WO2009016081A2

WO2009016081A2 - Benzoind0l-2-one derivatives for use in therapy

Info

Publication number: WO2009016081A2
Application number: PCT/EP2008/059664
Authority: WO
Inventors: Jacob Westman; Björn KULL; Patric Stenberg
Original assignee: Actar Ab
Priority date: 2007-08-02
Filing date: 2008-07-23
Publication date: 2009-02-05
Also published as: WO2009016081A3

Abstract

A compound of formula (I) wherein A is (C0-C1)-alkyl; R1 is (C1-C6)-alkyl when A is C1-alkyl and R1 is absent when A is C0-alkyl; R2 is optionally substituted mono- or polycyclic carbocyclyl or oxygen-containing heterocyclyl, R3 is H oroptionally substituted -S(O)2-NH-phenyl, and pharmaceutically acceptable salts thereof, for use as a medicament, e.g. in the treatment of inflammatory diseases and cancer.

Description

Compounds for use in therapy

Field of the invention

The present invention relates to benzo[cd]indol-2-one derivatives and to the use thereof in disease therapy. More particularly, the present invention relates to benzo[cd]indol-2- one derivatives for the treatment of inflammation related diseases. Even more particularly, the present invention relates to compounds acting on microsomal prostaglandin E synthase for the treatment and prevention of fever, pain and inflammation as well as cancer.

Background of the invention

The present invention relates to compounds which inhibit, regulate and/ or modulate the activity of microsomal prostaglandin E synthase, compositions which contain these compounds, and methods of using them to treat diseases and conditions such as pain, fever, inflammations and cancer, and the like in mammals.

The following is provided as background information only and should not be taken as an admission that any subject matter discussed or that any reference mentioned is prior art to the instant invention.

Prostaglandin (PG) E2 is produced in the mammalian body in a sequential action including liberation of arachidonic acid, conversion into PGG2/PGH2 by cyclooxygenase (Cox) -1 or Cox-2 and finally prostaglandin E synthase converts PGH2 into PGE2 (Fig IA). There exist three known enzymes that catalyze the latter reaction i.e. microsomal prostaglandin E synthase 1 (MPGESl), cytosolic prostaglandin E synthase (CPGES), and microsomal prostaglandin E synthase 2 (MPGES2). The latter two enzymes are constitutively expressed whereas MPGES 1 is inducible by proinflammatory cytokines. Initially, MPGES 1 was regarded as the enzyme predominantly coupled with Cox-2 activity. However; later results demonstrate that MPGES 1 can also catalyze the conversion of Cox- 1 derived PGH2 into PGE2. MPGESl possesses the highest catalytic efficiency of the known PGE synthases. The role of PGE2 as one of the most potent mediators of inflammation together with many in vitro reports on the presence of MPGESl in different models of inflammation suggested this enzyme to be an attractive drug target for development of new anti inflammatory drugs with fewer side effects than the currently available NSAIDs and selective Cox-2 inhibitors. The rationale being that MPGES 1 is predominantly expressed during inflammation and that other enzymes exist that mediate house keeping functions. NSAIDs constitute many drugs that inhibit Cox- 1 and Cox-2 with a continuum of different potencies on respective enzymes. They range from acetyl salicylic acid, being a preferred Cox- 1 inhibitor, to selective Cox-2 inhibitors, e.g. rofecoxib or celecoxib (Vioxx and Celebrex, respectively). Cox-1 inhibitors are cardio-protective by their capability to prevent thromboxane formation in platelets while deleterious vascular effects after prolonged usage of selective Cox-2 inhibitors have been reported, likely through the effect of Cox-2 dependent prostacyclin formation in endothelial cells. The ratio of thromboxane:prostacyline is diminished by Cox- 1 inhibitors but increased by Cox-2 inhibitors. Cox-1 inhibitors are also known to result in increased frequency of gastric bleedings and kidney function impairments. Cox-2 inhibitors also result in gastric side effects as well as negative changes in the body water-salt balance with problems of edema formation and hypertension as a consequence. This seems particularly a problem for rofecoxib.

