WO2002026743A1 - Novel pyridazine compounds for the treatment of diabetes - Google Patents

Novel pyridazine compounds for the treatment of diabetes Download PDF

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Publication number
WO2002026743A1
WO2002026743A1 PCT/SE2001/002097 SE0102097W WO0226743A1 WO 2002026743 A1 WO2002026743 A1 WO 2002026743A1 SE 0102097 W SE0102097 W SE 0102097W WO 0226743 A1 WO0226743 A1 WO 0226743A1
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Prior art keywords
pyridazine
amino
pyrano
compound
dihydro
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PCT/SE2001/002097
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French (fr)
Inventor
Emma Barker
Styrbjörn BYSTRÖM
Eric Desarbre
James Duffy
Stephen James
Charlotta Liljebris
Jessica Martinsson
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Biovitrum Ab
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Priority claimed from SE0003436A external-priority patent/SE0003436D0/en
Priority claimed from SE0101933A external-priority patent/SE0101933D0/en
Application filed by Biovitrum Ab filed Critical Biovitrum Ab
Priority to AU2001292480A priority Critical patent/AU2001292480A1/en
Publication of WO2002026743A1 publication Critical patent/WO2002026743A1/en

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    • 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
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D237/00—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings
    • C07D237/26—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings condensed with carbocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D237/00—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings
    • C07D237/26—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings condensed with carbocyclic rings or ring systems
    • C07D237/28—Cinnolines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems

Definitions

  • Novel pyridazine compounds for the treatment of diabetes are novel pyridazine compounds for the treatment of diabetes
  • the present invention is directed to novel compounds, to a process for their preparation, their use and pharmaceutical compositions comprising said novel compounds. These novel compounds are useful in therapy, particularly for the treatment of type 2 diabetes.
  • Phosphorylation on serine, threonine and tyrosine amino acid residues in downstream proteins forms the major output from growth factor and cytokine receptors, from which a cellular response is built.
  • a large number of growth factor and cytokine- regulated protein tyrosine kinases (PTKs) have been identified which can be integral parts of receptor proteins or cytosolic molecules (Al-Obeidi, FA, Wu, JJ & Lam, KS, Biopolym. Pept. Sci. Sect. 47, 197-223). These serve to phosphorylate proteins on tyrosine residues within specific primary amino acid sequences which, when phosphorylated, act as docking points for proteins which contain SH 2 domains. It is the docking of proteins to phosphorylated tyrosine residues which contributes to the activation of such proteins and the establishment of a signal transduction cascade.
  • Phosphotyrosines are returned to their free acid form by the action of protein tyrosine phosphatases (PTPs) (Zhang, ZY (1998) Crit. Rev. Biochem. Mol. Biol., 33, 1-52). Whilst a large number of PTKs has been identified (Hunter, T (1994) Se . Cell Biol. 5, 367-376), the number of PTPs identified to date is decidedly smaller (van Huijsduijnen, RH (1998) Gene 225, 1- 8).
  • PTPs protein tyrosine phosphatases
  • Loss of function in the receptor-like subfamily of PTPs leads to conditions such as heightened and reduced sensitivity to insulin (Ren, J- M, Li, P-M, Zhang, W-R, Sweet, LJ, Cline, G, Shulman, Gl, Livingston, IN &
  • mice in which the PTP PTP IB had been disrupted revealed that loss of function of this enzyme leads to enhanced insulin sensitivity and resistance to the development of obesity, thus revealing a therapeutic need for the development of specific PTP inhibitors (Elchelby, M, Payette, P, Michaliszyn, E, Cromlish, W, Collins, S, Loy, AL, Normandin, D, Cheng, A, Himms.Hagen, J, Chan, CC, Ramachandran, C, Gresser, MJ, Tremblay, ML & Kennedy, BP (1999) Science 283, 1544-1548; Klaman, LD, Boss, O, Peroni, OD, Kim, JK, Martino, JL, Zablotny, JM, Moghal, N, Lubkin, M, Kim, Y-B, Sharpe, AH, Stricker-Krongrad, A, Shulman, Gl, Neel, BG & Kahn, BB (2000) Mol.
  • the mechanism of insulin action depends critically upon the phosphorylation of tyrosine residues in several proteins in the insulin-signaling cascade. PTPs that dephosphorylate these proteins are important negative regulators of insulin action. Therefore, the use of specific PTP inhibitors may therapeutically enhance insulin action.
  • the anabolic effects of insulin are triggered through the activation of a variety of signal transduction cascades which lie downstream ofthe insulin receptor (Gustafson, T.A., Moodie, SA & Lavan, BE (1999) Rev. Physiol. Biochem. Pharmacol. 137, 71- 190).
  • the varieties of signals that are activated by insulin are thought to contribute to the range of effects that insulin controls.
  • each pathway is activated by a common series of biochemical reactions proximal to the insulin receptor.
  • the insulin receptor undergoes autophosphorylation on tyrosine residues when activated by insulin, and also phosphorylates other proteins, in particular, the insulin receptor substrate proteins (IRSs).
  • protein tyrosine phosphatase IB appears to be the major negative regulator ofthe insulin receptor in muscle and liver tissues (see for example Elechelby, M, Payette, P, Michalszyn, E, Cromlish, W, Collins, S, Loy, AL, Normandin, D, Cheng, A, Himms-Hagen, J, Chan, C-C, Ramachandran, C, Gresser, MJ, Tremblay, M & Kennedy, B P (1999) Science, 283, 1544-1548; Goldstein, BJ, Bittner- Kowalczyk, A, White, M F & Harbeck, M (2000) J.
  • PTP alpha may play a more dominant role in regulating the insulin receptor in adipose tissue (Calera, MR, Vallega, G & Pilch, PF (2000) J. Biol. Chem. 275 6308- 6312).
  • type 2 diabetes is characterized by a protracted period of insulin resistance.
