Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
  • A61K31/445—Non condensed piperidines, e.g. piperocaine
  • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
  • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• the invention provides the use of bicyclic pyridyl derivatives compounds in methods of treating disease.
• Fibroblast Growth Factors constitute a family of over twenty structurally related polypeptides that are developmentally regulated and expressed in a wide variety of tissues. FGFs stimulate proliferation, cell migration and differentiation and play a major role in skeletal and limb development, wound healing, tissue repair, hematopoiesis, angiogenesis, and tumorigenesis (reviewed in Ornitz, Novartis Found Symp 232: 63-76; discussion 76-80, 272-82 (2001 )).
• FGFs The biological action of FGFs is mediated by specific cell surface receptors belonging to the Receptor Protein Tyrosine Kinase (RPTK) family of protein kinases. These proteins consist of an extracellular ligand binding domain, a single transmembrane domain and an intracellular tyrosine kinase domain which undergoes phosphorylation upon binding of FGF.
• RPTK Receptor Protein Tyrosine Kinase
• FGFR1 also called Fig, fms-like gene, fit- 2, bFGFR, N-bFGFR or Cek1
• FGFR2 also called Bek-Bacterial Expressed Kinase-, KGFR, Ksam, Ksaml and Cek3
• FGFR3 also called Cek2
• FGFR4 also called Cek2
• FGFRs All mature FGFRs share a common structure consisting of an amino terminal signal peptide, three extracellular immunoglobulin-like domains (Ig domain I, Ig domain II, Ig domain III), with an acidic region between Ig domains (the "acidic box” domain), a transmembrane domain, and intracellular kinase domains (Ullrich and Schlessinger, Cell 61 : 203,1990 ; Johnson and Williams (1992) Adv. Cancer Res. 60: 1-41 ).
• the distinct FGFR isoforms have different binding affinities for the different FGF ligands.
• FGFR4 may play an important role in liver cancer in particular (PLoS One, 2012, volume 7, 36713).
• Other studies have also implicated FGFR4 or its ligand FGF19 in other cancer types including breast, glioblastoma, prostate, rhabdomyosarcoma, gastric, ovarian, lung, colon (Int. J.
• WO2014/144737 and WO2014/01 1900 also describe low molecular weight FGFR4 inhibitors. Given the numerous therapies available to an individual having a particular disease, a
• the present invention aims to address the need for more adapted and effective treatment regimens using FGFR4 inhibitors.
• the invention provides compounds, pharmaceutically acceptable salts thereof, pharmaceutical compositions thereof, which compounds are FGFR4 inhibitors, for use in methods of treating, preventing, or ameliorating cancers.
• the invention relates to methods of treating, preventing or ameliorating solid malignancies in a patient, wherein the solid malignancies are characterized by positive expression of certain biomarkers.
• the invention relates to methods of treating, preventing or ameliorating solid malignancies in a patient, wherein the solid malignancies are characterized by positive expression of certain biomarkers.
• the invention provides, in a first aspect, a compound of formula (I) or a pharmaceutically acceptable salt thereof
• V is selected from CH 2 , O, CH(OH);
• W is selected from CH 2 , CH 2 CH 2 , bond
• X is C(R X ) or N;
• Y is C(R Y ) or N;
• Z is CH or N; wherein when X is N, Y and Z are not N;
• R x is selected from hydrogen, halogen, haloC"i-C 3 alkyl, cyano, Ci-C 6 alkyl, hydroxyCrC 6 alkyl;
• R Y is selected from hydrogen, halogen, Ci-C 3 alkyl, CrC 6 alkoxy, hydroxyd-dalkoxy, NR Y R Y2 , cyano, Ci-C 3 alkoxyCi-C 3 alkoxy, Ci-C 3 alkoxy-haloCrC 3 alkoxy, di(Ci-C 3 alkyl)aminoCi-C 6 alkoxy, O- (CH 2 )o-i-R Y3 , CR Y6 R Y7 , S-Ci-C 3 alkyl, haloCi-C 6 alkoxy optionally substituted with hydroxy;
• R x and R Y together with the ring to which they are attached form a bicyclic aromatic ring system optionally further comprising one or two heteroatoms selected from N, O, or S, which ring system is optionally substituted with d-dalkyl;
• R Y is hydrogen
• R Y2 is selected from Ci-C 6 alkyl; hydroxyC"i-C 6 alkyl; haloCrC 6 alkyl optionally substituted with hydroxy; Ci-C 4 alkoxyCi-C 6 alkyl; haloCi-C 3 alkoxyCi-C 6 alkyl; (CH 2 )o-i-R Y4 ; di(d-dalkyl)aminod- C 6 alkyl substituted with hydroxy; bicycloC 5 -C 8 alkyl optionally substituted with hydroxyd-dalkyl; phenyl substituted with S(0) 2 -CH(CH 3 ) 2 ; C 2 -C 3 alkylsulfonic acid;
• R Y and R Y2 together with the N atom to which they are attached form a saturated or unsaturated non-aromatic 6-membered heterocyclic ring which may contain an O atom, which ring may be substituted once or twice by R Y5 ;
• R Y3 is selected from quinuclidinyl, a 4-, 5- or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S, or a 5- or 6-membered aromatic heterocyclic ring, which saturated or aromatic heterocyclic ring is optionally substituted with d-dalkyl and/or oxo;
• R Y4 is a 4-, 5- or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O, or S, which ring is optionally substituted with d-dalkyl;
• R Y5 is independently selected from d-dalkyl, hydroxy, di(d-dalkyl)aminod-C 3 alkyl,
• R Y5 attached at the same carbon atom form together with the carbon atom to which they are attached a 5-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S, which ring is substituted once or more than once with d-dalkyl;
• R YS and R Y7 together with the carbon atom to which they are attached form a 6-membered saturated or unsaturated non-aromatic heterocyclic ring comprising one heteroatom selected from N, O or S;
• R is selected from hydrogen; halogen; d-dalkyl; halod-dalkyl; hydroxyd-dalkyl; C 3 - dcycloalkyl; CH 2 NR 2 R 3 ; CH(CH 3 )NR 2 R 3 ; d-dalkoxyd-dalkyl; CH 2 C0 2 H; C(0)H; d-dalkoxy; a 5- or 6-membered saturated heterocyclic or aromatic heterocyclic ring comprising at least one heteroatom selected from N, O or S, which ring is optionally substituted once or more than once with a group independently selected from Ci-C 3 alkyl, haloC"i-C 3 alkyl, oxetanyl or oxo;
• R 2 is selected from d-C 3 alkyl, di(Ci-C 3 alkyl)aminoCi-C 3 alkyl;
• R 3 is selected from C C 3 alkyl, C(0)C C 3 alkyl, C(0)-CH 2 -OH, C(0)-CH 2 -0-CH 3 , C(0)-CH 2 - N(CH 3 ) 2 , S(0) 2 CH 3 ;
• R 2 and R 3 together with the N atom to which they are attached form a saturated 5- or 6- membered ring optionally comprising one additional heteroatom selected from N, N-oxide, O or S, which ring may be substituted once or more than once with R 4 ;
• R 4 is independently selected from Ci-C 3 alkyl, di(Ci-C 3 alkyl)amino, C(0)CH 3 , hydroxy;
• R 5 is selected from hydrogen or d-C 3 alkyl
• the terms “compounds of the present invention” or “compounds of the invention” or “compounds used in the present invention” refer to compounds of formula (I), (la), (la- 1 ) and salts thereof as defined herein, as well as all stereoisomers (including diastereoisomers and enantiomers), rotamers, tautomers, isomeric internal addition products and isotopically labeled compounds (including deuterium substitutions), as well as inherently formed moieties.
