WO2009098282A1

WO2009098282A1 - Low molecular weight 2,5-disubstituted thiophene derivatives and use thereof in therapy

Info

Publication number: WO2009098282A1
Application number: PCT/EP2009/051365
Authority: WO
Inventors: Johan Wannberg; Mathias Alterman; Patric Stenberg; Jacob Westman
Original assignee: Novasaid Ab
Priority date: 2008-02-06
Filing date: 2009-02-06
Publication date: 2009-08-13

Abstract

A compound of formula (I). The compound is useful for the treatment of disorders such as pain, fever, inflammation and cancer. A method for preparing the compound.

Description

LOW MOLECULAR WEIGHT 2 , 5-DISUBSTITUTED THIOPHENE DERIVATIVES AND USE THEREOF IN THERAPY

Field of the invention

The present invention relates to 2,5-disubstituted thiophene derivatives and to the use thereof in disease therapy. More particularly, the present invention relates to 2,5-disubstituted thiophene 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 inflammatory conditions, 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, inflammatory conditions 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 a sequential process including liberation of arachi- donic acid, conversion into PGG2/PGH2 by cyclooxygenase (Cox) -1 or Cox-2 and wherein finally prostaglandin E synthase converts PGH₂ into PGE₂ (Fig 1). 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 MPGESl is inducible by proinflammatory cytokines. Initially, MPGESl was regarded as the enzyme predominantly coupled with Cox-2 activity. However; later results demonstrate that MPGESl 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 is that MPGESl 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 MPGESl 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 2). 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 prostacycline. In that case, increased prostacycline 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 re- garding the physiological role of MPGESl. 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-I beta or cytokine induced fever. 4) Finally, as of today, gross histopatho logical 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 MPGESl 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.

Summary of the invention

There still is a need for new and efficacious drugs for the treatment of conditions such as fever, pain and inflammation or inflammatory conditions, as well as cancer, and one main object of the present invention is to provide such drugs.

Therefore, according to a first aspect, the invention provides a compound of formula

(I)

(I) wherein

R¹ is halogen, phenyl-(C2-C10)-alkenyl, phenyl-C(O)NH-, phenyl-NHC(O)- and phenyl-SO2-NH-, mono-, bi- or tricyclic, saturated or unsaturated, aromatic or non-aromatic (C3-C12)-carbocyclyl or (Cl-C12)-heterocyclyl containing one or more heteroatoms selected from N, O and S;

R² and R³ are independently selected from H (hydrogen), saturated or unsaturated, non- aromatic, mono-, bi- or tricyclic (C3-C12)-carbocyclyl or (Cl-C12)-heterocyclyl containing one or more heteroatoms selected from N, O and S; and (C2-C10)-alkyl, optionally substituted with one or more moieties R⁴; R⁴ is selected from saturated or unsaturated, aromatic or non-aromatic, bridged or non- bridged, mono-, bi- or tricyclic (C3-C12)-carbocyclyl or (Cl-C12)-heterocyclyl containing one or more heteroatoms selected from N, O and S; (Cl-ClO)-alkyl-OC(O)-, R¹²R¹³N-; or R² and R³ form, together with the N to which they are bound, a ring of formula (II)

II wherein n is 1, 2 or 3; X is selected from >CHNR⁶R⁷, >CHC(O)OR⁶, >CH-(CH₂)-NHC(O)OR⁶, and >CHC(O)NR⁶R⁷;

R⁵ is linked to an adjacent atom of the ring of formula (II) so as to form a saturated or unsaturated, aromatic or non-aromatic (C1-C6) carbocycle or heterocycle containing one or more heteroatoms selected from N, O and S; or

R⁵ is selected from -R⁸, -C(O)OR⁸ and -C(O)NR⁸R⁹ ;

R⁶ and R⁷, and R⁸ and R⁹, respectively, together with the N to which they are bound, form a saturated 5-, 6-, or 7-membered heterocyclyl, optionally fused with an aromatic or non-aromatic (C3-C12)-carbocyclyl or (Cl-C12)-heterocyclyl containing one or more het- eroatoms selected from N, O and S; or

R⁶, R⁷, R⁸ and R⁹ are independently selected from H; saturated or unsaturated, aromatic or non-aromatic, bridged or non-bridged, mono-, bi- or tricyclic (C3-C12)-carbocyclyl or (Cl- C12)-heterocyclyl containing one or more heteroatoms selected from N, O and S; and (Cl- C10)-alkyl optionally substituted with one or more moieties R¹⁰; each R¹⁰ is independently selected from (Cl-ClO)-alkoxy, (Cl-ClO)-alkylthio, saturated or unsaturated, aromatic or non-aromatic, mono-, bi- or tricyclic (C3-C12)-carbocyclyl or (C3-C12)-heterocyclyl containing one or more heteroatoms selected from N, O and S; or R¹²R¹³N; wherein in any of R^R¹⁰ any phenyl, carbocyclyl or heterocyclyl is optionally substi- tuted with one or more substituents R¹¹ ;

R¹¹ is selected from halogen, -CN, -CF3, saturated or non-saturated, aromatic or non- aromatic (C3-C10) carbocyclyl or (Cl-ClO) heterocyclyl optionally, substituted with halogen; (Cl-ClO)-alkyl optionally substituted with one or more saturated or non-saturated, aromatic or non-aromatic (C3-C10) carbocyclyl or (Cl-ClO) heterocyclyl; (Cl-Cβ)-alkoxy, (C1-C3)- alkylendioxy, (Cl-C6)alkyl-NH-SO₂-, (Cl-C6)alkylC(O)NH- and R¹²R¹³N,

R¹² and R¹³ are independently selected from H, (Cl-Cβ)-alkyl, saturated or non- saturated, aromatic or non-aromatic (C3-C10)-carbocyclyl, optionally substituted by (C1-C6)- alkyl; as well as pharmaceutically acceptable salts thereof, for use as a medicament. According to one aspect of the invention, a pharmaceutical composition is provided, comprising a therapeutically effective amount of a compound as defined herein above, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipi- ent. In one embodiment of the invention, the pharmaceutical composition is the treatment of a disorder selected from pain, fever, inflammation and inflammatory conditions and cancer.

According to one aspect of the invention, there is provided the use of a compound as defined herein above for preparing a medicament for the treatment of a disorder selected from pain, fever, inflammation and other inflammatory conditions and cancer. In one embodiment of the invention, the disorder is inflammation. In one embodiment of the invention, the disorder is rheumatoid arthritis. According to one aspect of the invention, there is provided a method of treatment of a disease selected from pain, fever, inflammation and other inflammatory conditions, and can- cer by administration of a therapeutically effective amount of a compound as defined herein above, or a pharmaceutically acceptable salt or prodrug thereof to a mammal in the need of such treatment.

