CROSS-REFERENCE TO RELATED APPLICATION

[001] This application claims priority to US Application No. 62/709,882, filed on February 5, 2018, the contents of which are incorporated herein in their entirety.

BACKGROUND OF THE INVENTION

[002] This invention relates to heterocyclic amide derivatives, or their pharmaceutically acceptable salts, pharmaceutical acceptable prodrugs, pharmaceutical compositions containing them, and their medical uses. The compounds of this invention have activity as prostaglandin E2 receptor antagonists, and these are useful in the treatment or alleviation of pain and inflammation and other inflammation-associated disorders, such as arthritis, treating or preventing disorders or medical conditions selected from pain, inflammatory diseases and the like.

[003] Prostaglandins are mediators of pain, fever and other symptoms associated with inflammation. Especially prostaglandin E2 (PGE2) is the predominant eicosanoid detected in inflammation conditions. In addition, PGE2 has also been implicated as an important constituent in the immunosuppressive environment created by many solid tumors (Whiteside, Expert Opinion in Biological Therapy, 2010. 10, 1019-1035), and inhibition of EP4 signaling by antagonists were shown to reduce tumor growth (Terada et al. Cancer Res. 2010, 70, 1606-1615) and tumor metastasis in tumor animal models (Yang et al. Cancer Res. 2006, 66, 9665-9672). PGE2 has also been shown to promote colorectal cancer stem cell expansion and metastasis in mice (Wang et al., Gastroentology, 2015, 1-12). The anti-tumor activity EP4 antagonists was also shown to improve or synergize with the anti-tumor effect of antibodies against checkpoint proteins, such as anti-CTLA4, anti-PD-Ll or anti-PD-1 (Bao et al. Journal for ImmunoTherapy of Cancer 2015, 3(Suppl 2):P35).

[004] This invention relates to a series of novel EP4 antagonists and methods for treating prostaglandin E2 mediated diseases, in particular for pain, inflammation and cancer immunotherapy, along with certain pharmaceutical compositions thereof. SUMMARY OF THE INVENTION

[005] The invention is directed to a series of novel heterobicyclic derivatives as EP4 receptor antagonists useful for the treatment of EP4 mediated diseases or conditions, such as acute and chronic pain, osteoarthritis, rheumatoid arthritis and cancer. Pharmaceutical compositions and methods of use are also included.

[006] In one aspect, the present invention provides compounds of Formula I or a pharmaceutically acceptable salt or prodrug thereof,

I

wherein:

each of A, B and C' is independently selected from N, CH and C(R);

-D-E-F- is -C(R ^C ) ₂ -N=C-, -N(R ^C )-C(R ^C )=C-, -S-N=C-, -0-N=C-, -N(R ^C )-N=C-, -C(R ^C ) ₂ -C(R ^C ) ₂ -N-, - C(R ^C ) =C(R ^C )-N-, -C(R ^C ) =N-N-, -N=N-N-, -0-C(R ^c )=C-, or -S-C(R ^C )=C-;

each of R ^la and R ^2a independently is H, Ci- ₆ alkyl, Ci- ₆ cyclolkyl, Ci- ₆ fluorocycloalkyl, or Ci- ₆ fluoroalkyl; or R ¹ and R ² , together with the carbon atom to which they are both attached to, form a three- to six-membered carbocyclic ring which is optionally substituted with R ^c , or form a three- to six-membered ring which contains one or two heteroatom(s) each independently being S, S(0) ₂ , S(0)(NH), O, or NR ^e , wherein R ^e is H, Ci _-6 alkyl, Ci- ₆ cyclolkyl, Ci- ₆ fluorocycloalkyl, Ci_ ₆ fluoroalkyl, aryl, heteroaryl, C(O) Ci- ₆ alkyl, C(0)aryl, S(0) ₂ alkyl, or S(0) ₂ aryl;

each R ^c independently is H, halogen, Ci _-4 alkyl, Ci _-4 fluoroalkyl, Ci _-4 alkoxy, Ci _-4 fluoroalkoxy, or acetyl;

each R independently is H, Ci _-4 alkyl, Ci _-4 fluoroalkyl, Ci _-4 alkoxy, Ci _-4 fluoroalkoxy, or acetyl;

Y is -CH ₂ -, -C(0)-, -C(S)-, or -S(0) ₂ -;

X is CH ₂ , O, or S;

W is NH or O; each of Ar ¹ and Ar ² , independently, is C3-6 cycloalkyl, aryl, heteroaryl, or heterocyclyl, or a fused analog of C3-6cycloalkyl, aryl, heteroaryl or heterocyclyl, and each of Ar ¹ and Ar ² , is optionally substituted with one to three R ^b groups;

each R ^b is independently H, halogen, Ci_ ₆ alkyl, Ci- ₆ alkoxyl, or Ci- ₆ fluoroalkyl;

M is a pharmaceutically acceptable salt or an ester prodrug group.

[007] I n some embodiments, the compounds of Formula I are of Formula la

wherein:

each of A, B and C' independently is N, CH, or C(R);

-D-E-F- is -C(R ^C ) ₂ -N=C-, -N(R ^C )-C(R ^C )=C-, -S-N=C-, -0-N=C-, -N(R ^C )-N=C-, -C(R ^C ) ₂ -C(R ^C ) ₂ -N-, - C(R ^C ) =C(R ^C )-N-, -C(R ^C ) =N-N-, -N=N-N-, -0-C(R ^c )=C-, or -S-C(R ^C )=C-;

each R ^b is independently H, halogen, Ci- ₆ alkyl, Ci- ₆ alkoxyl, or Ci- ₆ fluoroalkyl;

each of R ^la and R ^2a is independently H, Ci _-6 alkyl, Ci- ₆ cyclolkyl, Ci- ₆ fluorocycloalkyl, or Ci- ₆ fluoroalkyl, or

each of R ¹ and R ² , independently, is H, halogen, Ci _-6 alkyl, Ci _-6 alkoxyl, Ci- ₆ cyclolkyl, Ci- ₆ fluorocycloalkyl, Ci- ₆ fluoroalkyl, or SF5; or R ¹ and R ² , together with the carbon atom to which they are both attached to, form a three- to six-membered carbocyclic ring which is optionally substituted with Rc or a three- to six-membered ring which contains one or two heteroatom(s) each independently being S, S(0) ₂ , S(0)(NH), O, or NR ^e , wherein R ^e is H, Ci- ₆ alkyl, Ci- ₆ cyclolkyl, Ci_ ₆ fluorocycloalkyl, Ci- ₆ fluoroalkyl, aryl, heteroaryl, C(0)Ci- ₆ alkyl, C(0)aryl, S(0) ₂ alkyl, or S(0) ₂ aryl; each R ^c is independently H, Cl-4alkyl, Cl-4fluoroalkyl, Cl-4alkoxy, Cl-4fluoroalkoxy or acetyl; each R is independently H, halogen, Ci _-4 alkyl, Ci- ₄ fluoroalkyl, Ci- ₄ alkoxy, Ci- ₄ fluoroalkoxy, or acetyl;

X is CH ₂ , O, or S;

each R ^b is independently H, halogen, Cl-6alkyl, Cl-6alkoxyl, or Cl-6fluoroalkyl;

n is 0,1, 2, or 3.

[008] In some other embodiments, the compounds are of Formula lb:

lb

wherein:

-D-E-F- is -C(Rc)2-N=C-, -N(Rc)-C(Rc)=C-, -S-N=C-, -0-N=C-, -N(Rc)-N=C-, -C(Rc)2-C(Rc)2- N-, -C(Rc) =C(Rc)-N-, -C(Rc) =N-N-, -N=N-N-, -0-C(Rc)=C-, -S-C(Rc)=C-;

each of R1 and R2 is independently!-!, halogen, Cl-6alkyl, Cl-6alkoxyl, Cl-6cyclolkyl, Cl- 6fluorocycloalkyl, Cl-6fluoroalkyl, or SF5;

each Rb independently is H, halogen, Cl-6alkyl, Cl-6alkoxyl, or Cl-6fluoroalkyl;

each of Rla and R2a independently is H, Cl-6alkyl, Cl-6cyclolkyl, Cl-6fluorocycloalkyl, or Cl-6fluoroalkyl; or R1 and R2, together with the carbon atom to which they are both attached to, form a three- to six-membered carbocyclic ring which is optionally substituted with Rc or a three- to six-membered ring which contains one or two heteroatom(s) each independently being S, O or NRe, wherein Re is H or Cl-6alkyl;

each Rc independently is H, Cl-4alkyl, Cl-4fluoroalkyl, Cl-4alkoxy, Cl-4fluoroalkoxy or acetyl;

each R independently is H, halogen, Cl-4alkyl, Cl-4fluoroalkyl, Cl-4alkoxy, Cl- 4fluoroalkoxy or acetyl;

each Rb independently is H, halogen, Cl-6alkyl, Cl-6alkoxyl, or Cl-6fluoroalkyl; and n is 0,1, 2, or 3. [009] In still some other embodiments, the fused ring moeity compounds of Formula I is selected from the following heterocyclic moieties:

[010] Examples of the compounds of the present invention include

4-(l-(7-(4-(trifluoromethyl)phenoxy)-lH-indole-l-carboxam ido)cyclopropyl)benzoic acid;

4-(l-(7-(3-(trifluoromethyl)phenoxy)-lH-indole-l-carboxam ido)cyclopropyl)benzoic acid;

4-(2-(7-(3-(trifluoromethyl)phenoxy)-lH-indole-l-carboxam ido)propan-2-yl)benzoic acid;

(±)4-(l-(7-(3-(trifluoromethyl)phenoxy)-lH-indole-l-carb oxamido)ethyl)benzoic acid;

(±) 4-(l-(7-(4-(trifluoromethyl)phenoxy)-lH-indole-l-carboxamido )ethyl)benzoic acid;

(±) 4-(l-(4-(4-(trifluoromethyl)phenoxy)-lH-indole-3-carboxamido )ethyl)benzoic acid;

4-(l-(4-(4-(trifluoromethyl)phenoxy)-lH-indole-3-carboxam ido)cyclopropyl)benzoic acid;

4-(l-(4-(3-(trifluoromethyl)phenoxy)-lH-indole-3-carboxam ido)cyclopropyl)benzoic acid;

4-(l-(4-(3-(trifluoromethyl)phenoxy)benzofuran-3-carboxam ido)cyclopropyl)benzoic acid ; 4-(l-(4-(4-(trifluoromethyl)phenoxy)benzofuran-3-carboxamido )cyclopropyl)benzoic acid ;

(±) 4-(l-(4-(4-(trifluoromethyl)phenoxy)benzofuran-3-carboxamido )ethyl)benzoic acid;

(±) 4-(l-(4-(4-(trifluoromethyl)phenoxy)benzo[b]thiophene-3-carb oxamido)ethyl)benzoic acid; (±) 4-(l-(4-(3-(trifluoromethyl)phenoxy)benzo[b]thiophene-3-carb oxamido)ethyl)benzoic acid; 4-(l-(4-(3-(trifluoromethyl)phenoxy)benzo[b]thiophene-3-carb oxamido)cyclopropyl)benzoic acid;

4-(l-(4-(4-(trifluoromethyl)phenoxy)benzo[b]thiophene-3-carb oxamido)cyclopropyl)benzoic acid; 4-(l-(4-(4-(trifluoromethyl)phenoxy)benzo[d]isothiazole-3-ca rboxamido)cyclopropyl)benzoic acid;

4-(l-(4-(3-(trifluoromethyl)phenoxy)benzo[d]isothiazole-3-ca rboxamido)cyclopropyl)benzoic acid

(±) 4-(l-(4-(3-(trifluoromethyl)phenoxy)benzo[d]isothiazole-3-ca rboxamido)ethyl)benzoic acid; (±) 4-(l-(4-(4-(trifluoromethyl)phenoxy)benzo[d]isothiazole-3-ca rboxamido)ethyl)benzoic acid; (±) 4-(l-(4-(4-(trifluoromethyl)phenoxy)benzo[d]isoxazole-3-carb oxamido)ethyl)benzoic acid;

(±) 4-(l-(4-(3-(trifluoromethyl)phenoxy)benzo[d]isoxazole-3-carb oxamido)ethyl)benzoic acid;

4-(l-(4-(3-(trifluoromethyl)phenoxy)benzo[d]isoxazole-3-c arboxamido)cyclopropyl)benzoic acid;

4-(l-(4-(4-(trifluoromethyl)phenoxy)benzo[d]isoxazole-3-carb oxamido)cyclopropyl)benzoic acid;

4-(l-(7-(4-(trifluoromethyl)phenoxy)indoline-l-carboxamido)c yclopropyl)benzoic acid; and 4-(l-(7-(3-(trifluoromethyl)phenoxy)indoline-l-carboxamido)c yclopropyl)benzoic acid.

[Oil] As mentioned above, the invention also encompasses salts or prodrugs of compounds of Formula I. A prodrug can be a choice of an ester or amide of the compound of Formula I.

[012] In another aspect, the present invention provides pharmaceutical compositions each comprising an above-described compound and a pharmaceutically or physiologically acceptable carrier.

[013] The present invention also relates to a method for treating a subject suffering from a condition mediated by the action of PGE2 at EP4 receptors. The method includes administering to the subject in need thereof an effective amount of an above-described compound or pharmaceutical composition.

[014] In some embodiments, the condition is an inflammatory disease or cancer. Examples of the inflammatory disease include arthritis, acne vulgaris, asthma, autoimmune diseases, autoinflammatory diseases, Celiac disease, chronic prostatitis, colitis, diverticulitis, glomerulonephritis, hidradenitis suppurativa, hypersensitivities, inflammatory bowel diseases, interstitial cystitis, Mast Cell Activation Syndrome, mastocytosis, otitis, pelvic inflammatory disease, reperfusion injury, rheumatic fever, rheumatoid arthritis, rhinitis, sarcoidosis, and vasculitis.

[015] In some other embodiments, the condition is cancer and the method comprises administering to the subject in need thereof an effective amount of an above-described compound or composition, optionally in combination with a second therapeutic agent which is an antibody, a kinase inhibitor, IDO inhibitor, TDO inhibitor, STING activator, HDAC inhibitor, or an chemotherapeutic agent. Examples of a suitable antibody include those against CTCLA4, PDL1 and PD1.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[016] Abbreviations used herein have their conventional meaning within the chemical and biological arts.

[017] As used herein, the term "alkyl," by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e. unbranched) or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono-or polyunsaturated and can include di-and multivalent radicals, having the number of carbon atoms designated (i.e. C1-C10 means one to ten carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1-and 3-propynyl, 3-butynyl, and the higher homologs and isomers. Alkyl groups which are limited to hydrocarbon groups are termed "homoalkyl".

[018] As used herein, the term "fluoroalkyl" means alkyl as defined above wherein one or more hydrogen atoms have been replaced by fluoro atoms.

[019] As used herein, the term "alkylene" by itself or as part of another substituent means a divalent radical derived from an alkyl, as exemplified, but not limited, by -CH2CH2CH2CH2-, - CH2CH=CHCH2-, -CH2 CºCCH2-, -CH2CH2CH(CH2CH2CHB)CH2-. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.

[020] As used herein, the term "alkynyl" means carbon chains which contain at least one carbon-carbon triple bond, and which may be linear or branched or combinations thereof. Examples of alkynyl include ethynyl, propargyl, 3-methyl-l-pentynyl, 2-heptynyl and the like.

[021] As used herein, the term "cycloalkyl" means mono- or bicyclic saturated carbocyclic rings, each of which having from 3 to 10 carbon atoms. A "fused analog" of cycloalkyl means a monocyclic rings fused to an aryl or heteroaryl group in which the point of attachment is on the non-aromatic portion. Examples of cycloalkyl and fused analogs thereof include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, tetrahydronaphthyl, decahydronaphthyl, indanyl, and the like.

[022] As used herein, the term "alkoxy" means alkoxy groups of a straight or branched having the indicated number of carbon atoms. Ci- ₆ alkoxy, for example, includes methoxy, ethoxy, propoxy, isopropoxy, and the like.

[023] As used herein, the term "heteroalkyl," by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of at least one carbon atoms and at least one heteroatom selected from the group consisting of O, N, P, Si and S, and wherein the nitrogen, phosphorus, and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, P and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to,-CH2-CH2-0-CH3, -CH2-CH2- NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(0)-CH3, -CH2-CH2-S(0) 2-CH3, - CH=CH-0-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -0-CH3,-0-CH2-CH 3, and -CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and - CH2-0-Si(CH3)3. Similarly, the term "heteroalkylene" by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH2 -CH2- S -CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxo, alkylenedioxo, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(0)0R'- represents both-C(0)OR'- and -R'OC(O)-. As described above, heteroalkyl groups, as used herein, include those groups that are attached to the remainder of the molecule through a heteroatom, such as -C(0)R', -C(0)NR', -NR'R", -OR', - SR', and/or -S02R'. Where "heteroalkyl" is recited, followed by recitations of specific heteroalkyl groups, such as -NR'R" or the like, it will be understood that the terms heteroalkyl and--NR'R" are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term "heteroalkyl" should not be interpreted herein as excluding specific heteroalkyl groups, such as -NR'R" or the like.