Specific inhibition of MPGES 1 may overcome many of these side effects due to the fact that the balance among the prostaglandins will not primarily be influenced. Thus only the pro -inflammatory pressure during induced PGE2 formation will be targeted. The possibility also exists that an MPGES 1 inhibitor will possess enforced anti-inflammatory potential since Cox-2 generated anti-inflammatory prostaglandins such as cyclopentenones may increase due to shunting of PGH2 in macrophages (Fig IB). Such shunting will not occur in platelets (there is no evidence for PGE synthase activity in these cells). In endothelial cells there might occur a shunting upon activation since these cells become activated during inflammation which leads to high formation of PGE2 and prostacyclin. In that case, increased prostacyclin formation is expected, with protecting effects against vascular side effects.

Although the bulk of evidence already suggested MPGES 1 to constitute a drug target, the results using gene targeted knock out mice have provided unequivocal important data regarding the physiological role of MPGES l . 1) These mice develop significantly less arthritis symptoms in experimental models of arthritis (CIA and AIA). 2) These mice demonstrate less sensitivity to pain both induced in inflammatory settings and neuropathic settings. 3) These mice do not develop endotoxin, IL- lbeta or cytokine induced fever. 4) Finally, as of today, gross histopathological examinations of various organs including the GI tract, behavioural and reproductive parameters have not demonstrated any differences to the results obtained for wild type animals. However, in an ongoing study, the healing phase after heart damage caused by permanent ligation of one major coronary vessel in MPGES 1 knock out mice suggest impaired healing or remodeling of the heart. This must be compared with Cox-2 knock out mice where the heart spontaneously develop fibrosis and can not be used in such models. Also, in higher mammals, MPGES2 may take over this function since it is predominantly expressed in the heart.

WO2007042817 from Biolipox AB describes naphthalene disulfonamides as mPGES-1 inhibitors. However, tricyclic rings and monosulfonamide derivatives are not mentioned in this application.

Summary of the invention

According to a first aspect, the invention provides a compound of formula (I)

(I) wherein

A is (CO-C l)-alkyl;

Ri is (C l-C6)-alkyl when A is Cl -alkyl and R¹ is absent when A is CO-alkyl;

R² is mono- or polycyclic carbocyclyl or oxygen-containing heterocyclyl, optionally substituted by one or several moieties R⁴;

R³ is H or -S(O)₂-NH-phenyl, wherein the phenyl is optionally substituted with one or several moieties R⁵;

each R⁴ is independently selected from OH; (Cl -C6)-alkyl; C6-C10-aryl optionally substituted by OH; halogen; [ReC(O)][R⁷OC(O)]CH-; R^SC(O)-, R⁹OC(O)-, Rⁱ°C(O)NH-; R¹¹O-; nitro; R¹²S(O)2N-; and =O attached to a ring carbon atom of the carbocyclyl or heterocyclyl; each R5 is independently selected from (Cl-C6)-alkyl; Ri3C(O)NH-; Ri⁴O-; and halogen;

each one of R⁶-Rⁿ, R¹³ and R¹⁴ is independently selected from (C l -C6)-alkyl; optionally substituted with one or several halogens;

R¹² is heterocyclyl; optionally having =O attached to a ring carbon atom;

as well as pharmaceutically acceptable salts thereof, for use as a medicament.

In some embodiments A in formula (I) is CO-alkyl.

In some embodiments, R² is a mono-, bi-, tri- or tetracyclic carbocyclyl or oxygen- containing heterocyclyl, optionally substituted by one or several moieties R⁴ as defined herein above.

In some embodiments, R² is phenyl, naphthyl, e.g.

benzofuryl, e.g.

or a moiety of formula

wherein the phenyl, naphthyl, benzofuryl or the moiety of the above indicated formula optionally are substituted by one or several moieties R⁴ as defined herein above.

In some embodiments, R³ is -S(O)₂-NH-phenyl, wherein the phenyl is optionally substituted with one or several moieties R⁵ as defined herein above.

In other embodiments, R³ is H. In some embodiments, any (C l-C6)-alkyl in the compound of formula (I) is selected from (Cl-C4)-alkyl, more preferably from methyl, ethyl, n-propyl and iso-propyl.

In some embodiments, any halogen is selected from F and Cl.

In some embodiments of the invention, each R⁴ is independently selected from OH, CH₃-, C₃H₇-, naphtholyl, Cl, [CF₃C(O)] [(CHa)₂(CH)OC(O)]CH-; CH₃C(O)-, CH₃OC(O)-, CH₃CH₂OC(O)-, CH₃C(O)NH-; CH₃O-; CH₃CH₂O-; nitro; 2-oxo-l ,2-dihydro- benzo[cd]indole-6-sulfonamido; and =O attached to a ring carbon atom of the carbocyclyl or heterocyclyl.