According to one aspect, the invention relates to a method of preparing a compound of formula (I) as defined herein above. In one embodiment, the method comprises

(a) reacting 2-bromo-thiophene-2-carboxylic acid with thionyl chloride, reacting the 2- bromo-thiophene-2-carboxylic acid chloride obtained an amine NHR²R³ in the presence of a base in a suitable solvent so as to obtain a compound of formula (IV)

IV

(cl) reacting the compound of formula (IV) with R¹B(OH)₂ in the presence of bis(tri- teτt-butylphosphine)palladium(O); or

(c2) heating the compound of formula (IV) in the presence of a catalyst, such as CuI, a base such as CS₂CO₃, an N-H containing reactant/reagent, such as a primary amide or primary sulfonamide, or N-H containing heteroaromatic, such as pyrroles or indoles, and a solvent such as 1 ,4-dioxane or toluene, so as to obtain a compound of formula

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

In another embodiment, the method comprises (cl) reacting S-bromo-thiophene^-carboxylic acid ester, e.g. a (Cl-C3)-ester, with

R¹B(OH)₂ in the presence of bis(tri-tert-butylphosphine)palladium(0); or

(c2) heating 5-bromo-thiophene-2-carboxylic acid ester, e.g. a (Cl-C3)-ester, in the presence of a catalyst, such as CuI, a base such as CS2CO3, an N-H containing reac- tant/reagent, such as a primary amide or primary sulfonamide, or N-H containing heteroaro- matic, such as pyrroles or indoles, and a solvent such as 1,4-dioxane or toluene, and hydro lys- ing the ester function, so as to obtain a compound of formula (V)

V

(b) reacting the compound of formula (V) with NHR²R³ in the presence of o-(7- azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N- diisopropylethylamine so as to obtain a compound of formula (I)

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

Brief description of the drawings

Figure 1. Biosynthesis of prostaglandin E2 (A). Hypothetic effect of MPGESl blockage shunting into the anti- inflammatory PGD2 pathway and formation of cyclopentenons. Figure 2. Schematic representation of biochemical pathway in TBA-MDA Assay.

Figure 3. Net paw swelling (in 0.01 ml) of test animals at number of days after treatment with Complete Freund's Adjuvant (CFA).

Detailed description of the invention

In the structural formulae of various moieties in the compounds of the invention, the sign " ^~* ", as used herein, serves to indicate a covalent bond from the moiety in question to the point of attachment on the remaining part of the compound. For example the structural formula

represents an indolyl moiety having a methyl group in the 2-position and linked to the rest of the compound through a covalent bond to its N atom, viz. a 2 -methyl- 1 -indolyl moiety.

As used herein the term "Cn-Cm-", e.g. Cl-ClO or C1-C6, or any other combination where m > n, refers to a moiety having from n to m carbon atoms. Thus, a "(Cl-ClO)-alkyP' is an alkyl group having between one and ten carbon atoms, and a "(Cl-ClO)-alkoxy" is an alkoxy moiety having from one to ten carbon atoms in the alkyl moiety. The term "Cn-" refers to any specific moiety containing n carbon atoms.

Unless otherwise indicated or apparent from the context, any alkyl, alkenyl or alkynyl group as referred to herein may be branched or unbranched. This also applies said groups when present in moieties such as alkoxy groups etc.

It is contemplated that any branched, linear or cyclic moiety may be attached to another part of the molecule of formula (I) by a bond to any location on the moiety which is available for such binding. The term "saturated", as used herein, e.g. in respect of any moiety, indicates that the moiety contains no double or triple bonds. The term "unsaturated", on the other hand, means that the moiety contains one or several double or triple bonds.

The term "aromatic", as used herein, refers to an unsatured 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.

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 at- oms, but also at least one other atom in the ring structure, e.g. a nitrogen, sulphur or oxygen atom.

The term "cyclic", as used herein in respect of an atom, refers to an atom that is a member of at least one ring in a carbocycle or heterocycle. For example, pyridine contains five cyclic C and one cyclic N.

As used herein with respect to any carbocyclyl or heterocyclyl, the term monocyclic refers to a cyclic moiety containing only one ring. The term bicyclic refers to a cyclic moiety containing two rings, fused to each other, and the term tricyclic refers to a cyclic moiety containing three rings, fused to each other. A (Cl-ClO)-alkyl according to the invention may be selected e.g. from methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, heptyl, 2- methylhexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 3,3-dimethylpentyl, 3-ethylpentyl, 2,2,3-trimethylbutyl, octyl, nonyl, decyl etc. A (C3-C12)-carbocyclyl according to the invention may be e.g. cyclopropyl, cyclobu- tyl, cyclohexyl, cycloheptyl, cycloctyl, cyclononyl or cyclodecyl, or e.g. several of these fused together so as to form a polycyclic carbocyclyl, e.g. decaline.

An aromatic carbocyclyl according to the invention may be selected from phenyl or naphthyl. In a polycyclic (bi- or tricyclic) moiety according to the invention, each constituent monocycle may independently be selected from saturated, unsaturated and aromatic and non- aromatic carbo- and hetero cycles.

An aromatic heterocyclyl (i.e. a heteroaromatic moiety) according to the invention may be selected from e.g. a pyridyl, pyrrolyl, quinolinyl, furanyl, thienyl, oxadiazolyl, thiadiazolyl, thiazolyl, oxazolyl, pyrazolyl, triazolyl, tetrazolyl, tetrahydroquinolinyl, tetrazolyl, thiadiazolyl, thiazolyl, thiochromanyl, thienyl, triazolyl, isoxazolyl, isothiazolyl, isoquinolinyl, naphthyridinyl, imidazolyl, pyrimidinyl, phenazinyl, phenothiazinyl, phthalazinyl, indolyl, pyrazolyl, pyridazinyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinolizinyl, quinoxa- linyl, tetrahydroisoquinolinyl, pyrazinyl, indazolyl, indolinyl, indolyl, pyrimidinyl, thio- phenetyl, pyranyl, carbazolyl, chromanyl, cinnolinyl, acridinyl, quinolinyl, benzimidazolyl, benzodioxanyl, benzodioxepinyl, benzodioxolyl, benzo furanyl, benzothiazolyl, benzoben- zoxadiazolyl, benzothiazolyl, benzoxazinyl, benzoxazolyl, benzimidazolyl, benzo mor- pholinyl, benzoselenadiazolyl, benzothienyl, purinyl, cinnolinyl pteridinyl and the like. Examples of non-aromatic heterocyclyl according to the present invention may be selected from e.g. aziridinyl, azetidinyl, dihydropyranyl, dihydropyridyl, dihydropyrrolyl, di- oxolanyl, dioxanyl, dithianyl, dithiolanyl, imidazolidinyl, imidazolinyl, morpholinyl, oxetanyl, oxiranyl, pyrrolidinyl, pyrrolidinonyl, piperidyl, piperazinyl, piperidinyl, pyranyl, pyrazolidinyl, quinuclidinyl, sulfalonyl, 3-sulfolenyl, tetrahydrofuranyl tetrahydropyranyl tetrahydropyridyl, thietanyl, thiiranyl, thiolanyl, thiomorpholinyl, trithianyl, tropanyl and monosaccharide.

