JP3204971B2 - Inhibitor - Google Patents

Description

Description: FIELD OF THE INVENTION The present invention relates to the incorporation of serum protein, tumor necrosis factor (TNF).
It relates to compounds that are inhibitors of in vivo production.

BACKGROUND OF THE INVENTION Excessive or unregulated TNF production is associated with rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other arthritic conditions; sepsis, septic shock, endotoxin shock, Gram-negative Bacterial sepsis, toxic shock syndrome, adult respiratory distress syndrome, cerebral malaria, chronic pulmonary inflammatory disease, silicosis, pulmonary sarcoidosis, bone resorption disease,
Reperfusion injury, graft-versus-host reaction, allograft rejection, fever and muscle pain from infectious diseases such as influenza, cachexia associated with infections or malignancies, evil associated with acute immunodeficiency syndrome (AIDS) It is associated with mediating or exacerbating a number of diseases, including fluid, AIDS, ARC (AIDS-related syndrome), keloid formation, scar tissue formation, Crohn's disease, ulcerative colitis, or pyresis.

AIDS is caused by infection of T lymphocytes by the human immunodeficiency virus (HIV). For HIV, at least 3
Two types or strains: HIV-1, HIV-2 and HI
V-3 has been identified. As a result of HVI infection, the immunity mediated by T cells is reduced and infected individuals develop severe immunocompromised and / or abnormal tumors. In order for HIV to enter T lymphocytes, activation of T lymphocytes is required. Other viruses, such as HIV-1 and HIV-2, infect T lymphocytes following T cell activation, and expression and / or replication of such viral proteins is mediated or maintained by such T cell activation. Is done. Activated T lymphocytes are HI
Upon infection with V, the T lymphocytes must continue to be activated to allow HIV gene expression and / or HIV replication. Monokine, especially TNF
Is involved in activated T cell-mediated HIV protein expression and / or viral replication by playing a role in maintaining T lymphocyte activation. Thus, disruption of monokine activity, such as by inhibiting the production of monokines, particularly TNF, in HIV-infected individuals helps to limit the maintenance of T cell activation, thereby reducing HIV against uninfected cells. Reduce the progress of infectivity,
As a result, it delays or eliminates the progression of immunodeficiency caused by HIV infection. Monocytes, macrophages, and related cells such as Kupffer cells and glial cells have also been implicated in maintaining HIV infection. These cells are
Like T cells, they are targets for viral replication, and the level of viral replication depends on the activation state of the cells [Rosenberg et al., The Immunopathogenesis of HIV Infection, Advances in. The Immunopathogenesis of
HIV Infection, Advances in Immunology), Volume 57,
(1989)]. Monokines such as TNF have been shown to activate HIV replication in monocytes and / or macrophages [Poli et al., Proceedings of National Academy of Sciences (Proc. Natl. Acad. .Sci.), 87: 782-784 (1990).
Thus, inhibition of monokine production or activity helps limit HIV progression, as described above for T cells. Further research is needed on HIV in vitro
Identified α-THF-α as a common factor in the activation of E. coli and clarified the mechanism of action of nuclear factor κB, a nuclear regulatory protein found in the cytoplasm of cells [Osborn et al., PNAS (86) 2336]. −2340]. This evidence suggests that reduced TNF synthesis has an antiviral effect in HIV infection by reducing transcription and consequently viral production.

TNF has been implicated in various roles with respect to other viral infections, such as cytomegalovirus (CMV), influenza virus, adenovirus, and herpes virus, for reasons similar to those described herein.

The ability to control the adverse effects of TNF is facilitated by the use of compounds that inhibit TNF in mammals in need of such use. Excessive and / or uncontrolled TNF
TNF exacerbated or caused by production
There remains a need for compounds that are useful for treating mediated disease states.

SUMMARY OF THE INVENTION The present invention provides a TN effective for mammals, including humans, in need of treatment.
Administering an F-inhibitory amount of a compound of formula (I),
It relates to the use of a compound of formula (I) in inhibiting TNF production in a mammal. In particular, inhibition of TNF production is also useful for prophylactic or therapeutic treatment of any disease state in a mammal that is exacerbated or caused by excessive or unregulated TNF production.

The compound of the present invention of the formula (I) is a compound represented by the following structural formula and a pharmaceutically acceptable salt thereof.

Wherein R ₁ and R ₂ are each independently alkyl or-(CH
Be ₂₎ m _-A; m is a number from 0 to 3; A is an unsubstituted or substituted cyclic hydrocarbon group; R ₃ is halogen, nitro, or a -NR ₄ R _5; R ₄ And R ₅ independently together with hydrogen, alkyl, alkylcarbonyl or the nitrogen to which they are attached form an optionally substituted heterocyclic ring.

DETAILED DESCRIPTION OF THE INVENTION The compounds of formula (I) are also useful for treating viral infections. Here, the virus is susceptible to upregulation by TNF or in vivo.
Induces TNF production at o. Viruses intended for treatment herein are those that produce TNF as a result of infection or that are inhibited by a TNF inhibitor of formula (I), directly or indirectly, by reducing replication. It is sensitive to them. Examples of such viruses include HIV-1, HIV-2 and HIV-3, cytomegalovirus (CMV), influenza, adenovirus, and herpes groups such as, but not limited to, herpes zoster and herpes simplex. But not limited to these viruses.

The invention also relates, in particular, to the effective TNF of the compounds of formula (I).
A method of treating a mammal afflicted with human immunodeficiency virus (HIV), comprising administering an inhibitory amount to such mammal.

The compounds of formula (I) may also be used for veterinary treatment of non-human mammals in need of inhibiting TNF production. TNF-mediated diseases for therapeutic or prophylactic treatment in animals include those disease states as described above, but especially viral infections. Examples of such viruses include feline immunodeficiency virus (FIV) or other retroviral infections such as equine infectious anemia virus, goat arthritis virus, visnavirus, medevirus and other lentiviruses. However, the present invention is not limited to these.

The preferred method of the present invention comprises administering, in particular, an effective amount of a compound of formula (I) or, most preferably, a compound 1,3-di-cyclopropylmethyl-8-aminoxanthine or a pharmaceutically acceptable salt thereof. By the virus
Sensitive to upregulation by TNF or in vivo
Therapeutic or prophylactic treatment of viral infections that induces TNF production in E. coli.

The compounds of the present invention of formula (I) have the structural formula: Wherein R ₁ and R ₂ are each independently alkyl or
(CH ₂ ) _m- A group; m is a number from 0 to 3; A is an unsubstituted or substituted cyclic hydrocarbon group; R ₃ is halogen, nitro or —NR ₄ R ₅ ; R ₄ and R ₅ are Independently form hydrogen, alkyl, alkylcarbonyl or the nitrogen to which they are bonded to form an optionally substituted heterocyclic ring] and pharmaceutically acceptable compounds thereof. Salt.

Preferably, R ₁ and R ₂ together represents - (CH _{2) m} -A. Preferably, the group A is a C _3-8 cycloalkyl group, especially
_{Represents 3-6} cycloalkyl, preferably unsubstituted.
More preferably, A is a cyclopropyl or cyclobutyl group. Preferably, m is 0 or 1. Suitable optional substituents for any cyclic hydrocarbon include a C _1-6 alkyl group or a halogen atom.

R ₁ or preferred groups are C 1 -C 6 alkyl group regarding R _2, in particular methyl, ethyl, propyl or n-
Butyl. More preferably, it is n-butyl.

When R ₃ is halogen, a preferred substituent is bromine or chlorine.

R ₃ is -NR ₄ R _5, when R ₄ and R ₅ represent alkyl or alkylcarbonyl, preferably one of R ₄ or R ₅ is hydrogen.

Suitable heterocyclic groups include each ring having 5 to 7 ring atoms, which ring atoms may optionally comprise up to two further heteroatoms selected from O, N or S. Examples include a saturated or unsaturated heterocyclic group having a single ring or a condensed ring.

Preferred heterocyclic groups include monocyclic rings consisting of 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.