  • PTP protein concentrations are increased, which has led to the idea that elevations in the proteins contributes to the cause ofthe diabetic state (Ahmad, F, Azevedo, JL, Cortright, R, Dohm, G L & Goldstein, BJ (1997) J. Clin. Invest. 100 449-458).
  • the two most significantly elevated are PTP IB and LAR.
  • WO 96/40113 discloses heterocyclic nitrogen containing compounds, such as nitropyridine or nitrothiazole, capable of inhibiting protein tyrosine phosphatase activity.
  • WO 98/27065 discloses a class of compounds which are stated as being protein tyrosine phosphatase modulating compounds. These prior art compounds are however structurally distinct from the compounds claimed in the present patent application.
  • WO 97/08934 discloses aryl acrylic acid compounds of a certain structure, which compounds are stated as having protein tyrosine protease modulating activity. Also these prior art compounds are however structurally distinct from the compounds claimed in the present patent application.
  • WO 99/58519 discloses certain phenyl oxo-acetic acid compounds. These compounds are stated as being useful in the treatment of metabolic disorders related to insulin resistance and hyperglycemia. Also these prior art compounds are however structurally distinct from the compounds claimed in the present patent application.
  • WO 99/58521 discloses the use of ll-aryl-benzo[b]naphtho[2,3-d]furan and 11- aryl-benzo[b]naphtho[2,3-d]thiophene compounds to inhibit protein tyrosine phosphatase activity. Such compounds are disclosed as being useful to modulate or regulate signal transduction by inhibiting protein tyrosine phosphatase activity and to treat various disease states including diabetes mellitus.
  • the object ofthe present invention was to provide novel compounds for the specific inhibition of PTPs allowing the study og biological processes in which they are active. Furthermore, a second object ofthe current invention was to provide novel compounds having improved advantages over drugs currently used for the treatment of type 2 diabetes. It should be appreciated that the wording "improved advantages" is not necessarily defined as more potent compounds, but as compounds having improved advantages overall, including but not limited to also improved selectivity and less side- effects.
  • a halogen selected from the group consisting of fluoro, chloro, bromo, and iodo;
  • Y is O, S, or N-R x wherein R x is a straight or branched C ⁇ -C 6 alkyl
  • (c) -CO-A wherein A is phenyl, naphthyl, or a heteroaryl; said heteroaryl group having 5 to 10 ring atoms wherein at least one of said ring atoms is O, N, or S;
  • alkyl group is straight or branched
  • Q 1 and Q 2 is independently phenyl, naphthyl, or a heteroaryl; said heteroaryl group having 5 to 10 ring atoms wherein at least one of said ring atoms is O, N, or S; each of R a and R b is independently hydrogen, CrC 6 alkyl, or R a and R b together form a carbonyl group; and
  • R c is hydrogen, a straight or branched Ci-C ⁇ alkyl, or carbonyl; with the proviso that the compound 6H-pyrano[3,4-c]pyridazine-4-carbonitrile, 3- amino-2,8-dihydro-6,6-dimethyl-2-phenyl is excluded.
  • R 1 in accordance with the present invention are phenyl or naphthyl which is optionally and independently substituted by 1, 2, 3, 4 or 5 substituents selected from the group consisting of methyl; ethyl; straight, branched or cyclic propyl, butyl, pentyl, or hexyl; -CO-O-(CH 2 ) n -CH 3 wherein n is an integer 0, 1, 2, 3, 4, or 5; methoxy, ethoxy, propoxy, butyloxy, pentyloxy, or hexyloxy; -O-(CH ) ceremoni- phenyl where n is an integer 0, 1, 2, 3, 4, 5, or 6. .
  • R 1 is phenyl, optionally substituted with a straight or branched - alkyl (e.g., methyl, ethyl, n-propyl, or isopropyl), a straight or branched -Ce alkoxy (e.g., methoxy, ethoxy, or propoxy), nitro, CF 3 , halo (e.g., fluoro, chloro, or bromo; especially fluoro and chloro), cycloalkyl (e.g., cyclohexyl), heterocycloalkyl (e.g., heterocyclohexyl such as morpholino), aryl (e.g., phenyl), heteroaryl (e.g., furan),
  • a straight or branched - alkyl e.g., methyl, ethyl, n-propyl, or isopropyl
  • a straight or branched -Ce alkoxy
  • -CO-O-(Ci-C 4 alkyl) e.g., -CO-O-CH 3 or -CO-O-CH 2 CH 3
  • -N(R 2 )-CO-R 3 where R 2 is hydrogen, methyl, or ethyl, and R 3 is a straight or branched - alkyl (e.g., -NH-CO-CH 3 or -NH-CO-CH 2 CH 3 ).
  • R 1 are shown below:
  • n t and n 2 independently is an integer
  • n 3 is an integer 0, 1, 2, 3, 4, or 5;
  • Examples of X include an oxygen atom, a sulfur atom, a methylene group,
  • R 6 is benzyl, -CO-(CrC 4 alkyl), -CO-O-(C r C 4 alkyl), or
  • a preferred example of X is oxygen. Another preferred example
  • N— R 6 of X is /
  • R a , R b , and R c are hydrogen, methyl, or ethyl.
  • Examples of a compound ofthe present invention include compounds of formula la' wherein R 1 is phenyl, optionally substituted with a straight or branched Q- C 4 alkyl, a straight or branched Ci-C ⁇ alkoxy, nitro, CF 3 , fluoro, chloro, bromo, cyclohexyl, heterocyclohexyl, phenyl, -CO-O-(C ⁇ -C 4 alkyl), or -N(R 2 )-CO-R 3 , where R 2 is hydrogen, methyl, or ethyl, and R 3 is a straight or branched C C 4 alkyl; X is an
  • R 6 is benzyl, -CO- (C ⁇ -C 4 alkyl), or
  • R a , R b , and R c independently is hydrogen, methyl, or ethyl.