• V is selected from CH 2 , O, CH(OH);
• W is selected from CH 2 , CH 2 CH 2 , bond
• X is C(R X ) or N;
• Y is C(R Y ) or N;
• Z is CH or N
• R x is selected from hydrogen, halogen, haloCrC 3 alkyl, cyano, Ci-C 6 alkyl, hydroxyCrC 6 alkyl;
• R Y is selected from hydrogen, halogen, Ci-C 3 alkyl, CrC 6 alkoxy, hydroxyC-i-C 3 alkoxy, NR Y R Y2 , cyano, Ci-C 3 alkoxyCi-C 3 alkoxy, Ci-C 3 alkoxy-haloCrC 3 alkoxy, di(Ci-C 3 alkyl)aminoCi-C 6 alkoxy, CD- (CH 2 )o-i-R Y3 , CR Y6 R Y7 , S-Ci-C 3 alkyl, haloCi-C 6 alkoxy optionally substituted with hydroxy;
• R x and R Y together with the ring to which they are attached form a bicyclic aromatic ring system optionally further comprising one or two heteroatoms selected from N, O, or S, which ring system is optionally substituted with d-C 3 alkyl;
• R Y is hydrogen
• R Y2 is selected from Ci-C 6 alkyl; hydroxyC"i-C 6 alkyl; haloCrC 6 alkyl optionally substituted with hydroxy; Ci-C 4 alkoxyCi-C 6 alkyl; haloCi-C 3 alkoxyCi-C 6 alkyl; (CH 2 ) 0- i-R Y4 ; di(Ci-C 3 alkyl)aminoCr C 6 alkyl substituted with hydroxy; bicycloC 5 -C 8 alkyl optionally substituted with hydroxyC-i-C 3 alkyl; phenyl substituted with S(0) 2 -CH(CH 3 ) 2 ; C 2 -C 3 alkylsulfonic acid;
• R Y and R Y2 together with the N atom to which they are attached form a saturated or unsaturated non-aromatic 6-membered heterocyclic ring which may contain an O atom, which ring may be substituted once or twice by R Y5 ;
• R Y3 is selected from quinuclidinyl, a 4-, 5- or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S, or a 5- or 6-membered aromatic heterocyclic ring, which saturated or aromatic heterocyclic ring is optionally substituted with C-i-C 3 alkyl and/or oxo;
• R Y4 is a 4-, 5- or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O, or S, which ring is optionally substituted with Ci-C 3 alkyl;
• R Y5 is independently selected from Ci-C 3 alkyl, hydroxy, d C Csalky aminoC-i-Csalkyl, or
• R Y5 attached at the same carbon atom form together with the carbon atom to which they are attached a 5-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S, which ring is substituted once or more than once with Ci-C 3 alkyl;
• R YS and R Y7 together with the carbon atom to which they are attached form a 6-membered saturated or unsaturated non-aromatic heterocyclic ring comprising one heteroatom selected from N, O or S;
• R is selected from hydrogen; halogen; Ci-C 3 alkyl; haloCi-C 3 alkyl; hydroxyCi-C 3 alkyl; C 3 - Cecycloalkyl; CH 2 NR 2 R 3 ; CH(CH 3 )NR 2 R 3 ; Ci-C 3 alkox Ci-C 3 alk l; CH 2 C0 2 H; C(0)H; C C 3 alkoxy; a 5- or 6-membered saturated heterocyclic or aromatic heterocyclic ring comprising at least one heteroatom selected from N, O or S, which ring is optionally substituted once or more than once with a group independently selected from d-C 3 alkyl, haloC-i-C 3 alkyl, oxetanyl or oxo;
• R 2 is selected from Ci-C 3 alkyl, di(Ci-C 3 alkyl)aminoCi-C 3 alkyl;
• R 3 is selected from C C 3 alkyl, C(0)C C 3 alkyl, C(0)-CH 2 -OH, C(0)-CH 2 -0-CH 3 , C(0)-CH 2 - N(CH 3 ) 2 , S(0) 2 CH 3 ;
• R 2 and R 3 together with the N atom to which they are attached form a saturated 5- or 6- membered ring optionally comprising one additional heteroatom selected from N, N-oxide, O or S, which ring may be substituted once or more than once with R 4 ;
• R 4 is independently selected from Ci-C 3 alkyl, di(Ci-C 3 alkyl)amino, C(0)CH 3 , hydroxy;
• R 5 is selected from hydrogen or Ci-C 3 alkyl.
• R x is selected from hydrogen, halogen, haloC"i-C 3 alkyl, cyano, Ci-C 6 alkyl, hydroxyCrC 6 alkyl;
• R Y is selected from hydrogen, halogen, Ci-C 3 alkyl, CrC 6 alkoxy, hydroxyC-i-C 3 alkoxy, NR Y R Y2 , cyano, Ci-C 3 alkoxyCi-C 3 alkoxy, Ci-C 3 alkoxy-haloCrC 3 alkoxy, di(Ci-C 3 alkyl)aminoCi-C 6 alkoxy, O- (CH 2 )o-i-R Y3 , CR Y6 R Y7 , S-Ci-C 3 alkyl, haloCi-C 6 alkoxy optionally substituted with hydroxy;
• R x and R Y together with the ring to which they are attached form a bicyclic aromatic ring system optionally further comprising one or two heteroatoms selected from N, O, or S, which ring system is optionally substituted with d-C 3 alkyl;
• R Y is hydrogen
• R Y2 is selected from Ci-C 6 alkyl; hydroxyC"i-C 6 alkyl; haloCrC 6 alkyl optionally substituted with hydroxyl; Ci-C 4 alkoxyCi-C 6 alkyl; haloCi-C 3 alkoxyCi-C 6 alkyl; (CH 2 )o-i-R Y4 ; di(C C 3 alkyl)aminoCi- C 6 alkyl substituted with hydroxy; bicycloC 5 -C 8 alkyl optionally substituted with hydroxyC-i-C 3 alkyl; phenyl substituted with S(0) 2 -CH(CH 3 ) 2 ; C 2 -C 3 alkylsulfonic acid;
• R Y and R Y2 together with the N atom to which they are attached form a saturated or unsaturated non-aromatic 6-membered heterocyclic ring which may contain an O atom, which ring may be substituted once or twice by R Y5 ;
• R Y3 is selected from quinuclidinyl, a 4-, 5- or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S, or a 5- or 6-membered aromatic heterocyclic ring, which saturated or aromatic heterocyclic ring is optionally substituted with d-C 3 alkyl and/or oxo;
• R Y4 is a 4-, 5- or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O, or S, which ring is optionally substituted with Ci-C 3 alkyl;
• R Y5 is independently selected from Ci-C 3 alkyl, hydroxy, d C Csalky aminoC-i-Csalkyl, or
• R Y5 attached at the same carbon atom form together with the carbon atom to which they are attached a 5-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S, which ring is substituted with Ci-C 3 alkyl;
• R YS and R Y7 together with the carbon atom to which they are attached form a 6-membered saturated or unsaturated non-aromatic heterocyclic ring comprising one heteroatom selected from N. O or S;
• R is selected from hydrogen, halogen, Ci-C 3 alkyl, haloCi-C 3 alkyl, hydroxyCi-C 3 alkyl, C 3 - Cecycloalkyl, CH 2 NR 2 R 3 , CH(CH 3 )NR 2 R 3 , Ci-C 3 alkoxyCi-C 3 alkyl, CH 2 C0 2 H, C(0)H;
• R 2 is selected from CrC 3 alkyl, di(Ci-C 3 alkyl)aminoCrC 3 alkyl;
• R 3 is selected from C C 3 alkyl, C(0)C C 3 alkyl, C(0)-CH 2 -OH, C(0)-CH 2 -0-CH 3 , C(0)-CH 2 - N(CH 3 ) 2 , S(0) 2 CH 3 ;
• R 2 and R 3 together with the N atom to which they are attached form a saturated 5- or 6- membered ring optionally comprising one additional heteroatom selected from N, N-oxide, O or S, which ring may be substituted once or more than once with R 4 ;
• R 4 is independently selected from d-C 3 alkyl, di(CrC 3 alkyl)amino, C(0)CH 3 , hydroxy;
• a compound of formula (la-1 ) refers to:
• R Y is selected from hydrogen, halogen, Ci-C 3 alkyl, CrC 6 alkoxy, hydroxyCrC 3 alkoxy, NR Y R Y2 , cyano, Ci-C 3 alkoxyCi-C 3 alkoxy, Ci-C 3 alkoxy-haloCrC 3 alkoxy, di(Ci-C 3 alkyl)aminoCi-C 6 alkoxy, O- (CH 2 )o-i-R Y3 , CR Y6 R Y7 , S-Ci-C 3 alkyl, haloCi-C 6 alkoxy optionally substituted with hydroxy;
• R Y is hydrogen
• R Y2 is selected from Ci-C 6 alkyl; hydroxyC"rC 6 alkyl; haloCrC 6 alkyl optionally substituted with hydroxyl; Ci-C 4 alkoxyCi-C 6 alkyl; haloCi-C 3 alkoxyCi-C 6 alkyl; (CH 2 )o-i-R Y4 ; di(C C 3 alkyl)aminoCi- C 6 alkyl substituted with hydroxy; bicycloC 5 -C 8 alkyl optionally substituted with hydroxyCrC 3 alkyl; phenyl substituted with S(0) 2 -CH(CH 3 ) 2 ; C 2 -C 3 alkylsulfonic acid;
• R Y and R Y2 together with the N atom to which they are attached form a saturated or unsaturated non-aromatic 6-membered heterocyclic ring which may contain an O atom, which ring may be substituted once or twice by R Y5 ;
• R Y3 is selected from quinuclidinyl, a 4-, 5- or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S, or a 5- or 6-membered aromatic heterocyclic ring, which saturated or aromatic heterocyclic ring is optionally substituted with d-C 3 alkyl and/or oxo;
• R Y4 is a 4-, 5- or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O, or S, which ring is optionally substituted with d-C 3 alkyl;
• R Y5 is independently selected from d-C 3 alkyl, hydroxy, d C-i-Caalky aminoC-i-Caalkyl, or
• R Y5 attached at the same carbon atom form together with the carbon atom to which they are attached a 5-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S, which ring is substituted with Ci-C 3 alkyl;
• R YS and R Y7 together with the carbon atom to which they are attached form a 6-membered saturated or unsaturated non-aromatic heterocyclic ring comprising one heteroatom selected from N, O or S;
• R is selected from hydrogen, halogen, d-C 3 alkyl, haloCi-C 3 alkyl, hydroxyCi-C 3 alkyl, C 3 - Cecycloalkyl, CH 2 NR 2 R 3 , CH(CH 3 )NR 2 R 3 , Ci-C 3 alkoxyCi-C 3 alkyl, CH 2 C0 2 H, C(0)H;
• R 2 is selected from Ci-C 3 alkyl, di(Ci-C 3 alkyl)aminoCi-C 3 alkyl;
• R 3 is selected from C C 3 alkyl, C(0)C C 3 alkyl, C(0)-CH 2 -OH, C(0)-CH 2 -0-CH 3 , C(0)-CH 2 -
• R 2 and R 3 together with the N atom to which they are attached form a saturated 5- or 6- membered ring optionally comprising one additional heteroatom selected from N, N-oxide, O or S, which ring may be substituted once or more than once with R 4 ;
• R 4 is independently selected from Ci-C 3 alkyl, di(Ci-C 3 alkyl)amino, C(0)CH 3 , hydroxy;
• compounds of the invention where R is hydroxymethyl, CH 2 C0 2 H, 4-piperidinyl e.g. compounds (1-1 ), (I-2) and (I-5), may be in the form as depicted below (compounds (1-1 a), (l-2a) and (l-5a)).