Particularly exemplified compounds of formula (I) are: 1,3-di-n-butyl-8-nitroxanthine; 1,3-di-cyclopropylmethyl-8-nitroxanthine; 1,3-di-cyclobutyl Methyl-8-nitroxanthine; 1,3-di-cyclopentylmethyl-8-nitroxanthine; 1,3-di-cyclohexylmethyl-8-nitroxanthine; 1,3-di-n-butyl-8-aminoxanthine; 1,3-di-cyclopropylmethyl-8-aminoxanthine; 1,3-di-cyclobutylmethyl-8-aminoxanthine; 1,3-di-cyclopentylmethyl-8-aminoxanthine; 1,3-di- Cyclohexylmethyl-8-aminoxanthine; 1,3-di-cyclopropyl-8-aminoxanthine; 1,3-di-n-butyl-8-acetamidoxanthine; 1,3-di-n-butyl-8-chloroxanthine 1,3-di-n-butyl-8-bromoxanthine; 1,3-di-cyclopropylmethyl-8-chloroxanthine; 1,3-di-cyclohexyl-8-chloroxanthine; 1,3-di-cyclopropylmethyl-8-morpholinoxanthin; 1,3-di-n-butyl-8-pyrrolidinyl xanthine; 1,3-di- 1,3-di-cyclopropylmethyl-8-piperidinyl xanthine; 1,3-di-cyclohexylmethyl-8-piperidinyl xanthine; 1,3-di- Cyclohexylmethyl-8-bromoxanthine; and 1,3-di-cyclohexyl-8-nitroxanthine;
Or a pharmaceutically acceptable salt thereof.

Most preferred compounds of formula (I) for use in the method of the present invention are 1,3-di-cyclopropylmethyl-
8-aminoxanthine or a pharmaceutically acceptable salt thereof.

As used herein, the term "alkyl" group, when used alone or as part of another group (e.g., alkylcarbonyl), has 1 to 12 carbon atoms unless the chain length is limited. Containing a linear or branched group having an atom; methyl, ethyl, n-
Propyl, isopropyl, n-butyl, sec-butyl,
Examples include, but are not limited to, isobutyl, tert-butyl, and the like.

As used herein, the term "cyclic hydrocarbon" means a monocyclic or fused ring having from 3 to 8 carbon atoms, unless otherwise specified. Cyclic hydrocarbons may consist of up to 8 carbon atoms in each ring. As used herein, the term “cycloalkyl” or “cycloalkylalkyl” means that the terms “cyclic hydrocarbon” can be interchanged. Cycloalkyl and cycloalkyl-alkyl groups are meant to include, but are not limited to, cyclopropyl, cyclopropyl-methyl, cyclopentyl or cyclohexyl.

As used herein, the term “halo” refers to all halogens, ie, chloro, fluoro, bromo, and iodo.

The terms “inhibit the production of IL-1” or “inhibit the production of TNF” include: a) Inhibition of in vivo release of IL-1 by all cells, including but not limited to monocytes or macrophages. Respectively, in excess of in vivo IL in mammals, especially humans
Reducing -1 or TNF levels to or below normal levels; b) excess in vivo IL-1 or TNF in mammals, especially humans.
Down-regulating the level, at the translation or transcription level, to or below normal levels, respectively; or

The term "TNF-mediated disease or condition" refers to TNF
Is due to the production of TNF itself or to IL-1 or IL-
Any disease state played by TNF that releases other cytokines, such as, but not limited to, 6 is meant. That is, a disease state in which IL-1 is the major component and whose production or action worsens or is secreted in response to TNF is therefore considered a TNF-mediated disease state. Because TNF-β (also known as lymphotoxin) has strict structural homology to TNF-α (also known as cachectin), each also elicits a similar biological response and is associated with the same cellular receptor. Because they bind, TNF-α and TNF-β are both inhibited by the compounds of the present invention, and are thus collectively referred to as “TNF” unless otherwise specified. Preferably, TNF-α is inhibited.

As used herein, the term “cytokine” refers to any secreted polypeptide that is a molecule that affects the function of a cell and regulates the interaction between cells in an immune or inflammatory response. Cytokines include, but are not limited to, monokines and lymphokines, regardless of which cells produce them. That is, monokine generally refers to those produced and secreted by mononuclear cells such as macrophages and / or monocytes, but includes natural killer cells, fibroblasts, basophils, neutrophils, and endothelial cells. ,
Many other cells, such as brain astrocytes, bone marrow stromal cells, epidermal keratinocytes, and β-lymphocytes, produce monokines. Lymphokines usually refer to those produced by lymphocyte cells. Examples of cytokines according to the present invention include interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8
(IL-8), tumor necrosis factor-alpha (TNF-α) and tumor necrosis factor beta (TNF-β).

Inhibition of cytokines intended for use in the treatment of HIV-infected humans according to the present invention may include (a) initiating and / or maintaining T cell activation, and / or activating T cell mediated
Must be a cytokine associated with HIV gene expression and / or replication, and / or (b) any problem associated with cytokine-mediated diseases such as cachexia or muscle degeneration. A cytokine specifically desired to be inhibited is TNF-α.

Preparation Methods The preparation of the compounds of formula (I) can be performed by those skilled in the art according to the methods outlined herein.

The process for the preparation of the compound of formula (I) comprises the steps of formula (II): Wherein, R ₁ ^a is as R ₁ defined in relation to formula (I) or R,
₁ represents a group convertible to a compound represented by R ₂ ^a are as R ₂ or a convertible group, defined in relation to formula (I)], C-8 hydrogen of the compound of formula (II) in the group R ₃ ^a (wherein, R ₃ ^a is as previously defined in relation to formula (I)
R ₃ or is reacted with a reagent that can be displaced by representing) a group convertible; then, if desired, the following optional steps: (i) is group R ₁ ^a to R _1, and / or R converting the ₂ ^a to R _2; pharmaceutically acceptable salts of the (iii) where the compound of formula (I); (ii) reacting a compound of (I) further expression be converted to compounds of (I) Performing one or more of the steps of converting.

Suitable reagents for the C-8 hydrogen substituted with a group ₃ ^a compound of formula (II) is a reagent conventionally well known.
The reaction conditions for the replacement of the C-8 hydrogen in the compounds of formula (II) will, of course, depend on the particular reagent chosen, and in general the conditions employed will be those which are conventional with respect to the reagents used. One particular suitable reagent is a nitrating agent.

In one advantageous form of the method, the compound of formula (II) is reacted with a suitable nitrating agent to give a compound of formula (I) wherein R ₃ represents a nitro group, and then the nitro group is replaced with a halogen atom or converted into a group of formula -NR ₄ R ₅ defined above.

The compound of formula (II) has the formula (III): Wherein, R ₁ ^a is as R ₁ defined in relation to formula (I) or R _1,
Group convertible to, R ₂ ^a is as defined in relation to formula (I)
R ₂ or a group convertible thereto, A ₁ is —NO or —NHCHO, and A ₂ is —NHCH ₃ or NH ₂ ; provided that when A ₁ is —NO, A ₂ is — NHCH ₃ and A ₁ is -N
If it is HCHO, dehydration cyclization of A ₂ is a compound represented by is NH _2; then, optionally, R ₁
^a to R _1, and / or R ₂ ^a may be prepared by converting the R _2. The dehydration cyclization of the compound of formula (III) may be performed under any suitable conditions. Preferably, the conditions selected are those under which water formed is removed from the reaction mixture. Thus, the reaction is generally carried out between 100 ° C and 200 ° C.
C., for example in the range of 180.degree. C. to 190.degree.

In one embodiment of the method, in particular, A ₁ is -NO;
If A ₂ is -NHCH _3, the reaction while using a water separator remove water, water-immiscible solvent, such as toluene, at the reflux temperature of the solvent.

The suitable values regarding R ₁ ^a and R ₂ ^a, respectively, R ₁
And R ₂ , or a nitrogen protecting group such as a benzyl group.

R ₁ ^a and R ₂ ^a are each, may represent something other than R ₁ or R _2, the transformation from R ₁ ^a to R _1, and from R ₂ ^a to R ₂ are using suitable conventional methods You may go. For example, if R ₁ ^a (or R ₂ ^a) represents a nitrogen protecting group such as a benzyl group, the protecting group may be removed using an appropriate conventional methods such as catalytic hydrogenation, to obtain X- (CH ₂ ) _m -A (IV) wherein A and m are the same as defined for formula (I) and X is a suitable leaving group, for example bromide Or a halide such as iodide].

Protection of a reactive group or atom, such as a xanthine nitrogen atom, may occur at any suitable stage in the process.
Suitable protecting groups include those conventionally used in the art for individual groups or atoms to be protected, for example, a suitable protecting group for a xanthine nitrogen atom is a benzyl group. Such protecting groups are known to those skilled in the art and are described in Greene, T., Protective Groups in Organic Synthesis.
ynthesis), Willy Publishers, New York (1981). The contents are cited herein. Protecting groups may be prepared and removed using suitable conventional methods as described below.

For example, an N-benzyl protecting group can be prepared by treating a suitable compound of formula (II) with benzyl chloride in the presence of a base such as triethylamine. The N-benzyl protecting group is conveniently added at elevated temperature, in a suitable solvent such as ethanol, by catalytic hydrogenation over a suitable catalyst such as palladium on activated carbon, or at room temperature in dry benzene in anhydrous aluminum chloride. And can be removed.