  • Examples of a compound ofthe present invention also include compounds of formual la' wherein R 1 is
  • n 2 independently is an integer 0, 1, 2, 3, 4, or 5;
  • n is an integer 0, 1, 2, 3, 4, or 5;
  • n t and n 5 independently is an integer 0, 1, 2, 3, 4, or 5;
  • R 6 X is an oxygen atom; a sulfur atom; a methylene group; / , wherein R 6 is benzyl, -
  • each of R a , R b , and R c independently is hydrogen, methyl, or ethyl.
  • the compounds ofthe present invention are useful in therapy, particular for the treatment of type 2 diabetes mellitus.
  • a compound of formula I for the manufacture of a medicament for the treatment of type 2 diabetes mellitus.
  • a further aspect ofthe invention is the use of a compound of formula la (e.g., a compound of formula la'), for the manufacture of a medicament for the treatment of type 2 diabetes mellitus.
  • a compound of formula la e.g., a compound of formula la'
  • Still a further aspect ofthe invention is the use of a compound of formula lb, for the manufacture of a medicament for the treatment of type 2 diabetes mellitus.
  • a further aspect ofthe invention is a method for the treatment of a patient suffering from type 2 diabetes mellitus, whereby an effective amount of a compound according to formula I above, is administered to a patient in need of such treatment.
  • a further aspect ofthe invention is a method for the treatment of a patient suffering from type 2 diabetes mellitus, whereby an effective amount of a compound according to formula la (e.g., a compound of formula la') above, is administered to a patient in need of such treatment.
  • a compound according to formula la e.g., a compound of formula la'
  • a further aspect ofthe invention is a method for the treatment of a patient suffering from type 2 diabetes mellitus, whereby an effective amount of a compound according to formula lb above, is administered to a patient in need of such treatment.
  • an alkyl is a straight or branched hydrocarbon chain containing the indicated number of carbon atoms.
  • alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, n- pentyl, 2-methylpentyl, and n-hexyl.
  • cycloalkyl is meant a cyclic alkyl group containing the indicated number of carbon atoms. Some examples of cycloalkyl are cyclopropyl, cyclopentyl, cyclohexyl, adamantyl, and norbornyl. Heterocycloalkyl is a cycloalkyl group containing the indicated number of heteroatoms such as nitrogen, oxygen, or sulfur. Examples of heterocycloalkyl include piperidinyl, piperazinyl, tefrahydropyranyl, tefrahydrofuryl, and mo holinyl.
  • aryl is an aromatic group containing the indicated number of ring atoms.
  • examples of an aryl group include phenyl, naphthyl, phenanthryl, and anthracyl.
  • Heteroaryl is aryl containing the indicated number of heteroatoms such as nitrogen, oxygen, or sulfur. Some examples of heteroaryl are pyridyl, furanyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, and imidazolyl.
  • Each ofthe cycloalkyl, heterocycloalkyl, aryl, or heteroaryl groups described herein is optionally substituted with C 1 - 4 alkyl, C 3 . 6 cycloalkyl, 3-6 membered heterocycloalkyl, C 6 - ⁇ o aryl, 6-10 membered heteroaryl, C - 14 aralkyl, C ⁇ - 4 alkyl- heteroaryl with 6-10 ring atoms, Ci- 4 alkoxy, hydroxy, hydroxyl-C 1 - 4 alkyl, carboxyl, halo, halo-C M alkyl, amino, amino-C 1 .
  • an amino group can be unsubstituted, mono-substituted, or di- substituted. It can be substituted with groups such as C 1 - 4 alkyl, C 3 .
  • Compounds according to the present invention may be prepared by following the procedure reported by Norayan, Paronikian and Vartanyan, Khim. Geterotsikl.
  • Each of X, R a , R , R c , R , and n has the same definition as provided above.
  • Examples of a base that can be used in the reaction of Scheme I include sodium hydride and potassium t-butoxide.
  • Each of X, R a , R b , R c , and R 1 has the same definition as provided above.
  • Examples of a base that can be used in the reaction of Scheme I include sodium hydride and potassium t-butoxide.
  • NMR spectra were recorded on a Narian 400 MHz spectrometer, a Bruker Advance DPX 400 or a Bruker DRX 500 and chemical shifts are given in ppm using tetramethylsilane as an internal standard at 25°C.
  • HPLC analyses were performed using a Waters Xterra MS C18 column (100 x 4.6 mm, 5 ⁇ ) eluting with a gradient of 5% AC ⁇ in 95% water to 95% AC ⁇ in 5% water (0.2% TFA buffer) over 3.5 min. then 95% ACN in 5% water (0.2% TFA buffer) for a further 2.5 min.
  • IR spectra were recorded on a Perkin Elmer Spectrum 1000 FTIR spectrometer. Electrospray MS spectra were obtained on a Micromass platform LCMS spectrometer. The Biotage Quad 3 system was used for parallel flash purification. Silica gel column chromatography was performed using YMC gel, silica 120 A S-50 ⁇ m.
  • Tetrahydro-4H-pyran-4-one (1) (9.79g, 0.098mol) was then added, followed by ethyl formate (11.9 ml, 0.147 mol) and the reaction mixture stirred at room temperature overnight. The solvent was then removed under reduced pressure and the residue dissolved in water (40 ml). The resultant solution was added to a solution ofthe diazonium salt of aniline, prepared by the gradual addition of a solution of sodium nitrite (6.76 g, 0.098 mol) in water (20 ml) to a solution of aniline (9.11 g, 0.098 mol) in 2 M HC1 (98 ml, 0.196 mol) at -5 to 0°C.
  • HgSO 4 (0.86 g, 0.0029 mol), H 2 SO 4 (0.86 ml) and water (34 ml) at 85°C with stirring. Then HgSO 4 (0.251 g, 0.00073 mol) and H 2 SO (0.22 ml) in water (8.6 ml) were added over a period of one hour. In total the reaction mixture was heated for five hours at 85 to 100°C. This reaction mixture was then steam distilled. The product was then extracted with DCM, washed with water, dried (MgSO 4 ) and the solvent removed under reduced pressure to give a yellow liquid.
