CA2096623A1 - Tnf inhibitors - Google Patents

Abstract

Derivatives of 8-substituted xanthines are described which inhibit the production of TNF and are useful in the treatment of disease states mediated or exacerbated by TNF production.

Description

wO 92t09203 pcr/us91/o8734 f 2096~23 , TNF INHIBITORS
1 0 FI~LD OF lNYE~llON
The present invention relates to compounds which are inhibitors of the in vivo production of Tumor Necrosis Factor ( I NF), a serum protein.
BACKGROllND QF T~ INVEN~ON
Excessive or unregulated TNF production is implicated in mediating or 1 5 exacerbating a number of diseases including rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other a~thritic conditions; sepsis, septic shock, endotoxic shock, gram negative sepsis, toxic shock synd~me, adult respiratory distress syndrome, cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonaly sarcoidosis, bone resorption diseases, reperfusion injury, graft vs. host reaction, allograft Tejections, 2 0 fever and myalgias due to infection, such as influenza, cachexia secondary to infection or malignancy, cachexia, secondary to acquired immune deficiency syndrome (A~S), AIDS, ARC (AIDS relaud complex), keloid fonnation, scar tissue formation, Crohn's disease, ulcerative colitis, or pyresis.
AIDS results from the infection of T Iymphocytes with Human 2 5 Immunodeficiency Virus (HIV). At least three types or strains of ~V have been identified, i.e., HIV-l, HrV-2 and HIV-3. As a consequence of HrV infection, T-cell mediatedirnmunity is impaired and infected individuals manifest severe opportunistic infections andlor unusual neoplasms. HIV entry into the T Iymphocyte requires T lyrnphocyte activation.
C)ther viruses, such as HIV-l, HIV-2 infect T lyrnphocytes after T Cell activation and such 3 0 virus protein exp~ession and/o$ replication is mediated or maintained by such T cell activation. Once an activated T lyrnphocyte is infected with HIV, the T lymphocyte must continue to be maintained in an activated state to perrnit HIV gene expression and/or HIV
replication. MonQkines, specifically TNF, are implicated in activated T cell mediated HIV
protein expression ~ndJor virus replication by playing a role in maintaining T Iymphocyte 3 5 activation. Therefore, interfe~nce with monokine activity such as by inhibition of monoldne production, notably lNF, in an HIV-infected individual aids in limiting the maintenance of T

wo 92/09203 2 0 ~ PCI/US91/0873~

caused by HIV infection. Mo.- ocytes, macrophages, and related cells, such as kupffer and glial cells, have also been implicated in maintenance of the HIV infection. These cells, ~ike T-cells, are targets for viral replication and the level of viral replication is dependent upon t'ne activadon state of the cells. [See Rosenberg ~., The Immunopathogenesis of HIV
Infection, Advances in Immunology, Vol. 57, (1989)]. Monoxines, such as TNF, have 'oeen shown to activate HIV replication in monocytes and/or macrophages [See Poli, et al., Proc. Natl. Acad. Sci., 87:782-784 (l990)], therefore, inhibition of monokine production or activity aids in limiting HIV progression as stated above for T~ells. Additional studies have identified TMF-oc as a common factor in the activation of HIV in vitro and has provided a 1 0 clear mechanis n of action via the nuclear factor ~d?., a nuclear regulatory protein found in the cytoplasm of cells (Osborn, et al., PNAS (86) 2336-2340). This evidence suggests that a reduction of TNF synthesis may have an antivi~ al effect in ~V infections, by reducing the transcTiption and thus virus production.
TNF has also been implicated in various roles with oth~ viral nfections, such 1 5 as the cytomegalia virus (CMV), influenza virus, adenovirus, and the herpes family of viruses for similar reasons as those noted.
The ability to control the adverse affec~s of l'NF is furthered by the use of the compounds which inhibit TNF in mammals who aTe in need of such use. There remains a need fo; compounds which are useful in treating INF mediated disease states which are 2 0 exacer'oated or caused by the excessive and/or unregulated production of TNF.
Summarv of the Invention This invention relates to the use of the compounds of Formula (I) in inhibiting the production of TNF in a mammal, including humans, in need of such treatment, which method comprises administering to such mammal, an effective TNF2 5 inhibiting ams~unt of a compound of Fotmula (I). More specifically the innibition of the production of TNF is useful in the treatment, prophylactically or therapeutically, of any disease state in a rnarnmal which is exacerbated or caused by excessive or umegulated INF
pr~duction.
The compounds of the present invention of Formula (I) are represented by 3 0 the structuTe.
R, `N--I~X N
o r R2 (I) W0 92/09203 ~ 9 9 6 6 2 3 PCI /US91 /OX734 A is an unsubsdtuted or substituted cyclic hydrocarbon radical;
R3 is halogen, nitro, or -NR4Rs;
R4 and Rs are independently bydrogen, alkyl, alkylcarbonyl or together with the nitnogen to which they are attached forming an optionally substituted heterocyclic ring; and 5 the phannaceutically acceptable salts thereof.
DETAILED DESC RIPI ION OF THE INVENTION
1 he compounds of Formula (I) are also useful in ~he treatment of viral in~ections, where such viruses are sensitive to upregulation by TNE or will elicit TNF
production in vivo. The viruses contemplated for treatment herein are those that produce 1 0 TNF as a result of infection, or those which are sensitive to inhibition, such as by decreased replication, directly or indirectly, by the TNF inhibitors of Fonnula (1). Such viruses include, but are not limi~ed to; HIV-l, HIV-2 and HIV-3, Cytomegalovirus(CMV), Influenza, adenovirus and the Herpes group of vimses, such as but not limited to, He~pes Zoster and Herpes Simplex.
1 5 This invention more specifically relates to a method of treating a mammal, afflicted with a human immunodeficiency virus (HIV), which comprises administering to such mammal an effective TNF inhibiting arnount of a compound of Fonmula (I).
I he compounds of Formula (I) may also be used in association with the veterinary treatment of mammals, other than in humans, in need of inhibition of TNF
2 0 production. TNF mediated diseases for treatment, therapeutically or prophylactically, in animals include disease states such as those noted above, but in particular vi}al infections.
Examples of such viruses include, but are not limited to, feline immunodeficiency VilUS
(FIV) or other retroviral infection such as equine infectious anaemia virus, caprine arthritis virus, visna virus, maedi virus and olher lentiviruses.
2 5 A prefered method of this invention is the treatment, therapeutically or prophylactically, of viral infections, in particular where such viruses are sensitive to upregulation by TNF or will elicit TN~ production in vivo by administering an effective amount of a compound of Formula (I) or most preferably, the compound 1,3-di-cyclopropylmethyl-8-amino xanthine o~ a pharmaceutically acceptable salt theseo3 0 The compounds of the present invention of Fo~mula (I) are represented by the structure:
R1 ` N J~_ N
1 />--R3 r WO 92/09203 21~ 9 6 ~ PCI/US91/08734 ( Rl and R2 are each independently alkyl or a moiety of dhe formula - (CH2)m-A;
m is a number from 0 to 3;
A is an unsubsdtuted or substituted cyclic hydrocarbon radical;
R3 is halogen, nitro, or -NR4Rs;
S R4 and Rs are independendy hydrogen, alkyl, alkylcarbonyl or together with the nitrogen to which they are attached forrning an opdonally substituted heterocyclic ring; and the pharmaceudcally acceptable salts thereof.
~referably both Rl and R2 represent -(CH2)m-A. Preferably the A moiety represents a C3-8 cycloalkyl group, pardcularly a C3-6 cycloalkyl and preferably1 0 unsubsdtuted. More preferably A is a cyclopropyl or cyclobutyl moiety. Preferably m is zero or one. Suitable opdonal substituent groups for any cyclic hydrocarbon include a Cl 6alkyl moiety or halogen alom.
A preferred group for Rl or R2 is an aL1cyl group of 1 to 6 carbons, specifically methyl, ethyl, propyl or n-butyl. More preferred is n-butyl.
1 5 When R3 is halogen, the preferred subsdtution is bromine or chlorine.
When R3 is -NR4Rs, and R4 and Rs represent alkyl or alkyl~arbonyl, it is preferred that one of R4 or Rs is hydrogen.
Suitable heterocyclic groups include saturated or unsaturated heterocylic groups having single or fused rings, each ring having 5 to 7 ring atoms which ring atoms 2 0 optionally comprise up to two addidonal hetero atoms selected from O, N, or S.
Preferred heterocyclic groups include single rings comprising 5 to 7 ring atoms, more preferably 5 to 6 ring atoms,and most preferably 6 ring atoms. Preferred heterocyclic groups are pyrrolidinyl, piperidinyl, or morpholinyl rings.
Specifically exemplified compounds of Formula (I) are:
2 5 1,3-di-n-butyl-B-nitro xanthine;
1,3~i-cyclopropylmethyl-8-nitro xanthine;
1,3 di-cyclobutylmethyl-8-nitro xanthine;
1,3 di-cyclopentylmethyl-8-nitro xanthine;
1,3~i~yclohexylmethyl-B-nitro xanthine;
3 0 1,3 di-n-butyl-8-amino xanthine;
1,3 di-cyclop~pylmethyl-8-amino xanthine;
1,3~i-cyclobutylmethyl-8-amino xanthine;
1,3~i-cyclopentylmethyl-8-amino xanthine;
1,3~i-cyclohexylmethyl-8-amino xanthine;
3 5 1,3 di-cyclopropyl-8-amino xanthine;