As used herein the term "halogen" means a fluorine, chlorine, bromine or iodine, unless otherwise indicated or apparent from the context. In formula (I), R¹ is selected from halogen, e.g. Cl or Br; phenyl-(C2-C10)-alkenyl, e.g. phenyl-(C2-C6)-alkenyl, or phenyl-(C2-C4)-alkenyl; phenyl-C(O)NH-; phenyl-NHC(O)-; phenyl-SO2-NH-; and mono-, bi- or tricyclic, saturated or unsaturated, aromatic or non- aromatic (C3-C12)-carbocyclyl, e.g. (C6-C10)-carbocyclyl, or (Cl-C12)-heterocyclyl, e.g. (C3-C12)-heterocyclyl, containing one or more heteroatoms selected from N, O and S. In one embodiment of the invention, R¹ is mono-, bi- or tricyclic carbocyclyl or heterocyclyl, comprising at least one aromatic cycle.

In one embodiment of the invention, R¹ is mono-, bi- or tricyclic carbocyclyl or heterocyclyl, comprising at least one phenyl.

In one embodiment, when R¹ is heterocyclyl, it contains one or two heteroatoms. For example, R¹ may comprise 1 or 2 cyclic N and/or O and/or S.

In one embodiment, R¹ is selected from phenyl, naphthyl, 1,2-diazolyl, benzo morpholinyl, indolyl, benzimidazol, quinolinyl, benzothiophenyl and carbazolyl.

In another embodiment, R¹ is halogen, e.g. Cl or Br, in particular Br.

Any cyclic moiety in R¹ may be substituted with one or more, e.g. 2 or 3, groups R¹¹, as defined herein above. For example, in one embodiment, R¹¹ is selected from (Cl-C4)-alkyl, or from (Cl-C4)-alkoxy, halogen-substituted (Cl-C4)-alkyl, e.g. CF3-, (C1-C4)- alkylNHS(O)2-, (Cl-C4)-alkylC(O)NH-, C6H5-, CN-, and halogen, whereby any of the alkyl moieties in the groups R¹¹ may be selected from methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso -butyl and tert-butyl. In one embodiment, any cyclic moiety in R¹ is substituted with one or more groups R¹¹ selected from CH₃-, CH₃O-, CF₃-, (CH₃)₂CH- (CH₃)₃CNHS(O)₂-, CH₃C(O)NH-, (CH₃)₃C-, CH₃CH₂-, C₆H₅-, CN-, Cl-, and F-.

In one embodiment, R¹ is selected from Br- and from the following moieties:

In one embodiment of the invention, in the moiety NR²R³, R² and R³ are either independently selected from H, saturated or unsaturated, non-aromatic, mono-, bi- or tricyclic (C3-C12)-carbocyclyl, e.g. (C3-C8)-carbocyclyl, or (C5-C7)-carbocyclyl, such as C6- carbocyclyl, or (Cl-C12)-heterocyclyl, e.g. (C3-C9)-heterocyclyl, such as (C4-C5)- heterocyclyl, containing one or more heteroatoms selected from N, O and S; and (Cl-ClO)- alkyl, e.g. (Cl-C6)-alkyl, or (Cl-C4)-alkyl, e.g. methyl, ethyl or propyl, optionally substituted with one or more moieties R⁴, as defined herein above.

When any of R² and R³ is (C2-C6)-alkyl, it may be selected from e.g. ethyl, n-propyl, i- propyl, n-butyl, i-butyl, tert-butyl and 3,3-dimethylbutyl.

Any of R² and R³ may also be selected from e.g. cyclo-propyl, cyclo-butyl, cyclo- pentyl, cyclo-hexyl, cyclo-heptyl and cyclo-octyl.

In one embodiment, R² and/or R³ are independently selected from bicyclic heterocyclyl comprising phenyl fused with a heterocycle. As an example, the bicyclic heterocyclyl may comprise phenyl fused with a heterocycle comprising 1 or 2 heteroatoms independently selected from N, O and S.

In one embodiment, R² is H. In one embodiment, NR 2r R> 3 is selected from

R⁴ in the compound of formula (I), as defined herein above, is selected from saturated or unsaturated, aromatic or non-aromatic, bridged or non-bridged, mono-, bi- or tricyclic (C3- C12)-carbocyclyl, or (Cl-C12)-heterocyclyl containing one or more heteroatoms selected from N, O and S; (Cl-ClO)-alkyl-OC(O)-, e.g. (Cl-C6)-alkyl-OC(O)-, (Cl-C4)-alkyl- OC(O)-, or (Cl-C3)-alkyl-0C(0)-, and R¹²R¹³N-, wherein R¹² and R¹³ are as defined herein above.

In one embodiment, R⁴ is selected from monocyclic (C3-C8)-carbocyclyl, or (C5-C7)- carbocyclyl, e.g. C5-or C6 carbocyclyl, or (Cl-C12)-heterocyclyl, e.g. (C4-C8)-heterocyclyl, containing one or more heteroatoms selected from N, O and S.

In one embodiment, R⁴ is selected from piperidinyl, cyclohexenyl, phenyl, furanyl, tet- rahydrofuranyl, piperazinyl, cyclopentyl, tricyclo[3.3.1.1³'⁷]decyl, ethoxycarbonyl, pyridinyl, benzodioxanyl and dimethylamino.

In one embodiment, R⁴ is R¹²R¹³N-, wherein R¹² and R¹³ are independently selected from H, (Cl-C6)-alkyl, or (Cl-C4)-alkyl, e.g. (Cl-C3)-alkyl, such as methyl and ethyl, and saturated or non-saturated, aromatic or non-aromatic (C3-C8)-carbocyclyl, e.g. phenyl, optionally substituted by one or more groups selected from (Cl-Cβ)-alkyl, e.g. (Cl-C4)-alkyl, e.g. (Cl-C3)-alkyl, such as methyl and ethyl.

It should be noted that any cyclic moiety of R⁴ may be substituted with one or more groups R¹¹ as defined herein above. In one embodiment, any cyclic moiety of R⁴ is substituted with one or more groups R¹¹, selected from halogen, such as Cl, (Cl-C4)-alkyl, such as methyl; phenyl, optionally, substituted with halogen or methyl.

In one embodiment of the invention, R² and R³ form, together with the N to which they are bound, a ring of formula (II)

II

wherein R5, X and n are as defined herein above. The compound of formula (I) may then be represented by formula (I ')

r

wherein n, X, R¹ and R⁵ are as defined herein above. In one embodiment of the invention, in a compound of formula (Y), X is selected from

>CHC(O)OR⁶, and >CHC(O)NR⁶R⁷,.

In a particular embodiment X is >CHC(O)NR _> 6r R, 7 whereby a compound of the invention is represented by formula (I")

I"

wherein n, R¹, R⁵, R⁶ and R⁷ are as defined herein above.

In the compound of formula (I), R⁵ is selected from -R⁸, -C(O)OR⁸ and -C(O)NR⁸R⁹. In one embodiment, R⁵ is H or (Cl-C6)-alkyl, such as (Cl-C3)-alkyl, e.g. methyl, in particu- lar R⁵ is H. In another embodiment, R⁵ is linked to an adjacent atom of the ring of formula (II) so as to form a saturated or unsaturated, aromatic or non-aromatic (C3-C6)-carbocycle or (C1-C5)- heterocycle containing one or more heteroatoms selected from N, O and S, in particular an aromatic 6-membered carbocycle or heterocycle, e.g. phenyl, forming a fused ring system with the nitrogen containing ring of formula (II).