• d-C 6 alkyl refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to six carbon atoms, and which is attached to the rest of the molecule by a single bond.
• d-C 4 alkyl is to be construed accordingly.
• d-dalkyl examples include, but are not limited to, methyl, ethyl, n-propyl, 1 -methylethyl (iso- propyl), n-butyl, n-pentyl and 1 , 1 -dimethylethyl (f-butyl).
• hydroxyd-dalkyl refers to a radical of formula -R a -OH , wherein R a is C 1 -6 alkyl as defined above.
• examples of hydroxyd-dalkyl include, but are not limited to, hydroxy- methyl, 2-hydroxy-ethyl, 2-hydroxy-propyl, 3-hydroxy-propyl and 5-hydroxy-pentyl.
• C 3 -C 6 cycloalkyl refers to saturated monocyclic hydrocarbon groups of 3- 6 carbon atoms.
• Examples of C 3 -C 6 cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
• C"i-C 6 alkoxy refers to a radical of the formula -OR a where R a is a d- C 6 alkyl radical as generally defined above.
• R a is a d- C 6 alkyl radical as generally defined above.
• C"i-C 3 alkoxy is to be construed accordingly. Examples of d-dalkoxy include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, pentoxy, and hexoxy.
• C 1 -C 4 alkoxyC 1 -C 6 alkyl refers to a radical of the formula -R b -0-R a where R a is a C C 4 alkyl radical and R is a Ci-C 6 alkyl radical as defined above.
• R a is a C C 4 alkyl radical and R is a Ci-C 6 alkyl radical as defined above.
• d-dalkoxyd- C 6 alkyl is to be construed accordingly.
• the oxygen atom may be bonded to any carbon atom in either alkyl radical.
• C 1 -C 4 alkoxyC 1 -C 6 alkyl examples include, but are not limited to, methoxy- methyl, methoxy-ethyl, ethoxy-ethyl, 1 -ethoxy-propyl and 2-methoxy-butyl.
• Halogen or halo refers to bromo, chloro, fluoro or iodo.
• halogend-dalkyl or “halod-dalkyl” refers to CrC 6 alkyl radical, as defined above, substituted by one or more halo radicals, as defined above.
• halogend-Cealkyl include, but are not limited to, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1 -fluoromethyl-2-fluoroethyl, 3-bromo-2-fluoropropyl and 1 -bromomethyl-2-bromoethyl.
• halod-C 3 alkoxy refers to d-C 3 alkoxy as defined above, substituted by one or more halo radicals, as defined above.
• halod-C 3 alkoxy include, but are not limited to, trifluoromethoxy, difluoromethoxy, trifluoroethoxy.
• hydroxyd-dalkoxy refers to a d-dalkoxy radical as defined above, wherein one of the hydrogen atoms of the d-dalkoxy radical is replaced by OH.
• hydroxyd-dalkoxy include, but are not limited to, hydroxymethoxy, hydroxyethoxy.
• d-C 3 alkoxyd-C 3 alkoxy refers to a d-C 3 alkoxy radical as defined above, wherein one of the hydrogen atoms of the d -3 alkoxy radical is replaced by -O-d-dalkyl.
• C-i-CaalkoxyC-i-Caalkoxy include, but are not limited to, methoxymethoxy, ethoxymethoxy.
• d-Caalkoxy-haloC Caalkoxy refers to a haloCrC 3 alkoxy radical as defined above, wherein one of the hydrogen atoms of the haloCrC 3 alkoxy radical is replaced by - 0-C"i-C 3 alkyl.
• C-i-Caalkoxy-haloC-i-Caalkoxy include, but are not limited to,
• d C Caalky aminoC Cealkyl refers to a radical of the formula -R a1 - (R a2 )-Ra 2 where R a1 is a CrC 6 alkyl radical as defined above and each R a2 is a CrC 3 alkyl radical, which may be the same or different, as defined above.
• the nitrogen atom may be bonded to any carbon atom in any alkyl radical.
• the "diC-i-CaalkylaminoCrCealkyl” ma y b e substituted with hydroxy.
• d C Caalky aminoC Cealkoxy refers to a radical of the formula -R a1 -
• R a i is a CrC 6 alkoxy radical as defined above and each R a2 is a Ci-C 3 alkyl radical, which may be the same or different, as defined above.
• 6-membered saturated heterocyclic ring comprising one heteroatom selected from N, O or S includes piperidyl, tetrahydropyranyl and tetrahydrothiopyranyl.
• 6-membered unsaturated non-aromatic heterocyclic ring comprising one heteroatom selected from N, O or S includes, but is not limited to, tetrahydropyridinyl
• a 4-, 5-, or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S includes as examples, but is not limited to, azetidinyl, oxetanyl, pyrrolidyl, tetrahydrofuryl, tetrahydrothienyl, piperidyl, piperazinyl, tetrahydropyranyl, morpholinyl.
• 5-membered saturated heterocyclic ring includes as example, but is not limited to, pyrrolidine.
• a saturated 5- or 6- membered ring optionally comprising one additional heteroatom selected from N, O or S includes as examples, but is not limited to, pyrrolidine, oxazolidine, piperazine, morpholine, thiomorpholine rings.
• a "4-, 5- or 6-membered non-aromatic heterocyclic ring comprising at least one heteroatom selected from N, O or S” includes 4-, 5-, or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S as defined herein. It also includes 4-, 5-, or 6-membered unsaturated heterocyclic ring comprising at least one heteroatom selected from N, O or S.
• the term "bicyclic aromatic ring system optionally further comprising one or two heteroatoms selected from N, O or S” includes, but is not limited to, imidazopyridine and isothiazolopyridine.
• bicycloC 5 -C 8 alky refers to bicyclic hydrocarbon groups comprising 5 to 8 carbon atoms including, but not limited to, bicyclo[2.1.1]hexyl, bicyclo[1.1.1]pentyl,
• R Y , R x and R Y together, R Y2 , R Y3 , R Y4 includes unsubstituted or substituted once or twice.
• substituted as used herein, includes substituted once or twice, preferably once.
• substituent R 4 when referring to substituent R 4 , includes 2, 3, 4, 5, or 6 times. Preferably, it includes 2 or 3 times.
• solid malignancies refers to non-hematological malignancies.
• FGFR4 refers to fibroblast growth factor receptor 4, also known as CD334, JTK2, TKF (gene ID: 2264).
• FGF19 refers to fibroblast growth factor 19 (gene ID: 9965)
• KLB refers to the beta-klotho protein (gene ID: 152831 ).
• biomarkers of the invention refers to any of FGFR4, KLB and FGF19.
• FGFR4, KLB and/or FGF19 as described herein can be assessed by methods known to the skilled person such as e.g. RT-qPCR, Western blotting, ELISA,
• any positive expression in FGFR4, KLB and/or FGF19 can be assayed by detecting for expression of the RNA levels of FGFR4, KLB and/or FGF19 or detecting expression of the FGFR4, KLB and/or FGF19 protein product by methods known to the skilled person. It is within the reach of the skilled person to determine a positive expression of FGFR4, FGF19 and / or KLB.
• the positive expression in FGFR4, KLB and/or FGF19 can be assessed as described in the examples.
• test is used to refer to the act of identifying, screening, probing or determining, which act may be performed by any conventional means. For example, a sample may be assayed for the presence of a particular biomarker by using an ELISA assay, a Northern blot, imaging, etc. to detect whether a specific biomarker is present in the sample.
• ELISA assay a Northern blot, imaging, etc. to detect whether a specific biomarker is present in the sample.
• determining contemplate a transformation of matter, e.g., a transformation of a biological sample, e.g., a blood sample or other tissue sample, from one state to another by means of subjecting that sample to physical testing.
• a biological sample e.g., a blood sample or other tissue sample
• the terms “assaying” and “determining” are used to mean testing and/or measuring.
• the phrase “assaying a biological sample from the patient for the presence or the positive expression of FGF19 or FGF19 and KLB or FGFR4, FGF19 and/or KLB” and the like is used to mean that a sample may be tested (either directly or indirectly) for either the presence or absence of a given biomarker or for the level of a particular biomarker.
• selecting and “selected” in reference to a patient is used to mean that a particular patient is specifically chosen from a larger group of patients on the basis of (due to) the particular patient having a predetermined criteria.
• selecting refers to providing treatment to a patient having a particular disease, where that patient is specifically chosen from a larger group of patients on the basis of the particular patient having a predetermined criterion.
• selective administering refers to administering a drug to a patient that is specifically chosen from a larger group of patients on the basis of (due to) the particular patient having a predetermined criterion.
• selectively treating and selectively administering it is meant that a patient is delivered a personalized therapy based on the patient's particular biology, rather than being delivered a standard treatment regimen based solely on the patient having a particular disease.
• Selecting in reference to a method of treatment as used herein, does not refer to fortuitous treatment of a patient that has the biomarker, but rather refers to the deliberate choice to administer treatment to a patient based on the patient having positive expression of the biomarker.
• selective treatment differs from standard treatment, which delivers a particular drug to all patients, regardless of their biomarker.
• “likelihood” and “likely” is a measurement of how probable an event is to occur. It may be used interchangeably with “probability”. Likelihood refers to a probability that is more than speculation, but less than certainty. Thus, an event is likely if a reasonable person using common sense, training or experience concludes that, given the circumstances, an event is probable. In some embodiments, once likelihood has been ascertained, the patient may be treated (or treatment continued, or treatment proceed with a dosage increase) with the test compound. In one embodiment, the "likelihood” and “likely” denote a chance in percent of how probable an event is to occur.
• the phrase "increased likelihood" refers to an increase in the probability that an event will occur.