In some embodiments of the invention, each R⁵ is independently selected from CH₃-, CH₃O-, CH₃CH₂O-, CH₃C(O)NH-, and Cl.

In some embodiments of the invention, R² is selected from

In some embodiments of the invention, R³ is selected from

In some embodiments of the invention, A is C 1 -alkyl.

In some embodiments of the invention, R¹ is (Cl-C3)-alkyl.

In some embodiments of the invention, R² is carbocyclyl, e.g. a polycyclic carbocyclyl such as adamantyl.

In one embodiment of the invention, the compound of formula (I) is selected from

šnd

According to one aspect, the invention provides a compound according to formula (I), as defined herein above, or a pharmaceutically acceptable salt thereof, for use in the treatment of a disorder or disease selected from fever, pain, inflammation and cancer, in particular inflammation, e.g. the treatment or prevention of inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, psoriasis, asthma, chronic obstructive pulmonary disease, ulcers, inflammatory pain, fever, atheresclerosis, coronay artery disease, eczema, dermatitis, stroke, diabetes, autoimmune diseases, multiple sclerosis, arthritis or another malignancy as well as cancer; as well as the use thereof in the manufacturing of a medicament for the treatment of any of these diseases or disorders.

According to another aspect, the invention provides a pharmaceutical composition comprising a compound as defined herein above, together with at least one pharmaceutically acceptable excipient.

According to another aspect, the invention provides a method of treatment of a mammal sufffering from a disorder or disease selected from fever, pain, inflammation and cancer, in particular inflammation, e.g. the treatment or prevention of inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, psoriasis, asthma, chronic obstructive pulmonary disease, ulcers, inflammatory pain, fever, atheresclerosis, coronay artery disease, eczema, dermatitis, stroke, diabetes, autoimmune diseases, multiple sclerosis, arthritis or another malignancy as well as cancer.

Any further aspects are as defined in the claims.

Brief description of the drawings

Figure 1 illustrates the biosynthesis of prostaglandin E2. Hypothetic effect of MPGES l blockage - shunting into the anti-inflammatory PGD2 pathway and formation of cyclopentenons.

Figure 2 is a schematic representation of biochemical pathway in TBA-MDA Assay.

Detailed description of the invention

Some compounds of the present invention are commercially available from several sources such as ChemDiv, ChemBridge etc. The inventive compounds also may be synthesized by common methods, well known for the person skilled in the art. For example, in FR2018863 suitable synthetic methods are described. According to a first aspect, the invention provides a compound of formula (I)

(I) wherein A, R¹, R² and R³ are as defined herein above, or a pharmaceutically acceptable salt thereof, for use as a medicament.

By "Cn-alkyl" as used in the present description, wherein n is 0 or a postive integer integer is meant an alkyl moiety comprising n carbon atoms. For example, Cl -alkyl refers to a methyl group, unless otherwise apparent from the context. With reference to the moiety A, when A is Cl -alkyl, it comprises one carbon atom attached to the sulfonamido nitrogen, to R¹ and to R² and to one hydrogen atom, viz. the Cl -alkyl is a moiety -CH<.

By "CO-alkyl" as used in the present description is meant that the alkyl moiety in fact is absent, so that two moieties A and B linked to each other through a "C0-alkyl" moiety are attached directly to each other through a covalent single bond.

The sign " "~~~~~ " as used in the representation of various moieties denotes the bond through which the moiety is attached to the rest of the molecule. Thus, for example a moiety represented by the formula

is a phenyl radical substituted with a methyl in ortho position relative to the bond through which the phenyl radical is attached to the rest of the molecule.

In some embodiments A in formula (I) is CO-alkyl, i.e. the moiety represented by R² is attached directly to the sulfonamido nitrogen. In these embodiments, the compound of formula (I) may be represented by the structural formula wherein R² and R³ are as defined herein above.

As noted herein above, in the compound of formula (I), R² is mono- or polycyclic carbocyclyl or oxygen-containing heterocyclyl, optionally substituted by one or several, e.g. 1, 2 or 3, moieties R⁴.