  • Synthetic method la was used to prepare the hydrazone intermediate.
  • the method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.15 g, 0.0007 mol), DMSO (3ml), malononitrile (0.05g, 0.0007mol) and morpholine (0.064 ml, 0.0007 mol).
  • the reaction mixture was stirred at 80°C for 15 minutes.
  • the mixture was cooled and the solution poured into cold water (20 ml).
  • the resultant precipitate was collected by filtration, recrystallized from ethanol, then freeze- dried.
  • Synthetic method la was used to prepare the hydrazone intermediate.
  • the method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.5g, 0.0027mol), DMSO (1ml), malononitrile (0.18g, 0.0027mol) and morpholine (0.23ml, 0.0027mol).
  • the reaction mixture was stirred at 80°C for 15 minutes.
  • the mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (5:1 to 1:1), then freeze-dried.
  • Synthetic method la was used to prepare the hydrazone intermediate.
  • the method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.2 g, 0.0007 mol), DMSO (1ml), malonomtrile (0.05 g, 0.0007 mol) and mo ⁇ holine (0.06 ml, 0.0007 mol).
  • the reaction mixture was stined at 80°C for 15 minutes.
  • the mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (4:1 to ethyl acetate), then freeze-dried.
  • Synthetic method la was used to prepare the hydrazone intermediate.
  • the method described in Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.5 g, 0.0021 mol), DMSO (1ml), malononitrile (0.14 g, 0.0021 mol) and morpholine (0.18 ml, 0.0021 mol).
  • the reaction mixture was stined at 80°C for 15 minutes.
  • the mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (4:1 to ethyl acetate), then freeze-dried.
  • the starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3.
  • Synthetic method la, Scheme 1 was used to prepare the hydrazone intermediate.
  • the method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.256g, 0.000934mol) in DMSO(0.5ml). Malononitrile (0.062g, 0.000934mol) and mo ⁇ holine (0.08ml, 0.000934mol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (3:1 to 2:1).
  • the starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3.
  • Synthetic method la, Scheme 1 was used to prepare the hydrazone intermediate.
  • the method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone(0.350g, 0.00135mol) in DMSO(0.5ml). Malononitrile (0.089g, 0.00135mol) and mo ⁇ holine (0.12ml, 0.00135mol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane ethyl acetate (5:1 to 1:1).
  • the starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3.
  • Synthetic method la, Scheme 1 was used to prepare the hydrazone intermediate.
  • the method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.344g, 0.0013mol) in DMSO (1ml). Malononitrile (0.085g, 0.0013mol) and mo ⁇ holine (0.11ml, 0.0013mol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using hexane : ethyl acetate 4:1 as eluant.
  • the starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3.
  • Synthetic method la, Scheme 1 was used to prepare the hydrazone intermediate.
  • the method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.47 g, 0.0018 mol) in DMSO (0.5ml). Malononitrile (0.119 g, 0.0018 mol) and mo ⁇ holine (0.16ml, 0.0018mol) was added and the reaction mixture stined at 80°C for 15 minutes.
  • the starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3.
  • Synthetic method la, Scheme 1 was used to prepare the hydrazone intermediate.
  • the method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.291g, 0.001 lmol) in DMSO (1ml). Malononitrile (0.070g, 0.001 lmol) and mo ⁇ holine (0.09ml, 0.001 lmol) was added and the reaction mixture stirred at 80°C for 15 minutes.
  • the starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3.
  • Synthetic method la, Scheme 1 was used to prepare the hydrazone intermediate.
  • the method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.291g, 0.001 lmol) in DMSO (1ml). Malononitrile (0.084g, 0.0013mol) and mo ⁇ holine (0.11ml, 0.0013mol) was added and the reaction mixture stirred at 80°C for 15 minutes.
  • the starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3.
  • Synthetic method la, Scheme 1 was used to prepare the hydrazone intermediate.
  • the method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.39g, 0.0014mol) in DMSO (1ml). Malononitrile (0.094g, 0.0014mol) and mo ⁇ holine (0.12ml, 0.0014mol) was added and the reaction mixture stirred at 80°C for 15 minutes.
  • the starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3.
  • Synthetic method la, Scheme 1 was used to prepare the hydrazone intermediate.
  • the method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.197g, O.OOO ⁇ lmol) in DMSO (0.5ml).
  • Malononitrile (0.040g, 0.00061mol) and mo ⁇ holine (0.05ml, O.OOO ⁇ lmol) was added and the reaction mixture stirred at 80°C for 15 minutes.
  • the starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3.
  • Synthetic method la, Scheme 1 was used to prepare the hydrazone intermediate.
  • the method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.320g, 0.0012mol) in DMSO (1ml). Malononitrile (0.080g, 0.0012mol) and mo ⁇ holine (0.11ml, 0.0012mol) was added and the reaction mixture stined at 80°C for 15 minutes.
  • the starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3.
  • Synthetic method la, Scheme 1 was used to prepare the hydrazone intermediate.
  • the method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.190g, 0.00076mol) in DMSO (1ml). Malononitrile (0.050g, 0.00076mol) and mo ⁇ holine (0.07ml, 0.00076mol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane ethyl acetate (5:1 to 2:1) as eluant.
  • the starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3.
  • Synthetic method la, Scheme 1 was used to prepare the hydrazone intermediate.
  • the method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.381g, 0.0014mol) in DMSO (1ml). Malononitrile (0.094g, 0.0014mol) and mo ⁇ holine (0.12ml, 0.0014mol) was added and the reaction mixture was stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using hexane : ethyl acetate (2:1) as eluant.
  • the starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3.
  • Synthetic method la, Scheme 1 was used to prepare the hydrazone intermediate.
  • the method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.253g, O.OOlOmol) in DMSO (0.5ml). Malononitrile (0.068g, O.OOlOmol) and mo ⁇ holine (0.09ml, O.OOlOmol) was added and the reaction mixture stined at 80°C for 15 minutes.