• some methods herein allow prediction of whether a patient will display an increased likelihood of responding to treatment with the test molecule or an increased likelihood of responding better to treatment with the test molecule.
• the increased likelihood means that there is more than 50% chance, more than 60 % chance, more than 70 % or more than 80 % chance that an event will occur.
• a decreased likelihood means, that the chance is lower than 50%, lower than 60 %, lower than 70 % or lover than 80 %, respectively, that an event will occur.
• FGFR4 and KLB expression characterized by positive FGFR4 and KLB expression, or positive FGFR4 and FGF19 expression, or positive FGFR4, KLB and FGF19 expression.
• R x is selected from halogen, haloCrC 3 alkyl, cyano;
• R Y is selected from hydrogen, halogen, Ci-C 3 alkyl, CrC 6 alkoxy, haloCrC 3 alkoxy, hydroxyCr C 3 alkoxy, NR Y R Y2 , Ci-C 3 alkoxyCi-C 3 alkoxy, Ci-C 3 alkoxy-haloCi-C 3 alkoxy, O-(CH 2 ) 0 -i-R Y3 ;
• R Y is hydrogen and R Y2 is selected from Ci-C 6 alkyl, hydroxyCrC 6 alkyl, d-CsalkoxyC-i-Cealkyl, (CH 2 )o-i-R Y4 , haloC"i-C 6 alkyl optionally substituted with hydroxyl;
• R Y3 is a 4-, 5- or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S, which ring is optionally substituted with d-dalkyl;
• R Y4 is a 4-, 5-, or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S, which ring is optionally substituted with d-dalkyl;
• R is selected from hydrogen, halogen, d-C 3 alkyl, haloCi-C 3 alkyl, hydroxyCi-C 3 alkyl, C 3 - Cecycloalkyl, CH 2 NR 2 R 3 , CH(CH 3 )NR 2 R 3 ;
• R 2 is Ci-C 3 alkyl and R 3 is selected from C C 3 alkyl, C(0)-C C 3 alkyl or
• R 2 and R 3 together with the N atom to which they are attached form a saturated 5- or 6-membered ring optionally comprising one additional heteroatom selected from N, O or S, which ring may be substituted once or more than once with R 4 ;
• R 4 is independently selected from d-dalkyl, di(d-dalkyl)amino or two R 4 attached at the same carbon atom form an oxo group
• FGFR4 and KLB expression characterized by positive FGFR4 and KLB expression, or positive FGFR4 and FGF19 expression, or positive FGFR4, KLB and FGF19 expression.
• R Y is selected from hydrogen, halogen, d-dalkyl, CrC 6 alkoxy, haloC C 3 alkoxy, hydroxyd- dalkoxy, NR Y R Y2 , d-C 3 alkoxyd-C 3 alkoxy, d-dalkoxy-halod-C 3 alkoxy, O-(CH 2 ) 0 -i-R Y3 ;
• R Y is hydrogen and R Y2 is d-dalkyl, hydroxyd-dalkyl, d-dalkoxyd-dalkyl, (CH 2 ) 0 -i-R Y4 haloCrdalkyl optionally substituted with hydroxyl;
• R is a 4-, 5- or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S, which ring is optionally substituted with CrC 3 alkyl;
• R Y4 is a 4-, 5-, or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S, which ring is optionally substituted with CrC 3 alkyl;
• R is selected from hydrogen, halogen, Ci-C 3 alkyl, haloC-i-C 3 alkyl, hydroxyC-i-C 3 alkyl, C 3 - Cecycloalkyl, CH 2 NR 2 R 3 , CH(CH 3 )NR 2 R 3 ;
• R 2 is Ci-C 3 alkyl and R 3 is selected from C C 3 alkyl, C(0)-C C 3 alkyl or
• R 2 and R 3 together with the N atom to which they are attached form a saturated 5- or 6-membered ring optionally comprising one additional heteroatom selected from N, O or S, which ring may be substituted once or more than once with R 4 ;
• R 4 is independently selected from d-C 3 alkyl, di(CrC 3 alkyl)amino or two R 4 attached at the same carbon atom form an oxo group
• R Y is selected from NR Y R Y2 , Ci.C 3 alkoxyCi.C 3 alkoxy, O-(CH 2 ) 0 -i-R Y3 ;
• R Y is hydrogen
• R Y2 is selected from Ci-C 6 alkyl; hydroxyC"i-C 6 alkyl; haloCrC 6 alkyl optionally substituted with hydroxyl; Ci-C 4 alkoxyCi-C 6 alkyl; haloCi-C 3 alkoxyCi-C 6 alkyl; (CH 2 )o-i-R Y4 ; di(C C 3 alkyl)aminoCi- C 6 alkyl substituted with hydroxy; bicycloC 5 -C 8 alkyl optionally substituted with hydroxyC"i-C 3 alkyl; phenyl substituted with S(0) 2 -CH(CH 3 )2; C 2 -C 3 alkylsulfonic acid;
• R Y and R Y2 together with the N atom to which they are attached form a saturated or unsaturated non-aromatic 6-membered heterocyclic ring which may contain an O atom, which ring may be substituted once or twice by R Y5 ;
• R Y3 is selected from quinuclidinyl, a 4-, 5- or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S, or a 5- or 6-membered aromatic heterocyclic ring, which saturated or aromatic heterocyclic ring is optionally substituted with d-dalkyl and/or oxo;
• R Y4 is a 4-, 5- or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O, or S, which ring is optionally substituted with d-C 3 alkyl;
• R Y5 is independently selected from d-C 3 alkyl, hydroxy, di(d-dalkyl)aminod-dalkyl, or
• R Y5 attached at the same carbon atom form together with the carbon atom to which they are attached a 5-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S, which ring is substituted with d-dalkyl;
• R YS and R Y7 together with the carbon atom to which they are attached form a 6-membered saturated or unsaturated non-aromatic heterocyclic ring comprising one heteroatom selected from N, O or S;
• R is selected from hydrogen, halogen, d-dalkyl, haloC-i-C 3 alkyl, hydroxyd-dalkyl, C 3 - Cecycloalkyl, CH 2 NR 2 R 3 , CH(CH 3 )NR 2 R 3 , d-dalkoxyd-dalkyl, CH 2 C0 2 H, C(0)H;
• R 2 is selected from d-dalkyl, di(d-C 3 alkyl)aminod-C 3 alkyl;
• R 3 is selected from d-dalkyl, C(0)d-C 3 alkyl, C(0)-CH 2 -OH, C(0)-CH 2 -0-CH 3 , C(0)-CH 2 - N(CH 3 ) 2 , S(0) 2 CH 3 ;
• R 2 and R 3 together with the N atom to which they are attached form a saturated 5- or 6- membered ring optionally comprising one additional heteroatom selected from N, N-oxide, O or S, which ring may be substituted once or more than once with R 4 ;
• R 4 is independently selected from d-dalkyl, di(d-C 3 alkyl)amino, C(0)CH 3 , hydroxy;
• two R 4 attached at the same ring atom form an oxo group for use in the treatment of solid malignancies characterized by positive FGFR4 and KLB expression, or positive FGFR4 and FGF19 expression, or positive FGFR4, KLB and FGF19 expression.
• R Y is selected from NR Y R Y2 , Ci.C 3 alkoxyCi.C 3 alkoxy, O-(CH 2 ) 0 -i-R Y3 ;
• R Y3 is a 4-, 5- or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S, which ring is optionally substituted with CrC 3 alkyl;
• R Y is hydrogen and R Y2 is C C 6 alkyl, CrC 3 alkoxyCi-C 6 alkyl, (CH 2 ) 0 -rR Y4 ;
• R Y4 is a 4-, 5-, or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S, which ring is optionally substituted with CrC 3 alkyl;
• R is selected from hydrogen, halogen, Ci-C 3 alkyl, haloCi-C 3 alkyl, hydroxyCi-C 3 alkyl, C 3 -
• Cecycloalkyl CH 2 NR 2 R 3 , CH(CH 3 )NR 2 R 3 ;
• R 2 is Ci-C 3 alkyl and R 3 is selected from C C 3 alkyl, C(0)-C C 3 alkyl or
• R 2 and R 3 together with the N atom to which they are attached form a saturated 5- or 6-membered ring optionally comprising one additional heteroatom selected from N, O or S, which ring may be substituted once or more than once with R 4 ;
• R 4 is independently selected from d-C 3 alkyl, di(CrC 3 alkyl)amino or two R 4 attached at the same carbon atom form an oxo group for use in the treatment of solid malignancies characterized by positive FGFR4 and KLB expression, or positive FGFR4 and FGF19 expression, or positive FGFR4, KLB and FGF19 expression.