As used herein, the term "carbocyclyl" refers to a cyclic moiety containing only carbon atoms, while the term "heterocyclyl" refers to a cyclic moiety containing not only carbon atoms, but also at least one other atom in the ring structure. By "oxygen-containing heterocyclyl" is meant that this at least one other atom is an oxygen, i.e. the heterocyclyl contains carbon and oxygen as ring atoms.

The term "aromatic", as used herein, refers to an unsaturated cyclic moiety that has an aromatic character, while the term "non-aromatic", as used herein, refers to a cyclic moiety, that may be unsaturated, but that does not have an aromatic character.

In a polycyclic ring system, as referred to herein, the rings may be all saturated or some or all may be unsaturated, e.g. all aromatic. The rings may also be of different degrees of saturation, and one ring may be aromatic whereas the other is non-aromatic. The rings also may comprise different numbers of atoms, e.g. one ring being 5-membered and the other one being 6-membered. A polycyclic ring system may comprise only carbocycles or only heterocycles or carbocycles and heterocycles fused to each other, but in the two latter cases will be referred to as heterocyclyl. Thus, in a polycyclic cyclyl, each constituent monocycle may independently be selected from aromatic or non-aromatic (i.e. saturated or unsaturated) carbo- and heterocycles.

The carbocyclyl or oxygen-containing heterocyclyl may be saturated, unsaturated or aromatic, and may contain e.g. 1-4 rings. In some embodiments, R² is a mono-, bi-, tri- or tetracyclic carbocyclyl or oxygen-containing heterocyclyl. Each ring in the mono- or polycyclic carbocyclyl or oxygen-containing heterocyclyl may contain e.g. from 5 to 6 ring atoms, selected from carbon (C) and oxygen (O). Preferably, a heterocyclic ring may contain 1 or 2 oxygen atoms, e.g one oxygen and 4-5 carbon atoms.

In some embodiments, R² is an aromatic carbocyclyl or oxygen-containing heterocyclyl, e.g. phenyl, naphthyl, benzofuryl or

in particular phenyl, naphthyl or benzofuryl, more particularly phenyl.

R² is optionally substituted by one or several, e.g. 1, 2 or 3, moieties R⁴, independently selected from OH; (C 1 -C6) -alkyl; C6-C10-aryl optionally substituted by OH; halogen; [(C 1 -C6)-alkyl-C(O)] [(C 1 -C6)-alkyl-OC(O)]CH-; (C 1 -C6)-alkyl-C(O)-, (C 1 -Cδ)-alkyl-OC(O)-, (Cl-C6)-alkyl-C(O)NH-; (C l -C6)-alkyl-O-; nitro; heterocyclyl-S(O)₂N-, the heterocyclyl optionally having =O attached to a ring carbon atom; and =O attached to a ring carbon atom of the carbocyclyl or heterocyclyl, wherein any alkyl moiety optionally is substituted by a halogen, e.g. F. For example, each R⁴ may be independently selected from OH, CH₃-, C₃H₇-, naphtholyl, Cl, [CF₃C(O)] [(CHa)₂(CH)OC(O)]CH-; CH₃C(O)-, CH₃OC(O)-, CH₃CH₂OC(O)-, CH₃C(O)NH-; CH₃O-; CH₃CH₂O-; and the heterocyclyl optionally having =O attached to a ring carbon atom; nitro; and =O attached to a ring carbon atom of the carbocyclyl or heterocyclyl.

When R⁴ is heterocyclyl-S(O)₂N- optionally having =0 attached to a ring carbon atom, the heterocyclyl e.g. may be a polycyclic nitrogen-containing heterocyclyl , e.g. a tricyclic nitrogen-containing heterocyclyl, such as e.g l ,2-dihydrobenzo[cd]indolyl, optionally having an oxo (=0) attached to a ring carbon atom.

In general it is contemplated that any (C l-C6)-alkyl in the compound of formula (I) more preferably is selected from (C 1-C4) -alkyl, e.g. from methyl, ethyl, n-propyl and iso- propyl. By "(Cl-C6)-alkyl in the compound of formula (I)" is meant any alkyl moiety having from 1 to 6 carbon atoms, whether attached directly to a carbocyclyl or heterocyclyl, or being part of another moiety, such as attached to an oxygen in an alkoxy group, or e.g. attached to the carbon atom of a carbonyl function. It is contemplated that any alkyl moiety may be branched or unbranched.

