) 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-1,5-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 mrL 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-1,5- 525 dioate (1.3 g, 4.0 mmol), methanol (10 mL) and 4N hydrochloric acid (10 ml) is 526 heated at 80 0 C for one hour. The solvent is removed in vacuo to yield 2-fluoro-2- 527 (1,3-dioxoisoindolin-2-yl)-propane-1,3-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-1,3- 529 remove in vdicarboxylic acuoid anhydride which is xed with ammonia in methanol (35 m 2 M) 530 dicarboxylic acid anhydride which is mixed with ammonia in methanol (35 mL,2M -19-

O

Si31 and stirred at room temperature for 18 hours. The solvent is then removed in vacuo Si32 and the residue is stirred with methylene chloride (50 mL) for 10 minutes. The organic i33 layer is washed with water and brine (40 mL each), and dried over sodium sulfate. The C i34 solvent is removed in vacuo and the residue is heated at reflux with carbonyl diimidazole (0.65 g. 4 mmol) and dimethylaminopyridine (50 mg) in tetrahydrofuran D ;36 (30 mL) for 18 hours. 1,3-Dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)isoindoline is O 37 isolated extraction with ethyl acetate and further purified by chromatography.

N 38 EXAMPLE 0 39 A stirred mixture of L-glutamic acid dimethyl ester (2.0 g, 11.4 mmol) and phthalic anhydride (1.7 g, 11.4 mmol) in acetic acid (30 mL) is heated to refluxed for one hour.

141 The solvent is removed in vacuo to yield dimethyl 2-(1,3-dioxoisoindolin-2-yl)i42 pentane-1,5-dioate which is further purified through chromatography.

i43 To a stirred solution of dimethyl 2-(1,3-dioxoisoindolin-2-yl)-pentane-1,5-dioate ,44 (1.0 g, 3.3 mmol) and tetramethylethylene diamine (0.5 g, 4.3 mmol) in tetrahydrofuran (10 mL) is added 2.5 M n-butyl lithium (1.6 mL, 4 mmol,) at -79 0

C.

i46 After 30 minutes, N-fluorobenzenesulfonimide (1 g, 3.2 mmol) is added to the mixture i47 which then is allowed to reach room temperature. The solvent is removed in vacuo i48 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 sulfate. The solvent is removed in vacuo to yield dimethyl 2-(1,3-dioxoisoindolin-2- 551 yl)-2-fluoropentane-1,5-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 0 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 0 563 mmol, 2 M) at -78 0 C. After 30 minutes, t-butyl acrylate (0.42 g, 3.2 mmol) is added Z564 to the mixture which is allowed to reach room temperature. The solvent is removed in C~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 S 567 sulfate. The solvent is removed in vacuo to give tert-butyl 4-(1 ,3-dioxoisoifldolifl-2- S 568 yl)4-fluoro-4-ethoxycarboflylbutanoate which is further purified by column 569 chromatography.

S 570 A solution of tert-butyl 4-(1,3-dioxoioindolif--l2YI4fluoro- 4 c1 571 ethoxycarboflylbutafloate (1.1I g, 3 mmol) and trifluoroacetic acid (5 mL) in methylene 572 chloride (5 niL) is stirred for 18 hours and then with methylene chloride (50 niL) for 573 10 min. The organic layer is washed with water and brine (30 mfL each), and dried 574 over sodium sulfate. The solvent is removed in vacuo to yield 4-(1,3-dioxoisoifldolin- 575 2 -yl)-4-(ethoxycarbofyl)4fluorobutanoic acid which can be purified by 576 chromatography or used in the next step without further purification.

577 A mixture of 4-(1,3doosid n2y)4(thxcroy)4furbtni acid 578 (0.9 g, 2.8 nimol), carbonyl diimidazole (0.46 g, 2.8 mmol) and 579 dimethylaminopyrflidifle (0.68 g, 5.6 nimol) in tetrahydrofur an (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 mninutes. The organic layer is washed with water 582 and brine (40 niL each) and dried over sodium sulfate. The solvent is removed in 583 vacuo to give 1,-ix--26doo3furpprdn3y~sidln which is 584 further purified by column chromatography.

