Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/04—Ortho-condensed systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/08—Bronchodilators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection

Definitions

• mAChRs have been localized to smooth muscle in the trachea and bronchi, the submucosal glands, and the parasympathetic ganglia. Muscarinic receptor density is greatest in parasympathetic ganglia and then decreases in density from the submucosal glands to tracheal and then bronchial smooth muscle. Muscarinic receptors are nearly absent from the alveoli.
• Recent literature has focused on the non-neuronal cholinergic system in the lungs where there is an emerging literature supporting a role for muscarinic receptors in mediating immunomodulatory and inflammatory functions in respiratory diseases such as asthma and COPD.
• Many of the components for cholinergic signaling have been reported to be contained within inflammatory and resident cells of the lungs, including muscarinic receptor expression on lymphocytes, alveolar macrophages, mast cells and epithelial cells.
• acetylcholine is solely a neurotransmitter of the parasympathetic nervous system is currently being challenged as there is mounting evidence to suggest it has an integral role in host defense and airway inflammation.
• COPD nearly 50 million people in the U.S. alone smoke cigarettes, and an estimated 3,000 people take up the habit daily.
• COPD is expected to rank among the top five as a world- wide health burden by the year 2020.
• Inhaled anti-cholinergic therapy is currently considered the "gold standard" as first line therapy for COPD (Pauwels et al. 2001 Am. J. Respir. Crit. Care Med. 163: 1256-1276).
• relatively few anti-cholinergic compounds are available for use in the clinic for pulmonary indications.
• WO 2004/091482 describes a dimeric bicyclic amine derivative having anti-muscarinic receptor activity:
• JP-2002-20386-A (Ono Yakuhin Kogyo KK) published on 23 January 2002 discloses pyrazolopyridine compounds of the following inter alia formula:
• s3 is 0 or an integer having a value of 1 to 3;
• v 1 is 0 or an integer having a value of 1 to 3;
• n and n 5 are 0, 1 or 2;
• Re, Rf, and Rg are each independently selected at each occurrence from hydrogen, or C j ⁇ alkyl;
• R 2 is selected from the group consisting of a hydrogen atom, C j _4alkyl, C j _2fluoroalkyl, cyclopropyl, cyclobutyl, and (cyclopropyl)methyl-;
• R ⁇ is selected from the group consisting of an optionally substituted Czj. ⁇ cycloalkyl, or optionally substituted mono-unsaturated-C5_7cycloalkenyl, an optionally substituted heterocyclic group of sub-formula (aa), (bb) and (cc), and a bicyclic group of sub-formula (dd), or (ee);
• Rg is an optionally substituted C5-C7 membered ring containing one or two nitrogens, or a corresponding bicyclic ring containing one or two nitrogens;
• Ri 5 and Ri 6 are each independently selected at each occurrence from hydrogen, or C J .4 alkyl; Ri 7 is independently selected at each occurrence from the group consisting of optionally substituted Ci-4 alkyl, optionally substituted C3-7 cycloalkyl, optionally substituted C 3 _ 7 cycloalkylC 1 -4alkyl, optionally substituted aryl, optionally substituted arylCj -4alkyl, heterocyclic, optionally substituted heterocyclic, optionally substituted heterocyclicC J -4alkyl, optionally substituted heteroaryl, and optionally substituted heteroarylC j -4alkyl;
• R7 is selected from hydrogen, or an optionally substituted C 1.4 alkyl
• R5a is hydrogen or a C j _2alkyl; n or n 5 is 0, 1 or 2;
• R ⁇ is selected from the group consisting of a hydrogen atom, Cj _4alkyl, C j _2fluoroalkyl, cyclopropyl, cyclobutyl, and (cyclopropyl)methyl-;
• R ⁇ is selected from the group consisting of an optionally substituted Czj. ⁇ cycloalkyl, or optionally substituted mono-unsaturated-C5_7cycloalkenyl, an optionally substituted heterocyclic group of sub-formula (aa), (bb) or (cc), and a bicyclic group of sub-formula (dd), or (ee);
• R ⁇ cycloalkyl ring is deemed to be the connection point to the -NH- in formula (I), that is the ring atom connecting to the -NH- in formula (I)); and wherein, when R ⁇ is the optionally substituted heterocyclic group of sub-formula (aa), (bb) or
• R6 is NR7R8, or is a heterocyclic group of the subformula (ff), (gg), (hh), (ii), (jj), (kk), (11), (mm) or (nn):
• Ri 5 and Ri 6 are each independently selected at each occurrence from hydrogen, or C J .4 alkyl;
• Ri 7 is independently selected at each occurrence from the group consisting of optionally substituted Ci-4 alkyl, optionally substituted C3-7 cycloalkyl, optionally substituted C3_ 7 cycloalkylCi- 4alkyl, optionally substituted aryl, optionally substituted arylCj -4alkyl, heterocyclic, optionally substituted heterocyclic, optionally substituted heterocyclic i ⁇ alkyl, optionally substituted heteroaryl, and optionally substituted heteroarylC i -4alkyl;
• X is (C(Ri 3)) p , or (CReR e ) sl - X 2 -(CR f R f ) s2 ;
• X 2 is NRi3a, O, S(O)m, or C(O);
• s is O, or is an integer having a value of 1 or 2;
• si is 0 or an integer having a value of 1 to 2;
• s2 is 0 or an integer having a value of 1 to 2, provided that when Rg is a heterocyclic group of the subformulas (ff), (ii), (jj), (11), (mm) and (nn), and X 2 is NRi 3 a , O, or S(0)m and m is 0 or 1, then s2 is 1 or 2, or X is (CH(Ri 3))p;
• p is an integer having a value of 1 or 2;
• R8 is (CRdlRdl)t - NRi IRi 2 or (CRdI RdI )ti - RuJ
• X 1 is oxygen, or N-(R 43 ); R 43 is independently selected from hydrogen or Cj _4alkyl;
• Zl is C(O), S(O)q, HNC(O), or OC(O);
• Z2 is C(O), S(O)q, or a bond
• Y4 is independently selected at each occurrence from the group consisting of hydrogen, halogen,
• RI is selected from the group consisting of Ci ⁇ alkyl, -CH2-C 1 ⁇ fluoroalkyl, and -CH2CH2OH;
• Z is C(O), or S(O)q;
• Y4 is independently selected at each occurrence from hydrogen, halogen, C 1-4 alkyl or C 1-4 alkoxy, or two of the Y4 moieties together with the carbons to which they are attached form a 5-6 membered saturated, partially unsaturated or fully unsaturated C5-C6 ring;
• RI is selected from the group consisting of C ⁇ alkyl, -CH2-C 1 ⁇ fluoroalkyl, and -CH2CH2OH;
• R 2 is selected from the group consisting of a hydrogen atom, Cj _4alkyl, C j _ 2 fluoroalkyl, cyclopropyl, cyclobutyl, and (cyclopropyl)methyl-;
• R ⁇ is selected from the group consisting of an optionally substituted Czj. ⁇ cycloalkyl, an optionally substituted mono-unsaturated-C5_7cycloalkenyl, an optionally substituted heterocyclic group of sub-formula (aa), (bb) and (cc), and a bicyclic group of sub-formula (dd), and (ee);
• Y 1 , Y 2 and Y 3 are independently CH 2 or oxygen, provided that no more than one of Y 1 , Y 2 and
• R c is independently selected at each occurrence from hydrogen or C 1-4 alkyl
• Rl 1 and R12 are independently selected from hydrogen, or Ci-4 alkyl; and the asterix indicating the point of attachment to the phenyl ring; or a pharmaceutically acceptable salt thereof.
• Xi is oxygen, or N-(R 43 ); n is 1 to 4; n5 is an integer having a value of 1 to 4; R 43 is independently selected from hydrogen or Ci ⁇ alkyl;
• Y 1 , Y 2 and Y 3 are independently CH 2 or oxygen, provided that no more than one of Y 1 , Y 2 and
• R ⁇ ring carbon bonded to the -NH- group of formula (I); and any OH, methoxy, fluoroalkoxy, -CH 2 OH, -CH(Me)OH, -CH 2 CH 2 OH, -CH 2 NH 2 , or -C(O)OH substituent on a ring carbon of the C /j . ⁇ cycloalkyl is at the 3 -position of a R ⁇ cyclobutyl ring; or at the
• R6 is an optionally substituted C5-C7 membered ring containing one or two nitrogens, or a corresponding bicyclic ring containing one or two nitrogens;
• R9 is selected from the group consisting of hydrogen, optionally substituted C 1-6 alkyl, optionally substituted aryl, optionally substituted arylC j -2alkyl, optionally substituted heteroaryl, optionally substituted heteroaryl C j ⁇ alkyl, optionally substituted heterocyclic, optionally substituted heterocyclic Cj -2alkyl, and C(O) Cj -2alkyl;
• R9a is selected from the group consisting of hydrogen, optionally substituted C J -6 alkyl, optionally substituted aryl, optionally substituted arylC j -2alkyl, optionally substituted heteroaryl, optionally substituted heteroaryl C j ⁇ alkyl, optionally substituted heterocyclic, optionally substituted heterocyclic Cj-2alkyl, and C(O)C j-2alkyl;
• Rd is independently selected at each occurrence from the group consisting of hydrogen, hydroxy, optionally substituted Cj -6 alkyl, amino, optionally substituted aryl, optionally substituted arylCj-2alkyl, optionally substituted heterocyclic, optionally substituted heterocyclicC j -2alkyl, optionally substituted heteroaryl, optionally substituted heteroaryl
• Ri 5 and Ri 6 are each independently selected at each occurrence from hydrogen, or Cj .4 alkyl; Ri 7 is independently selected at each occurrence from the group consisting of optionally substituted
• X is (C(Ri 3))p, or (CReR e ) sl - X 2 -(CR f R f ) s2 ;
• X 2 is NRi3a, O, S(O)m, or C(O);
• s is O, or is an integer having a value of 1 or 2;
• si is O or an integer having a value of 1 to 2;
• s2 is O or an integer having a value of 1 to 2, provided that when Rg is a heterocyclic group of the subformulas (ff), (ii), (jj), (11), (mm) and (nn), and
• X 2 is NRi 3 a , O, or S(0)m and m is O or 1, then s2 is 1 or 2, or X is (CH(Ri 3))p;
• p is an integer having a value of 1 or 2;
• R4 and R5 are each
• R7 is selected from hydrogen, or an optionally substituted C 1.4 alkyl
• R8 is (CRdlRdl)t - NRi IRi 2 or (CRdI RdI )ti - RuJ RdI is independently at each occurrence selected from the group consisting of hydrogen, optionally substituted Cj -4 alkyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocyclic;
• Ri 4 is selected from the group consisting of C 1-4 alkyl, C3-C5 cycloalkyl, optionally substituted heterocyclic, and optionally substituted heteroaryl moiety;
• t is an integer having a value of 1 to 4;
• tl is 0 or an integer having a value of 1 to 4;
• Rl 1 and R12 are independently selected from hydrogen, or Ci-4 alkyl; or a pharmaceutically acceptable salt thereof.
• n' is an integer having a value of 1 or 2;
• n 2 is an integer having a value of 1 or 2;
• Y is O, S, SO 2 , or NR 10a ;
• R9 is selected from the group consisting of hydrogen, optionally substituted C 1-6 alkyl, optionally substituted aryl, optionally substituted arylC j -2alkyl, optionally substituted heteroaryl, optionally substituted heteroaryl Cj -2alkyl, optionally substituted heterocyclic, optionally substituted heterocyclic Cj -2alkyl, and C(O) Cj ⁇ alkyl;
• R9a is selected from the group consisting of hydrogen, optionally substituted C J -6 alkyl, optionally substituted aryl, optionally substituted arylC j -2alkyl, optionally substituted heteroaryl, optionally substituted heteroaryl Cj-2alkyl, optionally substituted heterocyclic, optionally substituted heterocyclic Cj ⁇ alkyl, and C(O)C j ⁇ alkyl;
• RI is selected from the group consisting of Cj_3alkyl, -CH2-C j _2fluoroalkyl, and -CH2CH2OH;
• R ⁇ is selected from the group consisting of an optionally substituted Czj. ⁇ cycloalkyl, optionally substituted mono-unsaturated-C5_7cycloalkenyl, an optionally substituted heterocyclic group of sub-formula (aa), (bb) and (cc), and a bicyclic group of sub-formula (dd), and (ee);
• Ar 1 and Ar 2 are independently, selected from the group consisting of an optionally substituted phenyl and an optionally substituted monocyclic heteroaryl;
• Rg is NR ⁇ Rg, or is a heterocyclic group of the subformula (ff), (gg), (hh), (ii), (jj), (kk), (11), (mm) or (nn):
• R-6 is an optionally substituted C5-C7 membered ring containing one or two nitrogens, or a corresponding bicyclic ring containing one or two nitrogens;
• Rl 3a is selected from hydrogen, C 1-2 alkyl
• RdI is independently at each occurrence selected from the group consisting of hydrogen, optionally substituted C 1-4 alkyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocyclic;
• Ri 4 is selected from the group consisting of C 1-4 alkyl, C3-C5 cycloalkyl, optionally substituted heterocyclic, and optionally substituted heteroaryl moiety;
• t is an integer having a value of 1 to 4;
• tl is 0 or an integer having a value of 1 to 4;
• Rl 1 and Ri 2 are independently selected from hydrogen, or Ci-4 alkyl; or a pharmaceutically acceptable salt thereof. This invention provides for a method of treating both a muscarinic acetylcholine receptor
• mAChR acetylcholine binds to an M3 mAChR and a phosphodiesterase type IV (PDE4) mediated disease, whereby the compound also binds to the PDE4 isotype, which comprises administering an effective amount of a compound of Formula (I), (II),(III), (IV), (V) or (VI), or a pharmaceutically acceptable salt thereof to a mammal in need thereof.