Compounds of formula (III) wherein A ₁ represents -NHCHO and R ₂ represents -NH ₂ are preferably compounds of formula (A) according to the following reaction scheme:
Can be prepared from 6-aminouracil: Wherein, R ₁ ^a and R ₂ ^a are as defined in relation to formula (II)].

Preferably, the reaction conditions used in the reaction scheme are:
Appropriate conventional conditions. In a preferred embodiment of the method,
The conversion of 6-aminouracil (A) via (B) and (C) to the corresponding compound of formula (III) and the cyclization of the compound of formula (III) to the compound of formula (II) , H. Bredereck and A. Edenhofer, Chem.
m. Berichte), 88, 1306-1312 (1955), all performed in situ.

6-Aminouracil of formula (A) is itself a buoy.
V. Papesch and EF Schroder, Journal of Organic Chemistry (J. Org. Chem.), 16, 1879-90 (195)
1), or Yozo Ohtsuka, Bulletin of the Chemical Society of Japan (Bull. Chem. Soc. Jap.), 1973, 46 (2), 506-9
It may be manufactured by the method described above.

Compounds of formula (III) wherein A ₁ represents -NO and A ₂ represents -NHCH ₃ may be conveniently prepared from 6-chlorouracil of formula (E) according to the following reaction scheme: Wherein, R ₁ ^a and R ₂ ^a is defined the same relation to formula (II)].

Suitably, the reaction conditions used in the just described scheme include suitable conventional conditions such as, for example, H. Goldner, G. Dietz and E. Karstens (E. Carstens, Liebigs Annalen der Chemi
e), 691, 142-158 (1965). The 6-chlorouracil of formula (D) may be prepared according to the method of Dietz et al.

When R ₃ represents a nitro group, another group R ₃
Suitable conversion to ^a include: (i) a conversion of the nitro group to a halogen atom; conversion of (iii) nitro group into a halogen atom; (ii) conversion of a nitro group to an amino group , then conversion of the halogen atom to group -NR ₄ R ₅ (wherein, R ₄
And R ₅ together with the nitrogen atom to which they are attached form an optionally substituted heterocyclic group); (iv) conversion of the nitro group to an amino group followed by an amino group Is alkylated and / or acylated to form a group -NR ₄ R
₅ wherein R ₄ represents hydrogen, alkyl or alkylcarbonyl, and R ₅ represents alkyl or alkylcarbonyl.

The nitro group can be converted to a halogen atom by using any convenient halogenating agent. One suitable halogenating agent is hydrogen halide, which is preferably reacted at elevated temperature under aqueous conditions, for example by using concentrated hydrochloric or concentrated hydrobromic acid in the range of 50 to 150 ° C.

Further suitable halogenating agents are phosphorous oxyhalides, such as phosphorous oxychloride, and in any suitable solvent such as dimethylformamide, preferably in the range, for example, from 50 ° C to 150 ° C. It can react at such high temperatures.

The nitro group is conveniently prepared by conventional reduction methods.
For example, it can be converted to an amino group by using tin powder and concentrated hydrochloric acid at room temperature or by using sodium dithionite in aqueous methanol at room temperature.

If R ₃ in a compound of formula (I) represents a halogen atom, the formula ^{(III): -HNR 4a R 5a} (III) [ wherein, R ^4a and R ^5a are each, R ₄ and in formula (I) R by reaction with a reagent represented by ₅ is the same as defined in, can be converted based -NR ₄ R _5.

The reaction between the compound of formula (I) and the compound of formula (III) at atmospheric or elevated pressure, at any temperature that produces a convenient rate of product formation, preferably in the range of 50 ° C to 180 ° C. At such high temperatures, it can be carried out in any suitable solvent such as toluene.

Suitable alkylation methods for use in the above transformations include those commonly used in the art, for example, the use of a base such as potassium carbonate in any convenient solvent such as acetonitrile or toluene. A method using a halide, preferably iodide, in the presence is mentioned.

Suitable acylation methods for use in the above transformations include those commonly used in the art, whereby the amino group is converted to an alkylcarbonylamino group by using a suitable acylating agent. Can be converted to For example, an amino group can be converted to an acetylamino group by using acetic anhydride at an elevated temperature.

Method of Treatment The compound of formula (I) or a pharmaceutically acceptable salt thereof may be present in humans or other mammals in excess or non-existence by such human cells, such as, but not limited to, monocytes and / or macrophages. It can also be used in the manufacture of a medicament for the prophylactic or therapeutic treatment of disease states aggravated by, or resulting from, regulated TNF production, especially those resulting from excessive or unregulated TNF production. . TNF to improve or prevent the condition
Administer the compound of formula (I) in an amount sufficient to inhibit TNF production, such that production is reduced to normal levels, or, in some cases, to subnormal levels and controlled. . Abnormal levels of TNF are produced in the present invention by 1) more than or equal to 1 picogram per ml of free (non-cell-bound) TNF; 2) any cell-associated TNF; or 3) TNF. The level of the presence of TNF mRNA above the basal level in a given cell or tissue.

Excessive or unregulated TNF production by monocytes and / or macrophages exacerbates the disease, and / or
Or there are several disease states related to causing. Examples of these include endotoxemia and / or toxic shock syndrome [Treacey
Et al., Nature, 330: 662-664 (1987); and Hinshaw et al., Circ. Schock, 30: 279-.
292 (1990)]; cachexia [Dezube
Et al., Lancet, 335 (8690): 662 (1990).
]; Adult respiratory distress syndrome in which TNF at a concentration of 12,000 pg / ml or more was detected in lung aspirate from ARDS patients [Millar et al., Lancet, 2 (8665): 712-714 (198)
9)]. ARDS recombinant TNF systemic infusion
[Ferrai-Baliviera et al., Archives of Surgery (Arch. Surg.), 124 (12): 1400-14.
05 (1989)]; AIDS virus replication of latent HIV in T-cells and macrophage lines can be induced by TNF [Folks et al., PNAS, 86: 2365-23.
68 (1989)]. The molecular mechanism for virus-induced activity activates a gene regulatory protein (NF-kB) found in the cytoplasm of cells that promotes HIV replication through binding to the viral regulatory gene sequence (LTR)
Suggested by TNF's capabilities [Osborn
Et al., PNAS, 86: 2336-2340 (1989)]. TNF in AIDS-related cachexia is suggested by high serum TNF from patients and high levels of spontaneous TNF production in peripheral blood monocytes [Wright et al., Journal of Immunology (J. Immunol. ), 141 (1): 99-104
(1988)]. TN in bone resorption disease including arthritis
In the case of F, it has been determined that when activated, leukocytes produce bone resorption activity, and data suggest that both TNF-α and TNF-β contribute to this activity [see, eg, Bertolini et al. Nature, 319: 516-
518 (1986) and Johnson et al., Endocrinology, 124 (3): 1442-1427.
(1989)]. TNF in vitro via stimulation of osteoblast formation and activation combined with inhibition of osteoblast function
It has been determined to stimulate bone resorption and inhibit bone formation in tro and in vivo. Although TNF can be involved in many bone resorption diseases, including arthritis, the most compelling link to the disease is the association between TNF production by tumors or host tissues and hypercalcemia associated with malignancy [ Calcified Tissue International (Calci.Tissue In
t.) (US) 46 (Suppl.) S3-10 (1990)]. Elevated serum TNF levels in graft versus host reactions are associated with major complications after acute allogeneic bone marrow transplantation [Horror
ller et al., Blood, 75 (4): 1011-1016.
(See 1990)]; cerebral malaria, a fatal severe neurotic syndrome associated with high blood levels of TNF, is the most severe complication occurring in malaria patients. A form of experimental cerebral malaria (ECM) that mimics certain features of human disease
Was prevented in mice by administering anti-TNF antibody. [See Grau et al., Imm. Review 112: 49-70 (1989)]. Serum TNF levels are directly related to disease severity and prognosis in patients with acute malaria attacks [Grau et al., New England Journal of Medicine (N. Engl. J. Med.); 320 (24): 1586-1
591 (1989)]. Other disease states in which TNF plays a role are areas of chronic pulmonary inflammatory disease. Silica particle deposition induced silicosis and the disease of progressive respiratory failure was caused by a fibrotic reaction. Antibodies to TNF completely blocked silica-induced pulmonary fibrosis in mice [Piget (Pi
guet et al., Nature, 344: 245-247 (199).
0)]. High levels of TNF production (in serum and in isolated macrophages) have been demonstrated in animal models of silica and asbestos-induced fibrosis [Bissonnette et al., Inflammatio.
n), 13 (3): 329-339 (1989)]. Alveolar macrophages from patients with pulmonary sarcoidosis have also been found to spontaneously release higher amounts of TNF than normal, macrophages from donors [Baughman]
Et al., Journal of Laboratory and Clinical Medicine (J.Lab.Clin.Med.), 115 (1): 36.
-42 (1990)]. TNF also follows reperfusion, referred to as reperfusion injury, and is also implicated in other acute disease states, such as the inflammatory response, which is a major cause of tissue damage following loss of blood flow [Vedder et al. PNAS, 87: 2643--2646. (199
0)]. TNF also alters endothelial cell properties, resulting in increased tissue factor pro-coagulant activity and suppression of the anticoagulant protein C pathway, and down-regulation of thrombomodulin expression. Have various precoagulant activities, such as (down-regulating) [Sherry et al., J. Cell Biol., 107: 1 1269.
-1277 (1988)]. TNF, along with its initial production (during the early stages of the inflammatory event), also holds promise for tissue damage in several important diseases, including but not limited to myocardial infarction, stroke and cardiovascular shock Pro-inflammatory ac
tivity). TNF-induced expression of adhesion molecules such as intercellular adhesion molecules (ICAM) or endothelial leukocyte adhesion molecules on endothelial cells (ELAM) is of particular importance [Munro
ro) et al., American Journal of Pathology (Am. J. Path.), 135 (1): 121-132 (1989)].