In a compound of formula (I), R³ is selected from H and -S(O)₂-NH-phenyl. When R³ is S(O)₂-NH-phenyl, the compound of formula (I) may be represented by the formula

wherein A, R¹ and R² are as defined herein above and n is 0 to 5, e.g. 0-2 and each R⁵ is independently selected from (Cl-C6)-alkyl; (Cl-C6)-alkyl-C(O)NH-; (Cl-Cό)-alkyl-O-; and halogen. For example, each R⁵ may be independently selected from CH3-, CH3O-, CH₃CH₂O-, CH₃C(O)NH-, and Cl.

Thus, in the embodiment where A is CO-alkyl and R³ is -S(O)2-NH-phenyl, the compound of formula (I) may be represented by the formula

wherein R², R⁵ and n are as defined herein above. For example, the compound of formula (I) may be selected selected from

In some embodiments of the invention, A is Cl-alkyl. In these embodiments, the compound of formula (I) may be represented by the formula

wherein R¹, R² and R³ are as defined herein above.

In some embodiments of the invention, R¹ is (Cl-C3)-alkyl, e.g. methyl.

In some embodiments of the invention, R² is carbocyclyl, such polycyclic saturated carbocyclyl, e.g. adamantyl. In some embodiments of the invention, R² is optionally substituted phenyl. For example, the compound of formula (I) may be selected selected from



In some other embodiments of the invention, R² is optionally substituted naphthyl. For example, the compound of formula (I) may be selected selected from

In still some other embodiments of the invention, R² is optionally substituted benzofuryl. For example, the compound of formula (I) may be selected selected from

According to one aspect, the invention provides a compound according to formula (I), as defined herein above, or a pharmaceutically acceptable salt thereof, for use in the treatment of a mammal suffering from a disorder or disease selected from fever, pain, inflammation and cancer, in particular inflammation, e.g. the treatment or prevention of inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, psoriasis, asthma, chronic obstructive pulmonary disease, ulcers, inflammatory pain, fever, atheresclerosis, coronay artery disease, eczema, dermatitis, stroke, diabetes, autoimmune diseases, multiple sclerosis, arthritis or another malignancy as well as cancer; as well as the use thereof in the manufacturing of a medicament for the treatment of any of these diseases or disorders.

The mammal preferably is a human, but it is contemplated that it may also be an animal, such as a farm animal, a domestic animal or a laboratory animal, e.g. cow, sheep, horse, cat, dog, rat, monkey, rabbit etc.

According to another aspect, the invention provides a pharmaceutical composition comprising a compound as defined herein above, together with at least one pharmaceutically acceptable excipient or carrier.

According to another aspect, the invention provides a method of treatment of a mammal sufffering from a disorder or disease selected from fever, pain, inflammation and cancer by administration to said mammal a compound as defined herein above, or a pharmaceutically acceptable salt thereof. According to one aspect, the invention provides the use of the inventive compounds, or salts thereof in the manufacture of a medicament for the treatment or prevention of inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, psoriasis, asthma, chronic obstructive pulmonary disease, ulcers, inflammatory pain, fever, atheresclerosis, coronay artery disease, eczema, dermatitis, stroke, diabetes, autoimmune diseases, multiple sclerosis, arthritis or another malignancy as well as cancer.

Any further aspects are as defined in the claims.

Materials and Methods

Prostaglandins detection kits were purchased from Cayman Chemicals and used according to the instruction of the manufacturer. In vitro toxicology assay kit, MTT based from Sigma, cat N - TOXl .

HPLC Assay

Earlier studies have demonstrated that prostaglandins can be separated by RP-HPLC and detected by UV spectrophotometry (Terragno et al. Prostaglandins 21(1), 101-12 (1981); Powell Anal. Biochem. 148(1), 59-69 (1985)). The molar extinction coefficient of PGE2 is 16,500 at 192.5 nm (Terragno et al. Prostaglandins 21 (1), 101-12 (1981)). The main products of PGH2 are PGF2α, PGE₂ and PGD₂. Using the described RP-HPLC conditions, the retention times were 19.0, 23.8 and 28.6 minutes for PGF2α, PGE₂ and PGD₂, respectively. 1 lβ-PGE₂ was used as the internal standard and 1 lβ-PGE₂ was eluted with a retention time of 25.3 min with almost baseline separation from PGE₂. In order to quantify PGE₂, a standard curve of PGE₂ was made. The curve was linear over the range from 0.9 pmol to 706 pmol (R2 = 0.9997, k = 0.0012). For quantification both the external standard and the internal standard technique were routinely used, the latter method accounting also for losses during preparation.