  • the starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3.
  • Synthetic method la, Scheme 1 was used to prepare the hydrazone intermediate.
  • the method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.138g, 0.00050mol) in DMSO (0.5ml). Malononitrile (0.033g, 0.00050mol) and mo ⁇ holine (0.044ml, 0.00050mol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using hexane : ethyl acetate (1:1) as eluant.
  • the starting material 2,2-dimethyl-tetraliydro-pyran-4-one (52) was prepared according to method 2, Scheme 3.
  • Synthetic method la, Scheme 1 was used to prepare the hydrazone intermediate.
  • the method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.349g, 0.0012mol) in DMSO (1ml). Malononitrile (0.077g, 0.0012mol) and mo ⁇ holine (0.101ml, 0.0012mol) was added and the reaction mixture stined at 80°C for 15 minutes.
  • the starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3.
  • Synthetic method la, Scheme 1 was used to prepare the hydrazone intermediate.
  • the method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.489g, 0.0017mol) in DMSO (1ml). Malononitrile (0.109g, 0.0017mol) and mo ⁇ holine (0.144ml, 0.0017mol) was added and the reaction mixture stined at 80°C for 15 minutes.
  • the starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3.
  • Synthetic method la, Scheme 1 was used to prepare the hydrazone intermediate.
  • the method according to Scheme 4 was undertaken to synthesize the pyridazine using the the hydrazone (0.304g, O.OOlOmol) in DMSO (1ml). Malonomtrile (0.069g, O.OOlOmol) and mo ⁇ holine (0.09ml, O.OOlOmol) was added and the reaction mixture stined at 80°C for 15 minutes.
  • the starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3.
  • Synthetic method la, Scheme 1 was used to prepare the hydrazone intermediate.
  • the method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.290g, 0.00087mol) in DMSO (1ml). Malononitrile (0.057g, 0.00087mol) and mo ⁇ holine (0.08ml, 0.00087mol) was added and the reaction mixture stined at 80°C for 15 minutes.
  • the starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3.
  • Synthetic method la, Scheme 1 was used to prepare the hydrazone intermediate.
  • the method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.220 g, 0.00071 mol) in DMSO (1 ml). Malononitrile (0.047 g, 0.00071 mol) and mo ⁇ holine (0.06 ml, 0.00071 mol) was added and the reaction mixture stined at 80°C for 15 minutes.
  • the starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3.
  • Synthetic method la, Scheme 1 was used to prepare the hydrazone intermediate.
  • the method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.300g, 0.00108mol) in DMSO (1ml). Malononitrile (0.072g, 0.00108mol) and mo ⁇ holine (0.094ml, 0.00108mol) was added and the reaction mixture stined at 80°C for 15 minutes.
  • the starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3.
  • Synthetic method la, Scheme 1 was used to prepare the hydrazone intermediate.
  • the method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.240g, 0.00077mol) in DMSO (1ml). Malononitrile (0.05 lg, 0.00077mol) and mo ⁇ holine (0.07ml, 0.00077mol) was added and the reaction mixture stined at 80°C for 15 minutes.
  • the starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3.
  • Synthetic method la, Scheme 1 was used to prepare the hydrazone intermediate.
  • the starting material 2,2-dimethyl-tetraliydro-pyran-4-one (52) was prepared according to method 2, Scheme 3.
  • Synthetic method la, Scheme 1 was used to prepare the hydrazone intermediate.
  • Example 78 The starting material 2,2-dimethyl-t
  • the starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3.
  • Synthetic method la, Scheme 1 was used to prepare the hydrazone intermediate.
  • the method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.201g, 0.00067mol) in DMSO (0.5ml). Malononitrile (0.045g, 0.00067mol) and mo ⁇ holine (0.02ml, 0.00067mol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by prep hplc and then freeze-dried to give the TFA salt ofthe pyridazine as a brown oil.
  • the starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3.
  • Synthetic method la, Scheme 1 was used to prepare the hydrazone intermediate.
  • the starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3.
  • Synthetic method la, Scheme 1 was used to prepare the hydrazone intermediate.
  • the starting material 2,2-dimethyl-tetrahydro- ⁇ yran-4-one (52) was prepared according to method 2, Scheme 3.
  • Synthetic method la, Scheme 1 was used to prepare the hydrazone intermediate.
  • the starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3.
  • a solution of 2,2-dimethyl-tetrahydro-pyran-4-one (3.00g, 23.4 mmol) and ethyl formate (3.0 ml, 37.2 mmol) in dry THF (30 mL) was placed under nitrogen.
  • K l BuO (2.63 g, 23.4 mmol) in small portions during 10 min. A yellow precipitate was formed almost immediately.
  • the mixture was stined at room temperature for 3 hours.
  • the solvent was then evaporated and the crude product 88 was dried in vacuum.
  • the crude material was dissolved in EtOH (18 mL) and divided into 6 portions and stored in freezer until used.
  • a solution ofthe diazoniumsalt of 2-trifluoromethyl-aniline was prepared by the gradual addition of a solution of sodium nitrite (273 mg, 3.95 mmol) in water (2 mL) to a solution of 2-trifluoromethyl aniline (637 mg, 3.95 mol) in 1.00 M HCl (7.90 mL, 7.90 mmol) at -5°C.
  • the cold ethanol solution (3 mL) ofthe the potassium salt of 5- (hydroxymethylene)-2,2-dimethyltetrahydro-4H-pyran-4-one 88 (3.90 mmol) was added dropwise and the resulting mixture was stined vigorously at -5° C for 1 h.
  • the intermediate 6,6-dimethyldihydro-2H-pyran-3,4-dione 3-[(4- ethylphenyl)hydrazone] was synthesized according to the modified method lc described in example 82, Scheme 5.