The compounds of formula (I) each have a viral infection, for example,
Mediated by excessive TNF production, such as caused by herpes virus, or viral conjunctivitis,
Or it may be used topically in the treatment or prevention of a local disease state that is exacerbated.

That is, the treatment of TNF-mediated diseases is not limited, for example, rheumatoid arthritis, rheumatoid spondylitis,
Osteoarthritis, gouty arthritis and other arthritic conditions; sepsis, septic shock, endotoxin shock, Gram-negative sepsis, toxic shock syndrome, adult respiratory distress syndrome, cerebral malaria, chronic lung inflammation, silicosis, pulmonary sarcoidosis Osteoporosis, reperfusion injury, graft-versus-host reaction, acute graft rejection, allograft rejection, fever and related syndromes), keloid formation, scar tissue formation, Crohn's disease, ulcerative colitis, pyresis (Pyresis) and diseases such as viral infections.

All compounds of formula (I) are prepared according to the process of the invention, ie
Useful in methods of inhibiting production by macrophages, monocytes or macrophages and monocytes, preferably in mammals, particularly humans, in need of treatment. Preferably, the method of the invention is used to preventably or therapeutically treat a TNF-mediated disease that is not mediated by a phosphodiesterase (PDE IV) enzyme. Preferably, the method of the present invention provides a disease other than a disease associated with multiple eosinophils,
For example, Mashler filed on March 23, 1989
schler) et al., UK Patent Application No. 8906792.0, the contents of which are incorporated herein by reference, such as reproductive skin disease states, ie, psoriasis, atopic dermatitis, non-specific dermatitis, It is used for primary irritant contact dermatitis, allergic contact dermatitis, or allergic diseases such as atopy, urticaria, eczema, rhinitis, seborrheic dermatitis, and livestock scabies. However, compounds of formula (I) may be used to treat diseases associated with the inhibition or mediation of PDE IV, or diseases associated with numerous eosinophils, cerebral ischemic events such as neuronal degeneration resulting from surgery or stroke. , Or another drug useful in the treatment of other diseases associated with bronchodilator activity, such as reversible airway obstruction or asthma.

Furthermore, in the present invention, many biological disease states
As in the case of production, interleukin-1 (IL-1)
Attributed to activity. A comprehensive list of IL-1 activities can be found in Dinarello [J. Clinical Immun.
ology), 5 (5), 287-297 (1985)]. It should be noted that some of these effects have been described by others as indirect effects of IL-1.

Interleukin-1 (IL-1) has been shown to mediate a variety of biological activities that may be important in other physiological conditions such as immunomodulation and inflammation [eg, Dinalello et al., Reviews of Infectious Diseases (Rev. Infect. Disease), 6,
51 (1984)]. The myriad known biological activities of IL-1 include activation of T helper cells, induction of fever, stimulation of prostaglandin and collagenase production, neutrophil chemotaxis, induction of acute phase proteins and And suppression of plasma iron levels. These disease states are also considered suitable disease states of TNF activity, and therefore the compounds of formula (I) are equally useful in their treatment, and the use of the compounds of formula (I) is particularly useful herein. It should not be considered simply limited to the TNF-mediated disease states described. Thus, the compounds of the present invention are efficacious in IL-1 mediated disease states because TNF and IL-1 act synergistically. TNF also may, in some cases, mediate the release of the monokine IL-1, and thus reducing TNF levels may be useful in treating disease states where IL-1 is a major component.

Therefore, the present invention provides a method for exacerbating or resulting from excessive or unregulated IL-1 production by human monocytes and / or macrophages, ie, IL
An effective TNF production inhibiting amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, useful for the prophylactic or therapeutic treatment of human disease states wherein -1 is a major component.

Methods for treating and monitoring humans infected with HIV exhibiting problems associated with immunodeficiency or cytokine-mediated diseases are taught by Hanna (WO 90/15534, December 27, 1990). In general, early treatment methods can mimic methods known to be effective in inhibiting TNF activity in other TNF-mediated disease states by compounds of formula (1). Treated individuals have T cell counts and
Periodic indicators of viremia, such as the T4 / T8 ratio and / or levels of reverse transcriptase and viral proteins, and / or the progression of problems associated with monokine-mediated diseases such as cachexia and muscle degeneration Checked. If no effect is seen after normal treatment, the dose of the monokine activity inhibitor is increased, for example by 50% per week.

In humans infected with HIV exhibiting problems associated with a monokine-mediated disease such as cachexia, treatment with an effective amount of an inhibitor of monokine activity first results in a slowing of the progression of the disease-related problem, This will result in a delay in disease progression. It is likely that the progression of the disease-related problems will eventually end and reverse, thereby improving the quality of life of HIV-infected individuals treated in this manner. Compounds of formula (I) may be used for immunodeficiency, immunodeficiency AIDS
Associated Syndrome (ARC) and Acute Immunodeficiency Syndrome (AIDS)
It is useful in methods of treating all disease states associated with HIV infection, such as those referred to as themselves. Compounds of formula (I) reduce or eliminate inflammatory-related damage / pathology caused by opportunistic (secondary) infections such as, but not limited to, Pneumocystic pneumonia or cytomegalovirus infection Would be useful to

Of course, the practical amount of the monokine activity inhibitor required for the therapeutic effect depends on the selected drug, desired administration route, HIV-
It will be appreciated by those skilled in the art that various values may be taken depending on the nature and severity of the infection, and the individual condition of the person infected with the HIV being treated, and ultimately at the discretion of the physician. The nature and extent of the condition to be treated, the optimal amount and interval of individual administration of the monokine activity inhibitor being treated;
Those skilled in the art will recognize that the mode of administration, route and site, and the individual patient to be treated, will be determined and that such optimum conditions may be determined by conventional techniques. Also, the optimal route of treatment, ie, the number of doses of the monokine (TNF) activity inhibitor administered per day for a given number of days, can be determined by one of ordinary skill in the art using routine routes of treatment determination testing. Is well understood by those skilled in the art.

Compounds of formula (I) may be orally administered in conventional dosage forms prepared by combining such agents with standard pharmaceutical carriers in amounts sufficient to produce therapeutic TNF activity inhibitory activity according to conventional procedures. It may be administered topically (if this route is active), topically, parenterally, or by inhalation.

The pharmaceutical carrier employed will depend on various well-known factors, such as the nature, amount and characteristics of the particular monokine activity inhibitor used, as well as the mode and route of administration desired. Can be readily determined by those skilled in the art. The carrier used may be that described elsewhere herein.

The compounds of formula (I) or pharmaceutically acceptable salts thereof, for use in treating humans and other mammals,
It is usually formulated as a pharmaceutical composition according to standard pharmaceutical practice.

The pharmaceutical compositions of the invention comprise an effective non-toxic amount of a compound of Formula (I) and a pharmaceutically acceptable carrier or diluent. Compounds of formula (I) may be prepared by conventional administration of a compound of formula (I) in an amount sufficient to produce TNF production inhibitory activity, each in accordance with conventional means, in combination with a standard pharmaceutical carrier. It is administered in the form. These means include mixing the ingredients, as appropriate for the desired preparation,
It can involve granulation and tableting or dissolving.