585 EXAMPLE 12 586 Tablets, each containing 50 mg of 1, 3 -dioxo-2(2,6dioxo3fluoropiperidin 3 -yl)- 587 isoindoline, can be prepared in the following manner: 589 -Constituents (for 1000 tablets) 589 590 1 ,3-dioxo-2-(2,6-dioxo- 4 591 3-fluoropiperidiI1-3-yi)- 592 50.0 g 593 lactose 50.7 g 594 Wheat starch 7.5 g 595 polyethylene glycol 6000 5.0 g 596 5. g -21- 597 magnesium 1.8 g 0 598 demineralized water q.s.

S599 O 500 The solid ingredients are first forced through a sieve of 0.6 mm mesh width. The 501 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 -0 603 is added to a boiling solution of the polyethylene glycol in 100 mL of water. The \O 604 resulting paste is added to the pulverulent substances and the mixture is granulated, if N 605 necessary with the addition of water. The granulate is dried overnight at 35 0 C, forced S606 through a sieve of 1.2 mm mesh width and compressed to form tablets of 0 607 approximately 6 mm diameter which are concave on both sides.

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

622 The other half of the starch is suspended in 40 mL of water and this suspension is 623 added to 100 mL of boiling water. The resulting paste is added to the pulverulent 624 substances and the mixture is granulated, if necessary with the addition of water. The 625 granulate is dried overnight at 35 0 C, forced through a sieve of 1.2 mm mesh width and 626 compressed to form tablets of approximately 6 mm diameter which are concave on 627 both sides.

628 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: 0 -22- 531 Composition (for 1000 tablets) 532 533 1-oxo-2-(2,6-dioxo- 0 534 3-fluoropiperidin-3-yl)- M 535 isoindoline 75.0 g 636 230.0 g 637 lactose 150.0 g S 638 talc 21.0 g S639 glycine 12.5 g 640 stearic 10.0 g 641 1.5 g (C 642 5% gelatin solution q.s.

1 6 4 3 0 644 All the solid ingredients are first forced through a sieve of 0.25 mm mesh width.

N 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 0 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 lactose...... 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 S672 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 a, 676 677 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: 679 Composition (for 1000 capsules) 680 681 1-oxo-2-(2,6-dioxo- 682 3-fluoropiperidin- 3 -yl)- 683 5-aminoisoindoline 100.0 g 684 microcrystalline 30.0 g 685 sodium lauryl sulfate 2.0 g 686 magnesium 8.0 g 687 688 The sodium lauryl sulfate is sieved into the 1-oxo-2-( 2 ,6-dioxo-3-fluoropiperidin-3- 689 yl)-5-aminoisoindoline through a sieve of 0.2 mm mesh width and the two components 690 are intimately mixed for 10 minutes. The microcrystalline cellulose is then added 691 through a sieve of 0.9 mm mesh width and the whole is again intimately mixed for 692 minutes. Finally, the magnesium stearate is added through a sieve of 0.8 mm width 693 and, after mixing for a further 3 minutes, the mixture is introduced in portions of 140 694 mg each into size 0 (elongated) gelatin dry-fill capsules.

695 EXAMPLE 17 696 A 0.2% injection or infusion solution can be prepared, for example, in the following 697 manner: 698 1,3-dioxo-2-(2,6-dioxo- 699 3-fluoropiperidin-3-yl)- 700 5-aminoisoindoline hydrochloride. 5.0 g 701 sodium 22.5 g 702 phosphate buffer pH 300.0 g 703 demineralized to 2500.0 mL 704 705 1, 3 -Dioxo-2-(2 ,6-dioxo-3-fluoropiperidin-3-yl)-5-aminoisoindoline hydrochloride is 706 dissolved in 1000 mL of water and filtered through a microfilter. The buffer solution is -24added and the whole is made up to 2500 mL with water. To prepare dosage unit forms, portions of 1.0 or 2.5 mL each are introduced into glass ampoules (each containing respectively 2.0 or 5.0 mg of imide).