Care must be taken when assaying PGE synthase with PGH₂. The substrate is very labile and decomposes non-enzymatically, with a half-life of about 5 min at 37°C, into a mixture of PGE₂ and PGD₂ with a E/ D ratio of abut 3. Also, the PGE synthase catalysis is very fast, which is why substrate depletion easily can occur within seconds thus preventing a quantitative analysis. After the reaction has been terminated, any remaining PGH₂ must also rapidly be separated from the products in order not to interfere with the results. In order to minimize non-enzymatic production of PGE₂, the substrate (PGH₂) was always kept on CO₂-ice (-78⁰C) until use and the enzyme reaction was performed at 0⁰C in the presence of PGH2 and reduced glutathione (GSH). A stop- solution was used, containing FeCb, which converted any remaining PGH2 into HHT. Also, the products are much more stable in organic solvents so we immediately extracted the sample after termination by solid phase extraction and kept the eluate in acetonitrile.

Protein samples were diluted in potassium inorganic phosphate buffer (0.1M, pH 7.4) containing 2.5 mM reduced glutathione (GSH). 4 μl PGH2, dissolved in acetone (0,284 mM) were added to Eppendorf tubes and kept on CU2-ice (-78⁰C). Prior to the incubation, both the substrate and samples were transferred onto wet-ice (or 37°C) for 2 min temperature equilibration. The reaction was started by the addition of the lOOμl sample to the tubes containing PGH2. The reaction was terminated by the addition of 400μl stop solution (25 mM FeCl₂, 50 mM citric acid and 2.7 μM 1 1 -β PGE₂), lowering the pH to 3, giving a total concentration of 20 mM FeCb, 40 mM citric acid and 2.1 μM 1 1-β PGE₂. Solid phase extraction was performed immediately using C18-chromabond columns. The samples were eluted with 500 μl acetonitrile and thereafter ImI H2O was added. In order to determine the formation of PGE₂ and 1 1-β PGE₂, an aliquot (150μl) was analyzed by RP-HPLC, combined with UV detection at 195 nm. The reverse-phase HPLC column was Nova-Pak C 18 (3.9 X 150 mm, 4 μm particle size) obtained from Waters and the mobile phase was water, acetonitrile and trifluoroacetic acid (72:28:0.007, by vol). The flow rate was 0.7 ml/min and the products were quantified by integration of the peak areas.

Thiobarbituric acid assay (TBA-MDA assay or Malondialdehyde assay)

Malondialdehyde is a product of lipid peroxidation and reacts with thiobarbituric acid forming a red product that absorbs at 535 nm (W.G. Niehaus, Jr and B. Samuelsson, Eur. J. Biochem 6, 126 (1968). The extinction coefficient of the TBA-MDA conjugate is 1.56 x 10⁵ M-^I cm-ⁱ (E.D. Wills. Biochem. J. 1 13, 315 ( 1969).

The method used for detection of inhibition of mPGES- 1 is based on the detection of the amount of remaining PGH2. This method was described more than 20 years ago by Basevich et al (Bioorg. Khim. 1983, 9(5), 658-665.

The assay used was a modified variant and used citric acid instead of the TCA-TBA-HCl reagent described in the original assay. In this assay recombinant, membrane-bound mPGES-1 was incubated with PGH2. The reaction was stopped by adding citric acid with a final pH of 3 and a large excess of FeC12 (20 mM) to convert any remaining PGH2 into MDA and 12-HHT. TBA reagent was finally added (0.67%) and the samples were heated at 80 ⁰C for 30 min. The absorbance of the conjugate was measured at 535 nm.

The product of mPGES- 1 (PGE2) was not measured directly in this assay, but rather the remaining substrate (PGH2) indirectly by adding FeCl2 that converts PGH2 into MDA and 12-HHT. As a positive control a known mPGES- 1 inhibitor, MK-886, was used and the new inhibitors were compared with the inhibition of MK-886 (% of MK-886 inhibition).

Red product ~530nM ( 1.56XlO⁵M ¹ cm-').