The pharmaceutical carrier employed can be, for example, either solid or liquid. Examples of solid carriers include lactose,
Examples include clay, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers include syrup, peanut oil, olive oil, polyethylene glycol, coconut oil, water and the like. Similarly, the carrier or diluent may include a time delay material well known in the art, such as glyceryl monostearate or glyceryl distearate alone or in combination with a wax.
elaymaterial).

The compounds of formula (I) and their pharmaceutically acceptable salts can be used in a wide variety of pharmaceutical forms. The preparation of a pharmaceutically acceptable salt is determined by the nature of the compound itself, and can be prepared by conventional techniques readily available to those skilled in the art. 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 form of a troche or lozenge. The amount of solid carrier can vary, but is preferably from about 25 mg.
It is about 1g. When a liquid carrier is used, the preparation will be in the form of a syrup, emulsion, soft gelatin capsule, sterile injectable solution such as an ampule or non-aqueous suspension. When the composition is in the form of a capsule, for example, conventional encapsulation using the above carriers in a hard gelatin capsule shell is suitable. When the composition is in the form of soft gelatin capsules, pharmaceutical carriers commonly used to prepare dispersants and suspensions include, for example, aqueous gums, celluloses, silicates or oils, It is placed in the shell of a soft gelatin capsule. Syrup preparations generally consist of a suspension or solution of the compound or salt in a liquid carrier such as, for example, ethanol, polyethylene glycol, coconut oil, glycerin or water, including the flavoring or coloring agent. .

The amount of the compound of formula (I) required for therapeutic effect in topical administration may, of course, vary with the compound selected, the nature and severity of the inflammatory condition, and the treatment to which the animal will be subjected. Specifically, at the discretion of the physician.

The term "parenteral" as used herein includes:
Intravenous, intramuscular, subcutaneous, intranasal, rectal, vaginal or intraperitoneal administration is included. Generally, parenteral forms for subcutaneous and intramuscular administration are preferred. Appropriate dosage forms for such administration may be prepared by conventional techniques.

A typical parenteral composition may optionally contain a parenterally acceptable oil, such as polyethylene glycol, polyvinylpyrrolidone, lecithin, peanut oil or sesame oil. It consists of a solution or suspension of the compound or salt in a sterile aqueous or non-aqueous carrier. A daily dosage regimen for the inhibition of TNF production by parenteral administration is from about 0.001 mg / kg to about 40 mg of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, calculated as the free base.
/ kg, preferably about 0.01 mg / kg to 20 mg / kg, is suitable.

The compounds of formula (I) may be administered orally. Suitably a daily dosage regimen for oral administration is from about 0.1 mg / kg to 1000 mg days. For administration, dosages will range from about 0.001 mg / kg to 40 mg / kg, preferably about 0.01 mg, of the compound of formula (I) or a pharmaceutically acceptable salt thereof, calculated as the free base.
Suitably の 20 mg / kg. The active ingredient may be administered one to six times a day to provide sufficient activity.

The compounds of formula (I) may be administered by inhalation.
"Inhalation" refers to intranasal and oral inhalation administration. Dosage forms suitable for such administration, such as, for example, aerosol preparations or metered-dose inhalers, may be prepared by conventional techniques. The daily treatment regimen for inhaled administration, calculated as the free base, is about 0.001 mg / kg to 40 mg / kg, preferably 0.01 to 20 mg / kg, of the compound of formula (I) or a pharmaceutically acceptable salt thereof. Appropriate.

A typical composition for inhalation is in the form of a solution, suspension or emulsion, which can be administered as a dry powder, or in the form of an aerosol with a conventional propellant, such as dichlorodifluoromethane or trichlorofluoromethane.

The composition is preferably in unit dosage form, such as a tablet, capsule or metered dose aerosol dosage, and the patient can administer a single dose to himself.

The compounds of formula (I) may be administered topically. By topical administration is meant non-systemic administration, including external application of the compound of formula (I) to the epidermis, oral vestibule, and instillation of such compound into the ears, eyes and nose, The compound does not enter the bloodstream significantly. Therefore, the compounds of formula (I) are inflammatory local disease states mediated or exacerbated by excessive TNF production, for example,
Other inflammatory skin conditions such as eczema, psoriasis, or sunburn; inflammatory eye conditions including conjunctivitis; pyresis, pain and other symptoms associated with inflammation; herpes or other topical virus Infections may be treated or prevented, respectively, by topical administration. A daily dosage regimen for topical administration is suitably from about 0.001 mg / kg to 100 mg / kg, preferably calculated as the free base, of a compound of Formula (I) or a pharmaceutically acceptable salt thereof, 0.1-20mg /
kg.

By systemic administration is meant oral, intravenous, intraperitoneal and intramuscular administration.

By topical administration is meant non-systemic administration, including external application of the compound of formula (I) to the epidermis, oral vestibule, and instillation of such compound into the ears, eyes and nose, The compound does not enter the bloodstream significantly.

While it is possible for the active ingredient to be administered alone as the raw chemical, it is preferable to present it as a pharmaceutical composition. Active ingredient is 0.001% of the formulation when administered topically
~ 10% w / w, for example from 1% to 2% by weight, but it may consist of as much as 10% w / w of the formulation,
Preferably it does not exceed 5% w / w and more preferably consists of 0.1% to 1% w / w.

The topical formulations of the present invention comprise the active ingredient, optionally with one or more acceptable carriers, and optionally, other therapeutic ingredients. The carrier must be "acceptable" in the sense that it can be compatible with the other ingredients of the formulation and is not harmful to its recipients.

Formulations suitable for topical administration include liquid or semi-liquid formulations suitable for penetrating the site of inflammation through the skin, such as liniments, lotions, creams, ointments or pastes, and eye, ear or nasal preparations. Drops suitable for administration are included.

The drops according to the invention may consist of sterile aqueous or oily solutions or suspensions, which contain bactericides and / or fungicides and / or other suitable preservatives, preferably surfactants. It can be prepared by dissolving the active ingredient in a suitable aqueous solution. Next, the obtained solution may be clarified by filtration and transferred to an appropriate container.
The container is then sealed and sterilized by autoclaving or maintaining at 98-100 ° C for half an hour. Alternatively, the solution may be sterilized by filtration and transferred to the container by aseptic technique. Examples of fungicides and fungicides suitable for inclusion in drops include phenylmercuric nitrate or phenylmercuric acetate (0.002
%), Benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.

Lotions according to the present invention include those suitable for application to the skin or eyes. The ophthalmic lotion may be composed of a sterile aqueous solution optionally containing a bactericide, and may be prepared by a method similar to the method of preparing drops. Lotions or liniments for application to the skin may further contain an agent to accelerate drying and cool the skin, such as alcohol or acetone, and / or a humectant such as glycerol or an oil such as castor oil or peanut oil. You may make it contain.

The cream, ointment or paste according to the present invention is a semi-solid preparation for external use containing an active ingredient. They consist of a finely divided or powdered active component, alone or as a solution or suspension in an aqueous or non-aqueous liquid, with or without an oil, using suitable machines. What is necessary is just to manufacture by mixing with an agent. Such bases include hard, soft or liquid paraffin, glycerol, beeswax, metal soaps; demulcents; oils of natural origin such as almond oil, corn oil, peanut oil, castor oil or olive oil; wool fat or derivatives thereof; Or a hydrocarbon such as a fatty acid such as stearic acid or oleic acid in combination with an alcohol such as propylene glycol or macrogol. The formulation may contain a suitable surfactant, for example, an anionic, cationic or nonionic surfactant such as a sorbitan ester or a polyoxyethylene derivative thereof. Also, suspending agents such as natural rubber, cellulose derivatives, or siliceous silicas
Inorganic substances such as caceous silicas) and other components such as lanolin may be included.

It will be appreciated by those skilled in the art that the form and nature of the pharmaceutically acceptable carrier or diluent will depend on the active ingredient with which it is to be combined, the amount of compound of formula (I), the route of administration and other well-known variables. Is what you understand.

The optimal amount and interval for individual administration of a compound of formula (I) or a pharmaceutically acceptable salt thereof will depend on the nature and extent of the condition being treated, the form, route and site of administration, and the individual patient being treated. Is determined by
That such optimal conditions can be determined by conventional techniques,
It is understood by those skilled in the art. The optimal treatment, ie the number of doses of the compound of formula (I) or pharmaceutically acceptable salt thereof administered per day for a given number of days,
One of skill in the art will also recognize that the skilled artisan can be identified using conventional treatment determination assays.