EXAMPLE 18 The efficacy of the 2-(2,6-dioxo-3-fluorpiperidin-3-yl)-isoindoline compound depicted below, as an inhibitor of TNFu production in lipopolysaccharide

(LPS)

stimulated human peripheral blood mononuclear cells (PBMCs), was determined as follows.

C' 712 \O 713 C'714 n 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 PBMCs were isolated from normal donors by Ficoll-Hypaque density centrifugation. Cells were then cultured in RPMI supplemented with 10% serum, 2mM L-glutamine, 10OU/mL penicillin and 100U/mL streptomycin. The compound was then assayed at half log dilutions starting at 50mg/ml. The compound was then applied to the PBMCs (106 cells/mL) in 96 well plates and incubated for one hour. Cells were then treated with Img/mL LPS from Salmonella Minnesota R595 (List Biological Labs, Campbell, CA). The cells were then further incubated at 37 0 C for 18-20 hours.

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

Vs o 734 The decrease in TNFca production as a function of the concentration of 735 compound added, allowed the determination of the ICso of the test compound. In this 0 Z 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 o for this particular fluorine substituted compound was 230nM.

CM 739 Given this result, it is expected that other substituted 2-(2,6-dioxo-3-fluorpiperidin-3- S740 yl)-isoindoline compounds function would also be effective TNFc reducing agents.

Claims (69)

1. A compound selected from an optical isomer of 2-(2,6-dioxo-3fluoropiperidin-3-yl)- isoindoline of the formulas: in which Y is oxygen or H 2 and each of R 1 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 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 compound according to claim 1, wherein R 1 R 2 R 3 and R 4 are hydrogen.

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

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

5. The compound according to claim 1, wherein R 4 is amino, and R 1 R 2 ¢q and R 3 are hydrogen. c n

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

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

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

9. The compound according to claim 1, wherein the compound is 1,3- dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4-aminoisoindoline.

10. The compound according to claim 1, wherein the compound is 1,3- dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-5-aminoisoindoline.

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

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

14. The compound according to claim 1, wherein the compound is 1,3- dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetramethoxyisoindoline. O 28 O O

15. The compound according to claim 1, wherein the compound is 1-oxo-2- Z (2,6-dioxo-3-fluoropiperidin-3-yl)-5-aminoisoindoline.

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

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

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

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

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

22. The A pharmaceutical composition comprising the compound according to any one of claims 1 to 21 and a pharmaceutically acceptable, diluent, excipient, carrier and/or adjuvant.

23. A method of reducing or inhibiting undesirable levels of TNFa in a mammal which comprises administering thereto and effective amount of a compound of any one of claims 1 to 21.

24. A method of reducing or inhibiting undesirable levels of NFK in a mammal which comprises administering thereto and effective amount of a compound of any one of claims 1 to 21.

25. A method of increasing cAMP levels in a mammal which comprises Z administering thereto and effective amount of a compound of any one of en claims 1 to 21. S26. A method of reducing or inhibiting undesirable phosphodiesterase Slevels in a mammal which comprises administering thereto and effective c amount of a compound of any one of claims 1 to 21. S 10 27. A method of treating a condition selected from the group consisting of Adult Respiratory Distress syndrome, chronic pulmonary inflammatory diseases, wasting, viral infections, 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 sclerosis, systemic lupus erythrematosis, ENL in leprosy, radiation damage, and hyperoxic alveolar injury which comprises administering thereto and effective amount of a compound of any one of claims 1 to 21.

28. The method of claim 27, wherein the viral infection is herpes, viral conjunctivitis, HIV, psoriasis and atopic dermatitis.

29. The method of claim 28, wherein the viral infection is caused by feline immunodeficiency virus, HIV, equine infectious anaemia virus, caprine arthritis virus, visna virus and maedi virus. A method of reducing or inhibiting undesirable level of inflammatory cytokines in a mammal, wherein the inflammatory cytokines are at least one member selected from the group consisting of TNFa, IL-1, IL-6 and IL-12, the method comprising administering to the mammal an effective amount of a compound selected from the group consisting of: 0s O S(a) a 2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline of the formula: O RO R2 1' R F H N N 3 C 0in which Y is oxygen or H 2 and each of R 1 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 acid addition salts of said 2-(2,6-dioxo-3-fluoropiperidin-3-yl)- isoindoline isomer which contain a nitrogen atom capable of being protonated.