• PDE4 phosphodiesterase type IV
• the present invention is directed to a concept of having dual pharmacophores in one molecule that retains potency across both pharmacological groups. Another aspect of the invention is that in addition to retaining dual pharmacological activity the compounds are developable for commercial activities.
• the compound may be administered to a mammal in needed thereof, suitably one to four times daily, and preferably either a once or a twice daily treatment.
• the compound is administered topically or by inhalation (via nose or mouth) for use in the treatment and/or prophylaxis of a disease for which either pharmacophore has previously been associated with treatment of a PDE4 or an M3 mediated disease.
• this will be an inflammatory and/or allergic disease, such as the treatment of COPD, asthma, adult respiratory distress syndrome, rhinitis, allergic rhinitis, atopic dermatitis, urticaria, allergic conjunctivitis, psoriasis, ulcerative colitis, or Crohn's disease.
• One or more specific compounds within the presently invented compounds may be suitable for use as dual PDE4/mAChR inhibitors via an inhaled route of administration.
• RMs suitably selected from Cj .3 alky 1, -CH 2 -C j_ 2 fluoroalkyl, or -CH2CH2OH.
• R ⁇ is suitably selected from Cj. 3alkyl, such as methyl, ethyl, n-propyl, or isopropyl.
• R 2 is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, Cj_2fluoroalkyl, cyclopropyl, cyclobutyl, or (cyclopropyl)methyl-.
• R 2 is methyl, ethyl, n-propyl, isopropyl, or n-butyl.
• R 2 is ethyl.
• R ⁇ is optionally substituted
• n ⁇ and n 2 independently are 1 or 2; and in which Y is O, S, SO2, or NR 10; where R ⁇ is a hydrogen atom (H), methyl, C(O)NH 2 , C(O)-methyl, or C(O)-C j fluoroalkyl.
• R ⁇ is the optionally substituted heterocyclic group of sub-formula (aa), (bb) or (cc)
• Ry is optionally substituted mono-unsaturated-C5_7cycloalkenyl
• the cycloalkenyl is optionally substituted on a ring carbon with one substituent being fluoro or methyl, and the R ⁇ ring carbon bonded to the -NH- group of formula (I) does not partake in the cycloalkenyl double bond.
• R ⁇ is the heterocyclic group of sub-formula (aa) and Y is NRI O
• RI O is not C(O)-methyl, or C(O)-C 1 fluoroalkyl
• R ⁇ is the heterocyclic group of sub-formula (bb) and Y is NR ⁇
• R ⁇ is not methyl
• R ⁇ is the heterocyclic group of sub-formula (cc)
• Y is O, S, SO2 or NR ⁇ wherein R ⁇ is H or methyl.
• any -C(O)NHR 24 or -C(O)R 25 substituent on a ring carbon is: at the 3-position of a R ⁇ cyclobutyl ring; or at the 3- or 4- position of a R 3 cyclopentyl ring; or at the 4-position of a R ⁇ cyclohexyl ring; or at the 3-, A-, 5- or 6- position of a R ⁇ cycloheptyl ring (wherein, in this connection, the 1 -position of the R ⁇ cycloalkyl ring is deemed to be the connection point to the -NH- in formula (I), that is the ring atom connecting to the -NH- in formula (I)).
• R ⁇ is optionally substituted Czj. ⁇ cycloalkyl, then any OH, methoxy, fluoroalkoxy,
• Y is O, S, SO 2 , or NR ⁇ a .
• Y is O.
• R ⁇ a is a hydrogen atom (H), methyl, C(O)NH 2 , C(O)-methyl, Or C(O)-CJfIuOTOaIlCyI.
• LINK is an optionally substituted heterocyclic or optionally substituted heteroaryl.
• s3 is O or an integer having a value of 1 to 3.
• vl is O or an integer having a value of 1 to 3.
• s4 is 0 or an integer having a value of 1 to 3, provided that the sum of s3 + vl + s4 is greater than 0.
• Re, Rf and Rg are each independently selected at each occurrence from hydrogen, or C j .4 alky 1.
• LINK heteroaryl ring is selected from an optionally substituted C5-C7 mono heteroaryl ring or an optionally substituted C8-C12 fused bicyclic heteroaryl ring. It is recognized that only one of the fused rings may be aromatic, and the other partially unsaturated or saturated and contain one or more additional heteroatoms, suitably one or two heteroatoms selected from oxygen, nitrogen or sulfur.
• Suitable examples of a LINK heteroaryl ring include, but are not limited to, furyl, pyranyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, imidazolyl, pyrazolyl, oxadiazolyl, oxathiadiazolyl, triazolyl, tetrazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and uracil.
• Suitable examples of a LINK bicyclic fused aromatic ring includes, but is not limited to, indolyl, isoindolyl, indazolyl, indolizinyl, azaindolyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, benzofuranyl, benzothiophenyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, cinnolinyl, purinyl, and phthalazinyl.
• Suitable examples of a LINK bicyclic fused ring system which contains a non-aromatic ring includes, but is not limited to, an indoline, indanyl, l,4-dioxino[2,3-c] pyrrole, 1,4- dioxino[2,3-c] furan, or l,4-dioxino[2,3-c] thiophene.
• the LINK heteroaryl is selected from pyridine (e.g. 2- pyridyl, 3-pyridyl, or 4-pyridyl), pyrimidine, furan, thienyl, pyrrole, l,4-dioxino[2,3-c] pyrrole, or benzimidazole.
• heteroaryl ring For all formulas herein, the terms “heteroaryl ring”, “heteroaryl moiety”, and “heteroaryl” may be used interchangeably herein.
• the LINK heteroaryl, heterocyclic or aryl rings may be optionally substituted by the moiety (Y4)n3, wherein Y4 is independently selected at each occurrence from hydrogen, halogen, hydroxy, C 1-4 alkyl or C 1-4 alkoxy. It should be noted that the Y4 moieties may be substituted on either ring of the fused bicyclic ring system.
• LINK is an optionally substituted aryl it is an optionally substituted phenyl or an optionally substituted naphthyl.
• LINK is an optionally substituted heterocyclic it is a C3-C7 monocyclic non-aromatic hydrocarbon ring containing at least one heteroatom selected from nitrogen, oxygen, sulphur or oxidized sulphur moieties, such as S(0)m, and m is 0 or an integer having a value of 1 or 2, or the heterocyclic is a fused, C8-C12 saturated or partially unsaturated ring system wherein one of the rings may be aromatic, or heteroaromatic. Each of the fused rings may have from four to seven ring atoms.
• heterocyclyl groups include, but are not limited to, the saturated or partially saturated versions of the heteroaryl moieties as defined above, such as tetrahydropyrrole, tetrahydropyran, tetrahydrofuran, tetrahydrothiophene (including oxidized versions of the sulfur moiety), azepine, diazepine, aziridinyl, pyrrolinyl, pyrrolidinyl, 2-oxo-l- pyrrolidinyl, 3 -oxo-1 -pyrrolidinyl, l,3-benzdioxol-5-yl, imidazolinyl, imidazolidinyl, indolinyl, pyrazolinyl, pyrazolidinyl, piperidinyl, piperazinyl, morpholino and thiomorpholino (including oxidized versions of the sulfur moiety).
• LINK is an optionally substituted heterocyclic
• R 5a is hydrogen, methyl or ethyl. In one embodiment of the invention, R 5a is hydrogen.
• n 5 is 0 or an integer having a value of 1 or 2. In one embodiment of the invention, n5 is 0 or 1.
• Z is selected from C(O), or S(O)q. In one embodiment of the invention, Z is selected from C(O).
• Xi is oxygen, or N-(R 43 ). In one embodiment of the invention Xi is N-(R 43 ).
• R4a is hydrogen, methyl or ethyl. In one embodiment of the invention for compounds of Formula (III), R4a is hydrogen.
• R 3 is hydrogen, methyl or ethyl.
• R 53 is hydrogen.
• n 5 is an integer having a value of 1 to 5.
• n5 is 1.
• Z is selected from C(O), or S(O)q, and q is 0 or an integer having a value of 1 or 2.
• Z is C(O).
• Z is S(O)q and Zl is OC(O).
• Link is -((CRgRg)s3-heterocyclic-(CRhRh))s4- or -((CRgRg)s3- heteroaryl -(CRhRh))s4-.
• the LINK heterocyclic and heteroaryl rings are as described above for Formula (I).
• s3 is O or an integer of 1 to 5. In one embodiment of the invention s3 is O.
• s4 isO or an integer of 1 to 5. In one embodiment of the invention s4 is O. In one embodiment of the invention s3 and s4 are both O.
• Rg is independently selected from hydrogen, hydroxy, Cl-6 alkoxy, Cl-6 alkyl, or a C3-6 cycloalkyl, or C3-C6 cycloalkyl Cl-4 alkyl, or two Rg moieties together with the carbon to which they are attached form a 3-6 membered saturated ring.
• R5a is hydrogen or a Ci _2alkyl. In one embodiment of the invention R5a is hydrogen.
• v is O or an integer of 1 to 5
• vl is O or an integer of 1 to 5.
• LINK is an optionally substituted C5-C7 cycloalkyl, optionally substituted aryl, optionally substituted heterocyclic or optionally substituted heteroaryl.
• LINK is an optionally substituted aryl it is an optionally substituted phenyl or naphthyl.
• LINK is an optionally substituted C5-C7 cycloalkyl it is suitably an optionally substituted cyclobutyl or cyclohexyl.
• LINK is an optionally substituted heterocyclic or heteroaryl it is as defined above for Formula (I).
• Y4 is independently selected at each occurrence from hydrogen, halogen, C 1-4 alkyl or C 1-4 alkoxy, or two of the Y4 moieties together with the carbons to which they are attached form a 5-6 membered saturated, partially unsaturated or fully unsaturated C5-C6 ring.
• n 3 is an integer having a value of 1 to 4.
• Z is selected from C(O), or S(O)q.
• q is 0 or an integer having a value of 1 or 2.
• LINK is an optionally substituted aryl, optionally substituted heterocyclic or optionally substituted heteroaryl.
• LINK is an optionally substituted aryl it is an optionally substituted phenyl or naphthyl.
• LINK is an optionally substituted heterocyclic or heteroaryl it is as defined above for Formula (I).
• Xi is oxygen, or N-(R 43 ). In one embodiment Xi is N-(R 43 ).
• R 43 is independently selected from hydrogen or Ci_4alkyl.
• R4a is hydrogen.
• R 53 is hydrogen, methyl or ethyl. In one embodiment of the invention R 53 is hydrogen.
• n 3 is an integer having a value of 1 to 4.
• both Z and Zl are C(O).
• Z is C(O), and Zl is S(O)q.
• Z is C(O), and Zl is HNC(O).
• Z is C(O), and Zl is OC(O).
• Z is S(O)q and Zl is C(O).
• Z is S(O)q, and Zl is HNC(O).
• Z is S(O)q and Zl is OC(O).
• Z 4 is selected from C(O), or S(O)q.
• Z is C(O).
• Z and Zl are C(O).
• Z4 is C(O), Z is C(O), and Zl is S(O)q.
• Z4 is C(O), Z is C(O), and Zl is HNC(O).
• Z4 is C(O), Z is C(O), and Zl is OC(O).
• Z4 is C(O), Z is S(O)q and Zl is C(O).
• Z4 is C(O), Z is S(O)q, and Zl is HNC(O).
• Z4 is C(O), Z is S(O)q and Zl is OC(O).
• Z4 is S(O)q, Z is C(O), and Zl is C(O).
• Z4 is S(O)q, Z is C(O), and Zl is HNC(O).
• Z4 is S(O)q, Z is C(O), and Zl is OC(O).
• Z4, Z and Zl are all independently S(O)q. All possible variations of Z4, Z and Zl are within the scope of the invention.
• Y4 is independently selected at each occurrence from the group consisting of hydrogen, halogen, Cl .4 alkyl or Cl .4 alkoxy, or two of the Y4 moieties together with the carbons to which they are attached form a 5-6 membered saturated, and a partially unsaturated or fully unsaturated C5-C6 ring.
• Link is - ((CRgRg) s3- optionally substituted heterocyclic-(CRhRh))s4- or -((CRgRg)s3 -optionally substituted heteroaryl -(CRhRh))s4-.
• LINK is an optionally substituted heterocyclic it is a C3-C7 monocyclic non-aromatic hydrocarbon ring containing at least one heteroatom selected from nitrogen, oxygen, sulphur or oxidized sulphur moieties, such as S(0)m, and m is O or an integer having a value of 1 or 2, or the heterocyclic is a fused, C8-C12 saturated or partially unsaturated ring system wherein one of the rings may be aromatic, or heteroaromatic.
• Each of the fused rings may have from four to seven ring atoms.
• heterocyclyl groups include, but are not limited to, the saturated or partially saturated versions of the heteroaryl moieties as defined above, such as tetrahydropyrrole, tetrahydropyran, tetrahydrofuran, tetrahydrothiophene (including oxidized versions of the sulfur moiety), azepine, diazepine, aziridinyl, pyrrolinyl, pyrrolidinyl, 2-oxo-l- pyrrolidinyl, 3 -oxo- 1 -pyrrolidinyl, l,3-benzdioxol-5-yl, imidazolinyl, imidazolidinyl, indolinyl, pyrazolinyl, pyrazolidinyl, piperidinyl, piperazinyl, morpholino and thiomorpholino (including oxidized versions of the sulfur moiety).