In still a further embodiment of the invention, R⁵ is -COOR⁸, wherein R⁸ is H or (Cl- C6)-alkyl, such as (Cl-C3)-alkyl, e.g. methyl or ethyl.

In still a further embodiment of the invention, R⁵ is -C(O)NR⁸R⁹, wherein R⁸ and R⁹ are independently selected from H; and (Cl-Cβ)-alkyl, e.g. (Cl-C3)-alkyl, such as methyl, op- tionally substituted with one or more moieties R¹⁰.

R⁶ and R⁷, together with the N to which they are bound, may form a saturated 5-, 6-, or 7-membered heterocyclyl, optionally fused with an aromatic or non-aromatic (C3-C12)- carbocyclyl or (Cl-C12)-heterocyclyl containing one or more heteroatoms selected from N, O and S; or may be independently selected from H; saturated or unsaturated, aromatic or non- aromatic, bridged or non-bridged, mono-, bi- or tricyclic (C3-C12)-carbocyclyl or (C1-C12)- heterocyclyl containing one or more heteroatoms selected from N, O and S; and (Cl-ClO)- alkyl optionally substituted with one or more moieties R¹⁰, as defined herein.

In one embodiment of the invention, when R⁶ and/or R⁷ are selected from monocyclic (C3-C12)-carbocyclyl, at least one of them is selected from (C3-C8)-cycloalkyl and phenyl.

In one embodiment, when R⁶ and/or R⁷ are (Cl-ClO)-alkyl, at least one of them is selected from (Cl-Cβ)-alkyl, in particular from (Cl-C4)-alkyl.

In one embodiment of the invention, at least one of R⁶ and R⁷ is H.

In one embodiment, R⁶ and/or R⁷ are selected from (Cl-C6)-alkyl, in particular (Cl- C4)-alkyl, e.g. methyl or ethyl, substituted with R¹⁰ as defined herein above.

In one embodiment, R¹⁰ is selected from saturated or unsaturated, aromatic or non- aromatic, mono- or bicyclic (C3-C10)-carbocyclyl, in particular phenyl, and (C3-C9)- heterocyclyl, containing one or more heteroatoms independently selected from N, O and S, e.g. 1 or 2 heteroatoms. In one embodiment, R¹⁰ is phenyl.

In one embodiment, when R¹⁰ is (Cl-ClO)-alkoxy or (Cl-ClO)-alkylthio, the alkyl portion thereof in particular is (Cl-C6)-alkyl, more particularly (Cl-C4)-alkyl. In one embodiment, R¹⁰ is selected from phenyl, butylthio, thiophenyl, pyridinyl, fu- ranyl, tetrafuranyl, perhydroazocinyl, azolidinyl, cyclohexenyl, diethyleneamino, methoxy, ethoxy, i-propoxy, indolyl, N-methyl-N-cyclohexylamino.

Any cyclic moiety in R¹⁰ may be substituted with one or more groups R¹¹. In the compounds as defined herein above, any carbocyclyl, including phenyl, or het- erocyclyl may be substituted with a moiety R¹¹, selected from halogen, preferably Cl and F, - CN, -CF₃, saturated or non-saturated, aromatic or non-aromatic (C3-C10)-carbocyclyl, preferably phenyl or (Cl-ClO)-heterocyclyl, preferably (C3-C6)-heterocyclyl, optionally substituted with halogen; (Cl-ClO)-alkyl, preferably (Cl-C6)-alkyl, optionally substituted with one or more saturated or non-saturated, aromatic or non-aromatic (C3-C10), carbocyclyl, preferably phenyl, or (Cl-ClO) heterocyclyl; (Cl-C6)-alkoxy, preferably (Cl-C4)-alkoxy, (C1-C3)- alkylendioxy, (Cl-C6)-alkyl-NH-SO₂-, preferably (Cl-C3)-alkyl-NH- SO₂-, (Cl- C6)alkylC(O)NH-, (Cl-C3)alkylC(O)NH- and R¹²R¹³N.

In one embodiment, when R¹¹ is selected from (Cl-C6)-alkyl, it more preferably is se- lected from (C 1 -C4)-alkyl.

In one embodiment, when R¹¹ is selected from (Cl-C4)-alkoxy, it more preferably is selected from (Cl-C3)-alkoxy, and preferably is methoxy or ethoxy.

In one embodiment, when R¹¹ is (Cl-ClO) substituted with phenyl, it is benzyl.

In one embodiment of the invention, in R¹²R¹³N, R¹² and R¹³ are independently se- lected from H, (Cl-C6)-alkyl, more preferably (Cl-C3)-alkyl, and phenyl, optionally substituted with R¹¹.

In one embodiment, when NR²R³ forms a cycle of formula (II), it is selected from any of the following moieties:

The compounds represented in the following table exemplify the invention. These compounds were synthesized following the schemes, general procedures and examples described below.

Example R¹ NR²R³ [M+H]⁺(ESI+)

172 422

The compounds may be synthesized by common methods well known for the person skilled in the art or by methods described herein below. Methods of synthesis

The compounds of Formula I of the invention can be prepared according to the synthetic routes outlined in Scheme 1 below and by following the methods outlined therein.

With reference to Scheme 1, the 5-bromo-thiophene-2-carboxamides (ii) may be pre- pared from S-bromo-thiophene^-carboxylic acid (i) according to General procedure A, whereafter compounds of formula (I) may be prepared from (ii) by displacement of the bromine according to General procedure Cl or General procedure C2.

Alternatively, compounds of formula (I) can be prepared by ester hydrolysis of (iv) (for example by stirring (iv) and NaOH in a solvent or solvent mixture, such as ethano I/water) followed by amide coupling according to General procedure B. Compounds (iv) may be prepared from 5-bromo-thiophene-2-carboxylic acid esters (iii) by displacement of the bromine according to General procedure Cl or General procedure C2.

A compound (ii) as obtained according to General procedure A, or a compound of formula (I), obtained by a sequence of steps as outlined herein above, may be further reacted at the NR²R³ function (i.e. "Functional group manipulation") to transform it into still another compound of (ii) or of formula (I), respectively. E.g. a compound wherein X is >CHC(O)OR⁶ may be converted to a compound wherein X is >CHC(O)NR⁶R⁷, by hydrolysis of the ester function (in case R⁶ is not H), followed by amide coupling according to General procedure B.

Scheme 1

General procedure A: Synthesis of amides from carboxylic acids via the acid chlorides

Thionyl chloride (0.5-1 mL/mmol) is added to S-bromothiophene-l-carboxylic acid (i) (1.0 equiv.) and the mixture is heated at 80⁰C for 1 h. Excess SOCl₂ is then removed by repeated co-evaporation with toluene. The resulting crude acid chloride (1.0 equiv.) is dissolved in dichloromethane (~5 mL/mmol) and added dropwise to a cool solution (0-10 ⁰C) of a primary or secondary amine (1.0 equiv.) and a base (1.5 equiv. e.g. triethylamine or N,N- diisopropylethylamine) in dichloromethane (~10 mL/mmol). The mixture is stirred at room temperature for about 1 h and then diluted with additional dichloromethane. The mixture is washed with 2M HCl (aq.) (2x) and sat. NaHCO₃ (aq.) (2x), dried with MgSO₄ and evapo- rated, to give (ii). If required, the product (ii) is purified by silica chromatography using ethyl acetate/isohexane or dichloromethane/methanol mixtures.