• R Y is selected from NR Y R Y2 , d.CaalkoxyC-i.Caalkoxy;
• R Y is hydrogen
• R Y2 is selected from Ci-C 6 alkyl; hydroxyC"i-C 6 alkyl; haloCrC 6 alkyl optionally substituted with hydroxyl; Ci-C 4 alkoxyCi-C 6 alkyl; haloCi-C 3 alkoxyCi-C 6 alkyl; (CH 2 )o-i-R Y4 ; di(CrC 3 alkyl)aminoCi- C 6 alkyl substituted with hydroxy; bicycloC 5 -C 8 alkyl optionally substituted with hydroxyCrCaalkyl; phenyl substituted with S(0) 2 -CH(CH 3 )2; C 2 -C 3 alkylsulfonic acid;
• R Y and R Y2 together with the N atom to which they are attached form a saturated or unsaturated non-aromatic 6-membered heterocyclic ring which may contain an O atom, which ring may be substituted once or twice by R Y5 ;
• R Y4 is a 4-, 5- or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O, or S, which ring is optionally substituted with Ci-C 3 alkyl;
• R Y5 is independently selected from d-C 3 alkyl, hydroxy, d C-i-Caalky aminoC-i-Caalkyl, or
• R Y5 attached at the same carbon atom form together with the carbon atom to which they are attached a 5-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S, which ring is substituted with Ci-C 3 alkyl;
• R is selected from hydrogen, halogen, d-C 3 alkyl, haloCi-C 3 alkyl, hydroxyCi-C 3 alkyl, C 3 - Cecycloalkyl, CH 2 NR 2 R 3 , CH(CH 3 )NR 2 R 3 , Ci-C 3 alkoxyCi-C 3 alkyl, CH 2 C0 2 H, C(0)H;
• R 2 is selected from Ci-C 3 alkyl, di(Ci-C 3 alkyl)aminoCi-C 3 alkyl;
• R 3 is selected from C C 3 alkyl, C(0)C C 3 alkyl, C(0)-CH 2 -OH, C(0)-CH 2 -0-CH 3 , C(0)-CH 2 - N(CH 3 ) 2 , S(0) 2 CH 3 ;
• R 2 and R 3 together with the N atom to which they are attached form a saturated 5- or 6- membered ring optionally comprising one additional heteroatom selected from N, N-oxide, O or S, which ring may be substituted once or more than once with R 4 ;
• R 4 is independently selected from d-C 3 alkyl, di(CrC 3 alkyl)amino, C(0)CH 3 , hydroxy;
• R Y is selected from NR Y R Y2 , Ci.C 3 alkoxyCi.C 3 alkoxy;
• R Y is hydrogen and R Y2 is selected from d-C 6 alkyl, Ci-C 3 alkoxyCi-C 6 alkyl, (CH 2 ) 0 -i-R Y4 ;
• R Y4 is a 4-, 5-, or 6-membered saturated heterocyclic ring comprising at least one heteroatom selected from N, O or S, which ring is optionally substituted with CrC 3 alkyl;
• R is selected from CH 2 NR 2 R 3 , CH(CH 3 )NR 2 R 3 ;
• R 2 is Ci-C 3 alkyl and R 3 is selected from C C 3 alkyl, C(0)-C C 3 alkyl or
• R 2 and R 3 together with the N atom to which they are attached form a saturated 5- or 6-membered ring optionally comprising one additional heteroatom selected from N, O or S, which ring may be substituted once or more than once with R 4 ;
• R 4 is independently selected from d-C 3 alkyl, di(CrC 3 alkyl)amino or two R 4 attached at the same carbon atom form an oxo group
• the compound of formula (I), (la) or (la-1 ) used in the present invention is selected from V-(5-cyanopyridin-2-yl)-7-formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2 -/)-carboxamide;
• A/-(5-cyanopyridin-2-yl)-7-formyl- 3,4-dihydro-1 ,8-naphthyridine-1 (2 -/)-carboxamide or a pharmaceutically acceptable salt thereof for use in the treatment of solid malignancies characterized by positive FGFR4 and FGF19 expression are provided.
• rhabdomyosarcoma gastric cancer, ovarian cancer, lung cancer or colon cancer characterized by positive FGFR4 and KLB expression.
• rhabdomyosarcoma gastric cancer, ovarian cancer, lung cancer or colon cancer characterized by positive FGFR4 and FGF19 expression.
• liver cancer breast cancer, glioblastoma, prostate cancer, rhabdomyosarcoma, gastric cancer, ovarian cancer, lung cancer or colon cancer characterized by positive FGFR4 and KLB expression.
• liver cancer breast cancer, glioblastoma, prostate cancer, rhabdomyosarcoma, gastric cancer, ovarian cancer, lung cancer or colon cancer characterized by positive FGFR4 and FGF19 expression.
• liver cancer breast cancer, glioblastoma, prostate cancer, rhabdomyosarcoma, gastric cancer, ovarian cancer, lung cancer or colon cancer characterized by positive FGFR4, KLB and FGF19 expression.
• a compound of formula (I), (la) or (la-1 ) or a pharmaceutically acceptable salt thereof for use in treating a patient having solid malignancies characterized in that the compound of formula (I) or a pharmaceutically acceptable salt thereof is to be administered to the patient on the basis of said patient having positive FGFR4 and KLB expression, or positive FGFR4 and FGF19 expression, or positive FGFR4, KLB and FGF19 expression.
• a compound of formula (I), (la) or (la-1 ) or a pharmaceutically acceptable salt thereof for use in treating a patient having solid malignancies characterized in that a.
• the patient is selected for treatment with a compound of formula (I), (la) or (la-1 ) or a pharmaceutically acceptable salt thereof on the basis of the patient having positive FGFR4 and KLB expression, or positive FGFR4 and FGF19 expression, or positive FGFR4, KLB and FGF19 expression; and
• a compound of formula (I), (la) or (la-1 ) or a pharmaceutically acceptable salt thereof for use in treating a patient having solid malignancies characterized in that a. A biological sample from a patient is assayed for positive FGFR4 and KLB expression, or positive FGFR4 and FGF19 expression, or positive FGFR4, KLB and FGF19 expression; and b. a compound of formula (I), (la) or (la-1 ) or a pharmaceutically acceptable salt thereof is administered to the patient on the basis of the biological sample from the patient having positive FGFR4 and KLB expression, or positive FGFR4 and FGF19 expression, or positive FGFR4, KLB and FGF19 expression.
• a compound of formula (I), (la) or (la-1 ) or a pharmaceutically acceptable salt thereof for use in treating a patient having solid malignancies comprising a.
• the biological sample is characterized by positive FGFR4 and KLB expression, or by positive FGFR4 and FGF19 expression, or by positive FGFR4, KLB and FGF19 expression, administering a compound of formula (I), (la) or (la-1 ) or a pharmaceutically acceptable salt thereof to the patient.
• the invention relates to a method of treating a patient having solid malignancies characterized by positive FGFR4 and KLB expression comprising administering to said patient a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.
• the invention relates to a method of treating a patient having solid malignancies characterized by positive FGFR4 and FGF19 expression comprising administering to said patient a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.
• the invention relates to a method of treating a patient having solid malignancies characterized by positive FGFR4, KLB and FGF19 expression comprising administering to said patient a therapeutically effective amount of a compound of the present invention or a
• the invention relates to a method of selectively treating a patient having solid malignancies, comprising selectively administering a therapeutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof to the patient on the basis of the patient having solid malignancies characterized by positive FGFR4 and KLB expression, or positive FGFR4 and FGF19 expression, or positive FGFR4, KLB and FGF19 expression.
• the invention relates to a method of selectively treating a patient having solid malignancies with a compound of the present invention or a pharmaceutically acceptable salt thereof comprising
• the invention relates to a method of selectively treating a patient having solid malignancies, comprising
• the invention relates to a method of selectively treating a patient having solid malignancies, comprising:
• the biological sample is selected from blood, plasma and tissue sample.
• the positive expression of FGF19 is assayed from a blood sample.
• the positive expression of FGF19 is assayed from a tissue sample.
• the positive expression of FGFR4 or KLB is assayed from a tissue sample.
• the methods of the invention include detecting expression of the biomarker gene product (i.e. FGFR4, KLB, FGF19 gene products) in a sample taken from a patient having solid malignancies and can be performed by detecting, for example, RNA transcribed from the biomarker gene such as mRNA, or polypeptides encoded by the biomarker gene.
• the level of expression of the biomarker can be used to predict whether a patient will likely respond to a compound as described herein. Those patients that have an increased level of expression of the biomarker compared to a control (referred to also herein as patients having a "positive expression of the biomarker") are selected for treatment with the compound described herein as it is predicted that such a patient has an increased likelihood of responding to such compounds. Any appropriate sample of cells taken from a patient having solid malignancies can be used.
• the sample of cells or tissue sample will be obtained from the subject with solid malignancies by biopsy or surgical resection.
• the sample taken from the patient having solid malignancies may be a blood sample.
• the sample of, for example tissue may also be stored in, e.g., RNAIater (Ambion; Austin Tex.) or flash frozen and stored at -80 °C for later use.
• the biopsied tissue sample may also be fixed with a fixative, such as formaldehyde,
• RNA or protein may also be extracted from a fixed or wax-embedded tissue sample or a frozen tissue sample. Once a sample of cells or sample of tissue is removed from the subject with cancer, it may be processed for the isolation of RNA or protein using techniques well known in the art and as described below.
• RNA from a biopsy taken from a patient with solid malignancies can include, for example, guanidium thiocyanate lysis followed by CsCI centrifugation (Chirgwin, et al., Biochemistry 18:5294-5299, 1979).
• RNA from single cells may be obtained as described in methods for preparing cDNA libraries from single cells (see, e.g., Dulac, Curr. Top. Dev. Biol. 36:245, 1998; Jena, et al., J. Immunol. Methods 190:199, 1996).
• the RNA population may be enriched for sequences of interest.
• Enrichment may be accomplished, for example, by random hexamers and primer-specific cDNA synthesis, or multiple rounds of linear amplification based on cDNA synthesis and template-directed in vitro transcription (see, e.g., Wang, et al., Proc. Natl. Acad. Sci. USA 86:9717, 1989; Dulac, et al., supra; Jena, et al., supra).
• the assaying method includes providing a nucleic acid probe comprising a nucleotide sequence, for example, at least 10, 15, 25 or 40 nucleotides, and up to all or nearly all of the coding sequence which is complementary to a portion of the coding sequence of a nucleic acid sequence of FGFR4, KLB and FGF19; obtaining a tissue sample from a mammal having a cancerous cell; contacting the nucleic acid probe under stringent conditions with RNA obtained from a biopsy taken from a patient (e.g., in a Northern blot, in situ hybridization assay, PCR etc); and determining the amount of hybridization of the probe with RNA.