. . . ~

1,3-di-n-butyl-8-bromo xanthine;
1,3-di-cyclopropylmethyl-8-chloro xanthine;
1,3~i-cyclohexyl-8-chloro xan~hine;
1,3-di-n-butyl-8-piperidino xanthine;
1,3-di-cyclopropylmethyl-8-morpholino xanthine;
1,3-di-n-butyl-8-pyrrolidinyl xanthine;
1,3-di-cyclop~,~pylmethyl-8-pyrrolidinyl xanthine;
1,3~i-cyclopropylmethyl-8-piperidinyl xanthine;
1,3-di-cyclohexylmethyl-8-piperidinyl xanthine;
1 0 1,3-di-cyclohexylrnethyl-8-bromo xanthine; and 1,3-di-cyclohexyl-8-nitro xanthine; or the pharmaceutically acceptable salts thereof.
The most preferred compound of Fonnula (I) for use in the methods of this invention is 1,3-di-cyclopropylmethyl-8-amino xanthine or a phannaceutically acceptable salt thereof.
1 5 By the tenn "alkyl" groups as used herein, alone or when used as part of another group (for example as in alkylcarbonyl) is meant to include both straight or branched chain radicals of 1 to 12 carbon atoms, unless the chain length is lirnited thereto, including, but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl. ten-butyl, and the like.
2 0 By the term "cyclic hydrocarbon", unless specified otherwise, as used herein is meant a single ring or a fused rings of 3 to 8 carbon atoms. Cyclic hydrocarbons may comprise up to 8 carbons in each ring. The terrn "cycloaLkyl" or "cycloaL~cyl 31kyl" as used herein is meant to be interchangeable with the term "cyclic hydrocarbon". Cycloalkyl and cycloaLkyl-alkyl groups are meant to include, but not limited to cyclopropyl, 2 5 cyclopropyl-methyl, cyclopentyl or cyclohexyl.
By the term "halo" as used herein is meant all halogens, i.e., chloro, fluoro, bromo and iodo.
By the term "inhibiting the production of IL-1" or "inhibiting the production of INF" is meant 3 0 a) a decrease of excessive in vivo IL-1 or TNF levels, respectively, in a mammal, specifically humans, to normal levels or below nonnal levels by inhibition of the in vivo release of IL-l by all cells, including but not limited to monocytes or macrophages;
b) a down regulation, at the translational or transcription level, of excessive in vivo IL 1 or TNF levels, respectively, in a mammal, specifically humans, to noq~nal levels or 3 5 below nonnal levels; or WO 92/09203 2 ~ 9 6 6 2 ~ PCI/US91/08734 By the term "TNF mediated disease or disease states" is meant any and all disease states in which TNF plays a role, either by production of TNF itself, or by TNF
causing another cytokine to be released, such as but not limited to IL. 1, or lL-6. A disease state in which IL-l, for instance is a major component, and whose production or action, is S exacerbated or secreted in response to TNF, would therefore be considered a disease state mediated by TNF. As TNF-B (also known as Iymphotoxin) has close structural homology with TNF-a (also known as cachectin) and since each induces similar biologic responses and binds to the same cellular receptor, both TNF-a and TNF-B are inhibited by the compounds of the present invention and thus are herein referred to collectively as ' TNF" unless 1 0 specifically delineated otherwise. Preferably TNF-a is inhibited.
By the term "cytokine" as used herein is meant any secreted polypeptide that affects the functions of cells, and is a molecule which modulates interactions between cells in the immune or inflammatory response. A cytokine includes, but is not lirnited to monokines and Iymphokines regardless of which cells produce them. For instance, a monokine is 1 5 generally referred to as being produced and secreted by a mononuclear cell, such as a macrophage and/or monocyte but many other cells produce monokines, such as na~ al killer cells, fibroblasts, basophils, neutraphils, endothelial cells, brain astrocytes, bone marrow stromal cells, epideral keratinocytes, and ~ Iymphocytes. Lymphokines are generally referred to as being produced by Iymphoctye cells. Examples of cytokines for the present 2 0 invention include, but are not lirnited to, Interleukin-1 (IL-1), Interleukin-6 (IL-6), Interleukin-8 (IL,8), Tumor Necrosis Factor-alpha (TNF-) and Tumor Necrosis Factor beta (TNF-B).
The inhibition of a cytokine, contemplated by the present invention, for use in the treatment of a HIV-infected human, must be a cytokine which is implica~ed in (a) the 2 5 initiation andlor maintenance of T cell activation and/or activated T cell-mediated HIV gene expression and/or replication, and/or (b) any cytokine-mediated disease associated problem such as cachexia or muscle degeneration. The cytokine specifically desiIed to be inhibited is TNF a.

3 0 MEI~ODS OF PREPARATION
The preparation of the compounds of Formula (I) can be carried out by one of skill in the art according to the procedures outlined herein .
A process for the preparation of a compound of formula (I), which process comprises reacting a compound of formula (II):

WO 92/09203 '~ ~ 0`~ 3 PCr/US91/08734 o Rta` J~H

o~NlN
R2a wherein Rla represen~s Rl, as defined in ~elation to formula (I), or a group convertible to Rl and R2a represents R2, as defined in relation to formula (1), or a group convertible thereto, w~th a Teagent capable of substituting the C-8 hydrogen of the compound of 5 ~ormula (II) with a group R3a wherein R3a represents R3, as defined above in relation to formula (I), or a group convertible thereto; and thereafter, if required carrying out one or more of the following optional steps:

(i) converting any group Rla to Rl and/or R2a IO R2;
1 0 (ii) converting a compound of formula (I) into a further cornpound of formula a);
(iii) converting a compound of formula a) into a phannaceutically acceptable salt.
Suitable reagents for substituting the C-8 hydrogen of the compound of 1 5 formula (II) with a group R3a are well known, conventional, reagents. The conditions of reaction for the substitution of the C-8 hydrogen of the compound of formula (II) will of course depend upon the particular reagent chosen, and in general the conditions used will be those which are conventional for the reagent used. One particularly suitable reagent is a nitrating agent.
2 0 In one convenient form of the above mentioned process the compound of formula (Il) is reacted with a suitable nitrating agent to provide a compound of fonnula (I) wherein R3 represents a nitro group and then converting the nitro group into a halogen atom or a group of the above defined formula -NR4RS.
A compound of formula aI) may be prepared by the dehydrating cyclizadon 2 5 of a compound of formula (m):
O
NJ~ 1 r R2a (m) wherein Rla represents Rl, as defined in reladon to formula (I), or a group convemble to Rl and R~a represents R2, as defined in reladon to forrnula (I), or a group WO 92/09~03 2 0 9 G 6 '' 3 ~Cr/US9~/08734 NH2; and thereafter, if required, converdng any group Rla to Rl and/or R2a to R2. The dehydradng cyclization of a compound of forrnula (m) may be calTied out under any suitable conditdons. Favourably the conditions chosen are these wherein the water formed is removed from the reaction mixture, thus the reaction is gendy carried out at an elevated temperature in the range of from 100C to 200C such as in the range of 180C
to 190C.
In one aspect of the process, especially when Al is -NO and A2 is -NHCH3, the reaction is carried out in a solvent immiscible with water, such as toluene, at the reflux temperature of the solvent, the water being removed using a water-separator.
1 0 Suitable values for R1a and R2a include R1 and R2 respecdvely or nitrogen protecting groups such as benzyl groups.
When R1a or R2a represents other than Rl or R2 respectively, the abovementioned conversions of Rla into Rl and R2a to R2 may be carried out using the appro~iate conventional procedure. For example when Rla (or R2a) represents a nitrogen 1 5 protecting group, such as a benzyl group, the protecting group may be removed using the appropriate conventional procedure, such as catalytic hydrogenation, and the resulting product reacted with a compound of formula (IV):
X-(CEI2)m-A (IV) wherein A and m are as defined in relation to formula (I) and X represents a suitable 2 0 leaving group, such as halide, for example bromide or iodide.
The protection of any reactive group or atom, such as the xanthine nitrogen atom may be carried out at any appropriate stage in the afo~ementioned process. Suitable protection groups include those used conventionally in the art for the particular group or atom being protected, for example suitable protecting groups for the xanthine nitrogen 2 5 atoms are benzyl groups. Such protecting groups are known to those sldlled in the art and are readily disclosed in Greene, T., Protective Groups in Organic Svn~h~;~, Wiley Publishers, NY (1981), the contents of which are hereby incorporated by refeIence.
Protec~ng groups may be prepared and removed using the appropriate, conventionalprocedures such as illustrated below:
3 0 For example, N-benzyl protec~ing groups may be prepa~d by treating the appr~riate compound of formula (II) with benzyl chlo¢ide in the presencc of a base such as triethylamine. The N-benzyl protection groups may be lemoved by catalytic hy~genation over a suitable catalyst, such as palladium on activated charcoal, in a suitable solvent, such as ethanol conveniently at an elevated temperature, or by treatment 3 5 with anhydrous aluminium chloride in dry benzene at ambient temperature.