General procedure B: Conversion of carboxylic acids to the corresponding amides using HATU as coupling reagent

Thiophene-2-carboxylic acid (iv) (1.0 equiv.) is dissolved in dichloromethane (~10 mL/mmol). To this solution is added HATU [o-(7-Azabenzotriazol-l-yl)-N,N,N',N'- tetramethyluronium hexafluorophosphate; CAS Registry Number: 148893-10-1] (1.0 equiv.), N,N-diisopropylethylamine (1.5 equiv.) and a primary or secondary amine (1.0 equiv.). The mixture is stirred at room temperature for about 4 h and the amide product purified by silica chromatography or preparative RP-LC-MS as indicated in the specific examples below to give (I).

General procedure Cl : Suzuki coupling conditions

A mixture of a 5-bromothiophene derivative (iii) or (ii) (1.0 equiv.), a boronic acid (3.0 equiv.), bis(tri-tert-butylphosphine)palladium(0) (0.10 equiv.), K₂CO₃ (3.0 equiv.), H₂O (~3 mL/mmol) and 1 ,2-dimethoxyethane (~10 mL/mmol) in a suitable microwave vial is irradi- ated to 120 ⁰C for 10 min. The reaction mixture is then diluted with dichloromethane (~30 mL/mmol) and the organic layer filtered through a pad of celite and MgSO₄. Solvents are evaporated under reduced pressure and the residue is purified by silica chromatography or preparative RP-LC-MS as indicated in the specific examples below to give (iv) or (I), respectively. General procedure C2: Cu-catalyzed coupling conditions

A mixture of a 5-bromothiophene derivative (iii) or (ii) (1.0 equiv), an N-H containing reactant/reagent (such as primary amides or primary sulfonamides or N-H containing het- eroaromatics, such as pyrroles or indoles), CuI (0.1 equiv), Cs₂CO₃ (0.1 equiv), N,N'- dimethylethylenediamine (2.0 equiv) and a solvent such as 1,4-dioxane or toluene in a suit- able microwave vial is irradiated to 120 ⁰C for 10-48 h. After cooling to room temperature, the reaction mixture is filtered through a pad of celite and silica and the combined filtrate is concentrated under reduced pressure. The residue is purified by silica chromatography or preparative RP-LC-MS as indicated in the specific examples below to give (iv) or (I), respec- tively.

EXAMPLES

The invention is illustrated by the following non-limiting examples: Example 44 1 -r5-(2-Methyl-indol- 1 -yl)-thiophene-2-carbonyll-piperidine-4-carboxylic acid butylamide

Step 1 : 5-(2-Methyl-indol-l-yl)-thiophene-2-carboxylic acid ethyl ester A screw cap reaction tube was charged with 5-bromo-thiophene-2-carboxylic acid ethyl ester (0.941 g, 4.00 mmol), 2-methylindole (0.525 g, 4.00 mmol), CuI (76 mg, 0.040 mmol), Cs₂CO₃ (2.6 g, 8.0 mmol), N,ΛT-dimethylethylenediamine (35 mg, 0.40 mmol) and toluene (4 mL). The tube was capped and the mixture stirred at 110 ⁰C for 20 h. After cooling to room temperature, the reaction mixture was filtered through a pad of celite (1 cm) and silica (2 cm) (pad eluted with 10 mL of isohexane/ethyl acetate 1 :1) and the combined filtrate was concentrated under reduced pressure. The residue was purified by reversed phase column chromatog- raphy (C18-silica, manual gradient of 50-100% acetonitrile in water). Product containing fractions were concentrated and the residue further purified by silica chromatography (5-10% ethyl acetate in isohexane). Pure fractions were concentrated and dried under vacuum to give the sub-title compound (515 mg, 45%).

Step 2: l-[5-(2-Methyl-indol-l-yl)-thiophene-2-carbonyll-piperidine-4-carboxylic acid ethyl ester

5-(2-Methyl-indol-l-yl)-thiophene-2-carboxylic acid ethyl ester (0.500 g, 1.75 mmol) was dissolved in ethanol (10 mL) and 1 M NaOH (aq.) (10 mL, 10 mmol) was added and the reaction was stirred at room temperature for 5 h. The reaction mixture was carefully evaporated to approx. half the volume and then acidified with 2 M HCl (aq.) (~10 mL) and ex- tracted with dichloromethane (3x40 niL). The combined organic layers were dried (MgSO₄) and evaporated to give crude 5-(2-Methyl-indol-l-yl)-thiophene-2-carboxylic acid. The acid was then converted to l-[5-(2-Methyl-indol-l-yl)-thiophene-2-carbonyl]-piperidine-4- carboxylic acid ethyl ester using piperidine-4-carboxylic acid ethyl ester (a compound of for- mula (I), wherein X is CHC(O)OH) according to General procedure B. Purification was performed by silica chromatography (0.8% methanol in dichloromethane) to give the subtitle compound of formula (I), wherein X is CHC(O)OEt (0.666 g, 96%).

Step 3: l-[5-(2-Methyl-indol-l-yl)-thiophene-2-carbonyll-piperidine-4-carboxylic acid butylamide 1- [5 -(2-Methyl-indol-l-yl)-thiophene-2-carbonyl] -piperidine-4-carboxylic acid ethyl ester 0.658 g, 1.66 mmol) was dissolved in ethanol (20 mL), 0.4 M NaOH (aq.) (20 mL, 8.3 mmol) was drop wise added and the reaction was stirred at room temperature for 1 h. The reaction mixture was carefully evaporated to approx. half the volume and then acidified with 2 M HCl (aq.) (~10 mL) and extracted with dichloromethane (3x50 mL). The combined organic layers were washed with brine (20 mL), dried (MgSO₄) and evaporated to give crude l-[5-(2- Methyl-indol-l-yl)-thiophene-2-carbonyl] -piperidine-4-carboxylic acid. The acid was then converted to l-[5-(2-Methyl-indol-l-yl)-thiophene-2-carbonyl]-piperidine-4-carboxylic acid butylamide according to General procedure B. Purification was performed by silica chromatography (1.5-2.5% methanol in dichloromethane) to give the title compound (0.617 g, 88%) of formula (I) wherein X is CHC(O)NH(C₄H₉). H NMR (400 MHz, CDCl₃): δ 7.54-7.50 (m, IH), 7.29 (d, IH), 7.27-7.24 (m, IH), 7.15-7.11 (m, IH), 6.96 (d, IH), 6.40 (t, IH), 5.54-5.48 (m, IH), 4.56-4.47 (m, 2H), 3.30-3.24 (m, 2H), 3.16-3.05 (m, 2H), 2.44-2.35 (m, 4H), 1.99- 1.91 (m, 2H), 1.88-1.76 (m, 2H), 1.53-1.45 (m, 2H), 1.40-1.30 (m, 2H), 0.93 (t, 3H).