31. The method of claim 30, wherein the inflammatory cytokine is TNFa.

32. A method of reducing or inhibiting undesirable levels of NFKR in a mammal which comprises administering thereto and effective amount of a compound the method comprising administering to the mammal an effective amount of a compound selected from the group consisting of: a 2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline of the formula: R O R 2 C F H R4 in which Y is oxygen or H 2 and O 31 each of R 1 R 2 R 3 and R 4 independently of the others, is hydrogen, 0 halo, alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, or amino and Z acid addition salts of said 2 -(2,6-dioxo-3-fluoropiperidin-3-yl)- isoindoline isomer which contain a nitrogen atom capable of being protonated.

33. A method of increasing cAMP levels in a mammal which comprises Sadministering thereto and effective amount of the method comprising N administering to the mammal an effective amount of a compound selected 3from the group consisting of: N 10 a 2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline of the formula: r 0 R F H 3R* C 11 0 R Y in which Y is oxygen or H 2 and each of R 1 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 acid addition salts of said 2-(2,6-dioxo-3-fluoropiperidin-3-yl)- isoindoline isomer which contain a nitrogen atom capable of being protonated.

34. A method of reducing or inhibiting undesirable phosphodiesterase levels in a mammal which comprises administering thereto and effective amount of the method comprising administering to the mammal an effective amount of a compound selected from the group consisting of: a 2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline of the formula: s 32 I R I o O R 2 II C H N N R3 C (NO RR 0 in which SY is oxygen or H 2 and each of R 1 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 acid addition salts of said 2-(2,6-dioxo-3-fluoropiperidin-3-yl)- isoindoline isomer which contain a nitrogen atom capable of being protonated.

35. A method of treating a condition selected from the group consisting of Adult Respiratory Distress syndrome, chronic pulmonary inflammatory diseases, wasting, viral infections, 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 sclerosis, systemic lupus erythrematosis, ENL in leprosy, radiation damage, and hyperoxic alveolar injury which comprises administering thereto and effective amount of the method comprising administering to the mammal an effective amount of a compound selected from the group consisting of: a 2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline of the formula: R 1 0 z 3 3 21O (N R O O R2 C F H N N R C SY is oxygen or H 2 and each of R 1 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 acid addition salts of said 2 2 6 -dioxo-3-fluoropiperidin- 3 -yl)- isoindoline isomer which contain a nitrogen atom capable of being protonated.

36. The method according to any one of claims 30 to 35, wherein R 2 R 3 and R 4 are hydrogen.

37. The method according to any one of claims 30 to 35, wherein R 1 R 2 R 3 and R 4 are the same and each is chloro, fluoro, methyl, or methoxy.

38. The method according to any one of claims 30 to 35, wherein R 3 is amino, and R 1 R 2 and R 4 are hydrogen.

39. The method according to any one of claims 30 to 35, wherein R 4 is amino, and R 1 R 2 and R 3 are hydrogen. The method according to any one of claims 30 to 35, wherein R 3 is methyl, and R 1 R 2 and R 4 are hydrogen.

41. The method according to any one of claims 30 to 35, wherein R 4 is methyl, and R 1 R 2 and R 3 are hydrogen. fn 34 S42. The method according to any one of claims 30 to 35, wherein the Scompound is 1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline S43. The method according to any one of claims 30 to 35, wherein the compound is 1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4-aminoisoindoline S44. The method according to any one of claims 30 to 35, wherein the Scompound is 1,3-dioxo-2-( 2 6 -dioxo-3-fluoropiperidin-3-yl)-5-aminoisoindoline S 10 45. The method according to any one of claims 30 to 35, wherein the compound is 1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7- tetrafluoroisoindoline.