WO 92/09203 2 ~9 S~ 2 ~ PCI/US91/08734 i- ri; . . - .
9!., . ~.
A compound of formula (m) wherein Al represents -NHCHO and R2 represents-NH2 may suitably be prepared from a 6-aminouracil of fonnula (A) according to the following neaction scheme:
R~a~ Jl~ NaNO2/HCOOH R~a~ J~ NO
~N X

R2a R2a (A ) (B ) reduce NO to NH2 with e.g. sodium dithionit~

R1a~ 1 NH- CHO R~a NH
formylate ~N J~X 2 R2a R2a (D ) (C ) 5 wherein Rla and R2a are as defined in relation to fonnula (II).
Suitably, the reaction conditions used in the above mentioned reaction scheme are appropriate conventional conditions. In a preferred aspect of the process, the conversion of the 6-aminouracil ~A), via (B) and (C), to the corresponding compound of formula (m) and the cyclization of the compound of formula (III) to the compound of 1 0 formula (lI) are all carried out i~L suitably by using an analogous procedure to that of H. Bredereck and A. Edenhofer, em. Berichte. 88, 1306-1312 (1955).
The 6-aminouracils of formula (A) may themselves be prepared by the me~hod of V. Papesch and E.F. Schroder, J. e. Che~n., 16, 1879-90 (1951), or Yozo Ohtsuka, Bull. ~hem. Soc. Jap.. 1973, 46(2), 506-9.
1 5 A compound of folmula (m) wherein A1 represents -NO and A2 represents -NHCH3 may conveniently be prepa~ed from a 6-chlo~uracil of formula (E), according to the fiollowing Ieaction scheme:

WO 92/09203 PCr/US91/08734 9~62~ i:

R~a~ NaNO2/HCOOH R a,~

R2a R2a (E ) / (F ) O ,/ Na NO2 R~a~ NO HCI
o r NH2 R2a (B ) wherein Rla and R2a are as defined in relation to formula (II).
Suiilably, the reaction conditions used in the last above mcntioned scheme are the appropriate conventional conditions, for example those used in the method of H.
S Goldner, G. Dietz and E. Carstens, Liebigs Annalen der Chemie, 691, 142-158 (1965).
The ~chlorouracil of fonnula ~D) may also be prepared according to the procedure of Dietz et ~.
When R3 repIesent a ni~o group, suitable conversions of the nitro group into another group R3a include the following:
1 0 (i) converting the nitro group into a halogen atom;
tii) converting the nitro group into an arnine group;
(iii) converting the nitro group into a halogen atom followed by conversion of the halogen atom into a group -NR4R5 wherein R4 and R5 together with the nitrogen atom to which they attached forrn an optionally substituted heterocyclic group; and (iv) converting the nitro group into an amino group and theIeaf~ allyla~ng and/or acylating the arnino group to provide a group -NR4R5 wherein R4 represents hydrogen, alkyl or alkylcarbonyl and R5 represents alkyl or alkylcarbonyl.
A ni~ g~up may be conver~ed into a halogen atom by using any convenient halogenating agen~ One sui~able halogenating agent is a hyd~gen halide, suitably reacted in aqueous ... ~ . . . . . . .. . .. .

2096~23 I"' '~ .~ i "

hydrobromic acid at an elevated temperature, for example in the rangc of from 50 to 150C.
A further suitable halogenating agent is a phosphorous oxyhalide, such as phosphorous oxychloride, which may be reacted in any suitable solvent, such as dimethylf~mamide,suitably at an elevated temperature for exarnple in the range of from 50C to 150C.
A nitro group may conveniently be converted into an amino group by conventional leduction methods for example by using tin powder and concentrated hydrochloric acid at ambient temperature or by using sodium dithionite in aqueous 1 0 methanol at ambient temperature.
When R3 in the compound of formula (I) represents a halogen atom it may be converted into a group -NR4R5 by reacting with a reagent of formula (m):
-HNR4aRSa (m) wherein R4a and R5a are as defined above as R4 and Rs in Fo~mula (I) 1 5 respectively.
The reaction between the compound of formula (I) and the compound of foImula (m) may be canied out in any suitable solvent, such as toluene, at any temperature pr~>viding a convenient rate of for nation of the product, but suitably at an elevated temperature, such as in the range of from 50 to 1 80C, at atmospheric or an elevated 2 0 pressure.
Suitable alkylation methods for use in the above mentioned conversions include those used conventionally in the art, for example methods using halides, preferably iodides, in the presence of a base such as potassium carbonate in any convenient solvent for example acetonitrile or toluene.
2 5 Suitable acylation methods for use in the above mentioned conversions include those used conventionally in the art, thus an amino group may be converted into an aL~cylcarbonyl amino group by using an appropriate acylating agent, for example an amino group may be converted to an acetylamino group by using acetic anhydride at elevated temperature.
3 0 MEI HODS OF TREATMEN~r The compounds of Fonnula (I) or a phannaceulically acceptable salt thereof can also be used in the manufacture of a medicament for the prophylactic or therapeutic treatment of any disease statc in a human, or other mammal, which is exacerbate,d or caused by excessive or or unregulated TNF production by such human's cell, such as but not limited 3 5 to monocytes and/or macrophages, especially caused by excessive or unregulated TNF