  • the hydrazone (0.526 g, 2.02 mmol) was dissolved in DMSO (2 mL) and malononitrile (170 mg, 2.57 mmol) and piperidine (255 ⁇ L, 2.58 mmol) was added.
  • the resulting mixture was heated at 80°C for 30 min.
  • the reaction mixture was diluted with CHC1 3 , washed with water, dried (MgSO 4 ) and evaporated.
  • the intermediate 6,6-dimethyldihydro-2H-pyran-3,4-dione [3-(3- carboxymethylphenyl)-hydrazone] was synthesized according to the modified method lc described in example 82, Scheme 5.
  • the hydrazone (0.461 g, 1.59 mmol) was dissolved in DMSO (2 mL) and malononitrile (126 mg, 1.91 mmol) and mo ⁇ holine (170 ⁇ L, 1.95 mmol) was added.
  • the reaction mixture was stined at room temperature for 1 h.
  • the reaction mixture was diluted with EtOAc and washed with water, then extracted with 1 M HCl (aq.) x 3.
  • the intermediate 6,6-dimethyldihydro-2H-pyran-3,4-dione 3- ⁇ [2-(4- mo ⁇ holinyl)phenyl] -hydrazone ⁇ was synthesized according to the modified method lc described in example 82, Scheme 5, with the addition of an extra equivalent of acid when forming the diazonium salt.
  • the hydrazone (0.428 g, 1.35 mmol) was dissolved in DMSO (10 mL) and malononitrile (187 mg, 2.83 mmol) and piperazinomethyl polystyrene resin (1.30g, 1.08 mmol/g, 1 eq.) was added.
  • the intermediate 6,6-dimethyldihydro-2H-pyran-3,4-dione 3-[(2- ethoxyphenyl)hydrazone] was synthesized according to the modified method lc described in example 82, Scheme 5.
  • the hydrazone (0.491 g, 1.78 mmol) was dissolved in DMSO (10 mL) and malononitrile (289mg, 4.37 mmol) and piperazinomethyl polystyrene resin (1.67 g, 1.08 mmol/g, 1 eq.) was added.
  • the reaction mixture was heated at 80°C for 12h.
  • the reaction mixture was diluted with CH 2 C1 2 and the resin was filtered and washed with CH 2 C1 .
  • Divinyl ketone (8.0 g, 0.083 mol) was added dropwise to a mixture of HgSO 4 (0.616 g, 0.0027 mol), H 2 SO 4 (0.616 ml) and water (24 ml) at 85°C with stirring. Then HgSO 4 (0.152 g, 0.00052 mol) and H 2 SO 4 (0.152 ml) in water (6 ml) were added over a period of one hour. In total the reaction mixture was heated for five hours at 85 to 100°C. This reaction mixture was then steam distilled. The product was then extracted with DCM, washed with water, dried (MgSO 4 ) and the solvent removed under reduced pressure to give a yellow liquid.
  • novel compounds according to the present invention may be administered orally, intranasally, transdermally, subcutaneously, parenterally, intramusculary, as well as intravenously. Oral administration is the prefened route.
  • Solid or fluid dosage forms can be prepared for oral administration.
  • Solid compositions such as compressed tablets, are prepared by mixing the compounds ofthe invention with conventional ingredients such as talc, magnesium stearate, dicalcium phosphate, magnesium aluminum silicate, calcium sulfate, starch, lactose, acacia, methyl cellulose, or functionally similar pharmaceutical diluents and carriers.
  • Capsules are prepared by mixing the compounds of this invention with an inert pharmaceutical diluent and placing the mixture into an appropriately sized hard gelatin capsule.
  • Soft gelatin capsules are prepared by machine encapsulation of a sluny ofthe compounds of this invention with an acceptable inert oil such as vegetable oil or light liquid petrolatum.
  • Syrups are prepared by dissolving compounds ofthe invention in an aqueous vehicle and adding sugar, aromatic flavoring agents and preservatives.
  • Elixirs are prepared using a hydroalcoholic vehicle such as ethanol, suitable sweeteners such as sugar or saccharin and an aromatic flavoring agent.
  • Suspensions are prepared with an aqueous vehicle and a suspending agent such as acacia, tragacanth, or methyl cellulose.
  • Parenteral solutions are prepared by dissolving the compounds ofthe invention in aqueous vehicle and filter sterilizing the solution before placing in a suitable sealable vial or ampule.
  • Parenteral suspensions are prepared in substantially the same way except a sterile suspension vehicle is used and the compounds ofthe present invention are sterilized with ethylene oxide or suitable gas before it is suspended in the vehicle.
  • a further aspect ofthe present invention is a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula I, la, la', and lb (e.g., a compound specified in the following Examples) above respectively together with a pharmacologically and pharmaceutically acceptable carrier.
  • a pharmacologically and pharmaceutically acceptable carrier e.g., a compound specified in the following Examples
  • pharmaceutically inert carriers which may be solid or liquid.
  • Solid form preparations include but is not limited to powders, tablets, dispersible granules, capsules etc. The skilled person within the formulation field will readily know which carrier to use for the specific circumstance when formulating a composition in accordance with the present invention.
  • salts ofthe compounds of formula I above may be formed from organic and inorganic acids.
  • examples of such salts are hydrochloride salts, tosylate salts, citrate salts, maleate salts, acetate salts, hydrobromide salts, malate salts, stearate salts, aluminium salts, lithium salts, calcium salts, and magnesium salts among others. This list should however not in any way be regarded as exhaustive.
  • the hydrochloride salts are the prefened salts ofthe invention.
  • Human PTP1B (amino acid residues 1-298, cloned from a human placental library), without the GST tag and thrombin cleavage site, was inserted into a pMB replicon and transformed into E. coli BL21(DE3), a strain containing a chromosomal copy ofthe gene for T7 RNA polymerase under control of a lacUN5 promoter.
  • PTP IB protein kinase
  • lysis buffer comprising 50mM Tris-HCl pH 7.5, 10% glycerol, lmM EDTA, 3mM DTT, 3mM MgCl 2 , and 0.2mg/ml lysozyme with lmg/ml D ⁇ Ase I.