46. The method according to any one of claims 30 to 35, wherein the compound is 1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7- tetrachloroisoindoline.

47. The method according to any one of claims 30 to 35, wherein the compound is 1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7- tetramethylisoindoline. 46. The method according to any one of claims 30 to 35, wherein the compound is 1,3-dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7- tetramethoxyisoindoline. 47. The method according to any one of claims 30 to 35, wherein the compound is 1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-5-aminoisoindoline

48. The method according to any one of claims 30 to 35, wherein the compound is 1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline

49. The method according to any one of claims 30 to 35, wherein the compound is 1-oxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4-aminoisoindoline tf~

050. The method according to any one of claims 30 to 35, wherein the Zcompound is 1 oo2 26dioo3f rpip i l 4567 tetrafluoroisoindoline.

51. The method according to any one of claims 30 to 35, wherein the compound is 1 -oxo-2-(2 ,6-d ioxo-3-fl u orop ipe rid i n-3-yl1)-4 ,5,6,7 tetrachloroisoindoline. ci 10 52. The method according to any one of claims 30 to 35, wherein the compound is 1 -oxo-2-( 2 ,6-d ioxo-3-fl uorop ipe rid i n-3-yl)- 4 ,5,6,7 tetramethylisoindoline.

53. The method according to any one of claims 30 to 35, wherein the compound is 1 -oxo-2-(2- i oxo-3-fl u orop ipe rid in-3.yl)- 4 ,5,6,7- tetramethoxyisoindoline.

54. The use of the compound selected from the group consisting of an optical isomer of 2-(2,6doo3lorpprdn3yi-sidln of the formulas R 2 F H 11 0 IA m 36 O R 1 0 0 O R C H ZN N cro a the aci a sd in which Y is oxygen or H2 ierhic c a n 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 reducing or inhibiting undesirable levels of TNFa in a mammal. The use of the compound selected from the group consisting of an optical isomer of 2-(2,6dioxo3fluoropiperidin-3-yl)-isoindoline of the formulas R' O R C F H R4 Y IA 4 Y Vs 37 R' O I R 2 I o z N Cc,3 C R 4 Y C IB 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, reducing or inhibiting undesirable levels of NFKR in a mammal.

56. 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 Rl O o F H II 0 R 4 Y IA or in which Y is oxygen or H 2 and each of R 1 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 for increasing cAMP levels in a mammal.

57. 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 IB in which Y is oxygen or H 2 and each of R 1 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 for reducing or inhibiting undesirable phosphodiesterase levels in a mammal.

58. 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 in which Y is oxygen or H 2 and each of R 1 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 for treating a condition selected from the group consisting of Adult Respiratory Distress syndrome, chronic pulmonary inflammatory diseases, wasting and viral infections. n O N

59. The use of claim 58, wherein the viral infection is herpes, viral conjunctivitis, HIV, psoriasis and atopic dermatitis. The use of claim 58, wherein the viral infection is caused by feline immunodeficiency virus, HIV, equine infectious anaemia virus, caprine arthritis Svirus, visna virus and maedi virus. (N

61. The use of a 2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline of the n formula: c R0 4 Y in which Y is oxygen or H 2 and each of R 1 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 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 reducing or inhibiting undesirable levels of inflammatory cytokines in a mammal.

62. The use of claim 61 wherein the inflammatory cytokines are at least one member selected from the group consisting of TNF-a, IL-1, IL-6 and IL-12.

63. The use of claim 62 wherein the inflammatory cytokine is TNF-a.

64. The use of a 2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline of the formula: in which Y is oxygen or H 2 and each of R 1 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 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 reducing or inhibiting undesirable levels of NFKII in a mammal. The use of a 2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline of the formula: in which Y is oxygen or H 2 and each of R 1 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 42 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 increasing cAMP levels in a mammal.

66. The use of a 2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline of the formula: in which Y is oxygen or H 2 and each of R 1 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 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 reducing or inhibiting undesirable phosphodiesterase levels in a mammal.