Wo s2/ns203 PCr/US9l/08734 subnormal levels, so as to ameliorate or prevent the disease state. Abnormal levels of TNF, for the present invention, constitute levels of 1) free (not cell bound) TNF, greater than or equal to 1 picogram per ml; 2) any cell associated TNF; or 3) the presence of TNF mRNA
above basal levels in cells or tissues in which TNF is produced.
There are several disease states in which excessive or unregulated TNF
production by monocytes and/or macrophages is implicated in e~cacerbating and/a~ causing the disease. These include endotoxemia andlor toxic shock syndrome [See Tracey et al., Nature 330:662-664 (1987); and Hinshaw et al., Cirç ShQck 30:279-292 (1990)~; cachexia [See, Dezube et al., Lançet,335 (8690):662 (1990)]; Adult Respiratory Distress Syn~me 1 0 where TNF concentration in excess of 12,000 pg/ml have been detected h pulmonary aspirates from ARDS pa~ents. [See, Millar et al., ~a~ 2(8665):712-714 (1989).
Systemic infusion of recombinant TNF resulted in changes typically seen in ARDS tSee, Ferrai-Baliviera et al., Arch. Sur~. 124(12):1400-1405 (1989)]; AIDS viral replication of latent HIV in T-cell and macrophage lines can be induced by TNF [See, FoLl~s et al., 1 5 PNAS 86:2365-2368 (1989)]. A molecular mechanism for the ViTUS inducing activity is suggested by INFs ability to activate a gene regulatory protein (NF-kB) found in the cytoplasm of cells, which promotes HIV replication through binding to a viral regulatory gene sequence (LTR) [See, Osborn et al., PNAS 86:233~2340 (1989)]. TNF in AIDS
associated cachexia is suggested by elevated serum TNF and high levels of spontaneous TNFproducdon in peripheral blood monocytes from patients [See, Wright et al-, L
nmunol. 141(1):99-104 (1988)]. TNF in Bone Resorption Diseases, including arthritis, wherein it has been determined that when activated, leukocytes will p~duce a bone-~easorbing activity, and data suggests that TNF-a and TNF-B both contribute to this activity. [See e.g., Bertolini et al., Natur_ 319:51~518 (1986) and Johnson et al., 2 5 Endoc~inology 124(3):1424-1427(1989)]. It has been detennined that lNF s~imulates bone resorption and inhibits bone formation in vitro and in vivo thrwgh stunulation of osteoclast formation and activation combined with inhibition of osteoblast function.
Although TNF may be involved in many bone resorption diseases, including arthritis, the most compelling link with disease i~ the association between p~duction of TNF by tumor 3 0 or host tissues and malignancy associated hypercalcemia [See, Calci. rlssue I~USl 46(Suppl.):S3-10 (1990)]. In Graft versus Host Reaction, increased serum TNF levels have been associated with major complication following acute allogenic bone marrow transplants [See, Holler et al., ~,75(4):1011-1016(1990)]; ce~ebTal malalia, which is a lethal hyperacute neurological syndrome associated vith high blood levels of lNF and is 3 5 the most severe complication occuring in malaria patients. A fonn of expenrnental cerebral wO 92/09203 ; ~ PCr/US91/08734 (1989)]. Levels of serum TNF correlated directly with the seventy of disease andprognosis in patients with acute malaria attacks [See Grau et al., N. En~l. J. Med.
320(24): 158~1591 (1989)]. Another disease state in which TNF plays a role is the area of chronic Pulrnonary Inflammatory Diseases. The deposition of silica particles leads to 5 silicosis, a disease of progressive respiratory failure caused by a fibrotic reaction. Antibody to TNF completely blocked the silica-induced lung fibrosis in mice [See Piguet et al., Nature, 344:245-247 (1990)]. High levels of TNF production (in the serum and in isolated macrophages) nave been demonstrated in animal models of silica and asbestos induced fibrosis [See Bissonnette et al., Inflammation 13(3):329-339 (1989)]. Alveolar 1 0 macrophages from pulmonary sarcoidosis patients have also been found to sponsaneously release massive quantities of TNF as compared with macrophages from no~nal donors ~See Baughman et al., J. Lab. C!in. Med. 115(1):36-42 (1990)]. TNF is also implicated in another acute disease state such as the inflammatory response which follows reperfusion, called ReperÇusion Injury and is a major cause of tissue damage after loss of blood flow 1 5 [See, Vedder et al., PNAS 87:2643-2646 (1990)]. TNF also alters the properties of endothelial cells and has various pro-coagulant activities, such as producing an increase in tissue factor pro-coagulant activity and suppression of the anticoagulant protein C pathway as well as down-regulating the expression of thrombomodulin [See, Sherry et al., J.Cell Biol. 107:11269- 1277 (1988)]. TNF also has pro-inflammatory activities which together 2 0 with its early production (duting the initial stage of an inflamma~ory event) make it a l~ely mediator of tissue injuty in several important disorders including but not limited to, myocardial infarction, stroke and circulatory shock. Of specific imp~ance rnay be ~
induced expression of adhesion molecules, such as intercellular adhesion molecule (ICAM) or endothelial leukocyte adhesion molecule (ELAM) on endothelial cells [See, Munro et al., 2 5 Am. J. Path. 135(1):121-132 (1989)].
The compounds of Formula (I) may also be used topically in the tleatment or prophylaxis of topical disease states mediated or exacerbated by excessive INF production, respectively, such as viral infections, such as those caused by the herpes viruses, or viral conjunctivitis, etc.
3 0 In short, the treatment of TNF mediated disease includes but is not limited to such diseases as rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty a~ritis and other arthritic conditions; sepsis, septic shock, endotoxic shock, grarn negative sepsis, toxic shock syndrome, adult respiratory distress syndrome, cerebral malaria, chronic pulmonary inflarnmatory disease, silicosis, pulmonary sarcoisosis, bone reso~ption diseases, 3 5 reperfusion injury, graft vs. host reaction, acute graft rejection, allograft rejections, fever and Wo 92/09203 2 0 9 ~ 6~:~ PCr/US91/08734 related complex), keloid formation, scar tissue formation, Crohn's disease, ulcerative colitis, pyresis, and viral infections.
All the compounds of Formula (I) are useful in the method of the subject invention, i.e. methods of inhibiting the production, preferably by macrophages, monocyoes or macrophages and monocytes in a mammal, specifically a human, in need of such treatment. The method of the invention is preferably used to treat, pr~phylacdcally or therapeutically, TNF mediated disease states which are not mediated by the phospho-diesoerase tPDEIV) enzyme. Preferably, the method of this invention is used in a disorder other than one associated with increased numbers of eosinophils, such as proliferadve skin 1 0 disease states, i.e. psoriasis, atopic dermatitds, non-specific dennitdtis, prirnary irritant contact dermatdtds, allergic contact dermitdds, or allergic disorders such æ atopy, udcaria, eczema, rhinitis, serborrheic dermatitis, and mange in domesdc animals, as disclosed in Maschler et al., Great Britain Patent Application No. 8906792.0 filed on March 23, l989, whose entire disclosure is incorporated herein by reference. The compounds of Formula (I) may, 1 5 however, be administered concurrently with another agents useful for the trea~rnent of diseases associated with the inhibition or mediation of PDE IV or associated with increased number of eosinophils, with neuronal degeneration resulting from cerebral ischaemic events, such as surgery or stroke, or with those diseases associated with bronchodilator activity such as reversible airways obstrucdon, or asthma.
2 0 In addition, the present invention attributes many of the biological disease states attributable to interleukin- 1 (lL- l ) activity as being attributable to that of TNF activity as well. A comprehensive listing of L- 1 activi~ies can be found in Dinarello, J. Clinical Immunology, 5 (5), 287-297 tl985). It should be noted that some of these effects have been described by others as indirect effects of IL-l.
2 5 Interleukin-l (IL-l) has been demonstrated to mediate a variety of biological activides thought to be important in immunoregulation and other physiological conditions such as inflammation [See, e.g., Dinarello et al., Rev. ~nfect. Disease, 6, 51 (1984)]. The myriad of known biological activities of ~-l include the activation of T helper oells, induction of fever, stimulation of prostaglandin or collagenase production, neutrophil 3 0 chemotaxis, induction of acute phase proteins and the suppression of plasma iron levels.
These disease states are also considered appropriate disease states of TNF activity and hence compounds of Formula (I) are also useful in their treatrnent as well, and the use of the compounds of Forsnula (I) should not be considered solely limited to the specifically described TNF mediated disease states herein. The compounds of the present invention 3 5 are, therefore, efficacious in the treatment of an IL-l mediated diseæ state as TNP and WO 92/09203 2 ~ 3 Ç 6 2 3 pcr/us91/o8734 ~ . .- . .