• LINK is an optionally substituted heterocycl
• LINK is a heteroaryl ring is selected from an optionally substituted C5-C7 mono heteroaryl ring or an optionally substituted C8-C12 fused bicyclic heteroaryl ring. It is recognized that only one of the fused rings may be aromatic, and the other partially unsaturated or saturated and contain one or more additional heteroatoms, suitably one or two heteroatoms selected from oxygen, nitrogen or sulfur.
• the ring nitrogen may be optionally substituted with a Cl -6 alkyl or a C(O)Rl 8 moiety, wherein Rl 8 is hydrogen,Cl-6 alkyl, C3-7 cycloalkyl, cycloalkylCl-4 alkyl, aryl or aryl Cl-4 alkyl.
• Rl 8 is hydrogen,Cl-6 alkyl, C3-7 cycloalkyl, cycloalkylCl-4 alkyl, aryl or aryl Cl-4 alkyl.
• the ring sulfur atom in a non-aromatic ring system may also be oxidized to a sulfinyl or sulfonyl derivative.
• Suitable examples of a LINK heteroaryl ring include, but are not limited to, furyl, pyranyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, imidazolyl, pyrazolyl, oxadiazolyl, oxathiadiazolyl, triazolyl, tetrazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and uracil.
• Suitable examples of a LINK bicyclic fused aromatic ring includes, but is not limited to, indolyl, isoindolyl, indazolyl, indolizinyl, azaindolyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, benzofuranyl, benzothiophenyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, cinnolinyl, purinyl, and phthalazinyl.
• Suitable examples of a LINK bicyclic fused ring system which contains a non-aromatic ring includes, but is not limited to, an indoline, indanyl, l,4-dioxino[2,3-c] pyrrole, 1,4- dioxino[2,3-c] furan, or l,4-dioxino[2,3-c] thiophene.
• s3 is 0 or an integer having a value of 1 to 5.
• s4 is 0 or an integer having a value of 1 to 5.
• Rg is independently selected from the group consisting of hydrogen, hydroxy, Ci-6 alkoxy, Ci-6 alkyl, or a C3-6 cycloalkyl, and C3-6 cycloalkyl C 1-4 alkyl; or two Rg moieties together with the carbon to which they are attached form a 3-6 membered saturated ring.
• the Ar2 ring is phenyl. In one embodiment of the invention, the ArI ring is a heteroaryl ring. In another embodiment the ArI ring is a pyridine ring.
• the ArI ring is a phenyl optionally substituted one or more times independently by halogen, alkyl, alkoxy, or cyano. In another embodiment the ArI ring is a phenyl optionally substituted one or more times independently by fluorine, chlorine, methyl, methoxy or cyano.
• the Ar2 ring is phenyl
• the ArI ring is a phenyl optionally substituted one or more times independently by halogen, alkyl, alkoxy, or cyano.
• the ArI ring is mono-substituted in the 5- or in the 6-position. If the ring is di- substituted it is in the 5 and 6 -position.
• the ArI ring is an optionally substituted phenyl ring.
• the phenyl ring is substituted one or more times, suitably 1 to 2 times, by halogen, cyano, or C 1-4 alkoxy.
• the ArI ring is a phenyl, optionally substituted in the 6-position by fluorine, or methoxy.
• R ⁇ is NR ⁇ Rg, or is a heterocyclic group of the subformula (fit), (gg), (hh), (ii), Qj), (kk), (11), (mm) or (nn):
• R-6 is an optionally substituted C5-C7 membered ring containing one or two nitrogens, or a corresponding bicyclic ring containing one or two nitrogens.
• R ⁇ is an optionally substituted C5-C7 membered ring containing one or two nitrogens, or a corresponding bicyclic ring containing one or two nitrogens
• the rings include variations of the ring nitrogen positions from subformulas (ff) to (nn).
• the nitrogens are at the 1-4 position
• other options include 1-3, or 1-2 nitrogens with similarly substituted Ra, Rb RbI, R9, etc. substituents.
• Rg is a heterocyclic group of the sub formula (ff), and s is 1 or 2.
• R ⁇ is a heterocyclic group of the sub formula (ff), s is 1 or 2, and Rb is independently selected from hydrogen, or methyl.
• R ⁇ is a heterocyclic group of the subformula (jj).
• tl is 0 or an integer having a value of 1 to 4.
• Rd 1 is independently at each occurrence selected from the group consisting of hydrogen, optionally substituted Cj -4 alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclic.
• R M when R M is a heteroaryl it is a monocyclic five- to seven- membered unsaturated hydrocarbon ring containing at least one heteroatom selected from oxygen, nitrogen and sulfur; or a fused C8-C12 aromatic ring comprising at least one heteroatom selected from oxygen, nitrogen and sulfur.
• heteroaryl rings include, but are not limited to, furyl, pyranyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, imidazolyl, pyrazolyl, oxadiazolyl, oxathiadiazolyl, triazolyl, tetrazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, uracil, indolyl, isoindolyl, indazolyl, indolizinyl, azaindolyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, benzofuranyl, benzothiophenyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, naphthyridin
• R M is an optionally substituted heteroaryl it is selected from an optionally substituted thiophenyl, optionally substituted pyridinyl, or an optionally substituted pyrimidinyl.
• R M is a heterocyclic it is a C3-C7 monocyclic non-aromatic hydrocarbon ring containing at least one heteroatom selected from nitrogen, oxygen, sulphur or oxidized sulphur moieties, such as S(0)m, and m is 0 or an integer having a value of 1 or 2, or the heterocyclic is a fused, C8-C12 saturated or partially unsaturated ring system wherein one of the rings may be aromatic, or heteroaromatic.
• Each of the fused rings may have from four to seven ring atoms.
• suitable heterocyclyl groups include, but are not limited to, the saturated or partially saturated versions of the heteroaryl moieties as defined above, such as tetrahydropyrrole, tetrahydropyran, tetrahydrofuran, tetrahydrothiophene (including oxidized versions of the sulfur moiety), azepine, diazepine, aziridinyl, pyrrolinyl, pyrrolidinyl, 2-oxo- 1 -pyrrolidinyl, 3-oxo-l- pyrrolidinyl, l,3-benzdioxol-5-yl, imidazolinyl, imidazolidinyl, indolinyl, pyrazolinyl, pyrazolidinyl, piperidinyl, piperazinyl, morpholino and thiomorpholino (including oxidized versions of the sulfur
• R M is an optionally substituted heterocyclic ring
• the ring is an optionally substituted piperidinyl, piperazinyl, optionally substituted oxohexahydro-lH-azepine, or an optionally substituted 3'-[(l-Azabicyclo-[2.2.2]oct-3- yi.
• R M is a C3-C5 cycloalkyl it is suitably selected from cyclopropyl, cyclopentyl, or cyclohexyl.
• R 14 is a Cl .4 alkyl it is ethyl, isopropyl, n-propyl, n-butyl, sec-butyl, or t-butyl.
• t is an integer having a value of 1 to 4. In one embodiment, t is 1 or 2.
• tl is 0 or an integer having a value of 1 to 4. In one embodiment, tl is 0, or 1. In another embodiment, tl is 0.
• Ri 1 and Ri 2 are independently selected from hydrogen, or C 1.4 alkyl.
• Rd is independently at each occurrence selected from the group consisting of hydrogen, optionally substituted C 1-4 alkyl, optionally substituted aryl, optionally substituted heteroaryl, and an optionally substituted heterocyclic moiety.
• Rd is an optionally substituted moiety, excluding hydrogen, the moiety may be substituted one or more times, suitably 1 to 4 times, independently by halogen, such as fluorine or chlorine, or a C ⁇ alkyl.
• Rd is independently hydrogen or methyl.
• R9 is hydrogen, optionally substituted
• R9 is an optionally substituted C 1 -6 alkyl, the alkyl is substituted one or more times, suitably 1 or 2 times independently by halogen, hydroxy, NRl 5Rl 6, C 1-4 alkoxy, S(0)qCi-4 alkyl. In one embodiment of the invention, R9 is hydrogen or methyl.
• R a is independently hydrogen, or methyl.
• R a is independently hydrogen, or methyl.
• Rb is independently selected from hydrogen or methyl.
• RbI is independently selected at each occurrence from hydrogen, halogen, optionally substituted C
• Rd is an optionally substituted C i -6 alkyl
• the alkyl is substituted one or more times, suitably 1 or 2 times independently by halogen, hydroxy, NR15R16, C 1-4 alkoxy, S(0)qCi-4 alkyl.
• R(J is hydrogen or methyl.
• R c is independently selected at each occurrence from hydrogen or C I -4 alkyl.
• RlO is independently selected from hydrogen or C i .4 alkyl.
• Ri 5 and Ri 6 are independently selected from hydrogen, or C 1-4 alkyl. In one embodiment of the invention Ri 5 and Ri 6 are hydrogen or methyl.
• Ri 7 is selected from optionally substituted
• Ci-4 alkyl optionally substituted C3-7 cycloalkyl, optionally substituted C 3 _ 7 cycloalkylCi-4alkyl, optionally substituted aryl, optionally substituted arylCi ⁇ alkyl, heterocyclic, optionally substituted heterocyclic, optionally substituted heterocyclicC i ⁇ alkyl, optionally substituted heteroaryl, optionally substituted heteroaryl Ci ⁇ alkyl.
• X is (C(Ri 3))p, or (CReReXr X 2 -
• X 2 is NRi 3 a , O, S(0)m, or C(O).
• Rl 3 a is selected from hydrogen, C 1-2 alkyl.
• Ri 3 is hydrogen.
• si is 0 or an integer having a value of 1 to 2. In one embodiment of the invention si is 0.
• s2 is 0 or an integer having a value of 1 to 2.
• Rg is a heterocyclic group of the subformulas (ff), (ii), (jj), (11), (mm) and (nn) and X 2 is NRi3 a , O, or S(0)m (and m is 0 or 1) then s2 is 1 or 2, or X is the group (CH(Ri3))p.
• q is 0 or an integer having a value of 1 or 2.
• n is an integer having a value of 1, 2 or 3.
• n 3 is an integer having a value of 1 to 3.
• m is 0 or an integer having a value of 1 , or 2.
• Y 4 is independently selected at each occurrence from hydrogen, halogen, or Cj .4 alkyl; C3-C7 cycloalkyl, or C3-C7 cycloalkyl C j .4 alkyl. In one embodiment of the invention Y 4 is hydrogen, and n 3 is 1.
• R5 is hydrogen, and n is 1.
• R4 is hydrogen, or C j _4alkyl. In another embodiment, R4 and R5 are both hydrogen, and n is 1.
• the present invention covers all combinations of particular and preferred groups described hereinabove. It is also to be understood that the present invention encompasses compounds in which a particular group or parameter, e.g. S(0)m, etc. may occur more than once. In such compounds it will be appreciated that each group or parameter is independently selected from the values listed. When any variable occurs more than one time in a formula, its definition on each occurrence is independent of its definition at every other occurrence.
• Particular compounds according to the invention include those mentioned in the examples and their pharmaceutically acceptable derivatives.
• pharmaceutically acceptable means a compound which is suitable for pharmaceutical and veterinary usage.
• Salts and solvates of compounds of the invention which are suitable for use in medicine are those wherein the counter- ion or associated solvent is pharmaceutically acceptable.
• salts and solvates having non-pharmaceutically acceptable counter- ions or associated solvents are within the scope of the present invention, for example, for use as intermediates in the preparation of other compounds of the invention and their pharmaceutically acceptable salts and solvates.
• the compounds of the present invention may be in the form of and/or may be administered as a pharmaceutically acceptable salt.
• suitable salts see Berge et al., J. Pharm. Sci., 1977, 66, 1-19.
• a pharmaceutical acceptable salt may be readily prepared by using a desired acid or base as appropriate.
• the salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent.
• Salts of the compounds of the present invention may, for example, comprise acid addition salts resulting from reaction of an acid with a nitrogen atom present in a compound of formula (I). Salts encompassed within the term "pharmaceutically acceptable salts" refer to non-toxic salts of the compounds of this invention.
• Pharmaceutically acceptable base salts include ammonium salts such as a trimethylammonium salt, alkali metal salts such as those of sodium and potassium, alkaline earth metal salts such as those of calcium and magnesium and salts with organic bases, including salts of primary, secondary and tertiary amines, such as isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexyl amine and N-methyl-D-glucamine.
• ammonium salts such as a trimethylammonium salt
• alkali metal salts such as those of sodium and potassium
• alkaline earth metal salts such as those of calcium and magnesium
• salts with organic bases including salts of primary, secondary and tertiary amines, such as isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexyl amine and N-methyl-D-glucamine.
• solvates refers to a complex of variable stoichiometry formed by a solute (in this invention, a compound of Formula (I), or a salt thereof) and a solvent.
• solvents for the purpose of the invention may not interfere with the biological activity of the solute.
• suitable solvents include water, methanol, ethanol and acetic acid.
• the solvent used is a pharmaceutically acceptable solvent.
• suitable pharmaceutically acceptable solvents include water, ethanol and acetic acid.
• the solvent used is water.
• a complex with water is known as a "hydrate”. Solvates of the compound of the invention are within the scope of the invention.
• prodrug means a compound which is converted within the body, e.g. by hydrolysis in the blood, into its active form that has medical effects.
• Pharmaceutically acceptable prodrugs are described in T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series; Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987; and in D. Fleisher, S. Ramon and H. Barbra "Improved oral drug delivery: solubility limitations overcome by the use of prodrugs", Advanced Drug Delivery Reviews (1996) 19(2) 115-130, each of which are incorporated herein by reference.
• Prodrugs are any covalently bonded carriers that release a compound of formula (I) in vivo when such prodrug is administered to a patient.