[024] As used herein, the term "cycloalkoxy" means cycloalkyl as defined above bonded to an oxygen atom, such as cyclopropyloxy.

[025] As used herein, the term "fluoroalkoxy" means alkoxy as defined above wherein one or more hydrogen atoms have been replaced by fluoro atoms.

[026] As used herein, the term "aryl" means mono- or bicyclic aromatic rings containing only carbon atoms. A "fused analog" of aryl means an aryl group fused to a monocyclic cycloalkyl or monocyclic heterocyclyl group in which the point of attachment is on the aromatic portion. Examples of aryl and fused analogs thereof include phenyl, naphthyl, indanyl, indenyl, tetrahydronaphthyl, 2,3-dihydrobenzofuranyl, dihydrobenzopyranyl, 1,4-benzodioxanyl, and the like.

[027] As used herein, the term "heteroaryl" means a mono- or bicyclic aromatic ring containing at least one heteroatom selected from N, O and S, with each ring containing 5 to 6 atoms. A "fused analog" of heteroaryl means a heteroaryl group fused to a monocyclic cycloalkyl or monocyclic heterocyclyl group in which the point of attachment is on the aromatic portion. Examples of heteroaryl include pyrrolyl, isoxazolyl, isothiazolyl, pyrazolyl, pyridyl, oxazolyl, oxadiazolyl, thiadiazolyl, thiazolyl, imidazolyl, triazolyl, tetrazolyl, furanyl, triazinyl, thienyl, pyrimidyl, pyridazinyl, pyrazinyl, benzoxazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, furo(2,3-b)pyridyl, quinolyl, indolyl, isoquinolyl, and the like.

[028] The aryl groups and heteroaryl groups referred to in the definitions of Arl and Ar2 are unsubstituted or are substituted by at least one substituent selected from the group consisting of substituents 0.

[029] The substituents are selected from the group consisting of halogen atoms, alkyl groups having from 1 to 4 carbon atoms, alkoxy groups having from 1 to 4 carbon atoms, haloalkyl groups having from 1 to 4 carbon atoms, haloalkoxy groups having from I to 4 carbon atoms, cyano groups, alkynyl groups having from 2 to 6 carbon atoms, alkanoyl groups having from 1 to 5 carbon atoms, cycloalkyl groups having from 3 to 7 ring atoms, heteroaryl groups, aryl groups, aralkoxy groups having from 7 to 10 carbon atoms, arylcarbonyl groups, two adjacent-x groups are optionally joined together to form an alkylene or an alkenylene chain having 3 or 4 carbon atoms, aminocarbonyl groups, alkenyl groups having from 2 to 5 carbon atoms, alkylthio groups having from 1 to 4 carbon atoms, aminosulfinyl groups, aminosulfonyl groups, hydroxy groups, hydroxyalkyl groups having from 1 to 4 carbon atoms, nitro groups, amino groups, carboxy groups, alkoxycarbonyl groups having from 2 to 5 carbon atoms, alkoxyalkyl groups having from 1 to 4 carbon atoms, alkylsulfonyl groups having from I to 4 carbon atoms, alkanoylamino groups having from 1 to 4 carbon atoms, alkanoyl(alkyl)amino groups having from 1 to 6 carbon atoms, alkanoylaminoalkyl groups having from 1 to 6 carbon atoms in both the alkanoyl and alkyl part, alkanoyl(alkyl)aminoalkyl groups having from 1 to 6 carbon atoms in both the alkanoyl and each alkyl part, alkylsulfonylamino groups having from 1 to 4 carbon atoms, mono-or di- alkylaminocarbonyl groups having from 1 to 6 carbon atoms, mono-or di-alkylaminosulfinyl groups having from 1 to 6 carbon atoms, mono-or di alkylaminosulfonyl groups having from 1 to 6 carbon atoms, aminoalkyl groups having from 1 to 4 carbon atoms, mono-or di-alkylamino groups having from 1 to 6 carbon atoms, mono-or di-alkylaminoalkyl groups having from 1 to 6 carbon atoms in each alkyl part, aralkyl groups having from 7 to 10 carbon atoms, heteroarylalkyl groups having from 1 to 4 carbon atoms in the alkyl part, heteroarylalkoxy groups having from 1 to 4 carbon atoms in the alkoxy part and alkylsulfonylamino groups having from I to 4 carbon atoms. [030] As used herein, the term "heterocyclyl" means mono- or bicyclic saturated rings containing at least one heteroatom selected from N, S and O, each of said ring having from 3 to 10 atoms in which the point of attachment may be carbon or nitrogen. A "fused analog" of heterocyclyl means a monocyclic heterocycle fused to an aryl or heteroaryl group in which the point of attachment is on the non-aromatic portion. Examples of "heterocyclyl" and fused analogs thereof include pyrrolidinyl, piperidinyl, piperazinyl, imidazolidinyl, 2,3-dihydrofuro(2,3- b)pyridyl, benzoxazinyl, tetrahydrohydroquinolinyl, tetrahydroisoquinolinyl, dihydroindolyl, and the like. The term also includes partially unsaturated monocyclic rings that are not aromatic, such as 2- or 4-pyridones attached through the nitrogen or N-substituted-(lH,3H)-pyrimidine- 2,4-diones (N-substituted uracils).

[031] As used herein, the term "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl," are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(Cl-C4)alkyl" is mean to include, but not be limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[032] As used herein, the term "prodrug" refers to an agent that is converted into the parent drug in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent is not. The prodrugs may also have improved solubility in pharmaceutical compositions over the parent drug. An example, without limitation, of a prodrug would be a compound of any of Formula I, which is administered as an ester (the "prodrug") to facilitate transmittal across a cell membrane where water solubility is detrimental to mobility but which then is metabolically hydrolyzed to the carboxylic acid, the active entity, once inside the cell where water-solubility is beneficial. A further example of a prodrug might be a short peptide (polyaminoacid) bonded to an acid group where the peptide is metabolized to reveal the active moiety.

Optical Isomers - Diastereomers - Geometric Isomers - Tautomers

[033] Compounds of Formula I contain one or more asymmetric centers and can thus occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. The present invention is meant to comprehend all such isomeric forms of the compounds of Formula I.

[034] Some of the compounds described herein contain olefinic double bonds, and unless specified otherwise, are meant to include both E and Z geometric isomers.

[035] Some of the compounds described herein may exist with different points of attachment of hydrogen, referred to as tautomers. Such an example may be a ketone and its enol form known as keto-enol tautomers. The individual tautomers as well as mixture thereof are encompassed with compounds of Formula I to Formula Ig.

[036] Compounds of the Formula I may be separated into diastereoisomeric pairs of enantiomers by, for example, fractional crystallization from a suitable solvent, for example MeOH or EtOAc or a mixture thereof. The pair of enantiomers thus obtained may be separated into individual stereoisomers by conventional means, for example by the use of an optically active amine as a resolving agent or on a chiral HPLC column.

[037] Alternatively, any enantiomer of a compound of the general Formula I may be obtained by stereospecific synthesis using optically pure starting materials or reagents of known configuration.

Salts

[038] The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethyl- aminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

[039] When the compound of the present invention is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p- toluenesulfonic acid, and the like. Particularly preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids.

[040] It will be understood that, as used herein, references to the compounds of Formula I are meant to also include the pharmaceutically acceptable salts.

Utilities

[041] The compounds of the invention are antagonists of the EP4 receptor and are therefore useful in treating a prostaglandin E2 mediated disease or condition.

[042] The invention also encompasses a method of treating cancer with an effective amount of a compound of the present invention or using a combination of an effective amount of a compound of the present invention with an effective amount of radiation; antibodies to cytotoxic t-lymphocyte antigen 4 (anti-CTLA4); antibodies to programmed death ligand 1 (anti-PDLI); antibodies to programmed cell death protein 1 (anti-PDl); indoleamine-2, 3-dioxygenase (IDO) inhibitors; tryptophan-2, 3-dioxygenase (TDO) inhibitors; and antimetabolites. These antibodies can be selected from, but not limited to, MDX-010 (ipilimumab, Bristol-Myers Squibb), CP- 675,206 (tremelimumab, Pfizer), MPDL3280A (Roche), MDX-1106 (nivolumab, Bristol- Myers Squibb), labrolizumab (Merck), and pembrolizumab (KEYTRUDA ^® , Merck).