Example 158 l-(5-Bromo-thiophene-2-carbonyl)-piperidine-4-carboxylic acid ethyl ester

5-Bromo-thiophene-2-carboxylic acid (2.07 g, 10 mmol) was converted to l-(5-Bromo- thiophene-2-carbonyl)-piperidine-4-carboxylic acid ethyl ester using piperidine-4-carboxylic acid ethyl ester (1.57 g, 10.0 mmol) according to General procedure A (no chromatography, 3.19 g, 92%). H NMR (400 MHz, CDCl₃): δ 7.03 (d, IH), 6.99 (d, IH), 4.33-4.26 (m, 2H), 4.16 (q, 2H), 3.21-3.07 (m, 2H), 2.64-2.54 (m, IH), 2.03-1.92 (m, 2H), 1.81-1.70 (m, 2H), 1.26 (t, 3H).

Example 159

1 -(5-Bromo-thiophene-2-carbonyl)-piperidine-4-carboxylic acid butylamide

l-(5-Bromo-thiophene-2-carbonyl)-piperidine-4-carboxylic acid ethyl ester (see exam- pie 195, 0.960 g, 2.77 mmol) was dissolved in ethanol (10 mL) and 1 M NaOH (aq.) (10 mL, 10 mmol) was dropwise added and the reaction was stirred at room temperature until complete hydrolysis was observed (45 min in this case). The reaction mixture was carefully evaporated to approx. half the volume and then acidified with 2 M HCl (aq.) (~10 mL) and extracted with dichloromethane (2x40 mL). The combined organic layers were washed with brine (20 mL), dried (MgSO₄) and evaporated to give crude l-(5-Bromo-thiophene-2- carbonyl)-piperidine-4-carboxylic acid. The acid was then converted to l-(5-Bromo- thiophene-2-carbonyl)-piperidine-4-carboxylic acid butylamide using butylamine according to General procedure B (no chromatography, 1.06 g, 103%). NMR (400 MHz, CDCl₃): δ 7.03 (d, IH), 6.99 (d, IH), 5.55-5.48 (m, IH), 4.45-4.35 (m, 2H), 3.29-3.21 (m, 2H), 3.09-2.96 (m, 2H), 2.40-2.31 (m, IH), 1.94-1.86 (m, 2H), 1.81-1.70 (m, 2H), 1.52-1.43 (m, 2H), 1.39-1.29 (m, 2H), 0.92 (t, 3H).

Example 160

1 -(5-Bromo-thiophene-2-carbonyl)-piperidine-4-carboxylic acid cyclopentylamide

l-(5-Bromo-thiophene-2-carbonyl)-piperidine-4-carboxylic acid (see example 196, 0.64 g, 2.0 mmol) was converted to l-(5-Bromo-thiophene-2-carbonyl)-piperidine-4-carboxylic acid cyclopentylamide using cyclopentylamine according to General procedure B (0.557 g, 72%). NMR (400 MHz, CDCl₃): δ 7.03 (d, IH), 6.99 (d, IH), 5.47-5.40 (m, IH), 4.46-4.36 (m, 2H), 4.24-4.15 (m, IH), 3.09-2.96 (m, 3H), 2.37-2.28 (m, IH), 2.04-1.95 (m, 2H), 1.93- 1.85 (m, 2H), 1.81-1.55 (m, 6H), 1.39-1.29 (m, 2H).

Example 161

1 -(5-Benzoylamino-thiophene-2-carbonyl)-piperidine-4-carboxylic acid butylamide

According to General procedure C2, l-(5-Bromo-thiophene-2-carbonyl)-piperidine-4- carboxylic acid butylamide (see example 196, 37.3 mg, 0.10 mmol), benzamide (14.5 mg, 0.12 mmol), CuI (3.8 mg, 0.020 mmol), Cs₂CO₃ (130 mg, 0.40 mmol) and NJf- dimethylethylenediamine (1.8 mg, 0.020 mmol) and 1,4-dioxane (1 mL) were added to a reac- tion vial. The mixture was flushed with N₂, the vial was capped and the reaction heated at 110 ⁰C over night (~17 h). The reaction was allowed to cool to room temperature and the mixture was filtered through celite and solvents evaporated. The residue was purified by preparative RP-LC-MS (C8 column 150x21.2 mm, using a 15 niL/min CH₃CN/H₂O gradient (0.05% HCOOH) with UV-triggered fraction collection (254 nm) and MS (ESI+) detection. Evapora- tion of pure fractions and drying under vacuum over night yielded the title compound. (11.3 mg, 27%). ¹H NMR (400 MHz, CD₃OD/CDC1₃ 1 : 1): δ 7.97-7.93 (m, 2H), 7.58-7.53 (m, IH), 7.51-7.46 (m, 2H), 7.20 (d, IH), 6.86 (d, IH), 4.51-4.44 (m, 2H), 4.23 (s, IH), 3.16 (t, 2H), 3.11-3.01 (m, 2H), 2.50-2.41 (m, IH), 1.89-1.66 (m, 4H), 1.51-1.42 (m, 2H), 1.37-1.27 (m, 2H), 0.91 (t, 3H). Example 164

1 -(5-Phenyl-thiophene-2-carbonyl)-piperidine-4-carboxylic acid butylamide

l-(5-Bromo-thiophene-2-carbonyl)-piperidine-4-carboxylic acid butylamide (see exam- pie 196, 37.3 mg, 0.10 mmol) was converted to l-(5-Phenyl-thiophene-2-carbonyl)- piperidine-4-carboxylic acid butylamide using phenylboronic acid according to General procedure Cl. The residue was purified by preparative RP-LC-MS (as described for example 200) to give the title compound (23.4 mg, 63%). H NMR (400 MHz, CDCl₃): δ 7.62-7.58 (m, 2H), 7.42-7.36 (m, 2H), 7.34-7.29 (m, 1H),7.25 (d, IH), 7.21 (d, IH), 5.62-5.54 (m, IH), 4.54-4.44 (m, 2H), 3.29-3.23 (m, 2H), 3.10-3.00 (m, 2H), 2.42-2.33 (m, IH), 1.95-1.88 (m, 2H), 1.85-1.73 (m, 2H), 1.52-1.44 (m, 2H), 1.39-1.29 (m, 2H), 0.92 (t, 3H).