  • the soluble protein was purified by ion exchange, hydrophobic interaction and gel exclusion chromatography for use in assays to identify PTP IB inhibitors.
  • the plasmid pGEX2K- SHP2 which encoded the catalytic domain of human SHP-2 (residues 252-529) was used to transform E.
  • coli cells After induction of protein expression, cells were lysed in PBS containing 1% Triton X100 and lysozyme (2mg/ml). Recombinant protein was purified by glutathione sepharose 4B chromatography followed by Superdex 200 size exclusion chromatography. Recombinant proteins were stored at -70°C until used. Recombinant T cell PTP (TCPTP) and LAR were purchased from New England Biolabs.
  • TCP T cell PTP
  • LAR were purchased from New England Biolabs.
  • Human PTP IB activity was measured using p-nitrophenol phosphate (pNPP) as substrate in a 96- well microtiter plate format.
  • pNPP p-nitrophenol phosphate
  • Standard assays were conducted at room temperature in a total volume of 0.2 ml that contains Hepes buffer (50 mM, pH 7.2), NaCl (50 mM), EDTA (1 mM), DTT (1 mM), bovine serum albumin (1 mg/ml), pNPP (1.25mM) and PTP1B (500ng/ml, 13.5nM).
  • a master plate was set up for each compound in which a stock solution of compound in DMSO (19mM or ImM) was diluted 1 to 10 with assay buffer in column 1 (giving a ImM or 100 ⁇ M concentration). Substances were subsequently diluted serially by two thirds in all columns across the plate.
  • the activity ofthe other PTPs was determined in a similar fashion except that the concentration of pNPP was varied according to the Km values for individual enzymes (0.6mM for TCPTP and 6.25mM for each of SHP-2 and LAR) and the buffer used for TCPTP was 25mM Tris-HCl pH 7.2.
  • L6 myocytes were cultured in ⁇ -MEM with 10% foetal bovine serum and antibiotics. Cells were differentiated into myotubes in 24-well plates by culturing for 10 days in medium containing 2% serum. The medium was refreshed on alternate days and 0.24mg/ml cytidine was included from day 7 to stop any remaining cycling cells. Cells were starved of serum overnight prior to use. Cells were pretreated with compound at approximately five times the IC 50 for inhibition of PTP IB for 30 minutes, prior to being stimulated with insulin (25nM) for five minutes.
  • Cells were lysed in buffer comprising 25mM Tris-HCl pH 7.4, 150mM NaCl, ImM EDTA, ImM EGTA, 1% Nonidet-40, 0.25% sodium deoxycholate, ImM sodium orthovanadate, lOmM ⁇ -glycerophosphate, 5mM sodium pyrophosphate and protease inhibitors. Cleared lysates were stored at - 70°C until used.
  • Insulin receptor phosphorylation was assessed using a lanthanide-based fluorescent assay (DELFIA).
  • DELFIA lanthanide-based fluorescent assay
  • An anti-insulin receptor antibody was captured on the wells of a 96-well plate using an anti-rabbit IgG antibody. After incubation with lysates containing between 100-250 ⁇ g protein which was consistent for all wells in a single experiment, phosphate on the receptor was detected with a biotinylated anti- phosphotyrosine antibody (PY99B from Santa Cruz) and europium-labelled streptavidin.
  • PY99B biotinylated anti- phosphotyrosine antibody
  • IC 50 values were determined for all compounds against each of four PTPs (PTP1B, SHP-2, LAR and TCPTP). Compounds were active across a wide range of concentrations from 100 nM to 50 ⁇ M.

Abstract

The present invention is directed to novel pyridazine compounds of the formula (I) as well as pharmaceutically and pharmaceutically acceptable salts, and hydrates thereof; to a process for their preparation, their use and pharmaceutical compositions comprising said novel compounds. These novel compounds are useful in therapy, particularly for the treatment of type 2 diabetes mellitus.

Description

Novel pyridazine compounds for the treatment of diabetes
TECHNICAL FIELD The present invention is directed to novel compounds, to a process for their preparation, their use and pharmaceutical compositions comprising said novel compounds. These novel compounds are useful in therapy, particularly for the treatment of type 2 diabetes.
BACKGROUND ART Phosphorylation on serine, threonine and tyrosine amino acid residues in downstream proteins forms the major output from growth factor and cytokine receptors, from which a cellular response is built. A large number of growth factor and cytokine- regulated protein tyrosine kinases (PTKs) have been identified which can be integral parts of receptor proteins or cytosolic molecules (Al-Obeidi, FA, Wu, JJ & Lam, KS, Biopolym. Pept. Sci. Sect. 47, 197-223). These serve to phosphorylate proteins on tyrosine residues within specific primary amino acid sequences which, when phosphorylated, act as docking points for proteins which contain SH2 domains. It is the docking of proteins to phosphorylated tyrosine residues which contributes to the activation of such proteins and the establishment of a signal transduction cascade.
The overall output from signal transduction cascades is derived from the balance between phosphorylation and dephosphorylation of proteins. Phosphotyrosines are returned to their free acid form by the action of protein tyrosine phosphatases (PTPs) (Zhang, ZY (1998) Crit. Rev. Biochem. Mol. Biol., 33, 1-52). Whilst a large number of PTKs has been identified (Hunter, T (1994) Se . Cell Biol. 5, 367-376), the number of PTPs identified to date is decidedly smaller (van Huijsduijnen, RH (1998) Gene 225, 1- 8). Despite the smaller number of enzymes in the PTP family available for investigation, a detailed understanding ofthe roles they play in signal transduction and disease has not been forthcoming. This is due in part to the lack of small molecule inhibitor molecules which are specific for members ofthe PTP family and which are permeable to the cell membrane and can thus be used in cell-based experiments. Furthermore, whilst experiments in transgenic animals can be and have been performed in which individual PTPs can be ablated, the effects ofthe loss of function of a specific enzyme may be masked by compensation by other members ofthe PTP family. Thus, the availability of small molecule inhibitors of PTPs would be very useful to the study of this important family of enzymes.