67. The use of a 2-(2,6-dioxo-3-fluoropiperidin-3-yl)-isoindoline of the formula: R 1 0 R21 C F H N N 'vII o^ 43 c in which Y is oxygen or H 2 and Z each of R 1 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 acid addition salts of said 2-(2,6-dioxo-3-fluoropiperidin-3-yl)- n isoindoline isomer which contain a nitrogen atom capable of being protonated NO to prepare a medicament for treating a condition selected from the group n consisting of Adult Respiratory Distress syndrome, chronic pulmonary inflammatory diseases, wasting, viral infections, septic shock, sepsis, c 10 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 sclerosis, systemic lupus erythrematosis, ENL in leprosy, radiation damage, and hyperoxic alveolar injury.

68. The use of any one of claims 54 to 67, wherein R 1 R 2 R 3 and R 4 are hydrogen.

69. The use of any one of claims 54 to 67, wherein R 1 R R 3 and R 4 are the same and each is chloro, fluoro, methyl, or methoxy. The use of any one of claims 54 to 67, wherein R 3 is amino, and R 1 R 2 and R 4 are hydrogen.

71. The use of any one of claims 54 to 67, wherein R 4 is amino, and R 1 R 2 and R 3 are hydrogen.

72. The use of any one of claims 54 to 67, wherein R 3 is methyl, and R 1 R 2 and R 4 are hydrogen. 44

73. The use of any one of claims 54 to 67, wherein R 4 is methyl, and R 1 R 2 and R 3 are hydrogen.

74. The use of any one of claims 54 to 67, wherein the compound is 1,3- dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yI)-isoindoline. The use of any one of claims 54 to 67, wherein the compound is 1,3- dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yI)-4-aminoisoindoline.

76. The use of any one of claims 54 to 67, wherein the compound is 1,3- dioxo-2-(2,6-d ioxo-3-fluoropiperid

77. The use of any one of claims 54 to 67, wherein the compound is 1,3- dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yI)-4,5,6,7-tetrafluoroisoindoline.

78. The use of any one of claims 54 to 67, wherein the compound is 1,3- dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetrachloroisoindoline.

79. The use of any one of claims 54 to 67, wherein the compound is 1,3- dioxo-2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4,5,6,7-tetramethylisoindoline. The use of any one of claims 54 to 67, wherein the compound is 1 ,3- d ioxo-2-(2 ,6-d ioxo-3-fluoropiperid in-3-yl)-4,5 ,6 ,7-tetramethoxyisoindoline.

81. The use of any one of claims 54 to 67, wherein the compound is 1-oxo- 2-(2 ,6-dioxo-3-fluoropiperidin-3-yI)-5-aminoisoindoline.

82. The use of any one of claims 54 to 67, wherein the compound is 1-oxo- 2-(2,6-d ioxo-3-fluo rop ipe rid in-3-yl)-iso ind ol ine.

83. The use of any one of claims 54 to 67, wherein the compound is 1-oxo- 2-(2,6-dioxo-3-fluoropiperidin-3-yl)-4-aminoisoindoline.

84. The use of any one of claims 54 to 67, wherein the compound is 1- oxo-2-(2,6-d ioxo-3-fl uorop ipe rid in-3-yi)-4,5,6,7-tetrafl uoro isoi ndol ine. The use of any one of claims 54 to 67, wherein the compound is 1 -oxo- 2-(2,6-dioxo-3-fluoropiperidin-3-yI)-4,5,6,7-tetrachloroisoindoline. INO86. The use of any one of claims 54 to 67, wherein the compound is 1-oxo- 2-(2,6-dioxo-3-fluoropiperidin-3-yI)-4,5,6,7-tetramethylisoindoline.

87. The use of any one of claims 54 to 67, wherein the compound is 1-oxo- 2-(2 ,6-d ioxo-3-fluoropiperid in-3-yl)-4,5,6 ,7-tetramethoxyisoindoline. DATED THIS THIRTIETH DAY OF NOVEMBER 2005 CELGENE CORPORATION BY PIZZEYS PATENT AND TRADE MARK ATTORNEYS