Example 185 l-r5-(2-tert-Butylsulfamoyl-phenyl)-thiophene-2-carbonyll-piperidine-4-carboxylic acid

l-(5-Bromo-thiophene-2-carbonyl)-piperidine-4-carboxylic acid cyclopentylamide (see example 197, 38.5 mg, 0.100 mmol) was converted to l-[5-(2- tert-Butylsulfamoyl-phenyl)- thiophene-2-carbonyl]-piperidine-4-carboxylic acid cyclopentylamide using 2-tert- Butylsulfamoyl-phenyl boronic acid according to General procedure Cl. The residue was purified by preparative RP-LC-MS (as described for example 200) to give the title compound (22.9 mg, 44%). H NMR (400 MHz, CDCl₃): δ 8.19 (dd, IH), 7.59-7.45 (m, 3H), 7.42 (d, IH), 7.26 (d, IH), 5.49 (br d, IH), 4.53-4.42 (m, 2H), 4.20(sext, IH), 3.99 (s, IH), 3.15-3.00 (m, 2H), 2.40-2.30 (m, IH), 2.04-1.87 (m, 4H), 1.84-1.73 (m, 2H), 1.70-1.55 (m, 4H), 1.40- 1.30 (m, 2H), 1.04 (s, 9H). Example 187

1 -[5-(2-Isopropyl-phenyl)-thiophene-2-carbonyll-piperidine-4-carboxylic acid cyclopentyla- mide

l-(5-Bromo-thiophene-2-carbonyl)-piperidine-4-carboxylic acid cyclopentylamide (see example 197, 38.5 mg, 0.100 mmol) was converted to l-[5-(2-Isopropyl-phenyl)-thiophene-2- carbonyl]-piperidine-4-carboxylic acid cyclopentylamide using 2-isopropylphenyl boronic acid according to General procedure Cl. The residue was purified by preparative RP-LC-MS (as described for example 200) to give the title compound (21.1 mg, 50%). H NMR (400 MHz, CDCl₃): δ 7.41-7.34 (m, 2H), 7.32-7.28 (m, IH), 7.25 (d, IH), 7.22-7.17 (m, IH), 6.90 (d, IH), 5.48 (br d, IH), 4.56-4.47 (m, 2H), 4.21(sext, IH), 3.27 (sept, IH), 3.11-3.00 (m, 2H), 2.40-2.30 (m, IH), 2.05-1.88 (m, 4H), 1.85-1.73 (m, 2H), 1.71-1.55 (m, 4H), 1.40-1.30 (m, 2H), 1.20 (d, 6H).

Example 204 l-[5-(5-tert-Butyl-2-methoxy-phenyl)-thiophene-2-carbonyll-piperidine-4-carboxylic acid butylamide

l-(5-Bromo-thiophene-2-carbonyl)-piperidine-4-carboxylic acid butylamide (see exam- pie 196, 37.3 mg, 0.10 mmol) was converted to l-[5-(5-tert-Butyl-2-methoxy-phenyl)- thiophene-2-carbonyl]-piperidine-4-carboxylic acid butylamide using 5-tert-butyl-2-methoxy- phenylboronic acid according to General procedure Cl. The residue was purified by preparative RP-LC-MS (as described for example 200) to give the title compound (19.5 mg, 43%). H NMR (400 MHz, CDCl₃): δ 7.64 (d, IH), 7.40 (d, IH), 7.31 (dd, IH), 7.25 (d, IH), 6.9? (d, IH), 5.59-5.52 (m, IH), 4.56-4.47 (m, 2H), 3.90 (s, 3H), 3.29-3.23 (m, 2H), 3.09-2.98 (m, 2H), 2.41-2.32 (m, IH), 1.95-1.87 (m, 2H), 1.84-1.73 (m, 2H), 1.53-1.43 (m, 2H), 1.39-1.28 (m, HH), 0.92 (t, 3H).

Example 127 {l-[5-(2-Isopropyl-phenyl)-thiophene-2-carbonyll-piperidin-4-ylmethyl|carbamic acid tert- butyl ester

Step 1 : [l-(5-Bromo-thiophene-2-carbonyl)-piperidin-4-ylmethyllcarbamic acid tert- butyl ester 5-Bromo-thiophene-2-carboxylic acid (51.8 mg, 0.25 mmol) was converted to [l-(5-

Bromo-thiophene-2-carbonyl)-piperidin-4-ylmethyl]-carbamic acid tert-butyl ester using piperidin-4-ylmethyl-carbamic acid tert-butyl ester according to General procedure A and purified by silica chromatography (isohexane/ethyl acetate gradient) (48.7 mg, 48%). Step 2: {l-[5-(2-Isopropyl-phenyl)-thiophene-2-carbonyll-piperidin-4-ylmethyl|- carbamic acid tert-butyl ester

[l-(5-Bromo-thiophene-2- carbonyl)-piperidin-4-ylmethyl]-carbamic acid tert-butyl ester (40 mg, 0.10 mmol) was converted to {l-[5-(2-Isopropyl-phenyl)-thiophene-2-carbonyl]- piperidin-4-ylmethyl} -carbamic acid tert-butyl ester using 2-isopropylphenylboronic acid according to General procedure Cl as described for example 200) to give the title compound (33.0 mg, 75%). The residue was purified by preparative RP-LC-MS-¹H NMR (400 MHz, CDCl₃): δ 7.41-7.34 (m, 2H), 7.32-7.29 (dd, IH), 7.24 (d, IH), 7.22-7.17 (m, IH), 6.89 (d, IH), 4.70-4.47 (m, 3H), 3.28 (sept, IH), 3.11-2.89 (m, 4H), 1.84-1.76 (m, 3H), 1.44 (s, 9H), 1.33-1.23 (m, 2H), 1.20 (d, 6H).

Biological test

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 as well as 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 Ia- bile and decomposes non-enzymatically, with a half- life of about 5 min at 37⁰C, into a mixture of PGE₂ and PGD₂ with a E/D ratio of about 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 O⁰C in the presence of PGH₂ and reduced glutathione (GSH). A stop-solution was used, containing FeCl₂, which converted any remaining PGH₂ into HHT. Also, the products are much more stable in organic solvents and therefore the sample was immediately extracted after termination by solid phase extrac- tion 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 CO₂-ice (-78⁰C). Prior to the incubation, both the substrate and samples were transferred onto wet-ice (or 37⁰C) for 2 min temperature equili- bration. 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 11-β PGE₂), lowering the pH to 3, giving a total concentration of 20 mM FeCl₂, 40 mM citric acid and 2.1 μM 11-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 11-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 Cl 8 (3.9 X 150 mm, 4 μm particle size) obtained from Waters and the mobile phase was water, acetonitrile and trifluoroace- tic 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 10E5 M-I cm-1 (E.D. Wills. Biochem. J. 113, 315 (1969).

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

The assay has been modified in that citric acid is used instead of the TCA-TBA-HCl reagent described in the assay. In this assay recombinant, membrane-bound mPGES-1 is incubated with PGH2. The reaction is 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 is finally added (0.67%) and the samples are heated at 80 ⁰C for 30 min. The absorb- ance of the conjugate is measured at 535 nm.

The product of mPGES-1 (PGE2) is not directly measured in this assay, but rather the remaining substrate (PGH2) indirectly by adding FeC12 that converts PGH2 into MDA and 12-HHT. As a positive control a known mPGES-1 inhibitor, MK-886, is used and the new inhibitors are compared with the inhibition of MK-886 (% of MK-886 inhibition).

Red product ~530nM (1.56XlO⁵M^"1 cm^"1)

Total Activity = A₅₃₀-A₅₆₀ / 1 min 0.265 (U/ml) χ 1.56x10⁵

0.05 Selected results from the TBA-MDA assay (IC50 in μM)

Fibroblast assay

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 uM 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. Results

Example PGE₂ %DMSO control

0.1 uM test comp. 1 uM test comp. 10 uM test comp.

44 26 28 9 41 66 49 4

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. 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 uM 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.