• Prodrugs are generally prepared by modifying functional groups in a way such that the modification is cleaved, either by routine manipulation or in vivo, yielding the parent compound.
• Prodrugs include, for example, compounds of this invention wherein hydroxy or amine groups are bonded to any group that, when administered to a patient, cleaves to form the hydroxy or amine groups.
• representative examples of prodrugs include (but are not limited to) acetate, formate and benzoate derivatives of alcohol and amine functional groups of the compounds of formula (I).
• esters may be employed, such as methyl esters, ethyl esters, and the like. Esters may be active in their own right and/or be hydro lysable under in vivo conditions in the human body. Suitable pharmaceutically acceptable in vivo hydrolysable ester groups include those which break down readily in the human body to leave the parent acid or its salt.
• halogen such as fluorine, chlorine, bromine or iodine
• hydroxy such as methoxy or ethoxy
• halosubstituted Ci-IO alkoxy such as methoxy or ethoxy
• S(0)m alkyl such as methyl thio, methylsulfmyl or methyl sulfonyl
• a ketone such as methyl thio, methylsulfmyl or methyl sulfonyl
• a ketone such as methyl thio, methylsulfmyl or methyl sulfonyl
• a ketone such as methyl thio, methylsulfmyl or methyl sulfonyl
• a ketone such as methyl thio, methylsulfmyl or methyl sulfonyl
• -C(O) ketone
• -C(O)Rg' aldehyde
• Ci-IO alkyl such CF2CF2H, or CF3
• an optionally substituted aryl such as phenyl, or an optionally substituted arylalkyl, such as benzyl or phenethyl, wherein these aryl containing moieties may also be substituted one to two times by halogen; hydroxy; hydroxy substituted alkyl; C 1-4 alkoxy; S(O) m Ci ,4 alkyl; amino, mono & di-substituted Ci ,4 alkyl amino; Ci ,4 alkyl, or
• Ci-iQalkyl or “alkyl” or “alkyl i-io” is used herein to mean both straight and branched hydrocarbon chain containing the specified number of carbon atoms, e.g. Ci_ioalkyl means a straight of branched alkyl chain of at least 1, and at most 10, carbon atoms, unless the chain length is otherwise limited.
• alkenyl as used herein include, but are not limited to ethenyl, 2-propenyl, 3-butenyl, 2-butenyl, 2-pentenyl, 3-pentenyl, 3- methyl-2-butenyl, 3-methylbut-2-enyl, 3-hexenyl, l,l-dimethylbut-2-enyl and the like.
• cycloalkyl refers to cyclic radicals, such as a non-aromatic hydrocarbon ring containing a specified number of carbon atoms.
• C3_7cycloalkyl means a non-aromatic ring containing at least three, and at most seven, ring carbon atoms.
• Representative examples of "cycloalkyl” as used herein include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl and the like.
• cycloalkenyl is used herein to mean cyclic radicals, such as a non-aromatic hydrocarbon ring containing a specified number of carbon atoms preferably of 5 to 7 carbons, which have at least one bond including but not limited to cyclopentenyl, cyclohexenyl, and the like.
• alkenyl is used herein at all occurrences to mean straight or branched chain radical of 2- 10 carbon atoms, unless the chain length is limited thereto, including, but not limited to ethenyl, 1-propenyl, 2-propenyl, 2 -methyl- 1 -propenyl, 1-butenyl, 2-butenyl and the like.
• aryl is used herein to mean phenyl, naphthyl, and indene.
• heteroaryl rings include, but are not limited to, furyl, pyranyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, imidazolyl, pyrazolyl, oxadiazolyl, oxathiadiazolyl, triazolyl, tetrazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and uracil.
• fused aromatic rings include, but are not limited to, indolyl, isoindolyl, indazolyl, indolizinyl, azaindolyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, benzofuranyl, benzothiophenyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, cinnolinyl, purinyl, and phthalazinyl.
• heterocyclic rings is used herein to mean a monocyclic three- to seven-membered saturated or non-aromatic, unsaturated hydrocarbon ring containing at least one heteroatom selected from nitrogen, oxygen, sulphur or oxidized sulphur moieties, such as S(0)m, and m is 0 or an integer having a value of 1 or 2.
• heterocyclic rings shall also refer to a fused C8-C12 saturated or partially unsaturated ring system wherein one of the rings may be aromatic, or heteroaromatic.
• Each of the fused rings may have from four to seven ring atoms.
• suitable heterocyclyl groups include, but are not limited to, the saturated or partially saturated versions of the heteroaryl moieties as defined above, such as tetrahydropyrrole, tetrahydropyran, tetrahydrofuran, tetrahydrothiophene (including oxidized versions of the sulfur moiety), azepine, diazepine, aziridinyl, pyrrolinyl, pyrrolidinyl, 2-oxo- 1 -pyrrolidinyl, 3-oxo-l-pyrrolidinyl, 1,3- benzdioxol-5-yl, imidazolinyl, imidazolidinyl, indolinyl, pyrazolinyl, pyrazolidinyl, piperidinyl, piperazinyl, morpholino and thiomorpholino (including oxidized versions of the sulfur moiety
• arylalkyl or “heteroarylalkyl” or “heterocyclicalkyl” is used herein to mean a C 1-4 alkyl (as defined above) attached to an aryl, heteroaryl or heterocyclic moiety (as also defined above) unless otherwise indicated.
• sulfinyl is used herein to mean the oxide S(O) of the corresponding sulfide, the term “thio” refers to the sulfide, and the term “sulfonyl” refers to the fully oxidized S (0)2 moiety.
• alkanoyl is used herein to mean C(O)Ci-IO alkyl wherein the alkyl is as defined above.
• the term "optionally” means that the subsequently described event(s) may or may not occur, and includes both event(s) which occur and events that do not occur.
• substituted refers to substitution with the named substituent or substituents, multiple degrees of substitution being allowed unless otherwise stated.
• the compounds of the Formulas herein may have one or more asymmetric carbon atom and may occur as racemates, racemic mixtures and as individual enantiomers or diastereomers. All such isomeric forms are included within the present invention, including mixtures thereof.
• Cis (E) and trans (Z) isomerism may also occur.
• the present invention includes the individual stereoisomers of the compound of the invention and where appropriate, the individual tautomeric forms thereof, together with mixtures thereof.
• Separation of diastereoisomers or cis and trans isomers may be achieved by conventional techniques, e.g. by fractional crystallisation, chromatography or H.P.L.C.
• a stereoisomeric mixture of the agent may also be prepared from a corresponding optically pure intermediate or by resolution, such as H.P.L.C. of the corresponding racemate using a suitable chiral support or by fractional crystallisation of the diastereoisomeric salts formed by reaction of the corresponding racemate with a suitable optically active acid or base, as appropriate.
• crystalline forms of the compounds of the Formulas herein may exist as polymorphs, which are included in the present invention.
• Exemplified compounds of the compounds of this invention include the racemates, or optically active forms of the compounds of the working examples herein, and pharmaceutically acceptable salts thereof.
• the compounds of this invention may be made by a variety of methods, including standard chemistry. Any previously defined variable will continue to have the previously defined meaning unless otherwise indicated. Illustrative general synthetic methods are set out below and then specific compounds of the invention are prepared in the working Examples.
• PBMC cell
• in vivo anti-inflammatory activity rat LPS neutrophilia
• Desired attributes of the molecule would be to maintain or improve the M3 pharmacophores potency with no or partial Ml agonism. Therefore, one embodiment of the invention are compounds which posses appropriately balanced pharmacology, and have desirable physicochemical properties, such as crystallinity, micronizability, and excipient compatibility. If the compounds are too administered by inhalation, then a low aqueous solubility would not suitable for a nebulized/ solution formulation.
• a compound of Formula (I) or a pharmaceutically acceptable salt thereof in therapy it will normally be formulated into a pharmaceutical composition in accordance with standard pharmaceutical practice.
• This invention also relates to a pharmaceutical composition comprising an effective amount of a compound of Formula (I) and a pharmaceutically acceptable carrier or diluent.
• Compounds of Formula (I), pharmaceutically acceptable salts thereof and pharmaceutical compositions incorporating such may conveniently be administered by any of the routes conventionally used for drug administration, for instance, orally, topically, parenterally or by inhalation.
• the compounds of Formula (I) may be administered in conventional dosage forms prepared by combining a compound of Formula (I) with standard pharmaceutical carriers according to conventional procedures.
• the compounds of Formula (I) may also be administered in conventional dosages in combination with a known, second therapeutically active compound.
• the pharmaceutical carrier employed may be, for example, either a solid or liquid.
• solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like.
• liquid carriers are syrup, peanut oil, olive oil, water and the like.
• the carrier or diluent may include time delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax.
• the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form or in the form of a troche or lozenge.
• the amount of solid carrier will vary widely but preferably will be from about 25mg. to about Ig.
• the preparation will be in the form of a syrup, emulsion, soft gelatin capsule, sterile injectable liquid such as an ampule or nonaqueous liquid suspension.
• Compounds of Formula (I) may be administered topically, that is by non-systemic administration. This includes the application of a compound of Formula (I) externally to the epidermis or the buccal cavity and the instillation of such a compound into the ear, eye and nose, such that the compound does not significantly enter the blood stream.
• systemic administration refers to oral, intravenous, intraperitoneal and intramuscular administration.
• Compounds of Formula (I) may be administered parenterally, that is by intravenous, intramuscular, subcutaneous intranasal, intrarectal, intravaginal or intraperitoneal administration.
• the subcutaneous and intramuscular forms of parenteral administration are generally preferred.
• Appropriate dosage forms for such administration may be prepared by conventional techniques.
• Compounds of Formula (I) may also be administered by inhalation, that is by intranasal and oral inhalation administration.
• Appropriate dosage forms for such administration such as an aerosol formulation or a metered dose inhaler, may be prepared by conventional techniques.
• the compounds may be delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydro fluoroalkane such as tetrafluoroethane or heptafluoropropane, carbon dioxide or other suitable gas.
• a suitable propellant e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydro fluoroalkane such as tetrafluoroethane or heptafluoropropane, carbon dioxide or other suitable gas.
• a suitable propellant e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a
• gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.
• Dry powder compositions for topical delivery to the lung by inhalation may, for example, be presented in capsules and cartridges of for example gelatine or blisters of for example laminated aluminum foil, for use in an inhaler or insufflator.
• Powder blend formulations generally contain a powder mix for inhalation of the compound of the invention and a suitable powder base (carrier/diluent/excipient substance) such as mono-, di- or poly-saccharides (e.g. lactose or starch).
• Each capsule or cartridge may generally contain between 20 ⁇ g- 1 Omg of the compound of formula (I) optionally in combination with another therapeutically active ingredient.
• the compound of the invention may be presented without excipients.
• the packing/medicament dispenser is of a type selected from the group consisting of a reservoir dry powder inhaler (RDPI), a multi-dose dry powder inhaler (MDPI), and a metered dose inhaler (MDI).
• RDPI reservoir dry powder inhaler
• MDPI multi-dose dry powder inhaler
• MDI metered dose inhaler
• reservoir dry powder inhaler By reservoir dry powder inhaler (RDPI) it is meant an inhaler having a reservoir form pack suitable for comprising multiple (un-metered doses) of medicament in dry powder form and including means for metering medicament dose from the reservoir to a delivery position.
• the metering means may for example comprise a metering cup, which is movable from a first position where the cup may be filled with medicament from the reservoir to a second position where the metered medicament dose is made available to the patient for inhalation.
• multi-dose dry powder inhaler is meant an inhaler suitable for dispensing medicament in dry powder form, wherein the medicament is comprised within a multi-dose pack containing (or otherwise carrying) multiple, define doses (or parts thereof) of medicament.
• the carrier has a blister pack form, but it could also, for example, comprise a capsule-based pack form or a carrier onto which medicament has been applied by any suitable process including printing, painting and vacuum occlusion.
• the formulation can be pre -metered (e.g. as in Diskus, see GB 2242134, US Patent Nos. 6,632,666, 5,860,419, 5,873,360 and 5,590,645 or Diskhaler, see GB 2178965, 2129691 and 2169265, US Patent No.s 4,778,054, 4,811,731, 5,035,237, the disclosures of which are hereby incorporated by reference) or metered in use (e.g. as in Turbuhaler, see EP 69715 or in the devices described in US Patents No. 6,321,747 the disclosures of which are hereby incorporated by reference).
• Turbuhaler see EP 69715 or in the devices described in US Patents No. 6,321,747 the disclosures of which are hereby incorporated by reference.
• the Diskus inhalation device comprises an elongate strip formed from a base sheet having a plurality of recesses spaced along its length and a lid sheet hermetically but peelably sealed thereto to define a plurality of containers, each container having therein an inhalable formulation containing a compound of Formula (I) preferably combined with lactose.
• the strip is sufficiently flexible to be wound into a roll.
• the lid sheet and base sheet will preferably have leading end portions which are not sealed to one another and at least one of the said leading end portions is constructed to be attached to a winding means.
• the hermetic seal between the base and lid sheets extends over their whole width.
• the lid sheet may preferably be peeled from the base sheet in a longitudinal direction from a first end of the said base sheet.
• the multi-dose pack is a blister pack comprising multiple blisters for containment of medicament in dry powder form. The blisters are typically arranged in regular fashion for ease of release of medicament there from.
• the multi-dose blister pack comprises plural blisters arranged in generally circular fashion on a disc-form blister pack.
• the multi-dose blister pack is elongate in form, for example comprising a strip or a tape.
• the multi-dose blister pack is defined between two members peelably secured to one another.