[043] In yet another aspect of the invention, the cancer treated is selected from the group consisting of breast cancers, cervical cancers, colorectal cancers, endometrial cancers, glioblastomas, head and neck cancers, kidney cancers, liver cancers, lung cancers, medulloblastomas, ovarian cancers, pancreatic cancers, prostate cancers, skin cancers and urinary tract cancers. In more particular aspects of the invention, provided are methods of

IB treating cancer and/or generating a memory immune response comprising administering a compound of Formula I.

[044] As used herein, the term "treating a prostaglandin E2 mediated disease or condition" means treating or preventing any chronic disease or condition that is advantageously treated or prevented selective EP4 antagonists. The term includes the relief of pain, fever and inflammation of a variety of conditions including rheumatic fever, symptoms associated with influenza or other viral infections, common cold, low back pain, neck pain, dysmenorrhea, headache, migraine, toothache, sprains and strains, myositis, neuralgia, synovitis, arthritis, including rheumatoid arthritis, degenerative joint diseases (osteoarthritis), gout, ankylosing spondylitis, bursitis, burns, injuries, and pain and inflammation following surgical procedures. In addition, such a compound may inhibit cellular neoplastic transformations and metastatic tumor growth and hence can be used in the treatment and/or prevention of cancer.

[045] As used herein, the term "Effective amount" or "treatment-effective amount" refers to an amount that is effective for treating a prostaglandin E2 mediated disease or condition or a cancer as noted through clinical testing and evaluation, patient observation, and/or the like. An "effective amount" can further designate an amount that causes a detectable change in biological or chemical activity. The detectable changes may be detected and/or further quantified by one skilled in the art for the relevant mechanism or process. Moreover, an "effective amount" can designate an amount that maintains a desired physiological state, i.e., reduces or prevents significant decline and/or promotes improvement in the condition. An "effective amount" can further refer to a therapeutically effective amount.

[046] As used herein, the term "cancer" as used herein may include cancers that are the result of genetically inherited mutations. Examples of such cancers include, but are not limited to, breast cancers, cancers which can be related to Li-Fraumeni syndrome, for example, childhood sarcomas, leukemias and brain cancers, cancers which can be related to Lynch syndrome, for example, colon cancers, bile duct cancers, brain cancers, endometrial cancers, kidney cancers, ovarian cancers, pancreatic cancers, small intestinal cancers, stomach cancers and ureter cancers, lung cancers, melanomas, prostate cancers, retinoblastomas, thyroid cancers and uterine cancers. Moreover, cancer can be the result of acquired mutations, for example, mutations resulting from diet, environment and/or lifestyle, or somatic mutations. Examples of such cancers may include, but are not limited to, adrenal cancer, adrenal cortex cancer, bladder cancer, brain cancer, primary brain cancer, glioma, glioblastoma, breast cancer, cervical cancer, colon cancer (non-limiting examples include colorectal carcinomas such as colon adenocarcinoma and colon adoma), endometrial cancer, epidermal cancer, esophageal cancer, gall bladder cancer, genitourinary cancer, head or neck cancer, kidney cancer, liver cancer, lung cancer (non-limiting examples include adenocarcinoma, small cell lung cancer and non-small cell lung cancer), lymphomas (non-limiting examples include B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma), melanoma, malignant melanoma, malignant carcinoid carcinoma, malignant pancreatic insulinoma, myeloma, multiple myeloma, ovarian cancer, pancreatic cancer (such as exocrine pancreatic carcinoma), prostate cancer, renal cell cancer, skin cancer, such as, in addition to others previously mentioned, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, thyroid follicular cancer, Wilms' tumor, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell lymphoma, Burkett's lymphoma, acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, promyelocytic leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, fibrosarcoma, habdomyosarcoma, astrocytoma, neuroblastoma, rhabdomyosarcoma, schwannoma, Kaposi's sarcoma, polycythemia vera, essential thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, soft-tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, seminoma, teratocarcinoma, osteosarcoma, xenoderoma pigmentoum, keratoctanthoma and retinoblastoma.

[047] Compounds of Formula I can also be used in combination with one or more chemotherapeutic agents such as:

i. an aromatase inhibitor,

ii. an antiestrogen, an anti-androgen (especially in the case of prostate cancer) or a gonadorelin agonist,

iii. a topoisomerase I inhibitor or a topoisomerase II inhibitor, iv. a microtubule active agent, an alkylating agent, an antineoplastic antimetabolite or a platin compound,

v. a compound targeting/decreasing a protein or lipid kinase activity or a protein or lipid phosphatase activity, a further anti-angiogenic compound or a compound which induces cell differentiation processes,

vi. a bradykinin I receptor or an angiotensin II antagonist,

vii. a cyclooxygenase inhibitor, a bisphosphonate, a rapamycin derivative such as everolimus, a heparanase inhibitor (prevents heparan sulphate degradation), e.g. PI 88, a biological response modifier, preferably a lymphokine or interferons, e.g. interferon if, an ubiquitination inhibitor, or an inhibitor which blocks anti-apoptotic pathways,

viii. an inhibitor of Ras oncogenic isoforms, e. g. H-Ras, K-Ras or N-Ras, or a farnesyl transferase inhibitor, e.g. L-744, 832 or DK8G557,

ix. a telomerase inhibitor, e.g. telomestatin,

x. a protease inhibitor, a matrix metalloproteinase inhibitor, a methionine aminopeptidase inhibitor, e.g. bengamide or a derivative thereof, or a proteosome inhibitor, e.g. PS 341,

xi. histone deacetylase inhibitors, e.g. Vorinostat, MG0103 or MS275, and

xii. kinase inhibitors.

[048] The pharmaceutical compositions of the present invention comprise a compound of Formula I as an active ingredient or a pharmaceutically acceptable salt, thereof, and may also contain a pharmaceutically acceptable carrier and optionally other therapeutic ingredients. The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases including inorganic bases and organic bases. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N- dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

[049] It will be understood that in the discussion of methods of treatment which follows, references to the compounds of Formula I are meant to also include the pharmaceutically acceptable salts.

[050] The EP4 antagonist, antibody and/or anti-metabolite can be administered to subjects by any suitable route, including orally (inclusive of administration via the oral cavity and further including administration via an orogastric feeding tube), intraperitoneally, parenterally, by inhalation spray, topically (i.e., both skin and mucosal surfaces, including airway surfaces), transdermally, rectally, nasally (including a nasogastric feeding tube), sublingually, buccally, vaginally or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intramuscular, intradermal, intravenous, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. In a particular embodiment, the EP4 antagonist, antibody and/or anti-metabolite is administered orally. In another particular embodiment, the EP4 antagonist, antibody and/or antimetabolite is administered intravenously.

[051] The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the technique described in the U.S. Patent 4,256,108; 4,166,452; and 4,265,874 to form osmotic therapeutic tablets for control release.

[052] Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredients is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.

[053] Aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethyl-cellulose, methylcellulose, hydroxypropylmethy-cellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene-oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, saccharin or aspartame.

[054] Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

[055] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.

[056] The pharmaceutical compositions of the invention may also be in the form of an oil-in- water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavouring agents.

[057] Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

[058] The compounds of the invention can also be administered intranasally or by inhalation, typically in the form of a dry powder (either alone, as a mixture, for example, in a dry blend with lactose, or as a mixed component particle, for example, mixed with phospholipids, such as phosphatidylcholine) from a dry powder inhaler or as an aerosol spray from a pressurised container, pump, spray, atomiser (preferably an I atomiser using electrohydrodynamics to produce a fine mist), or nebuliser, with or without the use of a suitable propellant, such as 1, 1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane. For intranasal use, the powder may comprise a bioadhesive agent, for example, chitosan or cyclodextrin.

[059] The pressurized container, pump, spray, atomizer, or nebuliser contains a solution or suspension of the compound(s) of the invention comprising, for example, ethanol, aqueous ethanol, or a suitable alternative agent for dispersing, solubilizing, or extending release of the active, a propellant(s) as solvent and an optional surfactant, such as sorbitan trioleate, oleic acid, or an oligolactic acid.

[060] Prior to use in a dry powder or suspension formulation, the drug product is micronized to a size suitable for delivery by inhalation (typically less than 5 microns).

[061] This may be achieved by any appropriate comminuting method, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure homogenization, or spray drying.