Total Activity = A₅₃₀-A₅₆₀ / 1 min 0.265 (U/ ml)

1.56xlO⁵ 0.05

Fibroblast assay

Expression of PGH2 levels in synovial fibroblasts from human RA patients (passage four) growing in 96 well tissue culture plates were induced with IL-I beta (10ng/ml) and TNFalfa (10ng/ml). Test compound at a concentration of 10, 1, 0.1 or 0 μM was added and the cells were further cultured for 24h.

After 24 hours, supernatants were collected and number of viable cells was evaluated using MTT test according to manufacturer's instructions. PGE2 levels in supernatants were measured by EIA according to manufacturer's instructions. Results were expressed as PGE2 levels in supernatants (and adjusted for MTT) and related to PGE2 levels in supernatants from cells which were induced without adding test compound. Since the test compound did not affect cell viability at any concentration tested, normalization for MTT did not contribute to observed differences in PGE2 content. For results see Table 1.

A549 assay

A549 lung carcinoma cells seeded at a density of 10, 000 cells/well were grown in 96 well tissue culture plates. TNFalfa (5ng/ml) and IL-lbeta (5ng/ml) was added and the cells were incubated for 16 hours for the induction of PGH2. Cells were washed in PBS and test compounds in at the appropriate concentration in HBSS/ 0.1% BSA were added. After 30 minutes incubation with test compounds, 10 μM arachidonic acid was added and cells were further incubated for 30 minutes. Supernatant was collected and analyzed for PGE2 content by EIA according to manufacturer's instructions. For some inventive compounds, 6-keto-PGFlalfa, a metabolite of prostacyclin, was analysed using EIA according to manufacturer's instructions.

Examples of active compounds of formula (I) (more than 50% inhibition compared to inhibition with MK-886 in an enzyme inhibition assay)



Table 1

The compounds according to formula (I) will be useful for treating various diseases such as pain, fever, inflammations and cancer. The treatment may be preventive, palliative or curative.

Examples of pharmaceutically acceptable addition salts for use in the pharmaceutical compositions of the present invention include those derived from mineral acids, such as hydrochlorid, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids. The pharmaceutically acceptable excipients described herein, for example, vehicles, adjuvants, carriers or diluents, are well-known to those who are skilled in the art and are readily available to the public. The pharmaceutically acceptable carrier may be one that is chemically inert to the active compounds and that has no detrimental side effects or toxicity under the conditions of use. Pharmaceutical formulations are found e.g. in Remington: The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995).

Prodrugs of the compounds of formula (I) may be prepared by modifying functional groups present on the compound in such a way that the modifications are cleaved, in vivo when such prodrug is administered to a mammalian subject. The modifications typically are achieved by synthesizing the parent compound with a prodrug substituent. Prodrugs include compounds of formula (I) wherein a hydroxy, amino, sulfhydryl, carboxy or carbonyl group in a compound of formula (I) is bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl, amino, or sulfhydryl group, respectively. Examples of prodrugs include, but are not limited to, esters and carbamates of hydroxy functional groups, esters groups of carboxyl functional groups, N-acyl derivatives, N-Mannich bases. General information on prodrugs may be found e.g. in Bundegaard, H. "Design of Prodrugs" pl-92, Elesevier, New York-Oxford (1985).

The composition according to the invention may be prepared for any route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperitoneal. The precise nature of the carrier or other material will depend on the route of administration. For a parenteral administration, a parenterally acceptable aqueous solution is employed, which is pyrogen free and has requisite pH, isotonicity and stability. Those skilled in the art are well able to prepare suitable solutions and numerous methods are described in the literature. A brief review of methods of drug delivery is also found in e.g. Langer, Science 249: 1527- 1533 (1990).

The dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to affect a therapeutic response in the mammal over a reasonable time frame. One skilled in the art will recognize that dosage will depend upon a variety of factors including the potency of the specific compound, the age, condition and body weight of the patient, as well as the stage /severity of the disease. The dose will also be determined by the route (administration form) timing and frequency of administration. In the case of oral administration the dosage can vary from about 0.01 mg to about 1000 mg per day of a compound of formula (I) or the corresponding amount of a pharmaceutically acceptable salt thereof.

The compounds of the present invention may be used or administered in combination with one or more additional drugs useful in the treatment of pain, fever, inflammations and cancer. The components may be in the same formulation or in separate formulations for administration simultaneously or sequentially. The compounds of the present invention may also be used or administered in combination with other treatment such as irradiation for the treatment of cancer.