• US Patent No.'s 5,860,419; 5,873,360 and 5,590,645 describe medicament packs of this general type.
• the device is usually provided with an opening station comprising peeling means for peeling the members apart to access each medicament dose.
• the device is adapted for use where the peelable members are elongate sheets which define a plurality of medicament containers spaced along the length thereof, the device being provided with indexing means for indexing each container in turn.
• the device is adapted for use where one of the sheets is a base sheet having a plurality of pockets therein, and the other of the sheets is a lid sheet, each pocket and the adjacent part of the lid sheet defining a respective one of the containers, the device comprising driving means for pulling the lid sheet and base sheet apart at the opening station.
• metered dose inhaler it is meant a medicament dispenser suitable for dispensing medicament in aerosol form, wherein the medicament is comprised in an aerosol container suitable for containing a propellant-based aerosol medicament formulation.
• the aerosol container is typically provided with a metering valve, for example a slide valve, for release of the aerosol form medicament formulation to the patient.
• the aerosol container is generally designed to deliver a predetermined dose of medicament upon each actuation by means of the valve, which can be opened either by depressing the valve while the container is held stationary or by depressing the container while the valve is held stationary.
• the valve may be a slide valve wherein the open/close mechanism comprises a sealing ring and receivable by the sealing ring a valve stem having a dispensing passage, the valve stem being slidably movable within the ring from a valve-closed to a valve-open position in which the interior of the valve body is in communication with the exterior of the valve body via the dispensing passage.
• the valve is a metering valve.
• the metering volumes are typically from 10 to 100 ⁇ l, such as 25 ⁇ l, 50 ⁇ l or 63 ⁇ l.
• the valve body defines a metering chamber for metering an amount of medicament formulation and an open/close mechanism by means of which the flow through the inlet port to the metering chamber is controllable.
• the valve body has a sampling chamber in communication with the metering chamber via a second inlet port, said inlet port being controllable by means of an open/close mechanism thereby regulating the flow of medicament formulation into the metering chamber.
• the valve may also comprise a 'free flow aerosol valve' having a chamber and a valve stem extending into the chamber and movable relative to the chamber between dispensing and non- dispensing positions.
• the valve stem has a configuration and the chamber has an internal configuration such that a metered volume is defined there between and such that during movement between is non-dispensing and dispensing positions the valve stem sequentially: (i) allows free flow of aerosol formulation into the chamber, (ii) defines a closed metered volume for pressurized aerosol formulation between the external surface of the valve stem and internal surface of the chamber, and (iii) moves with the closed metered volume within the chamber without decreasing the volume of the closed metered volume until the metered volume communicates with an outlet passage thereby allowing dispensing of the metered volume of pressurized aerosol formulation.
• a valve of this type is described in U.S. Patent No. 5,772,085. Additionally, intra-nasal delivery of the present compounds is effective.
• the means for applying a formulation of the present invention to the nasal passages is by use of a pre-compression pump.
• the pre-compression pump will be a VP7 model manufactured by Valois SA. Such a pump is beneficial as it will ensure that the formulation is not released until a sufficient force has been applied, otherwise smaller doses may be applied.
• Another advantage of the pre-compression pump is that atomisation of the spray is ensured as it will not release the formulation until the threshold pressure for effectively atomising the spray has been achieved.
• the VP7 model may be used with a bottle capable of holding 10-50ml of a formulation. Each spray will typically deliver 50-100 ⁇ l of such a formulation, therefore, the VP7 model is capable of providing at least 100 metered doses.
• the aerosol composition may be excipient free or may optionally contain additional formulation excipients well known in the art such as surfactants, e.g., oleic acid or lecithin and cosolvents, e.g. ethanol.
• Pressurized formulations will generally be retained in a canister (e.g. an aluminum canister) closed with a valve (e.g. a metering valve) and fitted into an actuator provided with a mouthpiece.
• Medicaments for administration by inhalation desirably have a controlled particle size.
• the optimum particle size for inhalation into the bronchial system is usually l-10 ⁇ m, preferably 2- 5 ⁇ m. Particles having a size above 20 ⁇ m are generally too large when inhaled to reach the small airways.
• the particles of the active ingredient as produced may be size reduced by conventional means e.g., by micronization.
• the desired fraction may be separated out by air classification or sieving.
• the particles will be crystalline in form.
• an excipient such as lactose is employed, generally, the particle size of the excipient will be much greater than the inhaled medicament within the present invention.
• Intranasal sprays may be formulated with aqueous or non-aqueous vehicles with the addition of agents such as thickening agents, buffer salts or acid or alkali to adjust the pH, isotonicity adjusting agents or anti-oxidants.
• Solutions for inhalation by nebulization may be formulated with an aqueous vehicle with the addition of agents such as acid or alkali, buffer salts, isotonicity adjusting agents or antimicrobials. They may be sterilised by filtration or heating in an autoclave, or presented as a non-sterile product.
• novel compounds of Formula (I) may also be used in association with the veterinary treatment of mammals, other than humans, in need of antagonism of a muscarinic receptor or a PDE4 enzyme.
• the treatment, therapeutically or prophylactically, in animals include disease states such as those noted herein in the Methods of Treatment section.
• treatment may include prophylaxis. It may also include reducing the symptoms of, ameliorating the symptoms of, reducing the severity of, reducing the incidence of, or any other change in the condition of the patient, which improves the therapeutic outcome.
• formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents, or those for inhalation may include carriers, such as lactose.
• Elevated levels of cyclic AMP has been shown to have anti-inflammatory activity in a range of immune cells including T-cells, macrophages and neutrophils as well as resident lung cells such as epithelial and airway smooth muscle cells. Elevated cAMP can also cause airway smooth muscle relaxation and may offer a further mechanism independent of M 3 receptor blockade to initiate bronchodilation.
• PDE4 inhibitors see: Kroegel C & Foerster M. Phophodiesterase-4 inhibitors as a novel approach for the treatment of respiratory disease: cilomilast. Expert Opin. Investig. Drugs 2007; 16: 109-124. Dastidar SG.
• compounds and pharmaceutical formulations according to the invention may be used in combination with or include one or more other therapeutic agents, for example selected from anti-inflammatory agents, other selective anticholinergic agents (particularly an Mi, M 2 , or M 1 ZM 2 receptor antagonist), ⁇ 2 -adrenoreceptor agonists, antiinfective agents (e.g. antibiotics, antivirals), or antihistamines.
• other therapeutic agents for example selected from anti-inflammatory agents, other selective anticholinergic agents (particularly an Mi, M 2 , or M 1 ZM 2 receptor antagonist), ⁇ 2 -adrenoreceptor agonists, antiinfective agents (e.g. antibiotics, antivirals), or antihistamines.
• the invention thus provides, in a further aspect, a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt, solvate or physiologically functional derivative thereof together with one or more other therapeutically active agents, for example selected from an anti-inflammatory agent (for example a corticosteroid or an NSAID), an anticholinergic agent, ⁇ 2 -adrenoreceptor agonist, an antiinfective agent (e.g. an antibiotic or an antiviral), or an antihistamine.
• an anti-inflammatory agent for example a corticosteroid or an NSAID
• an anticholinergic agent for example a corticosteroid or an NSAID
• an antiinfective agent e.g. an antibiotic or an antiviral
• PDE4 inhibitor e.g. an antibiotic or an antiviral
• Preferred combinations are those comprising one or two other therapeutic agents.
• the other therapeutic ingredient(s) may be used in the form of salts, (e.g. as alkali metal or amine salts or as acid addition salts), or prodrugs, or as esters (e.g. lower alkyl esters), or as solvates (e.g. hydrates) to optimize the activity and/or stability and/or physical characteristics (e.g. solubility) of the therapeutic ingredient.
• the therapeutic ingredients may be used in optically pure form.
• One suitable combination of the present invention comprises of compound of the invention together with a ⁇ 2 -adrenoreceptor agonist.
• ⁇ -adrenoreceptor agonists include salmeterol (which may be a racemate or a single enantiomer, such as the R-enantiomer), salbutamol, formoterol, salmefamol, fenoterol or terbutaline and salts thereof, for example the xinafoate salt of salmeterol, the sulphate salt or free base of salbutamol or the fumarate salt of formoterol.
• Long-acting ⁇ -adrenoreceptor agonists are preferred, especially those having a therapeutic effect over a 24 hour period, such as salmeterol or formoterol.
• Suitable long acting ⁇ -adrenoreceptor agonists include those described in WO02/66422A,
• WO02/270490 WO02/076933, WO03/024439, WO03/072539, WO 03/091204, WO04/016578, WO04/022547, WO04/037807, WO04/037773, WO04/037768, WO04/039762, WO04/039766, WOO 1/42193 and WO03/042160, whose disclosures are incorporated by reference herein.
• Preferred long-acting ⁇ 2 -adrenoreceptor agonists are: 3-(4- ⁇ [6-( ⁇ (2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl ⁇ amino) hexyl]oxy ⁇ butyl)benzenesulfonamide;
• Suitable anti-inflammatory agents include corticosteroids.
• Suitable corticosteroids which may be used in combination with the compounds of the invention are those oral and inhaled corticosteroids and their pro-drugs which have anti- inflammatory activity. Examples include methyl prednisolone, prednisolone, dexamethasone, fluticasone propionate, 6 ⁇ ,9 ⁇ -difluoro-17 ⁇ - [(2-furanylcarbonyl)oxy]- 11 ⁇ -hydroxy- 16 ⁇ -methyl-3-oxo-androsta- 1 ,4-diene- 17 ⁇ -carbothioic acid S-fluoromethyl ester, 6 ⁇ ,9 ⁇ -difluoro-l 1 ⁇ -hydroxy- 16 ⁇ -methyl-3-oxo-17 ⁇ -propionyloxy-androsta- l,4-diene-17 ⁇ -carbothioic acid S-(2-oxo-tetrahydro-furan-3S-yl) ester, 6 ⁇ ,9 ⁇ -
• Preferred corticosteroids include fluticasone propionate, 6 ⁇ ,9 ⁇ -difluoro-l l ⁇ -hydroxy-16 ⁇ -methyl-17 ⁇ -[(4-methyl-l,3- thiazole-5-carbonyl)oxy]-3-oxo-androsta- 1 ,4-diene- 17 ⁇ -carbothioic acid S-fluoromethyl ester and 6 ⁇ ,9 ⁇ -difluoro- 17 ⁇ -[(2-furanylcarbonyl)oxy]- 11 ⁇ -hydroxy- 16 ⁇ -methyl-3-oxo-androsta- 1 ,4-diene- 17 ⁇ -carbothioic acid S-fluoromethyl ester, more preferably 6 ⁇ ,9 ⁇ -difluoro-17 ⁇ -[(2- furanylcarbonyl)oxy]-l l ⁇ -hydroxy-16 ⁇ -methyl-3-oxo-androsta-l,4-diene-17 ⁇ -carbothioic acid S- fluoromethyl ester.
• Suitable NSAID 's include sodium cromoglycate, nedocromil sodium, leukotriene antagonists, inhibitors of leukotriene synthesis (for example, montelukast), iNOS inhibitors, tryptase and elastase inhibitors, beta-2 integrin antagonists and adenosine receptor agonists or antagonists (for example, adenosine 2a agonists), cytokine antagonists (for example, chemokine antagonists, such as a CCR3 antagonist) or inhibitors of cytokine synthesis, 5-lipoxygenase inhibitors, p38 inhibitors, and IKK2 inhibitors.
• cytokine antagonists for example, chemokine antagonists, such as a CCR3 antagonist
• Suitable other ⁇ 2 -adrenoreceptor agonists include salmeterol (for example, as the xinafoate), salbutamol (for example, as the sulphate or the free base), formoterol (for example, as the fumarate), fenoterol or terbutaline and salts thereof.
• An iNOS (inducible nitric oxide synthase inhibitor) is preferably for oral administration.
• Suitable iNOS inhibitors include those disclosed in WO93/13055, WO98/30537, WO02/50021, WO95/34534 and WO99/62875.
• Suitable CCR3 inhibitors include those disclosed in WO02/26722.
• Second generation antagonists which are non-sedating, have a similar structure- activity relationship in that they retain the core ethylene group (the alkylamines) or mimic the tertiary amine group with piperizine or piperidine.
• Exemplary antagonists are as follows:
• Ethanolamines carbinoxamine maleate, clemastine fumarate, diphenylhydramine hydrochloride, and dimenhydrinate.
• Ethylenediamines pyrilamine maleate, tripelennamine HCl, and tripelennamine citrate.
• Alkylamines chloropheniramine and its salts such as the maleate salt, and acrivastine.
• Piperidines Astemizole, levocabastine HCl, loratadine or its descarboethoxy analogue, and terfenadine and fexofenadine hydrochloride or another pharmaceutically acceptable salt.
• compositions comprising a combination as defined above together with a physiologically acceptable diluent or carrier represent a further aspect of the invention.
• the individual compounds of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations. Appropriate doses of known therapeutic agents will be readily appreciated by those skilled in the art.
• Desired attributes of the molecule would be to maintain or improve the M 3 pharmacophores potency with no or partial Mi agonism since agonism of the Mi receptor is usually counter- indicated. Another attribute is to decrease the dropoff between the PDE4 enzyme assay and inhibition reflected in the PBMC assay. Since both pharmacophores are in a single molecule, it is desirable to enhance intracellular inhibition of PDE4 reflected in the PBMC assay while retaining significant activity against the transmembrane M 3 receptor. In addition, in vivo efficacy and duration of action is not always reflected by in vitro measurements of activity, therefore other physiochemical properties of the molecules may be important for balanced efficacy at both targets.