[062] Capsules (made, for example, from gelatin or HPMC), blisters and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound of the invention, a suitable powder base such as lactose or starch and a performance modifier such as l-leucine, mannitol, or magnesium stearate. The lactose may be anhydrous or in the form of the monohydrate, preferably the latter. Other suitable excipients include dextran, glucose, maltose, sorbitol, xylitol, fructose, sucrose and trehalose.

[063] A suitable solution formulation for use in an atomizer using electrohydrodynamics to produce a fine mist may contain from log to 20 mg of the compound of the invention per actuation and the actuation volume may vary from 11 to 1001. A typical formulation may comprise a compound of formula (1), propylene glycol, sterile water, ethanol and sodium chloride. Alternative solvents which may be used instead of propylene glycol include glycerol and polyethylene glycol. [064] Suitable flavors, such as menthol and levomenthol, or sweeteners, such as saccharin or saccharin sodium, may be added to those formulations of the invention intended for inhaled/intranasal administration.

[065] Formulations for inhaled/intranasal administration may be formulated to be immediate and/or modified release using, for example, poly(DL-lactic-coglycolic acid (PGLA). Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.

[066] In the case of dry powder inhalers and aerosols, the dosage unit is determined by means of a valve which delivers a metered amount. Units in accordance with the invention are typically arranged to administer a metered dose or "puff" containing from 1 fig to 10 mg of the compound of formula (I). The overall daily dose will typically be in the range 1 lag to 10 mg which may be administered in a single dose or, more usually, as divided doses throughout the day.

[067] Compounds of Formula I may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycols.

[068] For topical use, creams, ointments, jellies, solutions or suspensions, etc., containing the compound of Formula I are employed. (For purposes of this application, topical application shall include mouth washes and gargles.)

[069] Dosage levels of the order of from about 0.01 mg to about 140 mg/kg of body weight per day are useful in the treatment of the above-indicated conditions, or alternatively about 0.5 mg to about 7 g per patient per day. For example, inflammation may be effectively treated by the administration of from about 0.01 to 50 mg of the compound per kilogram of body weight per day, or alternatively about 0.5 mg to about 3.5 g per patient per day, preferably 2.5 mg to 1 g per patient per day.

[070] The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for the oral administration of humans may contain from 0.5 mg to 5 g of active agent compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 95 percent of the total composition. Dosage unit forms will generally contain between from about 1 mg to about 500 mg of an active ingredient, typically 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, or 1000 mg.

Assays For Determining Biological Activity

[071] The compounds of Formula I can be tested using the following assays to determine their prostanoid antagonist or agonist activity in vitro and in vivo and their selectivity. The prostaglandin receptor activities demonstrated are DP, EP1, EP2, EP3, EP4, FP, IP and TP.

[072] Stable expression of prostanoid receptors in the human embryonic kidney (HEK) 293(ebna) cell line

[073] Prostanoid receptor cDNAs corresponding to full length coding sequences are subcloned into the appropriate sites of mammalian expression vectors and transfected into HEK 293(ebna) cells. HEK 293(ebna) cells expressing the individual cDNAs are grown under selection and individual colonies are isolated after 2-3 weeks of growth using the cloning ring method and subsequently expanded into clonal cell lines.

Prostanoid receptor binding assays

[074] Transfected HEK 293(ebna) cells are maintained in culture, harvested and membranes are prepared by differential centrifugation, following lysis of the cells in the presence of protease inhibitors, for use in receptor binding assays. Prostanoid receptor binding assays (for DPI, DP2 (CRTH2), EP1, EP2, EP3-III, EP4, FP, IP, and TP) are performed in 10 mM MES/KOH (pH 6.0) (EPs, FP and TP) or 10 mM HEPES/KOH (pH 7.4) (DPs and IP), containing 1 mM EDTA, 2.5-30 mM divalent cation and the appropriate radioligand. Synthetic compounds are added in dimethylsulfoxide which is kept constant at 1 % (v/v) in all incubations. The reaction is initiated by addition of membrane protein. Non-specific binding is determined in the presence of 10 mM of the corresponding non-radioactive prostanoid . Incubations are conducted for 60-90 min at room temperature or 30 °C and terminated by rapid filtration. Specific binding is calculated by subtracting non specific binding from total binding. The residual specific binding at each ligand concentration is calculated and expressed as a function of ligand concentration in order to construct sigmoidal concentration-response curves. The binding affinity of the compounds is determined by calculating the equilibrium inhibition constant (Ki) from the equation Ki=lnPt/l+[radioligand]/Kd where Kd is the equilibrium dissociation constant for the radioligand:receptor interaction and InPt is the inflection point of the dose-response curves.

[075] EP4 receptor binding assays were performed at MSD Pharma Service in Taiwan under the following assay conditions:

Source: Human recombinant Chem-1 cells

Ligand: 1 nM [ ³ H] Prostaglandin E ₂ (PGE ₂ )

Vehicle: 1% DM SO

Incubation Time/Temp: 2 hours @ 25 °C

Incubation Buffer: 10 mM MES, pH 6.0, 1 mM EDTA, 10 mM MgCI.

Non-Specific Ligand: 10 mM Prostaglandin E ₂ (PGE ₂ )

KD: 0.69 nM

Bmax: 4.S pmole/mg Protein *

Specific binding: 90%

Quantitation Method: radioligand binding

Significance Criteria: >50% maximum inhibition

Table 1. Inhibition of PGE2 Binding of Represented Compounds

Prostanoid receptor agonist and antagonist assays

[076] Whole cell second messenger assays measuring stimulation of intracellular cAMP accumulation in HEK-293(ebna)-hEP4 cells are performed to determine whether receptor ligands are agonists or antagonists. Cells are harvested and resuspended in HBSS containing 25 mM HEPES, pH 7.4. Incubations contain 0.5 mM IBMX (phosphodiesterase inhibitor, available from Biomol). Samples are incubated at 37°C for 10 min, the reaction is terminated and cAMP levels are then measured. Ligands are added in dimethylsulfoxide which is kept constant at 1 % (v/v; agonists) or 2% (v/v; antagonists) in all incubations. For agonists, second messenger responses are expressed as a function of ligand concentration and both EC50 values and the maximum response as compared to a PGE2 standard are calculated. For antagonists, the ability of a ligand to inhibit an agonist response is determined by carrying out dose-response curves in the presence of PGE2 agonist at a concentration corresponding to its EC70. IC50 values are calculated as the concentration of ligand required to inhibit 50% of the PGE2-induced activity.

Rat Paw Edema Assay

[077] The method is the same as described in Chan et al (J. Pharmacol. Exp. Ther. 274: 1531- 1537, 1995).

Acute Inflammatory Hyperalgesia Induced by Carrageenan in Rats

[078] The method is the same as described in Boyce et al (Neuropharmacology 33: 1609-1611, 1994).

Adjuvant-Induced Arthritis in Rats

[079] Female Lewis rats (body weight ~146-170 g) are weighed, ear marked, and assigned to groups (a negative control group in which arthritis was not induced, a vehicle control group, a positive control group administered indomethacin at a total daily dose of 1 mg/kg and four groups administered with a test compound at total daily doses of 0.10-3.0 mg/kg) such that the body weights were equivalent within each group. Six groups of 10 rats each are injected into a hind paw with 0.5 mg of Mycobacterium butyricum in 0.1 mL of light mineral oil (adjuvant), and a negative control group of 10 rats was not injected with adjuvant. Body weights, contralateral paw volumes (determined by mercury displacement plethysmography) and lateral radiographs (obtained under Ketamine and Xylazine anesthesia) are determined before (day -1) and 21 days following adjuvant injection, and primary paw volumes are determined before (day -1) and on days 4 and 21 following adjuvant injection. The rats are anesthetized with an intramuscular injection of 0.03 - 0.1 mL of a combination of Ketamine (87 mg/kg) and Xylazine (13 mg/kg) for radiographs and injection of adjuvant. The radiographs are made of both hind paws on day 0 and day 21 using the Faxitron (45 kVp, 30 seconds) and Kodak X-OMAT TL film, and are developed in an automatic processor. Radiographs are evaluated for changes in the soft and hard tissues by an investigator who was blinded to experimental treatment. The following radiographic changes are graded numerically according to severity: increased soft issue volume (0-4), narrowing or widening of joint spaces (0-5) subchondral erosion (0-3), periosteal reaction (0-4), osteolysis (0- 4) subluxation (0-3), and degenerative joint changes (0-3). Specific criteria are used to establish the numerical grade of severity for each radiographic change. The maximum possible score per foot was 26. A test compound at total daily doses of 0.1, 0.3, 1, and 3 mg/kg/day, indomethacin at a total daily dose of 1 mg/kg/day, or vehicle (0.5% methocel in sterile water) are administered per os b.i.d. beginning post injection of adjuvant and continuing for 21 days. The compounds are prepared weekly, refrigerated in the dark until used, and vortex mixed immediately prior to administration.