• Nucleic acids may be labeled during or after enrichment and/or amplification of RNAs.
• the step of assaying may comprise a technique as described below. Further examples of assaying are described in the examples.
• the biomarkers FGFR4, KLB and FGF19 can be assayed using any method known in the art such as reverse Transcriptase PCR (RT-PCR).
• RT-PCR reverse Transcriptase PCR
• This method includes isolating mRNA using any technique known in the art, e.g., by using a purification kit, buffer set and protease from commercial manufacturers, such as Qiagen.
• the reverse transcription step is typically primed using specific primers, random hexamers, or oligo-dT primers, depending on the circumstances and the goal of expression profiling and the cDNA derived can then be used as a template in the subsequent PCR reaction.
• TaqMan(R) RT-PCR can then be performed using, e.g., commercially available equipment.
• RT-PCR technique measures PCR product accumulation through a dual-labeled fluorigenic probe (e.g., using
• Real time PCR is compatible both with quantitative competitive PCR, where internal competitor for each target sequence is used for normalization, and with quantitative comparative PCR using a normalization gene contained within the sample, or a housekeeping gene for RT-PCR.
• quantitative competitive PCR where internal competitor for each target sequence is used for normalization
• quantitative comparative PCR using a normalization gene contained within the sample, or a housekeeping gene for RT-PCR.
• microarrays which include one or more probes corresponding to one or more of genes FGFR4, KLB and FGF19.
• the method includes the production of hybridization patterns of labeled target nucleic acids on the array surface.
• the resultant hybridization patterns of labeled nucleic acids may be visualized or detected in a variety of ways, with the particular manner of detection selected based on the particular label of the target nucleic acid.
• Representative detection means include scintillation counting, autoradiography, fluorescence measurement, calorimetric measurement, light emission measurement, light scattering, and the like.
• a TaqMan® Low Density Array (TLDA) card can be used which can include one or more probes corresponding to one or more of genes FGFR4, KLB and FGF19.
• TLDA TaqMan® Low Density Array
• the method of detection utilizes an array scanner that is commercially available (Affymetrix, Santa Clara, Calif.).
• the scanner is controlled from a system computer with an interface and easy-to-use software tools.
• the output may be directly imported into or directly read by a variety of software applications. Scanning devices are described in, for example, U.S. Pat. Nos. 5, 143,854 and 5,424,186.
• a control for comparison can be determined by one skilled in the art.
• a control is determined by choosing an expression value that serves as a cut-off value such that the value differentiates between those test samples that have increased biomarker expression, positive expression, from those that do not.
• the control can be a test sample taken from a healthy person or a sample such as a tumor sample where the biomarkers do not show expression or do not have increased expression above a normal/basal level.
• the expression of each biomarker is measured and can be converted into an expression value after normalization by the expression level of a housekeeping gene. These expression values then can be used to generate a score which is then compared against a cut-off to select which subjects have positive biomarker expression and therefore are likely to benefit from treatment with compound as described herein.
• the presence of a protein product encoded by the biomarkers can be assayed using any appropriate method known in the art and the level of protein product can be compared to a control.
• exemplary immunoassays include fluorescence polarization immunoassay (FPIA) 5 fluorescence immunoassay (FIA), enzyme immunoassay (EIA), nephelometric inhibition immunoassay (NIA), enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay (RIA).
• FPIA fluorescence polarization immunoassay
• FIA fluorescence immunoassay
• EIA enzyme immunoassay
• NIA nephelometric inhibition immunoassay
• ELISA enzyme-linked immunosorbent assay
• RIA radioimmunoassay
• An indicator moiety, or label group may be attached to the subject antibodies and is selected so as to meet the needs of various uses of the method that are often dictated by the availability of assay equipment and
• immunohistochemistry can be used.
• antibodies generated against the biomarkers of the invention can be used for visualizing for the presence of a protein of interest and can be labeled, for example, using a reporter molecule such as fluorophores, enzymes, biotin, chemiluminescent molecules, bioluminescent molecules, digoxigenin, avidin, streptavidin or radioisotopes.
• a reporter molecule such as fluorophores, enzymes, biotin, chemiluminescent molecules, bioluminescent molecules, digoxigenin, avidin, streptavidin or radioisotopes.
• the solid malignancies are from a cancer selected from liver cancer, breast cancer, glioblastoma, prostate cancer, rhabdomyosarcoma, gastric cancer, ovarian cancer, lung cancer, colon cancer.
• the invention relates to a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof and one or more
• the invention relates to a method of treating cancer, comprising selectively administering a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof to a patient in need thereof on the basis of said patient having positive FGFR4 and KLB expression, or positive FGFR4 and FGF19 expression, or positive FGFR4, KLB and FGF19 expression.
• the cancer is selected from liver cancer, breast cancer, glioblastoma, prostate cancer, rhabdomyosarcoma, gastric cancer, ovarian cancer, lung cancer, colon cancer.
• the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein.
• pharmaceutically acceptable carriers are sterile.
• the pharmaceutical composition can be formulated for particular routes of administration such as oral administration, parenteral administration, and rectal administration, etc.
• the pharmaceutical compositions of the present invention can be made up in a solid form (including without limitation capsules, tablets, pills, granules, powders or suppositories), or in a liquid form (including without limitation solutions, suspensions or emulsions).
• compositions can be subjected to conventional pharmaceutical operations such as sterilization and/or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and buffers, etc.
• the pharmaceutical compositions are tablets or gelatin capsules comprising the active ingredient together with one or more of:
• diluents e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine;
• lubricants e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or
• binders e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone;
• disintegrants e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and e) absorbents, colorants, flavors and sweeteners.
• the pharmaceutical compositions are capsules comprising the active ingredient only.
• Tablets may be either film coated or enteric coated according to methods known in the art.
• compositions for oral administration include an effective amount of a compound of the invention in the form of tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs, solutions or solid dispersion.
• compositions intended for oral use are prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions can 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 may contain the active ingredient in admixture with nontoxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
• excipients are, 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 are uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
• a time delay material such as glyceryl monostearate or glyceryl distearate can be employed.
• Formulations for oral use can 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 ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.
• an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
• water or an oil medium for example, peanut oil, liquid paraffin or olive oil.
• compositions are aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions.
• Said compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances.
• Said compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1-75%, or contain about 1-50%, of the active ingredient.
• compositions for transdermal application include an effective amount of a compound of the invention with a suitable carrier.
• Carriers suitable for transdermal delivery include absorbable pharmacologically acceptable solvents to assist passage through the skin of the host.
• transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound of the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin.
• compositions for topical application include aqueous solutions, suspensions, ointments, creams, gels or sprayable formulations, e.g., for delivery by aerosol or the like.
• topical delivery systems will in particular be appropriate for dermal application, e.g., for the treatment of skin cancer, e.g., for prophylactic use in sun creams, lotions, sprays and the like. They are thus particularly suited for use in topical, including cosmetic, formulations well-known in the art.
• Such may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.
• a topical application may also pertain to an inhalation or to an intranasal application. They may be conveniently delivered in the form of a dry powder (either alone, as a mixture, for example a dry blend with lactose, or a mixed component particle, for example with phospholipids) from a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray, atomizer or nebuliser, with or without the use of a suitable propellant.
• a dry powder either alone, as a mixture, for example a dry blend with lactose, or a mixed component particle, for example with phospholipids
• the pharmaceutical composition used in the present invention can be in unit dosage of about 1- 1000 mg of active ingredient(s) for a subject of about 50-70 kg, or about 1-500 mg or about 1-250 mg or about 1-150 mg or about 0.5-100 mg, or about 1-50 mg of active ingredients.
• the therapeutically effective dosage of a compound, the pharmaceutical composition, or the combinations thereof is dependent on the species of the subject, the body weight, age and individual condition, the disorder or disease or the severity thereof being treated. A physician, clinician or veterinarian of ordinary skill can readily determine the effective amount of each of the active ingredients necessary to prevent, treat or inhibit the progress of the disorder or disease.
• All starting materials, building blocks, reagents, acids, bases, dehydrating agents, solvents, and catalysts utilized to synthesise the compounds of the present invention are either commercially available or can be produced by organic synthesis methods known to one of ordinary skill in the art. Further, the compounds of the present invention can be produced by organic synthesis methods known to one of ordinary skill in the art as shown in the following examples.
• DIPEA V V-diisopropylethylamine, A/-ethyl-A/-isopropylpropan-2-amine
• DSC DSC measurements were performed using a DSC Q2000 (TA Instruments, New Castle, DE, USA) equipped with a DSC Refrigerated Cooling System (TA Instruments, New Castle, DE, USA). Data were treated mathematically using the resident Universal Analysis ® Software. Calibration for temperature and heat of fusion was carried out with indium as reference material. The samples were analyzed in open aluminium pans and scanned under a nitrogen purge with a heating rate of 10°C/min from 20 to 300 °C.
• Samples were absorbed on Isolute, or on silica gel, or applied as solutions.
• Example 3 A/-(5-cyanopyridin-2-yl)-7-formyl-3,4-dihydro-1 ,8-naphthyridine-1 (2 -/)-carboxamide.
• Example 27 V-(5-cyanopyridin-2-yl)-7-formyl-6-(hydroxymethyl)-3,4-dihydro-1 ,8-naphthyridine- 1 (2H)-carboxamide.
• the crude material was purified by normal phase chromatography (4 g silica gel cartridge, heptanes/EtOAc 100:0 to 0:100) followed by a reverse phase chromatography (13 g C18 cartridge, 0.1 % TFA in water/acetonitrile 80:20 to 0: 100).