• one embodiment of the invention are compounds which posses appropriately balanced pharmacology, and have desirable physicochemical properties, such as solubility, dissolution rate, permeability, crystallinity, micronizability, and excipient compatibility. If the compounds are administered by inhalation, then low aqueous solubility is generally not suitable for a nebulized/solution formulation.
• One embodiment of the invention is a display of sufficient antagonism at the M 3 receptor wherein p IC 5 O > 8.0 and a pA 2 > 8.0, as well as inhibition of the PDE4 enzyme with a pIC 5 o > 8.0 and cellular activity (as reflected in the PBMC assay) with a pIC 5 o> 7.0.
• compounds of Formula (I) are generally selective against agonism or partial agonism of the various muscarinic receptors (Mi, M 2 , M 3 ) and PDE4 > 100-fold vs. other PDEs.
• mAChR Muscarinic receptor
• PDE4 enzyme for the present invention
• CHO CHO cell lines.
• M 2 ACh receptor is co-expressed with the chimeric G protein, Gqi5, in CHO cells.
• Competition for [ 3 H]-N-methyl scopolamine (0.5 nM) binding is performed using crude CHO cell membranes using a Scintillation Proximity Assay (SPA). Atropine is run in every assay as the control. In the SPA assay membranes are preincubated with wheatgerm agglutinin beads (GE) in
• Cells are harvested by centrifugation at 1000 x g for 10 min at 4 0 C.
• the cell pellet is washed with Phosphate Buffered Saline (PBS) and quick frozen with liquid nitrogen.
• PBS Phosphate Buffered Saline
• the pellet is stored at - 80 0 C until the membrane preparation is made.
• the frozen pellet is thawed and re- suspended in cold hypotonic membrane buffer (40 mM Tris, pH 7.5, 1 mM MgSO 4 , 0.5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2.5 mg/L leupeptin, 0.1 mg/mL aprotinin) and incubated on ice for 5 min.
• cold hypotonic membrane buffer 40 mM Tris, pH 7.5, 1 mM MgSO 4 , 0.5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2.5 mg
• the cell suspension is homogenized in a 40 mL Dounce homogenizer and centrifuged at 2000 rpm at 4°C for 6 min to remove nuclei and cellular debris.
• the 2000 rpm pellet is resuspended in homogenization buffer and spun again at 2000 rpm for 6 min. This process is repeated two more times.
• the combined supernatant is collected and cell membranes are pelleted at 100000 x g for 1 hr at 4 0 C.
• the membrane pellet is resuspended in membrane buffer and aliquots stored at - 80 0 C. Protein concentration is quantified using the Bio-Rad protein assay reagent.
• the human M1-M3 receptors are cloned and stably expressed in Chinese Hamster Ovary (CHO) cells.
• the M2 receptors are co-expressed with the chimeric G protein, Gqi5.
• Method of Culture: CHO-Ml, CHO-Gqi5-M2 and CH0-M3 cells are cultured to confluence at 37°C in a humidified incubator with 5% CO 2 /95% air.
• CHO-Ml and CH0-M3 are cultured in Alpha MEM with nucleosides and L-glutamine and 10% fetal calf serum.
• Cells expressing the M2 receptor are cultured in DMEM/F12 media, supplemented with 200 mg/L G418 (geneticin), and 10% fetal calf serum.
• Assay Readout Calcium mobilization, monitored as change in cytosolic calcium concentration, is measured as change in 516 nm emission fluorescence intensity of cytosolic loaded Fluo-4, a green fluorescent calcium indicator which exhibits large (> 100-fold) fluorescence intensity increases on binding to calcium, the change in intensity being, therefore, directly related to cytosolic calcium levels.
• the emitted fluorescence from all 96 wells is measured simultaneously using a cooled CCD camera. Data points are collected every second. Maximal change in emission from each well after simultaneous addition of agonist or compound to each of the 96 wells is then exported to an excel spreadsheet. This data is then transferred to GraphPad Prism Version 4.03 for plotting of response to each treatment condition (ACh or compound).
• FLIPR Fluorometric Imaging Plate Reader, Molecular Devices, Sunnyvale CA, [Schroeder KS, Neagle, BD. FLIPR: a new instrument for accurate, high throughput optical screening. J. Biomol. Screen. 1996; 1 :75.]
• FLIPR Fluorometric Imaging Plate Reader, Molecular Devices, Sunnyvale CA, [Schroeder KS, Neagle, BD. FLIPR: a new instrument for accurate, high throughput optical screening. J. Biomol. Screen. 1996; 1 :75.]
• assay is used for the functional characterization of compounds against Ml, M2 (w/Gqi5) and M3 ACh receptors stably expressed in CHO cells.
• Receptor antagonist characterization (IC 50 determination), compounds tested for potency of inhibition of ACh induced muscarinic receptor activation: To evaluate antagonist potency of compounds against the Mi, M 2 and M 3 receptors, cell culture media is aspirated and replaced with 100 ⁇ L of dye load media [Eagles Minimal Essential Medium (EMEM) with Earl's salts and L- Glutamine, 0.1% BSA (Serologicals Corporation), 4 ⁇ M Fluo-4-acetoxymethyl ester fluorescent indicator dye (Fluo-4 AM, Molecular Probes, Eugene, OR) and 2.5 mM probenecid]. Cells are then incubated for 1 hour at 37°C.
• EMEM Eagles Minimal Essential Medium
• BSA Ses Corporation
• Fluo-4-acetoxymethyl ester fluorescent indicator dye Fluo-4 AM, Molecular Probes, Eugene, OR
• the dye load media is then aspirated off the cells and replaced with identical media without Fluo-4 AM and with 0.1% Gelatin (BSA removed) and 2.5 mM probenecid.
• Cells are incubated for 10 minutes at 37°C and then washed 3 times with KRH assay buffer [Krebs Ringer Henseleit (120 mM NaCl, 4.6 mM KCl, 1.03 mM KH 2 PO 4 , 25 mM NaHCO 3 , 1.0 mM CaCl 2 , 1.1 mM MgCl 2 , 11 mM Glucose, 20 mM HEPES (pH 7.4)) with 0.1% gelatin and 2.5 mM probenecid].
• KRH assay buffer KRH assay buffer
• the basal emission fluorescence is measured, then the cellular response to an ECgo concentration of ACh (3.3 nM against Mi, 10 nM against M 2 and 1.0 nM against M 3 ) prepared in KRH assay buffer with 0.1% BSA (no gelatin), is monitored in FLIPR for 90 seconds and then 50 ⁇ L of 100 ⁇ M ATP (assay concentration of 20 ⁇ M) is added to check cell viability (H. M. Sarau et a ⁇ , 1999. MoI. Pharmacol. 56, 657-663). Maximal change in emission from each well, vehicle or compound pretreated, after simultaneous addition of ACh to each of the 96 wells is then determined.
• the IC 50 is defined as the compound pretreatment concentration which inhibits 50% of the ACh induced response.
• a compound is believed to be active in this assay if it has an IC 5 O of between 33 uM and 10 nM or less.
• Exemplary compounds of Formula (I) which have been tested in this assay and found to be the most active can be found in Examples 125-138, 142-146, 148-150, 153, and 155-160.
• dye loaded (culture media is aspirated, replaced with 100 ⁇ L of dye load media and incubated for 1 hour at 37°C) and washed cells (washed three times with 100 ⁇ L KRH assay buffer) are treated with 150 ⁇ L of KRH assay buffer with 0.1% gelatin and 2.5 mM probenecid containing vehicle (0.01% DMSO), for control response, or appropriate concentration of antagonist (single concentration for each column of 12 wells, concentration determined from IC 5 O value) and incubated for 20 minutes at 37 0 C.
• Buffer is aspirated off and 150 ⁇ L of fresh KRH assay buffer with 0.1% gelatin and 2.5 mM probenecid containing vehicle (0.01% DMSO) or appropriate concentration of compound is added and incubated for 10 minutes at 37 0 C. Plates are then placed into FLIPR for fluorescent measurements. After determination of basal fluorescence emission, a concentration range of ACh (0.033-100,000 nM for M1/M3 and 0.33-1,000,000 nM for M2) is added to vehicle or compound treated (columns of 12 wells) cells to determine the shift of receptor potency in response to ACh in presence of compound.
• EMEM is aspirated and KRH assay buffer containing 0.1% gelatin with vehicle (0.01% DMSO) or antagonist is added to washout columns and incubated at 37 0 C for 20 minutes. Buffer with vehicle or compound is aspirated and cells retreated and incubated at 37 0 C for an additional 10 minutes. Buffer with vehicle or compound is then aspirated and cells washed 3 times with KRH assay buffer containing 0.1% BSA. KRH buffer (100 ⁇ L) containing 0.1% BSA is then added and cells incubated for 30 minutes at 37 0 C and washed 3 times. Cells are incubated for a further 30 minutes and washed 3 times, followed by a further 30 minute incubation.
• Dye load media is aspirated and cells are retreated with 150 ⁇ L of KRH assay buffer containing 0.1% gelatin and 2.5 mM probenicid for washout columns or KRH assay buffer containing 0.1% gelatin and 2.5 mM probenicid and vehicle or appropriate concentration of compound for no washout columns. Cells are incubated for 20 minutes at 37 0 C. Pretreatment buffer is aspirated and 150 ⁇ L of fresh KRH assay buffer with 0.1% gelatin and 2.5 mM probenecid is then added to washout columns and the same buffer containing vehicle (0.01% DMSO) or the appropriate concentration of antagonist is added to the no washout columns. Plates are incubated for 10 minutes at 37 0 C and plates placed into FLIPR where fluorescence is monitored.
• Receptor agonist characterization (EC 5 O determination): compounds tested to confirm no agonist potential at muscarinic receptors: To evaluate agonist potential of compounds and ACh potency for the Mi, M 2 and M 3 receptors, culture media is aspirated and replaced with 100 ⁇ L of dye load media. Cells are then incubated for 1 hour at 37°C. The dye load media is then aspirated off the cells and replaced with identical media without Fluo-4 AM and with 0.1% Gelatin (BSA removed) and 2.5 mM probenecid. Cells are incubated for 10 minutes at 37°C and then washed 3 times with 100 ⁇ L KRH assay buffer.
• 100 ⁇ L KRH assay buffer with 0.1% gelatin and 2.5 mM probenecid is added to wells of dye loaded and washed cells and plate warmed to 37 0 C for 10 minutes before being placed in FLIPR where dye loaded cells are exposed to excitation light (488 nm) from a 6 watt Argon Laser.
• the basal emission fluorescence is measured, then the cellular response to a concentration range of ACh or compound (50 ⁇ L of 3X in assay buffer) is monitored in FLIPR for 90 seconds and then 50 ⁇ L of 100 ⁇ M ATP (assay concentration of 25 ⁇ M) was added to check cell viability.
• the EC 5 O is the ACh or compound concentration required to obtain 50% the maximal response.
• Composition of the solution was (mM): NaCl (113.0), KCl (4.8), CaCl 2 (2.5), KH 2 PO 4 (1.2), MgSO 4 (1.2), NaHCO 3 (25.0) and dextrose (11.0) and equilibrated with 95% O 2 : 5% CO 2 and maintained at 37°C; meclofenamic acid (1 ⁇ M) was added to block endogenous cycloxygenase activity.
• trachea was removed from male Hartely guinea pigs (Charles River, Portage, MI; weight range 450-650 g). The epithelium of the trachea was removed and strips were cut, approximately 2 cartilage rings in width.
• Protocol A Tissues were suspended under an optimal resting tension of 1.5 g. After a 60 min equilibration period, the tissues were contracted with carbachol (1 ⁇ M) for the duration of the experiment. Upon reaching a sustained contraction isoproterenol (10 ⁇ M) was administered to maximally relax the tissue, and this change served as a reference. Isoproterenol exposure was halted and the carbachol-induced tension allowed to recover. Compounds and vehicle were infused at a single concentration per tissue until a sustained level of inhibition was attained. Compounds were infused for six hours and upon which the infusion of compounds and vehicle was halted. Carbachol-induced tension in tissues was then allowed to recover for 10 hours.
• a stock solution of 5% weight/volume of Tween 80 is made at least one day prior to dosing.
• the solution is made by dissolving 1 gram of Tween 80 in a total volume of 20 ml sterile saline.
• the stock 5% Tween solution is diluted 1 : 10 in sterile saline for a final concentration of 0.5% Tween.
• This solution is filtered through a 0.22 micron syringe filter to yield the final wet vehicle.
• Penh [(expiratory time / relaxation time)-l] x (peak expiratory flow / peak inspiratory flow) where relaxation time is the amount of time required for 70% of the tidal volume to be expired. Animals are returned to caging until the next noted exposure timepoint. Each animal's baseline airway parameter is used as its own control when determining the effect of ACh aerosol exposure.
• Human recombinant PDE4B in particular the 2B splice variant thereof (HSPDE4B2B), is disclosed in WO 94/20079 and also in M.M. McLaughlin et al., "A low Km, rolipram-sensitive, cAMP-specific phosphodiesterase from human brain: cloning and expression of cDNA, biochemical characterization of recombinant protein, and tissue distribution of mRNA", J. Biol. Chem., 1993, 268, 6470-6476.
• human recombinant PDE4B is described as being expressed in the PDE-deficient yeast Saccharomyces cerevisiae strain GL62. PDE4B expression is induced by the addition of 150 ⁇ M G1SO4.
• HSPDE4D3A Human recombinant PDE4D (HSPDE4D3A) is disclosed in P. A. Baecker et al., "Isolation of a cDNA encoding a human rolipram-sensitive cyclic AMP phosphodiesterase (PDE IV D )", Gene, 1994, 138, 253-256. Expression of human PDE4D in yeast, and subsequent preparation of the recombinant protein for assay was as described for PDE4B.