IL- 1 act in a synergistic manner. TNF as well mediates the release, in some instances, of the monokine ~-1, therefore a reduction in the levels of TNF may be useful in the treatment of a disease state wherein L-l is a major component.
The present invention also relates therefore, to an effective, TNF production inhibi~ing amount of a compound of Formula (I) or a pha.,l,aceutically acceptable salt thereof, useful in treating, prophylactically or therapeutically, any disease state in a human which is exacerbated or caused by excessive or unregulated IL- l production, i.e. where IL-1 is a major component, by such human's monocytes and/or macrophages.
The method of treatment and monitoring for an HIV-infected human 1 0 manifesting immune dysfunction or cytokine-mediated disease associated problems is taught in Hanna, W0 90/15~34, December 27, 1990. In general, an initial treatment regimen can be copied from that known to be effective in interfering with TNF activity for other TNF
mediated disease states by the compounds of Formula (l). Treated individuals will be regularly checked for T cell numbers and T4~r8 ratios and/or measures of viremia such as 1 5 levels of reverse transcriptase or viral proteins, andJor for progression of monokine-mediated disease associated problems such as cachexia or muscle degeneration. If no effect is seen following the normal t~atment regimen, then the amount of the monokine activity interfering agent administered is increased, e.g., by fifty percent per week.
In an HIV-infected human manifesting monokine-mediated disease 2 0 associated problems such as cachexia1 treatment with an effective amount of a monokine activity interfering agent will initially result in a slowing of the rate of the progression of the disease associated problem, thereby slowing disease progression. It is expected that the progression of the disease associated problem will eventually cease and reverse, thereby enhancing the quality of life of the HIV-infected individual treated in such a manner. The 2 5 compounds of Formula ~[) are useful in the method of treatment for all disease states associated with and HIV infection, such as immune abnormalities, immune dsyfunction AIDS Related Complex (ARC) and what is referred to as acquired immune defiency syndrome (All)S) itself. The compounds of Formula (I) will also be useful in reducing or elimina~ing the inflammation related damage/pathology caused by opportunistic (secondary) 3 0 infection, such as but not limited to Pneumocystic pneumonia, or cytomeglovirus infections.
It will be recognized by one of skill in the art that the actual amount of a monokine activi~y interfenng agent required for therapeutic effect will, of course, vary with the agent chosen, the route of administration desired, the nature and severity of the HIV-3 5 infection and the parncular condition of the HIV-infected human undergoing treatment, and WO 92/09203 PCr/US91/08734 209662~ f`
lG
interfering agent will k~ detennined by the nature and extent of the condidon being treated, the fonn, TDute and site of administration, and the particular padent being trated, and that such optimums can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment, i.e., the number of doses of S the monol~ne, TNF, activity interfenng agent given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment deteTmination tests.
The compounds of Formula (I) may be administered orally (when active by this route), topically, parenterally or by inhalation in conventional dosage fonns prepared 1 0 by combining such agent with standard pharmaceutical carTiers according to conventional procedures in an amount sufficient to produce therapeutic TNF activity interfeTing activity.
The phannaceutical carrier employed can be readily determined by one of skill in the art who will recogniæ that such determination will depend upon various well-known factors such as the nature, quantity and charæter of the particular monokine activity 1 5 interfering agent being employed and the forrn and rDute of administration desiTed. The carrieTs employed may be those described elsewhere herein.
In order to use a compolmd of the Folmula a) or a pharmaceutically acceptable salt theTeof fDr the treatment of humans and other marnmals it is normally formulated in accordance with standard pharmaceutical pTactice as a phaTmaceutical 2 0 . composition.
The pharmaceutical composition of the present invention will comprise an effective, non-toxic amount of a compound of Formula a) and a pharmaceutically acceptable carrier or diluent. The compounds of Formula a) are administered in conventional dosage fo~rns prepaTed by combining a compound of Formula (I) in an2 5 amount sufficient to produce TNF production inhibiting activity, respectively, with standard pharmaceutical carrieTs according to conventional prosedures. These procedures may involve mixing, granulalIing and compressing or dissolving the ingIedients as appropriate to the desi~ed preparation.
The pharmaceutical canier employed may be, for example, either a solid a~
3 0 liquid. Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pect~n, acacia, ma~esium stearate, stearic acid and the like. Exemplary of liquid caTriers are syT up, peanut oil, olive oil, polyethylene glycol, coconut oil, water and the like.
Similarly, the carrier or diluent may include time delay material well known to the art, such as glyceryl monostearate or glyceryl distearate alone or with a wax.
3 5 Compounds of Formula (I) and their phannaceutically acceptable salts can be employed in a wide variety of phalmaceu~cal forms. The preparadon of a phannaceutically acceptable salt will be determined by the nature of the compound itself, wo 92/09203 2 ~ 9 6 6 2 3 PCI/US91/08734 and can be prepared by r,onven~onal techniques readily available to one slcilled in the a Thus, if a solid carrier is used, the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form or in the fo~n of a troche or lozenge. Thc amount of solid carrier will vary widely but preferably will be from about 25 mg to about 1 grarn. When a S liquid carrier is used, the preparation will be in the forrn of a syrup, emulsion, soft gelatin capsule, sterile injectable liquid such as an ampule or nonaqueous liquid suspension.
Where the composition is in the forrn of a capsule, any routine encapsulation is suitable, for example using the aforementioned carriers in a hard gelatin capsule shell. Where the composition is in the folm of a soft gelatin shell capsule any phalmaceutical carrier routinely 1 0 used for preparing dispersions or suspensions may be considered, for example aqueous gums, celluloses, silicates or oils and are incorporated in a soft gelatin capsule shell. A
syrup formulation will gene~ally consist of a suspension or solution of the compound or salt in a liquid carrier for example, ethanol, polyethylene glycol, coconut oil. glycerine or water with a flavouring or colouring agent.
1 5 The amount of a compound of Forrnula (I) required for therapeutic effect on topical administration will, of course, vary with the compound chosen, the nature and severity of the inflammatory condition and the anirnal undergoing treatment, and is ultimately at the discretion of the physician.
The tenn 'parenteral' as used herein includes intravenous, intramuscular, 2 0 subcutaneous intranasal, intrarectal, intravaginal or intraperitoneal adrninistration. The subcutaneous and intrarnuscular forms of parenteral adrninistration are generally preferred.
Appropriate dosage forrns for such adrninistration may be prepared by conventional techniques.
Typical parenteral compositions consist of a solution or suspension of the 2 5 compound or salt in a slerile aqueous or non-aqueous carrier optionally containing a parenterally acceptable oil, for example polyethylene glycol, polyvinylpyrrolidone. Iecithin, arachis oil, or sesame oil. The daily dosage regisnen for inhibition of INF production, via pa~enteral administraticm is suitably about 0.001 mg/Kg to 40 mg/Kg, preferably about 0.01 mg/Kg to 20 mgt~g, of a compound of the forrnula (I) or a pharmaceutically 3 0 acoeptable salt thereof calculated as the free base.
The compounds of Fa~rnula (I) may be administcred orally. The daily dosage regimen for oral administration is suitably about .1 mg/lcg to lOOOmg day. For adrnir~istration the dosage is suitably about .OOlmgJkg to 40mg/lcg~ preferably about 0.01 to 20 mg/Kg of a compound of formula (I) or a pharrnaceutically acceptable salt thereof 3 5 calcula~ed as the free base. The active ing~dient may be ~ninistered from 1 to 6 times a d~y, sufficient to exhibit activity.

WO 92/09203 2 ~ 9 6 6 ~ 3 PCr/US91/08734~

The cornpounds of Formula (I) may also be administeTed by inhalation. By "inhalation" is meant intranasal and oral inhalation administration. AppTnpriate dosage forms for such administration, such as an aerosol fonnulation or a metered dose inhaler, may be prepared by conventional techniques. The daily dosage regimen for inhalation administration is suitably about .OOl mg~cg to 40rng/kg, preferably 0.01 to 20 mg/Kg of a compound of formula (I) or a pharmaceutically acceptable salt thereof calculated as the free base.
Typical compositions for inhalation are in the form of a solution, suspension or emulsion that may be administered as a dry powder or in the forrn of an ae~sol using a 1 0 conventional pT~pellant such as dichlorodifluoromethane or trichlorofluoromethane.
Preferably the composition is in unit dosage fo~n, for example a tablet, capsule or metered aerosol dose, so that the patient may administer to himself a single dose.
The compounds of Formula (I) may also be administered topically. By topical administration is meant non-systemic administration and includes the application of a 1 5 compound of Formula (I) externally to the epidennis, to the buccal cavity and instillation of such a compound into the ear, eye and nose, and where the compound does not significantly enter the blood stream. Thus, the compounds of Forrnula (I) may beadministered topically in the treatment or prophylaxis of inflammatory topical disease states mediated or exacerbated by excessive TNF production, respectively, such as eczema, 2 0 psoriasis or other inflammatory skin conditions such as sunburn; inflammatory eye conditions including conjunctivitis; pyresis, pain and other conditions associated with inflammation, herpes or other topical ViTal infections. The daily dosage regimen for topical administration is suitably about .001 mg/kg to lOOmgJkg, preferably 0.1 to 20 mg/Kg of a compound of formula (I) or a pharmaceutically acceptable salt thereof calculated as the free 2 5 base.
By systemic administration is meant oral, intravenous, intraperitoneal and intramuscular administration.
By topical administration is meant non-systemic administration and includes the application of a compound of Formula (I) extemally to the epidermis, to the buccal 3 0 cavity and instillation of such a compound in~o the ear, eye and nose, and where the compound does not significantly enter the blood stream.
While it is possible for an active ingredient to be administeTed alone æ the raw chemical, it is preferable to present it as a phannaceutical formulation. The active ingredient may comprise, for topical administration, from Q001% to 10% w/w, e.g. from 3 5 l% to 2% by weight of the formulation although it may comp~ise as much as 10% w/w but preferably not in excess of 5% w/w and more pTeferably from 0.l% to 1% wlw of the fonnulation. `
_ "

The topical foqmulations of the present invention comprise an active ingredient together with one or mose acceptable carrier(s) therefor and optionally any other therapeutic ingredient(s). The carner(s) must be 'acceptable' in the sense of being compatible with the other ing~dients of the forsnulation and not deleterious to the Npient thereof.
Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration thsough the skin to the site of inflasnmation such as liniments, lotions, creasns, ointlnents or pastes, and drops suitable for a~ministration to the eye, ear or nose.
Drops acco~ding to the present invention may comprise sterile aqueous or 1 0 oily solutions or suspensions and may be prepared by dissolving the active ingsedient in a suitable aqueous solution of a bacteTicidal and/or fungicidal agent and/or any other suitable preservative, and preferably including a surface active agent. The resulting solution may then be clarified by filtration, transfesred to a suitable containe~ which is then sealed and sterilized by autoclaving or maintaining at 98-100C for half an hour. Altesnatively, the 1 5 solution may be sterilized by filtration and transferred to the container by an asepdc technique. Exasnples of bactesicidal and fungicidal agents suitable for inclusion in the drops ase phenylmescusic nitrate or acetate (0.002%), benzallconium chloside (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for the psepas ation of an oily solution include glycesol, diluted alcohol and propylene glycol.
2 0 Lotions according to the present invention include those suitable for application to the skin or eye. An eye lotion may compsise a stesile aqueous solution optionally containing a bactesicide and may be psepared by methods similar to those for the preparation of drops. Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a 2 5 moisturizer such as glycerol or an oil such as castor oil or arachis oil.
Creams, ointments or pastes according to the present invention are se ni-solid fonnulanons of the active ingredient for external application. They may be made by mixing the active ingredient in finely divided or powdered farm, alone or in solution or suspension in zn aqueous or non-aqueous fluid, with the aid of suitable machinery, with a 3 0 g~easy or non-greasy basis. The basis may comprise hydrocarbons such as ha~, soft or liquid p~affin, glycerol, beeswax, a metallic soap; a mucilage; an oil of na~ral ongin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives, or a fatty acid such as stenc or oleic acid together with an alcohol such as propylene glycol or macrogols. The formula~ion may incospolate any suitable suIface ac~ve agent such as an anionic, cationic or 3 5 non-ionic surfactants such as sorbitan esters or polyoxyethylene derivatives thereof.
Suspending agents such as nanlral gums, oellulose de~ivatives or ino~ganic materials such as silicaceous silicas, and other in~edients such as lanolin, may also be included wO 92/09203 PCI/US91/08734 209~