Formulation Examples Formulations for pharmaceutical use incorporating the compounds of the present invention may be prepared in various forms and together with various excipients. Examples of liquid preparations are given below.

1. A solution containing a compound of formula (I) is prepared by dissolving the compound in water or other suitable carrier, with or without a preservative such as benzoic acid,
Transport desired amount of drug per use. The compound is present in an amount from about 10 μg to about 30 μg / ml carrier.

2. Solutions containing compounds of formula (I) are prepared by dissolving an amount of about 1 to about 10 mg of compound per ml of PEG 400, with or without BHA / BHT preservative. Alternatively, the solution can be filled into soft gelatin capsules to prepare a solid oral dosage form or used as a syrup.

3.1 A solid dosage form containing a compound of formula (I), such as 1,3-di-cyclopropylmethyl-8-aminoxatin, comprises 50 mg of the compound and various concentrations (mg) of mannitol,
Prepared by mixing hydroxypropyl methylcellulose, calipharm, starch 1500, and magnesium stearate (as a lubricant) to fill into appropriately sized capsules,
Or, if desired, the composition may be compressed into tablets. Formulations of various components are numbered 1-6 and
It is shown in the table.

Examples of utilities Example A Inhibitory effect of compounds of formula (I) on in vitro TNF production by human monocytes Section I: Assay mechanism Formula (I) on in vitro production of TNF by human monocytes
Was tested using the following protocol.

Colotta, R., et al., Journal of Immunology (J. Immunol.), 132 (2): 936 (198).
According to the method of 4), human peripheral blood monocytes were isolated and purified from blood bank buffy coat or platelet pheresis residue. Monocytes were plated in 24-well multi-dishes at a density of 1 × 10 ⁶ cells / ml of medium per well. After allowing the cells to attach for one hour, the supernatant was aspirated and fresh medium containing 1% fetal calf serum and 10 units / ml penicillin and streptomycin [RPMI-1640, Whitaker Biomedical, Whitaker, Calif.]・ Products (Whitaker
Biomedical Products)] was added. The cells were incubated for 45 minutes in the presence or absence of the test compound at a dose range of 1 nM-10 μM (compounds were dissolved in dimethylsulfoxide / ethanol and the final solvent concentration in the culture medium was 0.5% dimethylsulfoxide / 0.5%). % Ethanol). Then, bacterial lipopolysaccharide (Sigma Chemicals Co.)
E. coli 055: B5 [LPS]) at 100 ng / ml in 10 ml of phosphate buffered saline (PBS)
Cultures were incubated at 37 ° C. in a 5% CO ₂ incubator for 16-18 hours. After this incubation, the culture supernatant was removed from the cells and 3000 revolutions / min (rp
Cell debris was removed by centrifugation at m) and 0.05 ml of the supernatant was assayed for TNF activity using the radioimmunoassay described below.

Section II: Radioimmunoassay methods assay buffer related TNF activity, in pH7.4, 0.01M NaPO _4, 0.15M Na
Consisted of Cl, 0.025M EDTA and 0.1% sodium azide. Chen et al., Nature, 33.
Human recombinant TNF (rhTNF) obtained using the method of 0: 581-583 (1987) was iodinated by the modified chloramine-T method described in Section III below. Sample (50μ culture supernatant)
) Or rhTNF standard, polyclonal rabbit anti-rhTNF
(Genzym, Boston, Mass.)
e)) 1/9000 dilution and ¹²⁵ I-TNF8000 cpm were added in a final volume of 400 μl buffer and incubated overnight at 4 ° C (18 hours). Normal rabbit serum and goat anti-rabbit IgG (Calbiochem)
Were added dropwise to each other for maximum precipitation of anti-rh-TNF. Appropriate dilutions of normal rabbit serum (1/200) of carrier, appropriate dilutions of goat anti-rabbit IgG (1/4) and 25 units of heparin (Calbiochem) are precipitated and 200 μl of this conjugate per assay tube Was added and incubated at 4 ° C. overnight. The tubes were centrifuged at 2000 rpm for 30 minutes, the supernatant was carefully aspirated and the radioactivity on the pellet was measured on a Beckman Gamma 5500 counter. The logit-log linear conversion curve (lo
A git-log linear transformation curve) was used.
The concentration of TNF in the sample was read from a standard curve of rhTNF which was linear in the range of 157-20,000 pg / ml.

Section III: Radioiodination of rhTNF Frolik et al., Journal of Biological Chemistry, 259: 10995.
-11000 (1984) using the modified chloramine-T method.
Iodination of NF was performed. That is, 20MM Tris (pH 7.
5) 5 mg of rhTNF in 5 ml of 0.5 M KPO ₄ 15 ml and no carrier
Diluted with 10 ml of ¹²⁵ I (100 mCi / ml; INC). To start the reaction, 5 ml aliquots of 100 mg / ml (aqueous) chloramine-T solution were added. After 2 minutes at room temperature, another 5 ml aliquot was added and 1.5 minutes later the final chloramine-T
5 ml was added. The reaction was stopped and after 1 minute 50 ml sodium metabisulfite 20 ml, 120 mM potassium iodide 100 ml
And 200 ml of 1.2 mg / ml urea were added. The contents are mixed and the reaction mixture is passed through a pre-packed, equilibrated Sephadex G-25 column (PD 10 Pharmacia), phosphate buffered with 0.25% gelatin. Elution was performed with physiological saline (pH 7.4).
Fractions containing peak radioactivity were pooled and stored at -20 ° C. The specific activity of ¹²⁵ I-TNF was 80-100 mCi / mg protein. The biological activity of iodinated TNF has been determined by Neil, M.L. (Neale.ML), et al., European Journal of Cancer and Clinical Oncology (Eur.J.Can.Clin.Oncol.), 25 (1): 133-137 (19
89) Non-standard TN as measured by the L929 cytotoxicity assay
It turned out to be 80% of F's.

Section IV: Measurement of TNF-ELISA Winston et al., Current Protocols in Molecular Bio
logy), page 11.2.1, edited by Ausubel et al., (1
987) TNF levels were measured using the basic sandwich ELISA assay disclosed in John Willy and Sons (New York, USA). The ELISA method used the following murine monoclonal anti-human TNF antibody as a capture antibody and the following polyclonal rabbit anti-human TNF as a second antibody. For detection, a goat anti-rabbit antibody conjugated to peroxidase (Boehringer Mannheim, Indianapolis, Ind., Cat. # 605222) was added, followed by a substrate for peroxidase (containing 0.1% urea peroxide). 1 mg / ml orthophenylenediamine) was added. The TNF level in the sample was determined by E. Coli
Recombinant TNF produced in the United States (SmithKlein, King of Prussia, PA, USA)
Beecham Pharmaceuticals (SmithKline B
eecham Pharmaceuticals).

Section V: Production of Anti-Human TNF Antibodies: Kohler and Millstei
n), Nature, using a modification of the method of 256: 495 (1975), the entire content of which is incorporated herein by reference.
Monoclonal antibodies against human TNF were regulated from the spleen of BALB / c mice immunized with TNF. Polyclonal rabbit anti-human TNF antibodies were prepared by repeating immunization of New Zealand White (NZW) rabbits with recombinant human TNF emulsified in Freund's complete adjuvant (DIFCO, Ill.).

Results 1,3-Di-cyclopropylmethyl-8-aminoxanthine was approximately 0.005 μm in the in vitro TNF production assay system.
It has been determined that indicates an IC ₅₀ of M.

Example B of Utility Endotoxic Shock in D-gal-Sensitized Mice The protocol used to test the compounds of the methods of the present invention was essentially that of Galanos et al., Proceedings of・ National Academy of Sciences USA (Proc. Nat'l.Aca
d. Sci USA), 76: 5939-43 (1979), the contents of which are incorporated herein by reference. That is, D-gal (D (+) galactosidase) sensitizes various strains of mice to the lethal effects of endotoxin. Intravenous (iv) administration of D-gal (300-500 mg / kg) sensitizes mice to a dose of about 0.1 μg of lipopolysaccharide (LPS). That is, Charles River Laboratories (Stone Ridg, New York, USA)
e) Male C57BL / 6 mice, 6-12 weeks old, obtained from) were injected with D in 0.20-0.25 ml of pyrogen-free saline.
0.1 μg of LPC from Salmonella typhosa (Difco Laboratories, Detroit, Michigan, USA) mixed with (+)-gal (Sigma; 500 mg / kg) i.
v. Injected. The compound to be tested was administered at various times before or after iv injection of LPS / D-gal. In this model, control animals generally died 5-6 hours after injection of LPS, but occasionally between 24-48 hours.