Mouse syngeneic model for measurement of antitumor activity

[080] The assays described in the article by Spranger et al (Journal for ImmunoTherapy of Cancer 2014, 2:3) can be used to evaluate the synergistic effects of the compounds of the present invention in combination with an effective amount of antibodies to cytotoxic t-lymphocyte antigen 4 (anti-CTLA4); antibodies to programmed death ligand 1 (anti-PDLI); antibodies to programmed cell death protein 1 (anti-PDl); indoleamine-2, 3-dioxygenase (IDO) inhibitors; tryptophan-2, 3-dioxygenase (TDO) inhibitors.

METHOD OF SYNTHESIS

Scheme 1

Scheme 3:

Scheme 4

[081] In the above schemes, the designation Ar corresponds to Arl in Formula I and is optionally substituted as described herein.

Example 1: 4-(l-(7-(4-(trifluoromethyl)phenoxy)-lH-indole-l- carboxamido)cyclopropyl)benzoic acid

Scheme 1

Step 1: Methyl 4-(l-isocyanatocyclopropyl)benzoate

[082] To a solution of methyl 4-(l-aminocyclopropyl)benzoate (1.3 g, 6.87 mmol) and triphosgene (0.621g, 2.09 mmol) in toluene (30 mL) was added Et ₃ N (1.45 mL, 10.46 mmol) dropwise at 0 °C. This reaction was stirred at 0 °C for 30 min Then, stirred at room temperature for lh. TLC check reaction, the starting material was consumed by TLC, and filtration this suspension and concentration to give a 0.69 g of the title product as a pale-yellow solid, which was used for next step without further purification.

Step 2: l-Nitro-2-(4-(trifluoromethyl)phenoxy)benzene

[083] A solution of l-fluoro-2-nitrobenzene (1.41 g, 10 mmol), 4-(trifluoromethyl)phenol (1.63 g, 10 mmol) and k2C03 (2.76 g, 20 mmol) in DMF (25 ml) were stirred at 100 oC for 6h. TLC showed that all the starting material was consumed and a new spot were detected. 150 ml of water was added, then this mixture was extracted with EtOAc(40 ml*3), washed by brine, dried over Na2S04 and concentrated the crude was separated by silica gel column chromatography(EtOAc in hexane from 0 to 10%) to give 2.79 g of the title product as a pale yellow solid. MS (ES-API positive): 284 (M+l)+.

Step 3: 7-(4-(trifluoromethyl)phenoxy)-lH-indole

[084] To a solution of l-nitro-2-(4-(trifluoromethyl)phenoxy)benzene (2.79 g, 10 mmol) in THF (40 mL) was added dropwise EtMgBr (1 M, 30 mL) at -40 oC under N2 atmosphere. The resulting solution was stirred at -40 oC for lh. TLC showed that the starting material was consumed. And 30 mL of sat. NH4CI was added to quench this reaction, this mixture was extracted with EtOAc(30 mL*3), washed by brine, dried over Na2S04 and concentrated. This crude was purified via silica gel column (EtOAc in hexane from 0 to 7%) to give 0.91 g of the title product as a yellow solid. MS (ES-API positive): 278 (M+l)+.

Step 4: Methyl 4-(l-(7-(4-(trifluoromethyl)phenoxy)-lH-indole-l- carboxamido)cyclopropyl)benzoate

[085] To a solution of 7-(4-(trifluoromethyl)phenoxy)-lH-indole (0.1 g, 0.36 mmol) in THF (3 mL) was added n-BuLi (1.6 M, 0.24 mL) at 0 oC. After the reaction mixture was stirred at 0 oC for 15 min and for 10 min at room temperature, methyl 4-(l-isocyanatocyclopropyl)benzoate (0.078 g, 0.36) in 1 mL of THF was added. After stirred 5 min at room temperature, a solution of Sat. NH4CI (3 mL) and water (9 mL) was added to quench this reaction. The resulting solution was extracted with EtOAc(15mL*3), washed by brine, dried over Na2S04 and concentrated. The crude was purified via silica gel column (EtOAc in Hexane from 0 to 15%) to give 0.07 g of the title product as a white solid. MS (ES-API positive): 495 (M+l)+.

Step 5: 4-(l-(7-(4-(trifluoromethyl)phenoxy)-lH-indole-l-carboxamido )cyclopropyl)benzoic acid

[086] To a solution of methyl 4-(l-(7-(4-(trifluoromethyl)phenoxy)-lH-indole-l- carboxamido)cyclopropyl)benzoate (70 mg, 0.142 mmol) in THF (12 mL) was added LiOH (5.1 mg, 0.213 mmol) in water (6 mL) at room temperature. The resulting solution was stirred at room temperature overnight. The desired MS peak was detected by LCMS and most of the starting material was remained, also about 10% of the staring material was decomposed.

Another 3.1 mg of LiOH was added and stirred for another 2 days. The desired MS peak as a main peak was detected by LCMS and about 10 % of staring material was remained and 18% of decomposed component. 5 mL of the Sat. NH4CI was added to quench this reaction. This mixture was extracted with EtOAc (15mL*3), washed by brine, dried over Na2S04 and concentrated. This crude was mixture with about 500 mg of silica and separated via silica gel column (MeOH in DCM from 0 to 2%) to give 0.008 g of final product as a white solid.

^X H NMR (400 MHz, CDCI ₃ ): 0 8.61 (s, 1H), 8.03 (d, 1H), 7.87 (d, 2H), 7.57 (d, 2H), 7.51 (d, 1H), 7.24

(t, 1H), 7.14 (d, 2H), 7.01 (d, 2H), 6.89 (d, 1H), 6.68 (d, 1H), 1.25-1.36, (m, 4H).

MS (ES-API positive): 481 (M+l)+.

Example 2: 4-(l-(7-(4-(trifluoromethyl)phenoxy)indoline-l-carboxamido)c yclopropyl)benzoic acid

Scheme 2

Step l:7-(4-(trifluoromethyl)phenoxy)indoline

[087] To a solution of 7-(4-(trifluoromethyl)phenoxy)-lH-indole (0.1 g, 0.361 mmol) in HOAc (2 mL) was NaBH ₃ CN (0.045 g, 0.722 mmol) at room temperature. The reaction mixture was stirred at room temperature for overnight. TLC showed that all the starting material was consumed and one new spot was detected. Sat. Na ₂ CC>3 to quench this reaction and the resulting solution was extracted with EtOAc (10 mL*3), washed by brine, dried over Na ₂ S0 ₄ and concentrated to give 0.1 g of the title product as a pale yellow solid, which was used for next step without further purification.

MS (ES-API positive): 280 (M+l)+.

Step 2: methyl 4-(l-(7-(4-(trifluoromethyl)phenoxy)indoline-l-carboxamido) cyclopropyl) benzoate

[088] To a solution of 7-(4-(trifluoromethyl)phenoxy)indoline (0.1 g, 0.36 mmol) and methyl 4- (l-isocyanatocyclopropyl)benzoate (0.156 g, 0.716 mmol) in DCM (3 mL) was added Et3N (0.11 g, 1.07 mmol) at 0 oC. the reaction mixture was stirred overnight at room temperature. The desired MS peak was detected by LCMS and worked well. Sat. NH4CI solution was added to quench this reaction and the resulting solution was extracted with EtOAc(20mL*3), washed by brine, dried over Na2S04 and concentrated. The crude was separated via silica gel column (EtOAc in Hexane from 0 to 30%) to give 0.14 g of final product as a pale yellow solid.

MS (ES-API positive): 497 (M+l)+.