It will be recognized by one of skill in the art that the fosm and character of the pharrnaceutically acceptable carrier or diluen~ is dictated by the amount of active ingredient, a compound of Formula (I), with which it is to be combined, the route of ad~ninistration and other well-known variables.
S It will be recognized by one of skill in the art that the optimal quantity and spacing of individual dosages of a compound of For nu}a (I) or a phannaceutically acceptable salt thereof will be determined by the nature and extent of the condition being treated, the foq~n, route and site of administration, and the pa~icular patient being t,reated, and that such optimurns can be determined by conventional techniques. It will also be appreciated by one 1 0 of skill in the art that the optimal course of treatrnent, i.e., the number of doses of a compound of Fonnula (I) or a phannaceutically acceptable salt thereof given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment detesmination tests.
FORMULATION EXAMPLES
1 5 Forrnulations foq phannaceutical use incosposating compounds of the present invention can be psepared in various fns and with numerous excipients. Examples of liquid formulations are given below.
1. A solution containing a compound of Fonnula (I) is prepared by dissolving thecompound in water, or other suitable carrier, with or without a preservative, such as 2 0 benzoic acid, to deliver the desired amount of drug per use. The compound is present in an amount from about 1011g to about 30 llg/ per ml of carrier.
2. A solution containing a compound of Formula (I) is prepared by dissolving thecompound in an amount from about 1 to about l0mg per ml of PEG 400 with or without BHA/BHT preservatives. The solution can alternatively be filled into a soft gelatin capsule to 2 5 prspare a solid oral dosage form or used as a syrup.
3. A solid dosage fonn containing a compound of Formula (I), such as l,3~i-cyclopropylmethyl-8-amino xanthine has been prepared by mixing 50mg of the compound with various concentration (mg) of manni~ol, hydroxypropylmethylcellulose, calipharm, S~h lS00, and magnesium sterate (as a lubricant), to fill capsules of an appro~riate size or 3 0 the composition may, if desired, be compressed into tablets. V~ious fonnuladon of thè
i~g~ierlts are presented in Table l, numbered from 1 to 6s.

WO92/09203 2 0 9 6 ~ 2 3 PCI/US91/08734 ~a ~ O O O O O O
3 E. ~ ~ ~ ~ ~ ~

o o .~ ~
E

_ ~j; o ~Q ~;ia -O
X

~ _ _ _ _ E
C

o C~ C

, x E ~ 3 o o o o o. o ~ 6: o o o m ~ * * *

WO 92/09203 PCr/US91~08734 2~g~;~2~ ~' UTlLlTY EXAMPLES
I~xamyle A
Inhibitory Effect of compounds of Fnula (I) on in vitro TNF production by Human Monocytes Section I: Assav set-up The effects of compounds of Formula (I) on the in vitro producuon of TNF by human monocytes was examined using the following protocol.
Human peripheral blood monocytes were isolated and purified from either blood 1 0 bank buffy coats o~ plateletpheresis residues, according to the procedure of Colotta, R et al., J. Immunol., 132(2):936 (1984). The monocytes were plated at a density of 1 X 106 cells/ml medium/well in 24-well multi-dishes. The cells were allowed to adhere for 1 hour after which time the supernatant was aspirated and 1 ml fresh medium (RPMI-1640 (Whitaker Biomedical Products, Whitaker, CA) containing 1% fetal calf serum and 1 5 penicillin and streptomycin at 10 units/ml was added. The cells were incubated for 45 minutes in the presence or absence of test compounds at lnM-lOuM dose ranges (compounds were solubilized in Dimethyl- sulfoxide/Ethanol such that the final solvent concentration in the culture medi~n was 0.5% Dimethyl sulfoxide/0.5% Ethanol).
Bacterial lipapolysacchaIide (E. coli 055:B5 [LPS] from Sigma Chemicals Co.) was then 2 0 added at 100 ng/ml in 10 ml Phosphate Buffe~ed Saline (PBS) and cultures incubated for l~lg hours at 37~C in a 5% C02 incubator. At the end of the incubation peIiod, culture supernatants were removed from the oells, centnfuged at 3000 revolutions per minute (rpm) to remove cell debris and .05 ml of Ihe supernatant assayed for TNF activity using the radioimmunoassay described below.
Secdon ~: Radioimmunoassav pr~cedure for TN~ activitv The assay buffer consisted of O.OlM NaP04, 0.15M NaCl, 0.025M EDTA and 0.1% sodium a~ide ~t pH 7.4. Human reco~nbinant TNF (rhTNl:) obtained using the procedune of Chen et al., Nature, 330:581-583 (1987) was iodinated by a modified3 0 Chl~nine-T method described in Section m below. To samples (50 111 culture sup~natants) or rhTNF standards, a 1/9000 diludon of polyclonal rabbit anti-rhTN~
(Genzyrne, Boston, MA) and 8000 cpm of 125I-TNF was added in a final volume of 400 111 buf~er and incubated ove~ t (18 hours) at 4C. Ncqmal rabbit serum and goat anti-rabbit Ig(i (Calbiochem) were titrated against each other for ma~umum plecipiution of d~e 3 5 anti-~TNF. The appro~ate dilutions of c~riernolmal rabbit serum (1/200), goat anli-rabbi~ IgG (1/4) and 25 Units heparin (Calbiochem) were allowed to precipitate and 200 111 of ~is complex was added per assay tube and incubated overnigh~ at 4C. Tubes wae .

WO 92/09203 2 0 9 6 ~ 23 PCI~US91/08734 centrifuged for 30 minutes at 2000 rpm, supernatants werc carefully aspirated, and radioactivity associated with the pellets measured in a Beckman Gamma 5500 counter. The logit-log linear ~ansfol~nadon curve was used for the calculations. The concentrations of TNF in the sarnples was read from a standard curve of rhTNF that was linear in the 157 to 5 20,000 pg/ml range.

Section ~: Radioiodination of rhTNF
Iodination of rhTNF was performed using a modified chloramine-T method of Frolik et al., J. Biol. Chem.. 259:10995-11000 (1984). Briefly, S mg of rhTNF in S ml of 1 0 20MM Tris ph 7.5, was diluted with 15 ml of 0.5M KPO4 and 10 ml of carricr free 125I(lOOrnCUrnl;ICN). To initiau the reaction, a Sml aliquot of a 100mg/ml (aqueous) chloramine-T solution was added. After 2 minutes at room temperature, an additional 5 ml aliquot was added followed 1.5 minutes later by a final S ml addition of chlorarnine-T. The reaction was stopped 1 minute later by sequential addition of 20 ml of 50mM Sodium 1 5 Metabisulfiu, 100 ml of 120mM Potassium Iodide and 200 ml of 1.2 mg/ml Urea The contents were mixed and the reaction mixture was passed over a pre-paclced Sephadex G-25 column (PD 10 Pharmacia), equilibrated and eluted with Phosphate Buffered Saline pH 7.4 containing 0.25% gelatin. The peak radioactivity containing fractions were pooled and stored at -20~C. Specific activity of 125I-TNF was 8~100 mCi/mg protein. Biological 2 0 activity of iodinated TNF was measured by the L929 cytotoxicity assay of Neale, ML. et al., Eur. J. Can. Clin. Oncol., 25(1):133-137 (1989) and was found to be 80% that of unlabeled INF.
Section IV- Measurement of TNF- ELISA:
Levels of TNF were also measu~ed using a modification of the basic sandwich 2 5 ELISA assay method described in Winston et al., Curr~nt Protocols in Molecular Biolo~.
Page 11.2.1, Ausubel et al., Ed. (1987) John Wiley and Sons, New York, USA The ~ISA ernployed a mmine monoclonal anti-human TNF antibody, described below, as the capture antibody and a polyclonal rabbit anti-human TNF, described below, as the second antibody. For detection, a peroxidase-conjugated goat anti-rabbit antibody (Boe~inger 3 0 Ma~nheim, Indianopolis, Indiana, USA, Catalog # 605222) was added followed by a substrate forpe~oxidase (lmg/ml orthophenylenediamine with 0.1% urea peroxide). TNF
levels in sarnples were calculatcd from a standard curve generated with ~ecombinant human TNP prodblced in E. Coli (obtained from SmithKline Beecham Pharrnaceuticals, King of Prussia, PA, USA).
3 5 Section V: P~duction of anti-human TNF antibodies:
Monoclonal Mtibodies to human TNF were prepaied froqn spleens of BALB/c mice immuniæd with ~ecombinant human TNP using a modification of the method of , wo 9~/09203 PCr/US9t/08734 2~96~3 Kohler and Millstein, Nature 256:495 (1975), the entire disclosuIe of which is hereby incorporated by reference. Polyclonal rabbit anti-human TNF antibodies were prepaled by repeated immunization of New Zeland White (NZW) rabbits with recombinant human TNF
emulsified in complete Freund's adjuvant (DIFCO, IL., USA).
5 Results:
It has bcen detc,--lined that 1,3-di-cyclopropylrnethyl-8-arnino xanthine demonstrated an ICso of about 0.05 IlM in the in-vitro TNF production assay system.