Claims

1. A compound of formula (I)

(I) wherein

A is (CO-C l)-alkyl;

R¹ is (C l-C6)-alkyl when A is Cl -alkyl and R¹ is absent when A is CO-alkyl;

each R⁴ is independently selected from OH; (Cl -C6)-alkyl; C6-C10-aryl optionally substituted by OH; halogen; [ReC(O)][R^OC(O)]CH-; R»C(O)-, ReOC(O)-, RiOC(O)NH-; R¹¹O-; nitro; R¹²S(O^N-; and =O attached to a ring carbon atom of the carbocyclyl or heterocyclyl;

each R⁵ is independently selected from (Cl-C6)-alkyl; R¹³C(O)NH-; R¹⁴O-; and halogen;

each one of R⁶-Rⁿ, R¹³ and R¹⁴ is independently selected from (Cl-Cδ)-alkyl; optionally substituted with one or several halogens;

R¹² is heterocyclyl; optionally having =O attached to a ring carbon atom;

as well as pharmaceutically acceptable salts thereof, for use as a medicament.

2. A compound according to claim 1 , wherein A is CO-alkyl.

3. A compound according to claim 1 or 2, wherein R² is a mono-, bi-, tri- or tetracyclic carbocyclyl or oxygen-containing heterocyclyl, optionally substituted by one or several moieties R⁴ as defined in claim 1.

4. A compound according to claim 3, wherein R² is phenyl, naphthyl, benzofuryl or

optionally substituted by one or several moieties R⁴ as defined in claim 1.

5. A compound according to any one of the claims 1 -4, wherein R³ is -S(O)₂-NH-phenyl, wherein the phenyl is optionally substituted with one or several moieties R⁵ as defined in claim 1.

6. A compound according to any one of the claims 1 -5, wherein any (C l -C6)-alkyl is selected from methyl, ethyl, n-propyl and iso-propyl.

7. A compound according to any one of the claims 1 -6, wherein any halogen is selected from F and Cl.

8. A compound according to any one of the claims 1 -7, wherein each R⁴ is independently selected from OH, CH₃-, C₃H₇-, naphtholyl, Cl, [CF₃C(O)] [(CH₃J₂(CH)OC(O)]CH-; CH₃C(O)-, CH₃OC(O)-, CH₃CH₂OC(O)-, CH₃C(O)NH-; CH₃O-; CH₃CH₂O-; nitro; 2-oxo- l,2- dihydrobenzo[cd]indole-6-sulfonamido; and =O attached to a ring carbon atom of the carbocyclyl or heterocyclyl.

9. A compound according to any of the claims 1-8, wherein each R⁵ is independently selected from CH₃-, CH₃O-, CH₃CH₂O-, CH₃C(O)NH-, and Cl.

10. A compound according to any one of the claims 1-9, wherein R² is selected from

and

1 1. A compound according to any of the claims 1-10, wherein R³ is selected from

12. A compound according to claim 1, wherein A is Cl-alkyl.

13. A compound according to claim 12, wherein R¹ is (Cl-C3)-alkyl.

14. A compound according to claim 12 or claim 13, wherein R² is carbocyclyl.

15. A compound according to claim 14, wherein R² is adamantyl.

16. A compound according to claim 1, selected from



17. A compound according to any one of the claims 1 - 16, or a pharmaceutically acceptable salt thereof, for use in the treatment of a disorder or disease selected from inflammatory diseases, fever, atheresclerosis, coronay artery disease, eczema, dermatitis, stroke, diabetes, autoimmune diseases, multiple sclerosis, arthritis and cancer.

18. A compound according to claim 17, wherein the disorder or disease is an inflammatory disease.

19. The use of a compound according to any one of the claims 1-16, for the manufacturing of a medicament for the treatment of a disorder or disease selected from inflammatory diseases, fever, atheresclerosis, coronay artery disease, eczema, dermatitis, stroke, diabetes, autoimmune diseases, multiple sclerosis, arthritis and cancer.

20. A pharmaceutical composition comprising a compound according to any of the claims 1 - 16 together with at least one pharmaceutically acceptable excipient.

21. A method of treatment of a mammal sufffering from a disorder or disease selected from inflammatory diseases, fever, atheresclerosis, coronay artery disease, eczema, dermatitis, stroke, diabetes, autoimmune diseases, multiple sclerosis, arthritis and cancer, by administration to said mammal a compound according to any one of the claims 1 - 16.