• Luciferase-coupled PDE assay Inhibition of PDE4B and PDE4D are measured using a luminescence-coupled assay system developed by Cambrex. This assay system couples the formation of AMP, derived from PDE4-catalyzyed hydrolysis of cAMP, to the formation of ATP. The ATP is then used as a substrate for Luciferase and results in light as a signal output. When PDE is inhibited or inactive, no AMP is produced, the Luciferase is inactive, and no light signal is produced.
• This assay is used in a quenched assay format, where PDE4 enzyme (2.5 ⁇ L; ⁇ 120pM enzyme in 4OmM Tris-HCl, 1OmM MgCl 2 , ImM CHAPS, 0.01% BSA, pH 7.5.) and cAMP substrate (2.5 ⁇ L; 2 ⁇ M cAMP in 4OmM Tris-HCl, 1OmM MgCl 2 , ImM CHAPS, 0.01% BSA, pH 7.5.) are added sequentially to a 384 well assay plate (Greiner 784075) pre-stamped with 12.5-50 nL compound at the desired concentration.
• PDE4 enzyme 2.5 ⁇ L; ⁇ 120pM enzyme in 4OmM Tris-HCl, 1OmM MgCl 2 , ImM CHAPS, 0.01% BSA, pH 7.5.
• cAMP substrate 2.5 ⁇ L; 2 ⁇ M cAMP in 4OmM Tris-HCl, 1OmM MgCl 2
• TNF- ⁇ TNF-alpha
• PBMC cells peripheral blood mononuclear cells
• PBMC cells peripheral blood mononuclear cells
• heparinised human blood using 1% v/v Heparin Sodium 1000IU/ml Endotoxin Free, Leo Laboratories Ltd., Cashel Road, Dublin 12. Ireland, Cat No: PL0043/0149
• AccuspinTM System-Histopaque ® -1077 essentially (Sigma-Aldrich Company Ltd., The Old Brickyard New Rd, Gillingham Dorset SP8 4XT).
• About 20 ml of blood is overlaid onto 15ml Histopaque ® in
• AccuspinTM tubes The tube is then centrifuged at about 80Og for ca. 20 minutes. The cells are collected from the cell layer, washed by centrifugation (ca. 1300g, ca. 10 minutes) and resuspended in RPMI1640 medium (Low endotoxin RPMI1640 medium, Cat No: 31870, Invitrogen Corporation Invitrogen Ltd, 3 Fountain Drive, Inchinnan Business Park, Paisley PA4 9RF, UK) containing 10% foetal calf serum, 1% L-glutamine (Invitrogen Corporation, Cat No: 25030) and 1% penicillin/streptomycin (Invitrogen Corporation, Cat No: 15140).
• RPMI1640 medium Low endotoxin RPMI1640 medium, Cat No: 31870, Invitrogen Corporation Invitrogen Ltd, 3 Fountain Drive, Inchinnan Business Park, Paisley PA4 9RF, UK
• Viable cells are counted by trypan blue staining and diluted to 1x10" viable cells/ml.
• About 50 ⁇ l (about 5OuI) of diluted cells and about 75 ⁇ l (about 75ul) of LPS (ca. 1 ng/ml final; Sigma Cat No: L-6386) are added to the compound plate, which is then incubated at 37 0 C, 5% CO2, for about 20 hours.
• the supernatant is removed and the concentrations of TNF- ⁇ are determined by electrochemiluminescence assay using the Meso Scale Discovery (MSD) technology (Meso Scale Discovery, 9238 Gaither Road, Gaithersburg, Maryland 20877, USA).
• MSD Meso Scale Discovery
• MSD Human Serum Cytokine Assay Diluent (25 ⁇ l) Meso Scale Discovery, 9238 Gaither Road, Gaithersburg, Maryland 20877) is added to a 96-well High-Bind MSD plate pre-coated with anti- hTNF alpha capture antibody (MA6000) and then incubated for about 24 hours at 4°C to prevent non-specific binding.
• About 20 ⁇ l (ul) of supernatant from the PBMC plate are then transferred from columns 1-11 to columns 1-11 of the MSD plate using the Biomek FX.
• About 20 ⁇ l (ul) of TNF- ⁇ standard (Cat No.
• the XC50 module automatically constrains A, B or A and B if an acceptable unconstrained fit cannot be achieved. QC criteria are applied and fits are rejected where A ⁇ -40 or >30, B ⁇ 80 or >140 or the ratio of upper and lower confidence limits on C >10. The results for each compound are recorded as pIC 50 values (-C in the above equation).
• Compounds are considered active in this assay if they demonstrated a pIC 5 o of greater than 5 up to a pIC 5 o of 10 or greater, and were screened at concentrations up to 10 uM.
• Representative compounds of Formula (I) as described in Examples 125-131, 133-138, 142-145, and 147-160 were tested in the above assay and found to be the most active.
• LPS-induced pulmonary neutrophilia in rats effect of i.t. administered PDE4 inhibitors Pulmonary neutrophil influx is thought to be a significant component to the family of pulmonary diseases like chronic obstructive pulmonary disease (COPD) which can involve chronic bronchitis and/or emphysema (G.F. Filley, Chest. 2000; 117(5); 251s-260s).
• COPD chronic obstructive pulmonary disease
• the purpose of this neutrophilia model is to study the potentially anti- inflammatory effects in vivo of orally administered PDE4 inhibitors on neutrophilia induced by inhalation of aerosolized lipopolysaccharide (LPS), modeling the neutrophil inflammatory component(s) of COPD. See the literature section below for scientific background.
• mice Male Lewis rats (Charles River, Raleigh, NC, USA) weighing approximately 280-400 grams are pretreated with a single intratracheal dose (200 ⁇ l) of either 300 ⁇ g/kg, or 30 ⁇ g/kg, of the test compound suspended in 0.5% Tween 80 (Sigma-Aldrich, St Louis, MO, USA) in phosphate buffered saline or vehicle only.
• dose response curves may be generated using intratracheal doses of 300, 30 and 10 ⁇ g/kg, again administered in 0.5% Tween 80 (Sigma-Aldrich, St Louis, MO, USA) in phosphate buffered saline (200 ⁇ l per rat, 30 minutes prior to LPS exposure.
• the rats are exposed to aerosolized LPS (Serotype E. CoIi 026:B6 prepared by trichloroacetic acid extraction, Sigma- Aldrich, St Louis, MO, USA), generated from a nebulizer containing a 100 ⁇ g/mL LPS solution. Rats are exposed to the LPS aerosol at a rate of ca. 4 L/min for. 20 minutes. LPS exposure is carried out in a closed chamber with internal dimensions of roughly 45 cm length x 24 cm width x 20 cm height. The nebulizer and exposure chamber are contained in a certified fume hood.
• LPS Cerotype E. CoIi 026:B6 prepared by trichloroacetic acid extraction, Sigma- Aldrich, St Louis, MO, USA
• BAL Bronchoalveolar lavage
• 5 ml washes are performed to collect a total of 25 ml of BAL fluid.
• Total cell counts and leukocyte differentials are performed on the BAL fluids in order to calculate neutrophil influx into the lung.
• percent inhibition of neutrophil number, neutrophil percent, or both may be calculated and reported for that specific dose.
• percent neutrophil inhibitions of either neutrophil number or neutrophil percent at each dose cf.
• a sigmoidal dose- response curve (variable slope) usually using Prism Graph-Pad.
• the dose-response curve may also be used to calculate an ED50 value (in mg per kg of body weight) for inhibition by the test compounds of the LPS-induced neutrophilia.
• ED50 value in mg per kg of body weight
• Examples are listed as producing "significant” inhibition if the test compound demonstrated significant (p ⁇ 0.05, using a two tailed distribution and two sample equal variance students T test performed in Microsoft Excel) inhibition of either neutrophil number, neutrophil percent, or both, when dosed at either 300 or 30 ⁇ g/kg, 30 minutes prior to LPS aerosol exposure.
• the compounds of Formula (I), (II), (III), (IV), (V) and (VI) may be obtained by applying the synthetic procedures described herein.
• the synthesis provided is applicable to producing compounds of the Formulas herein having a variety of different Rj, R2, R3, Xl, Z, ArI, Ar2, and R5, etc. groups which are reacted, employing optional substituents which may be suitably protected, to achieve compatibility with the reactions outlined herein. Subsequent deprotection, in those cases as necessary, affords compounds of the nature generally disclosed. While a particular formula with particular substituent groups is shown herein, the synthesis is applicable to all formulas and all substituent groups herein.
• hydroxyl protecting groups include ether forming groups such as benzyl, and aryl groups such as tert-butoxycarbonyl (Boc), silyl ethers, such as t-butyldimethyl or t- butyldiphenyl, and alkyl ethers, such as methyl connected by an alkyl chain of variable link, (CR10R20V Amino protecting groups may include benzyl, aryl such as acetyl and trialkylsilyl groups. Carboxylic acid groups are typically protected by conversion to an ester that can easily be hydrolyzed, for example, trichloethyl, tert-buiyl, benzyl and the like.
• Compounds of formula (XI), wherein Rl, R ⁇ and R ⁇ are as defined herein, may be prepared from compounds of formula (XII), wherein RI , R ⁇ and R ⁇ are as defined herein and wherein i ⁇ is a leaving group such as a halogen atom, mesylate (methanesulfonate), tosylate (p- toluenesulfonate), or triflate (trifluoromethanesulfonate) (suitably a halogen atom such as a chlorine atom).
• the compounds of formula (XII), e.g. wherein X" is Cl, can be reacted with an azide salt such as sodium, lithium or potassium azide, in a suitable solvent such as dimethylsulfoxide such as dry DMSO, e.g. at a suitable temperature such as room temperature, to give compounds of formula (XI).
• an azide salt such as sodium, lithium or potassium azide
• a suitable solvent such as dimethylsulfoxide such as dry DMSO, e.g. at a suitable temperature such as room temperature
• Compounds of formula (XII), wherein RI , R ⁇ and R ⁇ and X ⁇ are as defined herein, can be prepared by reaction of compounds of formula (XIII), wherein RI , R ⁇ and R ⁇ are as defined herein, with a suitable reagent such as thionyl chloride (for when X" is Cl), oxalyl chloride (for when X" is Cl), methanesulfonyl chloride (for when X" is mesylate), or/> ⁇ r ⁇ -toluenesulfonyl chloride (for when i ⁇ is tosylate), preferably thionyl chloride.
• a suitable reagent such as thionyl chloride (for when X" is Cl), oxalyl chloride (for when X" is Cl), methanesulfonyl chloride (for when X" is mesylate), or/> ⁇ r ⁇ -toluenesulfonyl chloride
• Suitable conditions for when i ⁇ is Cl, include reacting with thionyl chloride in a suitable non-aqueous (e.g. anhydrous) aprotic organic solvent such as toluene, e.g. with heating to ca. 60-90 0 C for example ca. 85°C.
• Alternative conditions include reacting compounds of formula (XIII) with thionyl chloride and methanesulfonic acid in a suitable non-aqueous (e.g. anhydrous) aprotic organic solvent such as dichloromethane, e.g. at a suitable temperature such as room temperature.
• compounds of formula (XI) wherein Rl, R ⁇ and R ⁇ are as defined herein can be prepared directly from compounds of formula (XIII) wherein RI , R ⁇ and R ⁇ are as defined herein.
• compounds of formula (XI) may be prepared by reacting compounds of formula (XIII) with an azide salt, e.g. sodium azide, in the presence of a halogenating agent such as carbon tetrabromide and a phosphine such as triphenylphosphine under suitable conditions, such as N,N-dimethylformamide, e.g. at a suitable temperature such as between 0 0 C and room temperature (see e.g Toyota et. al. Journal of Organic Chemistry (2000), 65(21), 7110-7113).
• an azide salt e.g. sodium azide
• a halogenating agent such as carbon tetrabromide
• a phosphine such as triphenylphosphine
• suitable temperature such as between 0
• (IX) or a salt thereof e.g. HCl salt thereof, wherein RI , R ⁇ and R ⁇ are as defined herein and R ⁇ is as defined herein (in particular where R ⁇ is a hydrogen atom)
• RI , R ⁇ and R ⁇ are as defined herein and R ⁇ is as defined herein (in particular where R ⁇ is a hydrogen atom)
• XII a compound of formula (XII) or a salt thereof, wherein RI , R ⁇ and R ⁇ and X ⁇ are as defined herein, without first converting to an azide compound of formula (XI).
• X can in particular be a chlorine atom.
• a benzenesulfonate salt of the compound of formula (XII) can for example be used, in particular when RI and R ⁇ are ethyl and when Ry is, for instance, a tetrahydro-2H-pyran-4-yl.
• the compound of formula (XII) or the salt thereof wherein X" is a chlorine atom can for example be in the form of the benzenesulfonate salt, in particular when RI and R ⁇ are ethyl and when R ⁇ is for instance, a tetrahydro-2H-pyran-4-yl:
• a suitable reaction temperature can be from room temperature to the reflux temperature, e.g. about 50 to about 75 0 C, e.g. about 60 to about 70 0 C, e.g. 63-69 0 C or 64-68 0 C.
• Another reducing agent is di- ⁇ o-butylaluminium hydride (e.g. solution in toluene), in which case: a suitable solvent is dichloromethane and/or toluene, and/or a suitable reaction temperature can be about 0 0 C.
• Compounds of formula (XIV), wherein R ⁇ , R ⁇ and R ⁇ and X' are as defined herein, may be prepared by reaction of a compound of formula (XV) with an amine of formula R- ⁇ NH2, for example generally according to the method described by Yu et. al. in J. Med Chem., 2001, 44, 1025-1027.