UTILITY EXAMPLE B
1 0 Endotoxin Shock in D-~al-Sensitiæd Mice The p~otocol uscd to test the compound of the method of the subject invention was essentially as has been described in Galanos et al., Proc. Nat'l Acad. Sci USA, 76:593943 (1979) whose disclosure is herein incorporated by reference. Briefly, D-gal (D(+) Galactosidase) sensitizes various strains of mice to the lethal effects of endotoxin. The 1 5 administration of D-gal (30~5QOmglkg) intra-venously (i.v.) sensitizes the mice to doses of lipopolysaccharide (LPS) as low as O.lllg. Briefly, male C57BL/6 mice, obtained from Charles River Laboratories (Stone Ridge, New Yo~, USA) of 6-12 weeks of age wereinj~cted i.v. with 0.1 ~g of LPS from Salmonella tvphosa (Difco Laboratones, Detroit, Michigan, USA) admixed with D(~)-gal (Sigma; 500 mg/kg) in 0.2~0.25 ml pyrogen-free 2 0 saline. Compounds tO be tested were administered at vanous times prior to or following the i.Y. injection of LPS/D-gal. In this model, the control animals usually die 5-6 hr. following the injection of LPS, although on occasion deaths are seen between 24 and 48 hr.Measuwment of TNF Acnvitv Plasma levels of TNF wex measured using a modification of the basic sandwich 2 5 ELISA method described in Winston et al., Cunent Protocols in Molecular Biolo~v. Pg.
11~.1, Ausubel et al., Ed. (1987) John Wiley and Sons, New York, USA. The Elisa employed a harnpster monoclonal and-mouse TNF (Genzyme, Boston, MA, USA ) as thecapture antibody and a polyclonal rabbit anti-murine TNF (Genzyme, Boston, MA, USA ) as the detecting antibody. TN~ levels in mouse samples were calculated from a s~andard 3 0 curve generated with secombinant murine TNF (Genzyme, Boston, MA, USA). TNF
levels detennined by ELISA correlated with levels detected by the L929 bioassay of E~uff et.
al., J. Immunol. ~:1671-1677 (1980), with 1 Unit of activity in the biGassay cQIresponding to 70 picogrEuns (pg) of TNF in the ELISA. The ELISA detected levels of TNF down to 25 pg/ml.
3 5 esults:
It has been dete~mined that 1,3~i cyclopropylmethyl-8-amino xanthine also demonstrated a positive in-vivo response in the above noted utility model, having an EDso 20966~
wo 92/09203 PCr/US91/08734 f~ .
:- 25 forreducdon of serurn TNF of about O.lmg/kg intraperitoneally. The compound demonserates a 100% survival of the animals at this dose.

UllLlTY EXAMPLE C
~IV in vitro monocvte assav The effects of the compounds of Formula (I) on the in vitro inhibition of HIV
producdon of chIonically infected cells is examined using the following protocol.
~a~ion Qf ~V-infected ççll lines.
Clonal HIV-infected cell lines were derived by infecting a culture of the H9 1 0 T cell line with the HTLVII~B strain of human immunodeficiency v~us (HIV~) and cultunng ~e cells for S weeks dunng which a chronically-infected cell line developed Clones were derived ~om this culture by limiting dilution plating in a 1:1 mixture of RPMI
1640 + 15% fetal bovine serurn and H9 cell-conditioned medium. Clones were expanded to approximately 4 x 107 cells, aliquots were frozen and subsequent culh~res were assayed 1 5 for their producdon of HIV with and without stimulation by INF or other recombinant cytokines as described below.
Induction Qf HIV.
HIV induction was assayed by culturing clonal HIV- infected cell lines for four days in the plesence of the material to be tested for inducing acdvity. For meas~ement 2 0 of inhibition of ~V inducdon, cultured human monocytes were stimulated to produoe cytokines by treatment with lipopolysaccharide (LPS) in the p~eænce or abænce of test compounds for 18 hours. At the end of stimulation, supernatant medium from the monocyte cultures was collected, frozen in aliquots at -80 C, and the concentrations of INF, IL1-B, and ~-6 were determined in one aliquot by ELISA. The monocyte 2 5 superna~ants were then diluted into complete RPMI growth medium to provide an optimally inducing concentra~on of lNF in the case of the positive control (LPS s~nulated-) monocyte sample. Optimal induction was achieved at 10 to 100 units of l~F hnl (0.5 tO
5.0 ng/ml), depending upon the indicator cell line. Supernatants ~om expe~imentally-~eated monocyte cultures were diluted by Ihe same factor as that used for the po~itive 3 0 oon~ol in each expc~iment.
Following the four day experiment, culture supernatant ~uid (90 ~1) was removed from the HIV-infected cell line and added to 5% (v/v) Triton-X-100 (10 111; Sigma Chemical Campany) to liberate revcrse transcriptase from HIV particles and inac~vate the virus. Eight cultu~s were evaluated for each treatment in two oq more exp~iments.
3 5 Samples we~e sto~ed at -80 C until assayed forreverse ~anscnptase ac~vity.
B~Q t~scri~t~ assav.