• the product containing fractions were treated with sat. aq. Na 2 C0 3 , concentrated until the organic solvent had been removed extracted with DCM (3x).
• the combined organic layers were dried over Na 2 S0 4 , filtered and evaporated to give the title compound as a colorless resin. (UPLC-MS 3) t R 0.92 min; ESI-MS 384.1 [M+H] + .
• reaction mixture was quenched by addition of sat. aq. NH 4 CI, warmed to room temperature and extracted with EtOAc (2x). The combined organic layers were washed with brine, dried over Na 2 S0 4 , filtered and concentrated under reduced pressure.
• the residue was purified by normal phase chromatography (12 g silica gel cartridge, heptanes/EtOAc 100:0 to 0: 100) the 7-(dimethoxymethyl)-6-formyl- V-(5- (trifluoromethyl)pyridin-2-yl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide containing fractions were concentrated to give a white solid.
• Reference Intermediate 12 6-bromo-7-(dimethoxymethyl)-1 ,2,3,4-tetrahydro-1 ,8-naphthyridine.
• 7-(dimethoxymethyl)-1 ,2,3,4-tetrahydro-1 ,8- naphthyridine (intermediate 4, 1 14.6 g, 550.3mmol) in acetonitrile (2 I).
• NBS 103 g, 578 mol
• the resulting solution was stirred for 30 min at 25 °C.
• the resulting mixture was concentrated under vacuum and the residue was diluted with 1000 ml of diethylether.
• Example 39 V-(5-cyano-4-(2-methoxyethoxy)pyridin-2-yl)-7-formyl-6-(hydroxymethyl)-3,4-dihydro- 1 ,8-naphthyridine-1 (2H)-carboxamide.
• n-BuLi (1.6 M in hexane, 49.0 ml, 78 mmol) was added and the reaction mixture was stirred for 20 min, then again n-BuLi (1.6 M in hexane, 6.53 ml, 10.45 mmol) was added and the mixture was stirred for 10 min at - 78 °C.
• DMF (2.10 ml, 27.2 mmol) was added and the reaction mixture was stirred at -78 °C for 45 min, then it was allowed to warm to room temperature, poured into sat. aq. NH 4 CI and extracted twice with DCM.
• Example 49 (f?)- V-(5-cyano-4-((tetrahydrofuran-3-yl)oxy)pyridin-2-yl)-7-formyl-6-(hydroxymethyl)- 3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide.
• Example 50 (S)- V-(5-cyano-4-((tetrahydrofuran-3-yl)oxy)pyridin-2-yl)-7-formyl-6-(hydroxymethyl)- 3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide.
• Example 63 V-(5-cyano-4-(2-methoxyethoxy)pyridin-2-yl)-6-(difluoromethyl)-7-formyl-3,4-dihydro- 1 ,8-naphthyridine-1 (2H)-carboxamide.
• Example 80 V-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-(hydroxymethyl)-3,4- dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide.
• Example 83 V-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2- oxopiperazin-1-yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide.
• N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((4-methyl-2- oxopiperazin-1-yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide (6.5 g, 12.83 mmol) was placed in a 500ml 4-flask reactor. 49 ml of glacial acetic acid was added and the resulting suspension was stirred at 23 °C until a clear mixture was obtained.
• Example 92 N-(5-cyano-4-isopropoxypyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin-1- yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide.
• Tartrate salt with 1 :1 stoichiometry (mw 641.63): A solution of L-(+)-tartaric acid in acetone (0.1 M, 2.03 ml, 0.203 mmol) was added to a suspension of N-(5-cyano-4-isopropoxypyridin-2-yl)-7-formyl- 6-((4-methyl-2-oxopiperazin-1-yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide (100 mg, 0.203 mmol) in acetone (5 ml) at room temperature. The mixture was warmed to 55 °C, maintained at this temperature for 3 h and cooled slowly to room temperature.
• Tosylate salt with 1 :1 stoichiometry (mw 663.75): A solution of tosic acid in acetone (0.1 M, 2.03 ml, 0.203 mmol) was added to a suspension of N-(5-cyano-4-isopropoxypyridin-2-yl)-7-formyl-6-((4- methyl-2-oxopiperazin-1-yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide (100 mg, 0.203 mmol) in acetone (5 ml) at room temperature. The mixture was warmed to 55 °C, maintained at this temperature for 3 h and cooled slowly to room temperature.
• Citrate salt with 1 :1 stoichiometry (mw 683.68): A solution of citric acid in acetone (0.1 M, 2.03 ml, 0.203 mmol) was added to a suspension of N-(5-cyano-4-isopropoxypyridin-2-yl)-7-formyl-6-((4- methyl-2-oxopiperazin-1-yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide (100 mg, 0.203 mmol) in DCM (2 ml) at room temperature. The mixture was warmed with a bath at 65 °C, maintained at this temperature for 10 min and slowly cooled to 5 °C. The white preciptate that formed was collected, acetone (5 ml) and EtOH (1 ml) were added and the mixture heated at 50 °C for 3 h. The mixture was cooled to 5 °C, filtered and dried to give the title compound.
• reaction mixture was evaporated and applied to a 24 g RediSep silica column as a DCM solution and purified by normal phase chromatography, eluting with a gradient from DCM to 10% MeOH in DCM. Product containing fractions were combined and evaporated to give the title compound as an off-white solid.
• UPLC-MS 6 t R 0.92; ESI-MS 538.7 [M+H] + .
• Phenyl chloroformate (3.89 ml, 31.0 mmol) was added drop wise to a mixture of 6-amino-4- isopropoxynicotinonitrile (intermediate 97, 2.5 g, 14.1 1 mmol) and pyridine (2.51 ml, 31.0 mmol) in THF (100 ml) at room temperature.
• the reaction mixture was stirred for 12 h at room temperature, additional pyridine (2.51 ml, 31.0 mmol) added, before stirring for an additional 12 h and then partitioned between EtOAc and saturated aqueous NaHC0 3 solution.
• the organic layer was washed with saturated brine, dried over MgS0 4 and evaporated.
• the residue was triturated with Et 2 0 and the product obtained by filtration as a beige solid.
• UPLC-MS 7 t R 1.09; ESI-MS 298.2 [M+H] + .
• the reaction mixture was stirred for 21 h at room temperature and additional sodium triacetoxyborohydride (2.6 g, 9.74 mmol) was added. After a further 4 h stirring at room temperature, again additional sodium triacetoxyborohydride (1.3 g, 4.87 mmol) was added and the reaction maintained at 4 °C for 2.5 days. The reaction mixture was then warmed to room temperature, saturated aqueous NaHC0 3 solution added, the mixture extracted with DCM (3x), the combined organic layers dried over Na 2 S0 4 and evaporated. The residue was applied to a 120 g RediSep® silica column as a DCM solution and purified by normal phase chromatography, eluting with a gradient from DCM to 10% MeOH in DCM.
• Example 101 N-(5-cyano-4-(2-methoxyethoxy)pyridin-2-yl)-7-formyl-6-((4-methyl-2-oxopiperazin- 1-yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide.
• Citrate salt with 1 :1 stoichiometry (mw 699.68): A solution of citric acid (0.1 M, 1.97 ml, 0.197 mmol) was added to a suspension of N-(5-cyano-4-(2-methoxyethoxy)pyridin-2-yl)-7-formyl-6-((4- methyl-2-oxopiperazin-1-yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide (100 mg, 0.197 mmol) in acetone (5 ml) at room temperature. The mixture was stirred at 55 °C for 3 h, cooled slowly to room temperature, the white precipitate collected by filtration and dried under vacuum to give the title compound.
• Phenyl chloroformate (4.93 ml, 39.3 mmol) was added drop wise to a mixture of 6-amino-4-(2- methoxyethoxy)nicotinonitrile (intermediate 20, 3.45 g, 17.86 mmol) and pyridine (6.35 ml, 79 mmol) in THF (100 ml) at room temperature.
• the reaction mixture was stirred for 5 h at room temperature and then partitioned between EtOAc and saturated aqueous NaHC0 3 solution, the organic layer washed with saturated brine, dried over MgS0 4 and evaporated. The residue was triturated with EtOAc and the product obtained by filtration as a white solid.
• Example 205 (R)-N-(5-cyano-4-((1-methoxypropan-2-yl)oxy)pyridin-2-yl)-7-formyl-6-((4-methyl-2- oxopiperazin-1-yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide.
• Citrate with 1 :1 stoichiometry (mw 713.71 ): A solution of citric acid in acetone (0.1 M, 2.0 ml, 0.200 mmol) was added to a suspension of (f?)-N-(5-cyano-4-((1-methoxypropan-2-yl)oxy)pyridin-2-yl)-7- formyl-6-((4-methyl-2-oxopiperazin-1-yl)methyl)-3,4-dihydro-1 ,8-naphthyridine-1 (2H)-carboxamide (100 mg, 0.192 mmol) in acetone (4 ml) at room temperature.
• the mixture was warmed to 55 °C, maintained at this temperature for 2.5 h with sonication and then cooled slowly to room temperature. After standing 18 h at 5 °C the solid was collected by filtration, washed with acetone and then dried under vacuum to give the title compound.
• Phenyl chloroformate (1.53 ml, 12.2 mmol) was added drop wise to a mixture of (R)-6-amino-4-((1- methoxypropan-2-yl)oxy)nicotinonitrile (intermediate 146, 1.37 g, 5.55 mmol) and pyridine (0.99 ml, 12.2 mmol) in THF (60 ml) at 0 °C. The reaction mixture was stirred for 12 h at room temperature and additional pyridine (0.98 ml, 12.2 mmol) and phenyl chloroformate (1.53 ml, 12.2 mmol) were added.