Measuring TNF activity Winston et al., Current Protocols in Molecular Bio
logy), page 11.2.1, edited by Ausubel et al., (1
987) Plasma levels of TNF were measured using a variation of the basic sandwich ELISA method disclosed in John Willy and Sons (New York, USA). The ELISA method uses hamster
Monoclonal anti-mouse TNF (Genzyme, Boston, Mass., USA) and polyclonal rabbit anti-murine TNF as detection antibody
(Genzyme, Boston, Mass., USA) was used. Levels of TNF in mouse samples were calculated from a standard curve generated with recombinant murine TNF (Genzyme, Boston, Mass., USA). TNF levels measured by ELISA were
ff) et al., Journal of Immunology (J. Immuno
l.), 125: 1671-1677 (1980) and correlate with the levels detected by the L929 bioassay. One unit of activity in the bioassay corresponds to 70 picograms (pg) of TNF in the ELISA. ELISA dropped to 25 pg / ml
The level of TNF was detected.

Results 1,3-Di-cyclopropylmethyl-8-aminoxanthine shows a positive in vivo response in the utility model described above and has an ED ₅₀ for intraperitoneal reduction of serum TNF of about 0.1 mg / kg. Was measured. The compound exhibits 100% animal survival at this dose.

Utility C HIV In Vitro Monocyte Assay The following protocol is used to study the effect of compounds of formula (I) on the in vitro inhibition of HIV production in chronically infected cells.

Isolation of HIV-infected cell line HTL V _IIIB strain of human immunodeficiency virus (HIV _IIIB )
Clonal HIV-infected cell lines were induced by infecting a culture of the H9 T cell line and culturing the cells for 5 weeks, during which the chronically infected cell line was expanded. RPM
Clones were derived from this culture by limiting dilution plating in a 1: 1 mixture of I1640 + 15% fetal calf serum and H9 cell conditioned medium. The clone is about 4 × 10 cells
Increased to ⁷ . Aliquots were frozen and then their cultures with and without stimulation with TNF or other recombinant cytokines, as described below.
Assayed for HIV production.

Induction of HIV HIV induction was assayed by culturing the clonal HIV-infected cell line for 4 days in the presence of the substance to be tested for inducing activity. To measure inhibition of HIV induction, cultured human monocytes were stimulated to produce cytokines by treatment with lipopolysaccharide (LPS) for 18 hours in the presence or absence of test compounds. After stimulation, the supernatant medium from the monocyte cultures is collected, frozen in aliquots at -80 ° C, and TNF in one aliquot is detected by ELISA.
The concentrations of IL1-β and IL-6 were measured. The monocyte supernatant was then diluted in complete RPMI growth medium to provide a positive control (LP
The optimal induced concentration of TNF was obtained in the case of monocyte samples (S stimulated). Optimal induction depends on the indicator cell line, TNF 10-10
Achieved at 0 units / ml (0.5-50 ng / ml). In each experiment, supernatant from experimentally treated monocyte cultures was diluted by the same factors used for the positive control.

After 4 days of experiment, the culture supernatant (90μ) was removed from the HIV infected cell line and 5% (v / v) Triton
-X-100 (10μ; Sigma Chemical Company (S
igma Chemical Company)) to remove reverse transcriptase from HIV particles and inactivate the virus. Eight cultures were evaluated for each treatment in more than one experiment. Samples were stored at -80 C until assayed for reverse transcriptase activity.

Reverse Transcriptase Assay HIV reverse transcriptase was assayed by a variation of the microtiter assay of Goff et al. (J. Virol, 38: 239-248, 1981).
NA45 membrane filter on a dot-blot apparatus (Schleicher and Schleicher and
Scheull)), and autoradiography or AM
The incorporation of ³² P-dTTP into the polynucleotide on the oligo-A: poly-dT template: primer was measured by filtering the reaction products using a BIS quantitative instrument or both. Reverse transcriptase assays were performed twice for all samples.

Statistical Methods The statistical significance of the results was calculated using the COMPARE function of the RS / Explore software package.

Results: Most clonal HVI-infected cell lines show TNF in culture medium
Express high levels of HIV in response to RPMI 1640 + 10%
Ten clone H9 cell lines were cultured four times for 4 days with or without 5 ng rTNF / ml fbs. After the experiment, the reverse transcriptase in the culture medium was measured, and HIV
Determined the level of production. 8 out of 10 cell lines tested
Individuals produced high levels of HIV when cultured in the presence of TNF. Certain clone, cell line 3.7, responded to TNF in a typical manner known to those skilled in the art. Cell line 3.7 was selected and used to evaluate induction in response to monocyte supernatant.

Clone HIV-infected cells not only respond to TNF,
Expresses high levels of HIV in response to supernatants from LPS-stimulated cultured human monocytes (not controls). The cell line 3.7 was cultured for 4 days in a medium supplemented with 5 ng of TNF-α per ml of a liquid derived from cultured human monocytes or a liquid derived from cultured human monocytes stimulated with LPS. The level of HIV produced was measured by measuring the level of reverse transcriptase in.

Cell line 3.7 was selected because it reproducibly induces HIV in response to recombinant TNF. The results here were obtained using cell lines 4,7,3, U-1 and ACH-2. Commercially available cell lines are also useful in this assay.

Supernatants from cultured human monocytes stimulated with LPS in the presence of inhibitors of TNF synthesis have lower HIV activation activity compared to supernatants from monocytes stimulated with LPS in the absence of inhibitors Having. Control human monocytes, human monocytes stimulated with LPS, and a compound of formula (I), 1,3-di-cyclopropylmethyl-
HI in media supplemented with supernatant from human monocytes stimulated with LPS in the presence of 10 μM 8-aminoxanthine for 4 days.
The V-infected clonal cell line 3.7 was cultured.

This assay shows that compounds of formula (I) as inhibitors of TNF inhibit HIV induction, if present, by LPS-stimulated, LPS-stimulated monocyte supernatants. In particular,
1,3-Di-cyclopropylmethyl-8-aminoxanthine is (% +/- error) +/- (75-5) at a concentration of 10 [mu] M.
^* Showed HIV inhibition.

^* Percent inhibition in parentheses is from a representative experiment. The actual inhibition in any of the experiments may vary, depending on the monocyte donor, the dose response curve of the test HIV infected T cell line, and the dilution of the monocyte supernatant.

Example D of Utility Inhibition of Influenza Virus-Induced TNF In Vivo The effect of compounds of formula (I) on virus-induced TNF production in vivo was studied using the following protocol.

Mouse: Charles Rive Laboratories
r Laboratories), (Balb / cx C57B / 6) F ₁ (CB6F ₁ )
I purchased a mouse. At the time of arrival, mice were 4-10 weeks of age. Mice for the lower to the LD ₅₀ measurements was 8 to 14 weeks of age.

Virus production: Influenza A virus strain A / PR / 8/34 (H1N1 subtype) was grown in the allantoic cavity of fertilized eggs at 10 weeks.
After incubating the eggs for 48 hours, they were frozen for at least 2-1 / 2 hours before harvesting allantoic fluid. The pooled allantoic fluid is centrifuged (2000 rpm, 15 minutes, 4
C), cells were removed and then aliquoted for storage at -70C.

In Vitro Virus Titration Virus was quantified in an in vitro microassay using Madin-Darby canine kidney (MDCK) cells to establish a 50% tissue culture infectious titer (TCID ₅₀ ). Serial dilutions of virus or lung homogenate (medium +) were added to round-bottom microtiter wells containing adherent MDCK cells.
2.5 μg / ml trypsin) was added (4 each). 37 ℃
After incubation for 5 days (6% CO ₂ ), 50 μl of 0.5% chick red blood cells were added per well, and after 1 hour at room temperature, aggregation was measured. TCID ₅₀ doses were calculated using the SAS Version 5 program for a 50% effective dose (ED ₅₀ ) evaluation for dual dose-response assays [SAS / Statuser's Guide, Volume 2, SAS
The Institute (SAS Institute), Cary, NC (1985) and Applied Categorical Data Analysis
Data Analysis), Marcal Dekker Inc., publisher, New York, NY].

In vivo virus challenge: Freshly thawed virus was serially diluted 10-fold in sterile PBS with 0.05-1% bovine serum albumin (10 ^-1 -10 ^-8 ); dilutions were on ice until use Maintained. Methoxyfluorane impregnated on paper towels (methophane; Pittman Moore Company (Pitt)
by brief exposure to man Moore Co.)), it was anesthetized CB6F ₁ mice were intranasally challenged with virus 50μ. The same dose as 2LD ₅₀ was used in these experiments.