Step 3: 4-(l-(7-(4-(trifluoromethyl)phenoxy)indoline-l-carboxamido)c yclopropyl)benzoic acid

[089] To a solution of methyl 4-(l-(7-(4-(trifluoromethyl)phenoxy)indoline-l- carboxamido)cyclopropyl)benzoate (0.14 g, 0.282 mmol) in THF/MeOH (1/1/, 20 mL) was added a solution of UOH.H20 (0.036 g, 0.845mmol) in water (10 mL) at room temperature. The resulting solution was stirred at room temperature for 5h, The desired MS peak was detected by LCMS and most of the starting material was remained. And another 36 mg of the UOH.H20 was added and stirred at room temperature overnight. The LCMS shows this reaction was completed. IN HCI solution was added to quench this reaction and adjust the pH about 5-6, then the organic solvents were removed under reduced pressure to give a suspension. The solid was filtrated, washed by water and dried by oil pump to give 0.101 g of the product as a white solid. ^X H NMR (400 MHz, CDCI ₃ ): 0 7.87 (d, 2H), 7.55 (d, 2H), 7.18 (d, 1H), 7.14 (d, 2H), 7.09 (t, 1H), 6.99 (s, 1H), 6.90 (t, 3H), 4.28 (t, 2H), 3.09 (t, 2H), 1.27-1.36(m, 4H).

MS (ES-API positive): 483 (M+l)+.

Example 3: 4-((7-(4-(trifluoromethyl)phenoxy)indoline-l-carboxamido)met hyl)benzoic acid

Scheme 3

Step 1: 4-nitrophenyl 7-(4-(trifluoromethyl)phenoxy)indoline-l-carboxylate

[090] To a solution of 7-(4-(trifluoromethyl)phenoxy)indoline (0.1 g, 0.358 mmol) and 4- nitrophenyl carbonochloridate (0.072 g. 0.358 mmol) in THF (2 mL) was added pyridine (0.030 g, 0.358 mmol) at 0 °C. The resulting solution was stirred at room temperature under N2 atmosphere for 5h. TLC showed that the starting material was consumed. Sat. NH4CI was added to quench this reaction and the resulting solution was extracted with EA (15 mL*3), washed by brine, dried over Na ₂ S0 ₄ and concentrated to give the crude title product as a pale yellow solid, which was used for next step without further purification.

Step 2: methyl 4-((7-(4-(trifluoromethyl)phenoxy)indoline-l-carboxamido)met hyl)benzoate

[091] To a solution of crude 4-nitrophenyl 7-(4-(trifluoromethyl)phenoxy)indoline-l- carboxylate (0.B61 mmol) in pyridine (5 mL) was added methyl 4-(aminomethyl)benzoate hydrochloride (0.360 g, 1.79 mmol) and DBU (0.270 g, 1.79 mmol) at room temperature. The reaction mixture was stirred at room temperature for 2 days. TLC showed that all the starting material was consumed and one new spot was detected. And LCMS showed this reaction worked very well. IN HCI solution to quench this reaction and the resulting solution was extracted with EA(15mL*3), washed by brine, dried over Na ₂ S0 ₄ and concentrated, this crude was separated via silica gel column (EtOAc in Hex from 0 to 20%) to give 0.088 g of the title product as a white solid. MS (ES-API positive): 471 (M+l)+.

Step 3: 4-((7-(4-(trifluoromethyl)phenoxy)indoline-l-carboxamido)met hyl)benzoic acid

[092] To a solution of methyl 4-((7-(4-(trifluoromethyl)phenoxy)indoline-l- carboxamido)methyl)benzoate (0.088 g, 0.187 mmol) in THF/MeOH (1/1/, 12 mL) was added a solution of UOH.H2O (0.079 g, 1.87 mmol) in water (6 mL) at room temperature. The resulting solution was stirred at 50 °C for 5 h, The LCMS shows this reaction was completed. IN HCI solution was added to quench this reaction and adjust the pH about 3-4, then the organic solvents were removed under reduced pressure to give a suspension. A lot of the solid was appeared, then, put it in the ultrasonic clearing machine for 5 min. Collection the solid by filtration washed by water and dried by oil pump to give 0.054 g of the title product as a white solid. ^X H NMR (400 MHz, DMSO-d6): 0 12.83 (s, 1H), 7.82 (d, 2H), 7.61 (d, 2H), 7.39 (t, 1H), 7.22 (d, 2H), 7.14 (d, 1H), 7.03 (t, 1H), 6.96 (d, 2H), 6.90 (d, 1H), 4.23 (d, 2H), 4.00 (t, 2H), 3.11 (t. 2H). MS (ES- API positive): 457 (M+l)+.

Example 4: 4-(l-(l-methyl-4-(4-(trifluoromethyl)phenoxy)-lH-indole-3- carboxamido)cyclopropyl)benzoic acid

Example 5: (S)-4-(l-(l-methyl-4-(4-(trifluoromethyl)phenoxy)-lH-indole- 3- carboxamido)ethyl)benzoic acid

Scheme 4

Step 1: 4-(4-(trifluoromethyl)phenoxy)-lH-indole

[093] A solution of lH-indol-4-ol (3.20 g, 24 mmol), l-fluoro-4-(trifluoromethyl)benzene (3.58 g, 21.8 mmol) and CS2CO3 (10.66 g, 32.7 mmol) in DMSO (40 mL) were stirred at 150 oC for 3h. TLC showed that most of the starting material was consumed and the desired product was detected. BOO mL of water was added to quench this reaction and his mixture was extracted with EtOAc (70 mL*3), washed by brine, dried over Na ₂ S0 ₄ and concentrated. The crude was purified via silica gel column (EtOAc in Hex from 0 to 15%) to give 0.95 g of the title product as a brown solid.

MS (ES-API positive): 278 (M+l)+.

Step 2: l-methyl-4-(4-(trifluoromethyl)phenoxy)-lH-indole

[094] To a solution of 4-(4-(trifluoromethyl)phenoxy)-lH-indole (0.30 g, 3.047 mmol) in THF (5 mL) was added NaH (0.062 g, 1.6 mmol, 60%) at 0 °C under N ₂ atmosphere. The resulting solution was stirred at 0 °C for 20 min, then Mel (0.08 mL, 1.3 mmol) was added and stirred at room temperature for 1 h. TLC showed that this reaction worked well. Sat. NH ₄ CI solution was added to quench this reaction and the resulting solution was extracted with EtOAc (10 mL*3), washed by brine, dried over Na ₂ S0 and concentrated to give 0.315 g of the title product as a brown oil, which was used for the next step without further purification.

MS (ES-API positive): 292 (M+l)+.

Step 3: l-methyl-4-(4-(trifluoromethyl)phenoxy)-lH-indole-3-carboxyl ic acid

[095] To a solution of l-methyl-4-(4-(trifluoromethyl)phenoxy)-lH-indole (0.295 g, 1.013 mmol) in DCM (5 mL) was added TFAA (0.319 g, 1.52 mmol) at 0 °C under N ₂ atmosphere. The resulting solution was stirred at 0 °C for 20 min, then at room temperature for another 1 h. LCMS shows this reaction worked well and all the staring material was converted to the intermediate. The solvents were removed under reduced pressure. The obtained crude was used for next step without further purification. To this crude in MeOH (3 mL) was added a solution of NaOH (10 M, 3 mL). This mixture was stirred at 90 °C for 3 h. The LCMS shows this reaction was completed. This reaction was cooled to room temperature and HCI solution (10%) was added to adjust pH=l- 2. The resulting solution was extracted with EA (20 mL*3), washed by brine, dried over Na ₂ S0 and concentrated to give 0.29 g of the title product as a white solid.

MS (ES-API positive): 336 (M+l)+.

Step 4: methyl 4-(l-(l-methyl-4-(4-(trifluoromethyl)phenoxy)-lH-indole-3- carboxamido)cyclopropyl)benzoate

[096] To a solution of l-methyl-4-(4-(trifluoromethyl)phenoxy)-lH-indole-3-carboxyl ic acid (0.032 g, 0.095 mmol), methyl 4-(l-aminocyclopropyl)benzoate (0.022 g, 0.114 mmol) and HATU (0.054 g, 0.143 mmol) in DMF(3 mL) was added NMM (0.019 g, 0.19 mmol) at room temperature. The resulting solution was stirred at room temperature for 3 h. The desired MS peak was detected by LCMS, but most of the starting material was remained. Another 10 eq. of B was added and stirred at 60 °C for 3 days. LCMS shows this reaction was completed. IN HCI solution was added to quench this reaction and adjust pH= 3-4. Then, the resulting solution was extracted with EtOAc (10 mL*3), washed by brine, dried over Na ₂ S0 ₄ and concentrated. This crude product was separated via silica gel column (MeOH in DCM from 0 to 3%) to give 0.040 g of the title product as a pale yellow solid.

MS (ES-API positive): 509 (M+l)+.