• the reaction is preferably carried out in the presence of a base such as triethylamine or N,N-diisopropylethylamine, and/or in an organic solvent such as ethanol, dioxane, 1 -methyl-2- pyrrolidinone (NMP) or acetonitrile.
• the reaction may require heating e.g. to ca. 60-180 0 C, for example at 115°C:
• R ⁇ is a N-aminocarbonyl-piperidinyl or N-aminocarbonyl-pyrrolidinyl group
• the urea-forming reagent may be benzyl isocyanate (followed later by debenzylation e.g. reductive debenzylation), or preferably the urea- forming reagent is tri(Ci _4alkyl)silyl isocyanate such as a tri(Ci _2alkyl)silyl isocyanate, preferably trimethylsilyl isocyanate.
• the conversion of the compound (XIVa) or salt thereof to the compound (XIV) may be carried out in the presence of a suitable base such as N,N-diisopropylethylamine, in a suitable solvent such as dichloromethane or chloroform, at a suitable temperature such as at room temperature or at the reflux temperature of the solvent.
• a suitable base such as N,N-diisopropylethylamine
• a suitable solvent such as dichloromethane or chloroform
• Compound (XIVa), wherein R ⁇ , R% X ⁇ and ⁇ r are as defined herein, or the salt thereof can be prepared from compound (XIVb) below, wherein wherein Rl, R% ⁇ 7 and rr are as defined herein and Prot is a suitable nitrogen protecting group such as (tert-butyloxy)carbonyl, by removal of the nitrogen protecting group.
• suitable nitrogen protecting group such as (tert-butyloxy)carbonyl
• suitable acidic conditions such as with hydrogen chloride (e.g. 4M) in a suitable solvent such as 1,4-dioxane:
• the reaction is optionally carried out in the presence of a base such as triethylamine or NN- diisopropylethylamine , optionally in a suitable organic solvent such as acetonitrile, at a suitable temperature such as 60-100 0 C (e.g. 80-90 0 C).
• a base such as triethylamine or NN- diisopropylethylamine
• a suitable organic solvent such as acetonitrile
• Compounds of formula (XVIII), wherein R ⁇ and X ⁇ are as defined herein, may be prepared by reaction of a dialkyl malonate of formula (XIX), wherein X ' is as defined herein, with magnesium chloride and a suitable base such as triethylamine, in a suitable solvent such as acetonitrile, at a suitable temperature such as 5-10 0 C, followed by addition of an acid chloride of formula (XX), for example propanoyl chloride, at a suitable temperature such as between 10 0 C and room temperature.
• a dialkyl malonate of formula (XIX) wherein X ' is as defined herein
• magnesium chloride and a suitable base such as triethylamine
• a suitable solvent such as acetonitrile
• Suitable conditions include treatment suitable acidic conditions such as hydrogen chloride in a suitable solvent such as 1,4-dioxane at a suitable temperature such as room temperature.
• Compounds for formula XXXVIII, wherein RI and R ⁇ , n ⁇ and Proc are as defined herein, may be prepared from compounds for formula XXXIX, wherein RI and R ⁇ , n ⁇ and Proc are as defined herein.
• Suitable conditions include reaction of compounds of formula XXXIX with an azide such as sodium azide and a halogenating agent such as carbon tetrabromide, in the presence of a suitable phosphine such as triphenylphosphine, in a suitable solvent such as NN,- dimethylformamide, at a suitable temperature such as between 0 0 C and room temperature.
• Compounds of formula (XXXIX), wherein RI and R % rr and Proc are as defined herein, may be prepared from compounds of formula (XL), wherein R ⁇ and R ⁇ , rr, Proc and X ' are as defined herein, by reduction with a suitable reducing agent such as lithium borohydride, in a suitable solvent such as a mixture of tetrahydrofuran and methanol, at a suitable temperature such as at the reflux temperature of the solvent.
• a suitable reducing agent such as lithium borohydride
• Compounds of formula (XL), wherein R ⁇ and R ⁇ , rr, Proc and X' are as defined herein, may be prepared from compounds of formula (XV), wherein RI, R2, and X ⁇ are as defined herein, by reaction of a compound of formula (XV) with an amine of formula (XLI), wherein Proc and ⁇ r are as defined herein.
• the reaction is preferably carried out in the presence of a base such as triethylamine or N,N-diisopropylethylamine, and/or in an organic solvent such as ethanol, dioxane, 1 -methyl-2-pyrrolidinone (NMP) or acetonitrile.
• the reaction may require heating e.g. to ca. 60- 180 0 C, for example at 120 0 C:
• Scheme 1 describes the general synthesis of the compounds 1-4.
• Compound 1-1 and serine ethyl ester 1-2 are treated with a coupling agent such as DCC, EDC, HATU, HBTU, with or without the addition of HOBt, in the presence of a tertiary amine such as triethyl amine or diisopropyl ethyl amine in a solvent such as methylene chloride or DMF to give 1-3.
• Intermediate 1-3 is then treated with (methoxycarbonyl-sulfamoyl) triethyl ammonium hydroxide, inner salt in an organic solvent such as THF and is heated at 55 0 C for 2-6 h to give 1-4.
• Intermediate 1-4 is treated with a strong organic base, such as DBU in a mixture of organic solvents including CCU, pyridine and CH 3 CN at room temperature to give 1-5.
• a coupling agent such as DCC, EDC, HATU, HBTU,
• Scheme 2 describes the general synthesis of the compounds 2-4.
• an aqueous base e.g. NaOH, LiOH
• the resulting carboxylic acid (Z CO 2 H) and a suitable protected, where necessary, Ari-Ar 2 amine 2-2 is treated with a coupling agent such as DCC, EDC, HATU, HBTU, with or without the addition of HOBt, in the presence of a tertiary amine such as triethyl amine or diisopropyl ethyl amine in a solvent such as methylene chloride or DMF.
• a suitable protecting group is needed when R 6 contains a primary or secondary amine.
• the resulting intermediate 2-3 is then deprotected in a method defined by the nature of the protecting group used. In the case of an acid labile amine protecting group like Boc, deprotection can be achieved using a strong acid such as TFA in a solvent such as dichloromethane to give 2-4.
• Scheme 3 describes an alternate synthesis of compounds 3-6.
• an aqueous e.g. NaOH, LiOH in an organic solvent such as methanol, ethanol or dioxane.
• intermediate 4-4 Reductive amination of the resulting intermediate 4-3 with a suitably protected R ⁇ -H using a reducing reagent such as NaBH 3 CN in methanol or NaBH(OAc) 3 in dichloroethane or DMF gives intermediate 4-4.
• Intermediate 4-4 is then deprotected in a method defined by the nature of the protecting group used.
• an acid labile amine protecting group like Boc
• deprotection can be achieved using a strong acid such as TFA in a solvent such as dichloromethane to give 4-5.
• Scheme 4A describes the general synthesis of the compounds 4A-5.
• Intermediate 4A -1 and 2- propenoic acid is treated with a coupling agent such as DCC, EDC, HATU, HBTU, with or without the addition of HOBt, in the presence of a tertiary amine such as triethyl amine or diisopropyl ethyl amine in a solvent such as methylene chloride or DMF to give 4A -2.
• a solution of intermediates 4A -2 and 4A -3 in water and organic solvent such as DMF are treated with a palladium catalyst (e.g., PdCl 2 (dppf)), a quaternary amine salt (e.g., BU 4 NI) and a tertiary amine base (e.g., triethylamine, diisopropylethylamine) in a microwave reactor to give 4A -4.
• the resulting intermediate 4A -4 is then deprotected in a method defined by the nature of the protecting group used. In the case of an acid labile amine protecting group like Boc, deprotection can be achieved using a strong acid such as TFA in a solvent such as dichloromethane to give 4A -5.
• a palladium catalyst e.g., PdCl 2 (dppf)
• a quaternary amine salt e.g., BU 4 NI
• Scheme 5 describes the general synthesis of the compounds 5-4.
• an aqueous base e.g. NaOH, LiOH
• a suitable protecting group is needed when R 6 contains a primary or secondary amine.
• the resulting intermediate 5-3 is then deprotected in a method defined by the nature of the protecting group used. In the case of an acid labile amine protecting group like Boc, deprotection can be achieved using a strong acid such as TFA in a solvent such as dichloromethane to give 5-4.
• Scheme 7 describes the general synthesis of the compounds 7-4.
• an aqueous base e.g. NaOH, LiOH
• Scheme 8 describes the general synthesis of the compounds 8-3.
• the intermediate chloride 8-1 is treated with the piperazine 8-2 in the presence of an organic tertiary amine base (e.g., triethylamine, diisopropylethyl amine) or an inorganic base (e.g., NaHC ⁇ 3, Na 2 COs) in an organic solvent such as THF to give intermediate 8-3.
• an organic tertiary amine base e.g., triethylamine, diisopropylethyl amine
• an inorganic base e.g., NaHC ⁇ 3, Na 2 COs
• Scheme 9 describes the general synthesis of the compounds 9-4.
• an aqueous base e.g. NaOH, LiOH
• a suitable protecting group is needed when R 6 contains a primary or secondary amine.
• the resulting intermediate 9-3 is then deprotected in a method defined by the nature of the protecting group used. In the case of an acid labile amine protecting group like Boc, deprotection can be achieved using a strong acid such as TFA in a solvent such as dichloromethane to give 9-4.
• Scheme 10 describes the general synthesis of the compounds 10-4.
• the intermediates 10-1 and 10- 2 are combined in an organic solvent such as toluene and treated with a palladium catalyst (e.g., PdO AC 2 ) with a phosphine ligand (e.g., BiPhPCy 2 ) in the presence of an inorganic base (e.g., CS 2 CO3) and a quaternary amine salt (e.g., BnNBusBr) and heated in a microwave reactor to give 10-3.
• the resulting intermediate 10-3 is then deprotected in a method defined by the nature of the protecting group used.
• an acid labile amine protecting group like Boc
• deprotection can be achieved using a strong acid such as TFA in a solvent such as dichloromethane to give 10-4.
• a suitable protecting group is needed when R 6 contains a primary or secondary amine.
• the resulting intermediate 11-3 is then deprotected in a method defined by the nature of the protecting group used. In the case of an acid labile amine protecting group like Boc, deprotection can be achieved using a strong acid such as TFA in a solvent such as dichloromethane to give 11-4.
• a suitable protecting group is needed when R 6 contains a primary or secondary amine.
• the resulting intermediate 12-3 is then deprotected in a method defined by the nature of the protecting group used. In the case of an acid labile amine protecting group like Boc, deprotection can be achieved using a strong acid such as TFA in a solvent such as dichloromethane to give 12-4.
• a variety of reverse phase columns e.g., Luna 5u C 18(2) 100A, SunFireTM C 18, XBridgeTM C 18 were used in the purification with the choice of column support dependent upon the conditions used in the purification.
• the compounds are eluted using a gradient of acetonitrile and water.
• Neutral conditions used an acetonitrile and water gradient with no additional modifier
• acidic conditions used an acid modifier, usually 0.1 % TFA (added to both the acetonitrile and water)
• basic conditions used a basic modifier, usually 0.1 % NH 4 OH (added to the water).
• Analytical hplc was run using an Agilent system with variable wavelength UV detection using reverse phase chromatography with an acetonitrile and water gradient with a 0.05 or 0.1 % TFA modifier (added to each solvent).
• LC-MS was determined using either a PE Sciex Single Quadrupole LC/MS API- 150 or a Waters.
• the compound is analyzed using a reverse phase column, e.g., Thermo Aquasil/Aquasil C 18, Acquity UPLC C 18, Thermo Hypersil Gold eluted using an acetonitrile and water gradient with a low percentage of an acid modifier such as 0.02% TFA or 0.1 % formic acid.
• Cartridges or columns containing polymer based functional groups can be used as part of compound workup.
• the "amine” columns or cartridges are used to neutralize or basify acidic reaction mixtures or products. These include NH2 Aminopropyl SPE-ed SPE Cartridges available from Applied Separations and diethylamino SPE cartridges available from United Chemical Technologies, Inc.
• Heating of reaction mixtures with microwave irradiations was carried out on a Smith Creator (purchased from Personal Chemistry, Forboro, MA, now owned by Biotage), an Emrys Optimizer (purchased from Personal Chemistry) or an Explorer (purchased from CEM, Matthews, NC) microwave.
• the organic layer was separated and the aqueous layer was extracted with CHCI3 (150 mL x 2).
• NaBH 4 (2.4 g, 64.3 mmol) was added cautiously to a solution of NiCl 2 (2.8 g, 21.6 mmol), BoC 2 O (9.6 g, 44.0 mmol) and 5-bromo-2-methylbenzonitrile (4.2 g, 21.4 mmol) in EtOH (150 mL) at 0 0 C within 0.5 h, then stirred for 40 min. After the reaction had subsided, the mixture was left to stir at room temperature for 0.5 h. Then the solvent was removed and the residue was dissolved in AcOEt and a saturated solution of NaHC ⁇ 3, then filtered and washed with AcOEt. The combined organic layers were washed with brine and dried over Na 2 SO 4 .
• Pd(OAc) 2 (102.0 mg, 0.45 mmol, 0.03 eq.), PPh 3 (476.4 mg, 1.82 mmol, 0.12 eq.), K 2 C ⁇ 3 (3.14 g, 22.7 mmol, 1.50 eq.) and 2-[(3-bromo-5-methylphenyl)methyl]- lH-isoindole- l,3(2H)-dione (5.00 g, 13.1 mmol, 1.00 eq.) were suspended in anhydrous 1,4-dioxane (30 mL) under nitrogen.

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