wo 92/09203 PCr/US91/08734 2û96~f~3 ,-~V reverse ~ranscriptase was assayed by a modified version of the microtiter assay of Goff et al. a. Virol. 38:239- 248,1981). Incorporation of 32P-dTTP
into polynucleotide on an oligo- A:poly-dT template:primer was measured by filtering the reaction products with an NA45 membrane filter (Schleicher and Scheull) on a dot-blot apparatus, and either autoradiography or AMBIS quantitation or both. Duplicate reverse transcriptase assays were perfo~ned on all samples.
Statis~cal methods.
Statistical sig~uficance of results was calculated using the COMPARE
functions of the RS/Explore software package.
1 0 RESVI,TS:
Most clonal HIV-infected cell lines express increased levels of HlV in response to TNF in the culture medium. Ten clonal H9 cell lines we~e cultured in quadruplicate for four days with or without 5 ng of rTN~ Iml of RPMI 1640 + 10% fbs. At the end of the experiment, reverse transcriptase in the culture medium was measuIed to detennine the 1 5 level of HIV production by each culture. Eight out of the ten cell lines tested produced increased levels of HIV when cultured in the presence of TNF. A particular clone, cell line 3.7 respondW to TNF in a typical fashion, known to one skilled in the art. The cell line 3.7 was chosen an used to evaluate induction in response to monocyte supernatants.
Clonal HIV-infected cells express increased levels of HIV not only in response 2 0 to TNF but also to supernatant fluid from LPS-stimulated, but not control, cultured human monocytes. Cell line 3.7 was cultured for four days in medium supplemented with either recombinant TNF-a at 5ng/ml, fluid from cultured human monocytes or fluid from cultuIed human monocytes that were stimulated with LPS, and the levels of HIV p~duced were measured four days later by determining reverse transcriptase levels in the supernatant fluid.
2 5 Cell line 3.7 was chosen because it reproducibly induced HlV in response to recombinant TN~. The results here were also obtained with cell lines 4,7,3~U-1 and ACH-2. Comme~ially available cell lines are also useful in this assay.
Supernatants from cultuIed human monocytes s~mulated with LPS in the p~esence of inhibitors of INF synthesis have a reduccd HIV-activating activity compared 3 0 to supernatants from monocytes stimulated with LPS in the absence of inhibitors.
HIV-infected clonal OEll line 3.7 was cultured for four days in medium supplemented with supematantsi from con~ol human monocytes, human monocytes stimulated with LPS, and human monocytes stimulated with LPS in the presence of lOIlM a compound of Formula (I), a 1,3~i-cyclopropylsnethyl-8-amino xanthine.
3 5 This assay demonstrates that the compounds of Fonnula a), as inhibitors of TNP will inhibit HIV induction by LPS-stimulated monocyte <supcrnataots if present WO 92/09~03 2 0 9 `6 6 ~ ~ Pcr/usg1/08734 during LPS- st~mulation. In particular, 1,3-di-cycloprDpylmethyl-8-amino xanthinc exhibited an HIV Inhibition of (%+/-Error)+/- (75-5)* at a concentration of 10 ~* The percent inhibition in parenthesss comes from a representadve experiment. Actual inhibition in any experiment may vary depending upon the monocyte donor, dose response curve of the test HIV- infected T cell line, and dilution of monocyte supernatant.
UTILITY EXAMPLE D
In vivo inhibition ~f influenza virus-induced TNF
The effects of the compounds of Formula (I) on virus induced TNF production in vivo was examined using the following protocol.
Mice:
Age-matched, female specific pathogen free (Balb/c x C57B/6) Fl (CB6Fl) 1 5 mice were purchased from Charles River Laboratories. Mice were 4-10 weeks old at amval. Mice used for LD50 determinations were between 8-14 weeks ol~
Vin~s P~duction:
The type A influenza virus strain A/PR/8/34 (HlNl subtypc) was p~pagated in the allantoic cavity of 10 day old feriilized eggs. After incubating eggs for 48 2 0 hours, they were refrigerated for at least 2- l/2 hours before harvesting allantoic fluid.
Pooled allantoic fluid was centrifuged (2000 rpm, 15 min, 4C) to remove cells, and then divided into aliquots for storage at -70C.
In Vitro Virus rltration:
Virus was quantitated in an in vitro microassay using Madin-Darby canine 2 5 kidney (MDCK) cells to establish the 50% tissue culture infectious dose (TCn~;o). Serial dilutions of virus or lung homogenate (in medium plus 2.5 ug/ml trypsin) was added (in quadru~licate) to round-bottomed microtiter wells containing adherent MDCK cells. After S
days incubation at 37C (6% C02), 50 ul of 0.5% chick sed blood cells wele added per wc~, aT~d agglutination was read after 1 hour at room temperature. The TCID50 dosc was 3 0 calculated using the SAS version 5 program for 50% effective dose (ED50) estimation far a Binary Dose-Response Assay, (SAS/Statuser's Guide, Vol. 2, SAS Instimte, Cary, NC
(1985) and "Applied Catego~ical Data Analysis", Marcal Dekker Inc., Publishers, N.Y., N.Y.).
~ VivQ Yi~u~ Challe~:
3 5 Freshly thawed virus was serially diluted in tenfold steps (10-1-10-8) in sterile PBS with .05-1~ bovine serum alb~nin; dilutions weTe kept on ice until use.
CB~ n~ice were anes~esized by b~ief exposme to methoxyfluorane (metofane; Pinman _ Wo 92/09203 PCr/US91/08734_ 2~9~623 Moore Co.) soaked paper towels and were challenged intranasally with 50 ul virus. A dose equivalent to 2 LD50 was used in these experiments.
CoLlection of Samples from Virus Challenged Mice:
Sera: Mioe were bled from the orbital venous plexus using a heparinized pasteur pipet. Blood fT~m 3-4 mice was pooled and centrifuged at 15K for 15 minutes; plasma was divided into aliquots and frozen at 20C.
Lung Homogenates: Lungs from mice that were infected intranasally 3 days earLierwere removed aseptically and placed in vials (l lung per vial) containing l micron glass beads (Biospec Products, Bartlesville, OK) (about l/4 full) and 1 ml Eagles minimal 1 0 essential medium with penicillin and streptomycin. The lungs were homogenized for 1 minute using a mini-bead beater (Biospec Products); the vials were then centrifuged at 3000 rpm for 15 min at 4C, and the lung supernatants were frozen at 20C.
Bronchoalveolar Washes: Mice were euthanized by cervical dislocation and wet down with alcohol. Spleen was removed tO expose diaphragm. The diaphragm was cut to 1 5 collapse lungs, and rib cage cut away to expose trachea. The trachea was snipped about 3-5 mm above the lungs and 1 ml PBS was injected into the lungs through a blunt-end 19 gauge needle. The fluid was recovered into the synnge (~ 60-80% starting volume), spun at 2000 rpm for 15 min. to remove cells and debris. Aliquots of supernatants were frozen at 20 prior to assay.
2 0 DosinE with a compound of Fonnula (1): A compound of Formula ~I), a I,3-di-cyclopropylmethyl-g-amino xanthine (hereinafterreferred to as compound (1)) was initially dissolved in DMSO/EtOH and brought up to volume with FBS/saline to equal a finalconcentration of 1 mg/ml in 5% DMSO, 5% Etoh, 40% FBS and 50% normal saline.
Injections were administered ip (0.2 ml per mouse) to equal 0.2 mgs per 20 gms mouse (10 2 ~ mg/kg); or other doses as noted below.
TNF Elisa Assav: The TNF Elisa assay is the same as that described in Utility Example B above.
Bationale and Overall obiec~ive: There is a lack of an ~V animal model for testing the astivity of inhibitors of the HIV virus, and further one which could be readily monitored 3 0 in y}yQ. ~itial n:ports in the literature demonstrate that influenza-infected monocytes p~duce TNF and therefore lead to the choice of the influenza model as one which is useful for in vivo monitoring of the compounds of Pormula (I) as inhibitors of TNP.
Mouse influenza model: Bnef Description. Human influenza viruses Teplicate in mouse lungs, but do not cause overt disease. However, pathogenicity for mice can be 3 5 increased by serial passage in mouse lungs. The mouse-adapted viluses cause a lethal pneumonia, and not an upper respirato~y tract infection as in uncomplicated human influenza In murine influenza, virus replication is restricted to the lung, and is wO 92~09203 2 0 9 ~ ~ 2 3 PCI/US9l/08~34 ; ~ 7 accompanied by a massive inflammatory cell infilttate. It is well documented that pulmonary interferon levels rise dunng murine influenza t~Wyde, et. al) and a reccnt report documented non~uan~tative increases (by bioassay) in both IL-l andTNF levels in bronchoalveolar washes of influenza infected mice (Vacheron, et al).
RESULTS:
vivo TNF producdon.
In an ini~al study, blood and bronchoalveolar wash (BAW) samples were obtained from mice infected inttanasally with A/PRJ8/34 virus (2 LD50) at .5, 1, and 6 hours, and on days 1, 3, 5, 7, 9 and 14 post-infection. Analysis of pooled samples (3 mice 1 0 per g~oup) by TNF Elisa assay conf~nned that TNF was produced in the lung, but no TNF
was detected in the blood at any timepoint. In a follow-up experiment, BAW were assayed individually (n=5 mice per group), and lung homogenates were also prepared for TNF
analysis and virus titradons. The results demonstrate that TNF levels were increased on days 2 through 7 post-infection, whereas virus replication was evident by 24 hours.
1 5 Although virus titers began to decline after day 3, TNF levels remained elevate~i The results suggest that vIrus replication triggered events (ie-inflasnmatory cell infiltrate) leading to local TNF production, and that pulrnonary TNF levels were sustained as the virus was being cleared from the lung.
Effect of Comwlmd tl) on Influenza induced TNF P~duc~ion.
2 0 . A treatment p~tocol was designed based on the kinetics of TNF production seen in the lung. Treatment was initiated on day 1, which preceded any detectable rise in lung TNF. Mice were administered compound (1) at 10 mg/kg ip daily, and BAW
performed 2 hours after the last injection. Samples were taken on days 2 or 3 post-infection. TNF levels in BAW were significantly reduced on both days 2 and 3 in 2 5 comparison to untreated controls also, widh a maximal reduction of 67% (n=3 of 3 experirnents). INF levels were significandy reduced in lung homogenates on day 3 only (n=1 of 1 experiments) (not shown). A dose titration of compound (1) demonstrated that dle compound was active (~ 50% reduction of BAW TNE;) at 1 mg~g, and no effect was seen at 0.1 mg/kg.
CONCLU~IONS:
These studies demonstrate that therapeutic administration of the compounds of Formula (I) can reduce virus-induced TNF production in vivo. The data also suggest dhat ~he compound will be effective at reducing TNF levels in tissue as well as in dhe circulation.
ffect of Com~ound (1) on Influenza Virus Ti~ers in ~Mousç Lun~, wo 92/09203 PCr/US9l/0873~
~2~9~2~ ~i Virus titers were significantly reduced on days 2 through 5 in mice treated with1-10 mg~g compound (1) on days 1,2 and 3 post infection .
Conclusion: These data provide evidence that treatment with the compounds of Fonnula (I) reduces lung virus titers, and thus may be directly beneficial in human influenza S infection.
;

Effect of compound (1~ on Su~vival in a Lethal Challenge Model of Murine Influenza.
In mice given a lethal intranasal challenge dose of A/PR~/34 influenza virus, ther~ was a significant improvement in survival in mice tleated with 10 mg/kg compound ~1) 1 0 daily, but survival in groups of mice treated with lower doses, or treated with 0.1 -10 mg/kg during days 1, 2, and 3 post infection, was not significantly different than vehicle treated controls.
Conclusion: Treatment of influenza challenged mice with compound (1) at doses shown to reduce lung TNF levels resulted in a moderate improvement in survival 1 5 which was sigr~ificant when mice were dosed daily with 10 mg/kg. Thus, the reduction of TNF was not detrimental in this infection. Although the effects on survival were not dramatic, it is possible that more significant benefit might be demonstrated by measuring earlier endpoints such as clinical symptoms, but this is not readily done in the mouse model, where the infection is confined to the lower respiratory tract. Treatment with the 2 0 compounds of Fonnula (I) therefore suggest reduced morbidity and/or mortality in hwnan influenza, where the levels of nasal virus shedding generally correlate with clinical symptoms.

The a~ove description fully discloses the invention including preferrçd embodiments 2 5 thereof. Modificat~ons and improvements of the embodiments specifically disclosed herein are within the scope of the following claims. Without further elaboration, it is believed that one sldlled in the are can, using the preceding descIiption, utilize t`he present invention to its fullest extent. Therefore the Examples herein are to be construed as merely illustrative and not a limitation of the scope of the present invention in any way. The embodiments of the 3 0 invention in which an exclusive property or privilege is claimed a~e defined as follows.

Claims

SUBSTITUTE
REMPLACEMENT
SECTION is not Present Cette Section est Absente