Sample collection from virus challenged mice Serum: Blood was collected from the orbital venous plexus of mice using a heparinized Pasteur pipette. Blood was pooled from 3-4 mice and centrifuged at 15K for 15 minutes; plasma was aliquoted and frozen at -20 ° C.

Lung Homogenate: For the first 3 days, aseptically remove lungs from intranasally infected mice and 1 micron glass beads (BioSpec ™, Bartlesville, Okla.)
Products (Biospec Products) (about 1/4 full) and 1 ml of Eagle's minimum essential medium containing penicillin and streptomycin were placed in vials (one lung per vial). Lungs were homogenized using a mini-beads beater (Biospec Products) for 1 minute; the vials were then centrifuged at 3000 rpm for 15 minutes at 4 ° C. and lung supernatants were frozen at −20 ° C.

Bronchoalveolar lavage: Mice were euthanized by cervical dislocation and moistened with alcohol. The spleen was removed to expose the diaphragm. The diaphragm was cut off, the lungs were crushed, and the rib cage was excised to expose the bronchi. The bronchi were dissected about 3-5 mm above the lung and 1 ml of PBS was injected into the lung via a blunt 19 gauge needle. This fluid was collected back into the syringe (〜60-80% of the initial volume) and spun at 2000 rpm for 15 minutes to remove cells and debris. Prior to the assay, an aliquot of the supernatant was frozen at -20 ° C.

Administration of a compound of formula (I): a compound of formula (I), 1,3-
First, di-cyclopropylmethyl-8-aminoxanthine (hereinafter, referred to as compound (1)) is dissolved in DMSO / EtOH, adjusted to a predetermined volume with FBS / saline, and adjusted to 5% DMSO, 5%.
1m per ml of% Etoh, 40% FBS and 50% normal saline
g to a final concentration. 0.2 mg (10 mg / kg) per 20 g of mouse
The injection was administered ip to match (0.
2 ml); or other doses as described below.

TNF Elisa Assay: The TNF Elisa assay is identical to that described in Example B of Utility above.

Theoretical Interpretation and Overall Objective: There are no HIV animal models to test the activity of inhibitors of the HIV virus and no animal models that can be easily monitored in vivo. Early reports in the literature reported that influenza-infected monocytes produce TNF, and thus the influenza model as useful for in vivo monitoring of compounds of formula (I) as inhibitors of TNF. Is selected.

Mouse Influenza Model: Brief Description Human influenza virus replicates in the lungs of mice but does not cause overt disease. However, the pathogenicity for mice is increased by continuous passage in the lungs of mice. The mouse-compatible virus produces lethal pneumonia but does not cause upper respiratory tract infection as in simple human influenza. In murine influenza, viral replication is restricted to the lungs, with massive inflammatory cell infiltration. It is well documented that pulmonary interferon levels are elevated during murine influenza (Wyd
e) et al.), recent reports demonstrated a non-quantitative increase (by bioassay) in IL-1 and TNF levels in bronchoalveolar lavage fluid of influenza infected mice (Vacheron et al.).

Results: In vivo TNF production In the first study, the A / PR / 8/34 virus (2LD ₅₀ ) was given intranasally at 0.5, 1 and 6 hours and at days 1, 3, 5, 7, 9 and 14 Blood and bronchoalveolar lavage (BAW) samples were taken from infected mice. Pooled samples from TNF Elia assay (3 mice per group)
Analysis confirmed that TNF was produced in the lungs, but no TNF was detected in the blood at any time point. In subsequent experiments, BAW was assayed individually (5 mice per group) to prepare lung homogenates for TNF analysis and virus titration. The result is TN
F levels increase 2-7 days after infection, indicating that viral replication was evident by 24 hours. Virus titers began to decline after 3 days, but TNF levels remained elevated. The result is that virus replication is localized TNF
It suggests that it caused an event that induced production (ie, inflammatory cell infiltration) and that pulmonary TNF levels were maintained when the virus was removed from the lung.

Effect of Compound (1) on Influenza-Induced TNF Production The treatment protocol was designed based on the kinetics of TNF production found in the lung. Treatment was started on day 1 preceded by any detectable elevation of TNF in the lungs. Mice were dosed ip with compound (1) daily at 10 mg / kg and BAW was performed 2 hours after the last injection. TNF levels in BAW were significantly reduced on days 2 and 3 compared to untreated controls, with up to 67% reduction (n = 3 of 3 experiments). In lung homogenate, only TN on day 3
F levels were significantly reduced (n =
1) (not shown). Dose titration of compound (1) showed that the compound was active at 1 mg / kg (〜50% reduction in BAW TNF), but no effect was seen at 0.1 mg / kg.

Discussion: These studies show that therapeutic administration of compounds of formula (1)
Figure 4 shows that virus-induced TNF production in vivo can be reduced. The data suggests that the compound is effective in reducing TNF levels in tissues and in the circulation.

Effect of compound (1) on influenza virus titers in mouse lung Virus titers are significant on days 2-5 in mice treated with compound (1) 1-10 mg / kg 1, 2 and 3 days post infection Has dropped.

Discussion: These data demonstrate that treatment with compounds of formula (I) reduces lung viral titers and is therefore directly useful in human influenza infection.

Effect of Compound (1) on Survival in a Murine Influenza Lethal Challenge Model Induced by A / PR / 8/34 Influenza Virus in Mice Administered Daily with Compound (1)
There was a significant improvement in survival in mice treated with 10 mg / kg, but survival in groups of mice treated with lower doses, or at 0.1-10 mg / kg for 1, 2 and 3 days post-infection. Survival in the group of treated mice was not significantly different from vehicle-treated controls.

Discussion: Treatment of mice challenged with influenza with a dose of compound (1) that showed a decrease in pulmonary TNF levels resulted in a modest improvement in survival that was significant when mice received 10 mg / kg daily. occured. Thus, TN
Reduction of F was not detrimental in this infection. Although the effect on survival was not dramatic, a more significant benefit could be shown by measuring early endpoints such as clinical symptoms. However, this is not easily done in a mouse model where infection is limited to the lower respiratory tract. Thus, treatment with a compound of formula (I)
It is suggested that the level of escaping nasal virus generally reduces morbidity and / or mortality in human influenza, which is correlated with clinical symptoms.

The foregoing generally describes the invention including preferred embodiments. Modifications and improvements of the embodiments specifically described herein are within the scope of the following claims. Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. Accordingly, the examples herein should be construed as merely illustrative and in no way limit the scope of the invention. The embodiments of the invention in which exclusive rights and privileges are claimed are defined as follows.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification code FI A61P 31/12 A61P 31/12 171 171 31/18 31/18 37/00 37/00 37/02 37/02 37/04 37 / 04 43/00 111 43/00 111 // C07D 473/06 C07D 473/06 (56) References JP-A-2-273676 (JP, A) JP-A-2-19327 (JP, A) 2-502542 (JP, A) Allergol. Immunopa thol. , Vol. 11, No. 5 (1983) p. 321-327 Biochem. Biophys. Res. Commun. , Vol. 155, No. 3 (1988) p. 1230-1236 (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 31/522 C07D 473/06 CA (STN) CAOLD (STN) CAPLUS (STN) REGISTRY (STN)

Claims (6)

(57) [Claims] 1. A mammal in need of inhibiting tumor necrosis factor (TNF) production, characterized in that it contains 1,3-di-cyclopropylmethyl-8-aminoxanthine or a pharmaceutically acceptable salt thereof. A pharmaceutical composition for inhibiting the production thereof. 2. The pharmaceutical composition according to claim 1, which is for oral administration, parenteral administration or inhalation administration. 3. The method according to claim 1, wherein the mammal is an acute immunodeficiency syndrome (AIDS),
DS-related syndrome (ARC) or HIV-related disease, cachexia associated with AIDS or cancer, cachexia associated with cancer, adult respiratory distress syndrome, bone resorption, graft-versus-host reaction, acute graft rejection, The pharmaceutical composition according to claim 1, which is a human suffering from Crohn's disease, ulcerative colitis, septic shock, endotoxin shock, gram-negative bacterial sepsis or toxic shock syndrome. (4) a sufficient amount of 1,3-- to inhibit TNF production;
Comprising di-cyclopropylmethyl-8-aminoxanthine or a pharmaceutically acceptable salt thereof,
A pharmaceutical composition for treating a viral infection in a mammal. 5. The pharmaceutical composition according to claim 4, wherein the viral infection is a lentivirus infection including feline immunodeficiency virus (FIV), equine infectious anemia virus, goat arthritis virus, visna virus and medevirus. 6. The pharmaceutical composition according to claim 1, wherein the TNF is TNF-α.