• Methylene blue staining proliferation assay The effect of compounds on cell proliferation is assessed using HuH-7 hepatocellular carcinoma cells obtained from the Japanese Collection of Research Bioresources Cell Bank (Cat# JCRB0403) and cultured in the vendor-recommended medium (DMEM high glucose (Amimed Cat# 1-26F01-I), 10% foetal calf serum (Invitrogen Cat# 16140-071 ), 1 mM sodium pyruvate (Amimed Cat# 5-60F00-H), 1x Penicillin/Streptomycin (Amimed Cat# 4-01 F00-H)) at 37°C in a humidified 5% C02 incubator.
• DMEM high glucose Amimed Cat# 1-26F01-I
• 10% foetal calf serum Invitrogen Cat# 16140-071
• 1 mM sodium pyruvate Amimed Cat# 5-60F00-H
• Penicillin/Streptomycin Amimed Cat# 4-01 F00-H
• 5000 cells/well were seeded in 96-well tissue culture plates (TPP Cat# 92696) in a total media volume of 100 ⁇ /well and increasing compound dilutions or DMSO were added 24 hours thereafter in triplicates. 72 hours after compound addition, cells were fixed by adding 25 ⁇ _ ⁇ / ⁇ of 20% glutaraldehyde (Sigma Aldrich Cat# G400-4) and incubated for 10 minutes at room temperature. Cells were washed three times with H 2 0, 200 ⁇ Uwe ⁇ and stained with 100 ⁇ _ ⁇ / ⁇ 0.05% methylene blue (ABCR GmbH Cat# AB1 17904) for 10 minutes at room temperature.
• Hep 3B2.1-7 hepatocellular carcinoma cells FU97 gastric cancer cells, JHH7 hepatocellular carcinoma cells or JHH6 hepatocellular carcinoma cells is assessed as above.
• Hep 3B2.1-7, FU97, JHH7 or JHH6 are obtained from the Japanese Collection of Research Bioresources Cell Bank.
• Hep 3B2.1-7 cells are cultured in EMEM + 10%FCS + 1 mM Na pyruvate + 2mM L-glutamine and 2000 cells / well are seeded for the proliferation assay.
• FU97 cells are cultured in DMEM high glucose + 10%FCS + 1 mM Na pyruvate + 4mM L-glutamine + 1x ITS and 2000 cells / well are seeded for the proliferation assay.
• JHH6 and JHH7 cells are cultured in William's E + 10% FCS + 2mM L-glutamine and 3000 cells are seeded for the proliferation assay.
• CCG CellTiter Glo
• HuH-7 hepatocellular carcinoma cells obtained from the Japanese Collection of Research Bioresources Cell Bank (Cat# JCRB0403) and cultured in the vendor-recommended medium (DMEM high glucose (Amimed Cat# 1-26F01-I), 10% foetal calf serum (Invitrogen Cat# 16140- 071 ), 1 mM sodium pyruvate (Amimed Cat# 5-60F00-H), 1x Penicillin/Streptomycin (Amimed Cat# 4-01 F00-H)) at 37°C in a humidified 5% C02 incubator.
• DMEM high glucose Amimed Cat# 1-26F01-I
• 10% foetal calf serum Invitrogen Cat# 16140- 071
• 1 mM sodium pyruvate Amimed Cat# 5-60F00-H
• 1x Penicillin/Streptomycin Amimed Cat# 4-01 F00-H
• Compound-mediated suppression of cell proliferation/viability is assessed by quantification of cellular ATP levels using the CellTiter-Glo (CTG) reagent (Promega, Cat# G7573). Briefly, cells are seeded at 3 ⁇ 00 cells/well/80 ⁇ fresh medium into tissue-culture-treated 96-well plates (Costar Cat#3904), followed by addition of 20 ⁇ medium containing compound dilutions at 5-fold their final intended concentration. Dose-response effects are assessed by 3-fold serial dilutions of the test compound, starting at 10 ⁇ .
• CCG CellTiter-Glo
• the effect of inhibitors on cell viability is quantified following addition of 50 ⁇ CTG and luminescence measurement (integration time: 500ms) as per vendor manual, using a correspondingly equipped multi-mode plate reader (M200Pro, TECAN, Switzerland).
• integration time 500ms
• M200Pro multi-mode plate reader
• the assay background value determined in wells containing medium, but no cells, is subtracted from all data points.
• the number of viable cells is assessed relative to that observed at the time of compound addition using a separate cell plate (day 0).
• the effect of a particular test compound concentration on cell proliferation/viability is expressed as percentage of the background- and day 0-corrected luminescence reading obtained for cells treated with vehicle only (DMSO, 0.1 % f.c), which is set as 100%, whereas the luminescence reading for wells containing medium only, but no cells, is set as -100% .
• Compound concentrations leading to half- maximal growth inhibition (GI50) are determined using standard four parameter curve fitting (XLfit 5.2., IDBS, UK).
• the compounds can be tested in the following in vivo assay.
• In vivo assay In vivo assay
• Tumor response was quantified by calculating the change in tumor volume (endpoint minus starting value) as the T/C, i.e. (ATVoldrug/ATVolvehicle x 100). In the case of tumor regression, the tumor response was quantified by the percentage of regression of the starting tumor volume, i.e. (ATVoldrug/ATVoldayO x 100).
• Statistical analysis was performed by comparing the treatment groups to the vehicle control group at endpoint using Kruskal-Wallis followed by Dunn's post hoc test. At least one compound of the invention showed 70% tumour regression.
• FGF19 protein levels are measured using the human FGF-19 DuoSet DY969 from R&D Systems following the indications of the manufacturer.
• the capture antibody was diluted in PBS to a working dilution of 4 ⁇ g/mL and used to coat a 96-well plate (Costar #2592) with 100 ⁇ /well at room temperature overnight. Plates were washed 6 times with 400 ⁇ /well of PBS/0.05% Tween20 and blocked by adding 300 ⁇ /well of assay diluent (1 % BSA in PBS) for 2 hours at room temperature. Plates were washed 6 times with 400 ⁇ /well of PBS/0.05% Tween20.
• Cell lines were lysed using MPER lysis buffer (Pierce #78501 ) supplemented with Complete protease inhibitor tablets (Roche #1 1836145001 ) and PhosStop phosphatase inhibitor tablets (Roche #04906837001 ) on ice for 30 minutes. Lysates were clarified by centrifugation at 12000 x g for 15 minutes and protein concentration was determined using the DC protein assay reagents (Bio Rad # 500-01 16) and a BSA standard. Cell lysates were diluted in PBS/1 %BSA to add 100 ⁇ g/weW and 10 ⁇ g/well in 100 ⁇ . A 7-points standard ranging from 1000 pg/mL to 15.625 pg/mL was prepared.
• Samples and standards were added onto the coated plate, covered with a plate sealer and incubated for 2 hours on a plate mixer followed by 4 washings with 400 ⁇ /well of PBS/0.05% Tween20.
• the detection antibody diluted to a working concentration of 100 ng/mL in PBS/1 % BSA, was added in 100 ⁇ /well and incubated at room temperature for 2 hours on a plate mixer followed by 4 washings with 400 ⁇ l/well of PBS/0.05% Tween20.
• streptavidin solution diluted in PBS/1 % BSA was added onto the plate and incubated at room temperature, protected from light for 20 minutes followed by 3 washing with 400 ⁇ /well of
• the optical density of the plate was determined using a microplate reader set at 450nm.
• FGFR4 protein levels are quantified by performing sandwich-type capture ELISA on cell lysate. Cells were lysed as above.
• 96-well ELISA plates (N UNC #4371 1 1 ) were pre-coated with mouse anti-FGFR4 mAb (R&D Systems #MAB685), 100 ⁇ each diluted 1 : 100 in PBS without Ca2+/Mg2+.
• 150 ⁇ 3% MSD blocker A (MesoScale Discovery #R93BA-4) in TBS complemented with 0.05% Tween-20 (TBST-T) was added for 1 hour at room temperature.
• the wells were washed with 3 changes of 200 ⁇ TBST-T.
• Equal aliquots of the protein lysates were added to two pre-coated 96-well ELISA plates. Additional wells to assess the assay background were incubated with 100 ⁇ lysis buffer/MSD Blocker A 1 % (3: 1 ) buffer mix. After an overnight incubation at 4 °C, wells were washed with 3 changes of 200 ⁇ TBS-T. ELISA-plates pre-coated with the capture antibody were then incubated for 1 .5 hours with 100 ⁇ rabbit anti-FGFR4 mAb (Cell Signaling Technology #8562) diluted 1 : 1000 in 1 % MSD blocking buffer at room temperature on an orbital shaker.
• KLB protein levels are quantified by western blot.
• Cells were lysed as above. 50 ⁇ g of cell lysates were loaded onto 4-12% gradient NuPAGE Bis-Tris gels (Invitrogen #WG1402BX10) and blotted onto PVDF membranes. Filters were blocked in 5% milk for 1 hour at room temperature. The primary anti-human Klotho- ⁇ antibody (R&D Systems #AF5889) was used at 2 ⁇ g/mL. Signal detection was done using a secondary anti-goat-HRP antibody (Sigma #A5420).
• ⁇ -tubulin was used as an internal control, and the detection was performed with anti- -tubulin antibody (Sigma clone 2.1 ) followed by a secondary anti-mouse-HRP antibody (Amersham NA931 ). Membranes were imaged using the Fusion FX7 imaging system, and KLB levels were expressed as % of ⁇ - tubulin levels. The detection was done with super-signal west dura substrate (Thermo Signal, #34076).
• the compounds of the invention can be measured in a JHH6 cell proliferation assay (described above).
• a compound of the invention measured in this assay showed an IC50 > 10000 nM.

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