Step 5: 4-(l-(l-methyl-4-(4-(trifluoromethyl)phenoxy)-lH-indole-3- carboxamido)cyclopropyl)benzoic acid

[097] To a solution of methyl 4-(l-(l-methyl-4-(4-(trifluoromethyl)phenoxy)-lH-indole-3- carboxamido)cyclopropyl)benzoate (0.40 g, 0.079 mmol) in THF/MeOH (1/1/, 6 mL) was added a solution of UOH.H2O (0.033g, 0.790 mmol) in water (3 mL) at room temperature. The resulting solution was stirred at room temperature for overnight The LCMS shows this reaction was completed. IN HCI solution was added to quench this reaction and adjust the pH about 3-4, then the organic solvents were removed under reduced pressure to give a suspension. This mixture was extracted with EtOAc (15 mL*3), washed by brine and concentrated. This crude was solved with 1.5 mL of CH ₃ CN and put it in the ultrasonic clearing machine for 5 mins. Collection the solid by filtration and dried by oil pump to give 13.7 mg of the title product as a white solid. ^X H NMR (400 MHz, DMSO-d6): 0 8.48 (brs, 1H), 8.00 (s, 1H), 7.80 (d, 2H), 7.72 (d, 2H), 7.47 (d, 1H), 7.32 (t, 1H), 7.23 (d, 2H), 7.18 (d, 2H), 6.85 (d, 1H), 3.98 (s, 3H), 1.20-1.29 (m, 4H).

MS (ES-API positive): 495 (M+l)+.

Step 6: methyl (S)-4-(l-(l-methyl-4-(4-(trifluoromethyl)phenoxy)-lH-indole- 3- carboxamido)ethyl)benzoate

[098] To a solution of l-methyl-4-(4-(trifluoromethyl)phenoxy)-lH-indole-3-carboxyl ic acid (0.055 g, 0.164 mmol), methyl (S)-4-(l-aminoethyl)benzoate (0.147 g, 0.820 mmol) and HATU (0.125 g, 0.328 mmol) in DMF (3 mL) was added NMM (0.050 g, 0.492 mmol) at room temperature. The resulting solution was stirred at 60 oC for overnight. LCMS shows that this reaction worked well and all the starting material was consumed. IN HCI solution was added to quench this reaction and adjust pH= 3-4. Then, the resulting solution was extracted with EtOAc (10 mL*3), washed by brine, dried over Na ₂ S0 ₄ and concentrated. This crude product was separated via silica gel column (MeOH in DCM from 0 to 3%) to give 0.062 g of the title product as a pale yellow solid.

MS (ES-API positive): 497 (M+l)+.

Step 7: (S)-4-(l-(l-methyl-4-(4-(trifluoromethyl)phenoxy)-lH-indole- 3- carboxamido)ethyl)benzoic acid

[099] To a solution of methyl (S)-4-(l-(l-methyl-4-(4-(trifluoromethyl)phenoxy)-lH-indole- 3- carboxamido)ethyl)benzoate (0.062g, 0.125 mmol) in THF/MeOH (1/1/, 10 mL) was added a solution of UOH.H20 (0.053 g, 1.25 mmol) in water (5 mL) at room temperature. The resulting solution was stirred at 50 °C for 5 h. The LCMS shows this reaction was completed. IN HCI solution was added to quench this reaction and adjust the pH about 5-6, then the organic solvents were removed under reduced pressure to give a suspension. This mixture was extracted with EA (15 mL*3), washed by brine and concentrated. This crude was solved with 1.5 mL of CH ₃ CN and put it in the ultrasonic clearing machine for 5 min. Collection the solid by filtration and washed by 1 mL of CH ₃ CN, then dried by oil pump to give 21.9 mg of the title product as a white solid.

^X H NMR (400 MHz, DMSO-d6): 0 12.79 (s, 1H), 8.23 (d, 1H), 7.97 (s, 1H), 7.75 (d, 2H), 7.68 (d, 2H), 7.45 (d, 1H), 7.27 (t, 1H), 7.25 (d, 2H), 7.08 (d, 2H), 6.78 (d, 1H), 5.01 (dt, 1H), 3.87 (s, 3H), 1.29 (d, 3H).

MS (ES-API positive): 483 (M+l)+. Example 6: 4-(l-(4-(4-(trifluoromethyl)phenoxy)-lH-indole-3- carboxamido)cyclopropyl)benzoic acid

Example 6

Scheme 5

Step 1: 4-(4-(trifluoromethyl)phenoxy)-lH-indole-3-carbaldehyde

[0100] To a solution of DMF (2 mL) was added POCI3 (0.10 mL, 1.08 mmol) at 0 oC. The resulting solution was stirred at 0 °C for 20 min and stirred at room temperature for 20 min. Then 4-(4- (trifluoromethyl)phenoxy)-lH-indole (0.20 g, 0.72 mmol) in DMF (2 mL) was added and this mixture was stirred at room temperature for 2 h. TLC shows all the starting material was consumed. The desired MS peak was detected by LCMS. This mixture was poured to 25 mL of Sat. Na ₂ C0 ₃ solution and stirred for another 5 min. And the resulting solution was extracted with EA (20 mL*3), washed by brine, dried over Na ₂ S0 ₄ and concentrated. The crude was separated via silica gel column (EtOAc in Hexane from 0 to 20%) to give 0.11 g of the title product as a white solid. MS (ES-API positive): 306 (M+l)+.

Step 2: 4-(4-(trifluoromethyl)phenoxy)-lH-indole-3-carboxylic acid

[0101] To a solution of 4-(4-(trifluoromethyl)phenoxy)-lH-indole-3-carbaldehyde (0.11 g, 0.361 mmol) in acetone (3 mL) was added a solution of KMn04 (O.llg, 0.721 mmol) in water (1.5 mL) at room temperature. The resulting solution was stirred at room temperature for 4h. The desired MS peak was detected by LCMS and worked well. 0.2 mL of H2O2 (10%) was added to quench this reaction and filtered. And the acetone was removed, then the pH was adjusted to 1-2 and 40 mL of EtOAc was added, washed by brine, dried over Na ₂ S0 ₄ and concentrated. This crude was separated via silica gel column (MeOH in DCM from 0 to 6%) to give 0.073 g of the title product as brown solid.

MS (ES-API positive): 322 (M+l)+.

Step 3: methyl 4-(l-(4-(4-(trifluoromethyl)phenoxy)-lH-indole-3- carboxamido)cyclopropyl)benzoate

[0102] To a solution of l-methyl-4-(4-(trifluoromethyl)phenoxy)-lH-indole-3-carboxyl ic acid (0.073 g, 0.227 mmol), methyl 4-(l-aminocyclopropyl)benzoate (0.217 g, l.Mmmol) and HATU (0.173 g, 0.454 mmol) in DMF (3 mL) was added NMM (0.046 g, 0.454 mmol) at room

temperature. The resulting solution was stirred at 50 °C for 5 h. LCMS shows this reaction was completed. IN HCI solution was added to quench this reaction and adjust pH= 3-4. Then, the resulting solution was extracted with EtOAc (10 mL*3), washed by brine, dried over Na ₂ S0 ₄ and concentrated. This crude product was separated via silica gel column (MeOH in DCM from 0 to 3%) to give 0.079 g of the title product as a pale yellow solid.

MS (ES-API positive): 495 (M+l)+.

Step 4: 4-(l-(4-(4-(trifluoromethyl)phenoxy)-lH-indole-3-carboxamido )cyclopropyl)benzoic acid

[0103] To a solution of methyl 4-(l-(4-(4-(trifluoromethyl)phenoxy)-lH-indole-3- carboxamido)cyclopropyl)benzoate (0.079 g, 0.160 mmol) in THF/MeOH (1/1/, 6 mL) was added a solution of LiOH.EhO (0.062 g, 1.60 mmol) in water (6 mL) at room temperature. The resulting solution was stirred at 50 °C for 5 h. The LCMS shows this reaction was completed. IN HCI solution was added to quench this reaction and adjust the pH about 3-4, then the organic solvents were removed under reduced pressure to give a suspension. This mixture was extracted with EA (15 mL*3), washed by brine and concentrated. This crude was separated by reversed-phase column (CH ₃ CN in H ₂ 0 (0.1% HCOOH) from 0 to 70%) and lyophilized to give 0.0415 g of the title product as a white solid.

^X H NMR (400 MHz, DMSO-d6): 0 11.94 (s, 1H), 8.51 (s, 1H), 7.92 (d, 1H), 7.70 (d, 2H), 7.65 (d, 2H), 7.40 (d, 1H), 7.22 (t, 1H), 7.06 (d, 2H), 7.04 (d, 1H), 6.78 (d, 1H), 1.05-1.22 (m, 4H). MS (ES-API positive): 481 (M+l)+.