Results

In-vivo results

LPS Air Pouch Model of Acute Inflammation in the Rat

(Adapted from "Models of Inflammation: Carrageenan Air Pouch in the Rat" current protocols in pharmacology (1998) 5.6.1-5.6.6)

8-12 weeks old Dark Agouti rats were anesthetized with isofluorane and 20 ml of sterile filtered air was injected to each rat subcutaneously into the intracapsular area of the back (20μm sterile syringe filter, 20ml syringe, 23-G/l-in needle). The air pouches were allowed to mature for 24h.

On the day of the experiment, rats were anesthetized and injected intra-peritoneally with 1 mL test compound (Example 44) dissolved in 90% PEG400 /10% DMSO resulting in a 75mg/kg dose of test compound or PEG/DMSO vehicle alone. 30 minutes post administration of test compound or vehicle, an intra-pouch injection with 2ml of a solution of LPS 5μg/ml in sterile PBS (2ml syringe, 20G/l-in needle) was made.

6h post-LPS injection the rat has been killed by CO₂ inhalation. A second 2ml intra- pouch injection of lavage solution (5.4mM EDTA in sterile PBS, freshly prepared from a 54mM EDTA sterile filtered stock solution) (10ml syringe, 18G/1.5in needle) given. The pouch was immediately drained of lavage fluid and the effect of test compound relative vehicle control on the inflammatory reaction was assessed by analyzing PGE2 content in the the pouch exudate. PGE2 was measured by EIA (Cayman Chemicals) according to the manufacturers instructions. Performing this test on compound (Example 44) resulted in a 57% reduction in inducible PGE2 formation relative to vehicle control.

Adjuvant-Induced Arthritis

Lewis-derived male rats weighing 205 ± 15 g were used. Test compound (Example 44) at 50 mg/kg was administered intra-peritoneally for 5 consecutive days. A well-ground suspension of killed Mycobacterium tuberculosis (0.3 mg in 0.1 mL of light mineral oil; Complete Freund's Adjuvant, CFA) was administered in a single dose into the subplantar region of the right hind paw 1 hour following the first dose of test substance (denoted day 1). Right hind paw volume was measured by plethysmo meter and water cell (25 mm Diameter) on day 0 (before CFA treatment), and on days 1, 5, 8, 11, 14 and 18 after CFA treatment of right paw (with CFA). For CFA-injected right paw volume, the paw volume on days 1, 5, 8, 11, 14 and 18 was compared to that on day 0. Results are illustrated in Fig. 3.

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 hy- drochlorid, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, suc- cinic, 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).

The composition according to the invention may be prepared for any route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperi- toneal. 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 treat- ment of cancer.

Claims

1. A compound of formula (I)

(I) wherein

R² and R³ are independently selected from H (hydrogen), saturated or unsaturated, non- aromatic mono-, bi- or tricyclic (C3-C12)-carbocyclyl or (Cl-C12)-heterocyclyl containing one or more heteroatoms selected from N, O and S; and (C2-C10)-alkyl, optionally substituted with one or more moieties R⁴; R⁴ is selected from saturated or unsaturated, aromatic or non-aromatic, bridged or non- bridged, mono-, bi- or tricyclic (C3-C12)-carbocyclyl or (Cl-C12)-heterocyclyl containing one or more heteroatoms selected from N, O and S; (Cl-ClO)-alkyl-OC(O)-, R¹²R¹³N-; or R² and R³ form, together with the N to which they are bound, a ring of formula (II)

(H) wherein n is 1, 2 or 3;

X is selected from >CHNR⁶R⁷, >CHC(O)OR⁶, >CH-(CH₂)-NHC(O)OR⁶, and >CHC(O)NR⁶R⁷;

R⁵ is selected from -R⁸, -C(O)OR⁸ and -C(O)NR⁸R⁹ ; R⁶ and R⁷, and R⁸ and R⁹, respectively, together with the N to which they are bound, form a saturated 5-, 6-, or 7-membered heterocyclyl, optionally fused with an aromatic or non-aromatic (C3-C12)-carbocyclyl or (Cl-C12)-heterocyclyl containing one or more het- eroatoms selected from N, O and S; or R⁶, R⁷, R⁸ and R⁹ are independently selected from H; saturated or unsaturated, aromatic or non-aromatic, bridged or non-bridged, mono-, bi- or tricyclic (C3-C12)-carbocyclyl or (Cl- C12)-heterocyclyl containing one or more heteroatoms selected from N, O and S; and (Cl- C10)-alkyl optionally substituted with one or more moieties R¹⁰; each R¹⁰ is independently selected from (Cl-ClO)-alkoxy, (Cl-ClO)-alkylthio, satu- rated or unsaturated, aromatic or non-aromatic, mono-, bi- or tricyclic (C3-C12)-carbocyclyl or (C3-C12)-heterocyclyl containing one or more heteroatoms selected from N, O and S; or R¹²R¹³N; wherein in any of R^R¹⁰ any phenyl, carbocyclyl or heterocyclyl is optionally substituted with one or more substituents R¹¹; R¹¹ is selected from halogen, -CN, -CF3, saturated or non-saturated, aromatic or non- aromatic (C3-C10) carbocyclyl or (Cl-ClO) heterocyclyl optionally, substituted with halogen; (Cl-ClO)-alkyl optionally substituted with one or more saturated or non-saturated, aromatic or non-aromatic (C3-C10) carbocyclyl or (Cl-ClO) heterocyclyl; (Cl-Cβ)-alkoxy, (C1-C3)- alkylendioxy, (Cl-C6)alkyl-NH-SO₂-, (Cl-C6)alkylC(O)NH- and R¹²R¹³N; R¹² and R¹³ are independently selected from H, (Cl-C6)-alkyl, saturated or non- saturated, aromatic or non-aromatic (C3-C10)-carbocyclyl, optionally substituted by (C1-C6)- alkyl; as well as pharmaceutically acceptable salts thereof, for use as a medicament.

2. A compound according to claim 1, wherein R² is hydrogen.

3. A compound according to claim 1, wherein R² and R³, together with the N to which they are bound, form a ring of formula (II).

4. A compound according to claim 3, wherein R⁵ is H.

5. A compound according to claim 3 or claim 4, wherein X is >CHC(O)NR⁶R⁷.

6. A compound according to any one of the claims 3-5, wherein n is 2.

7. A compound according to any one of the claims 1-6, wherein R¹ is mono-, bi- or tricyclic carbocyclyl or heterocyclyl, comprising at least one aromatic cycle.

8. A compound according to claim 7, wherein R¹ comprises at least one phenyl.

9. A compound according to any one of the claims 1-8, wherein R¹ is a heterocyclyl containing at least one cyclic N.

10. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of the claims 1-9, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

11. A compound according to any one of the claims 1-9, for use in the treatment of a disorder selected from pain, fever, an inflammatory condition and cancer.

12. Use of a compound according any one of the claims 1-9, for preparing a medicament for the treatment of a disorder selected from pain, fever, an inflammatory condition and cancer.

13. The use according to claim 12, wherein the disorder is an inflammation.

14. The use according to claim 12, wherein the disorder is rheumatoid arthtritis.

15. A method of treatment of a disease selected from pain, fever, inflammation and cancer by administration of a therapeutically effective amount of a compound according to claim 1 or a pharmaceutically acceptable salt thereof to a mammal in the need of such treatment.