A role for the PTP family of proteins in ontogeny and disease is now becoming clearer (Li, L & Dixon, JE (2000) Sem. Immunol. 12, 75-84). Thus, experiments with gene knockouts in transgenic animals has revealed that the motheaten phenotype of mice in which cells ofthe haematopoietic lineage undergo hyper-proliferation is due to the loss of normal SHPTP1 function (Schultz, LD, Schweitzer, PA, Rajan, TV, Yi, T & Hile, IN (1993) Cell 73, 1445-1454). Loss of function in the receptor-like subfamily of PTPs leads to conditions such as heightened and reduced sensitivity to insulin (Ren, J- M, Li, P-M, Zhang, W-R, Sweet, LJ, Cline, G, Shulman, Gl, Livingston, IN &
Goldstein, BJ (1998) Diabetes 47, 493-497), stunted growth and neurological disruption (Elchelby. M, Wagner, J, Kennedy, TE, Lanctot, C, Michaliszyn, E, Itie, A, Drouin, J & Tremblay, ML (1999) Nature Genet. 21, 330-333) and blockages in T cell maturation (Kishihara, K, Penninger, J, Wallaca, VA, Kundig, TM, Kawai, K, Wakeham, A, Timms, E, Pfeffer K, Ohashi, PS & Thomas PL (1993) Cell 74, 143-156).
The recent descriptions of mice in which the PTP PTP IB had been disrupted revealed that loss of function of this enzyme leads to enhanced insulin sensitivity and resistance to the development of obesity, thus revealing a therapeutic need for the development of specific PTP inhibitors (Elchelby, M, Payette, P, Michaliszyn, E, Cromlish, W, Collins, S, Loy, AL, Normandin, D, Cheng, A, Himms.Hagen, J, Chan, CC, Ramachandran, C, Gresser, MJ, Tremblay, ML & Kennedy, BP (1999) Science 283, 1544-1548; Klaman, LD, Boss, O, Peroni, OD, Kim, JK, Martino, JL, Zablotny, JM, Moghal, N, Lubkin, M, Kim, Y-B, Sharpe, AH, Stricker-Krongrad, A, Shulman, Gl, Neel, BG & Kahn, BB (2000) Mol. Cell. Biol. 20, 5479-5489). The mechanism of insulin action depends critically upon the phosphorylation of tyrosine residues in several proteins in the insulin-signaling cascade. PTPs that dephosphorylate these proteins are important negative regulators of insulin action. Therefore, the use of specific PTP inhibitors may therapeutically enhance insulin action.
The anabolic effects of insulin are triggered through the activation of a variety of signal transduction cascades which lie downstream ofthe insulin receptor (Gustafson, T.A., Moodie, SA & Lavan, BE (1999) Rev. Physiol. Biochem. Pharmacol. 137, 71- 190). The varieties of signals that are activated by insulin are thought to contribute to the range of effects that insulin controls. However, each pathway is activated by a common series of biochemical reactions proximal to the insulin receptor. Thus, the insulin receptor undergoes autophosphorylation on tyrosine residues when activated by insulin, and also phosphorylates other proteins, in particular, the insulin receptor substrate proteins (IRSs). It has now become widely accepted that the resistance to insulin that is a feature of type 2 diabetes results in part from dysfunctions in signal transduction activated by the insulin receptor, in particular in steps early in the signaling cascades which are common to different pathways (Virkamaki, A, Ueki, K & Kahn, R C (1999) J. Clin. Invest. 103, 931-943; Kellerer, M, Lammers, R & Haring, H-U (1999) Exp. Clin. Endocrinol. Diabetes 107, 97-106). The signals which emanate from the insulin receptor are switched off by the returning ofthe insulin receptor and other components ofthe signal transduction cascades to their basal, non-active states. For the insulin receptor and the IRS proteins, this is achieved by dephosphorylation of phosphotyrosine residues. It is now becoming clear that different PTPs may regulate the insulin receptor in different tissues, but the number of candidate enzymes which do this is small (Walchi, S., Curchod, M-L.,
Pescini Gobert, R., Arkinstall, S. & Hooft van Huijsduijnen, R. (2000) J. Biol. Chem. 275, 9792-9796). Thus, protein tyrosine phosphatase IB (PTP1B) appears to be the major negative regulator ofthe insulin receptor in muscle and liver tissues (see for example Elechelby, M, Payette, P, Michalszyn, E, Cromlish, W, Collins, S, Loy, AL, Normandin, D, Cheng, A, Himms-Hagen, J, Chan, C-C, Ramachandran, C, Gresser, MJ, Tremblay, M & Kennedy, B P (1999) Science, 283, 1544-1548; Goldstein, BJ, Bittner- Kowalczyk, A, White, M F & Harbeck, M (2000) J. Biol. Chem. 275, 4283-4289). By contrast, PTP alpha may play a more dominant role in regulating the insulin receptor in adipose tissue (Calera, MR, Vallega, G & Pilch, PF (2000) J. Biol. Chem. 275 6308- 6312).
The development of type 2 diabetes is characterized by a protracted period of insulin resistance. In human subjects who are obese and insulin resistant, PTP protein concentrations are increased, which has led to the idea that elevations in the proteins contributes to the cause ofthe diabetic state (Ahmad, F, Azevedo, JL, Cortright, R, Dohm, G L & Goldstein, BJ (1997) J. Clin. Invest. 100 449-458). The two most significantly elevated are PTP IB and LAR. Considering that loss of LAR activity is associated with insulin resistance and diabetes (Ren, J-M, Li, P-M, Zhang, W-R, Sweet, LJ, Cline, G, Shulman, Gl, Livingston, JN & Goldstein, BJ (1998) Diabetes 47 493- 497), these data support the concept that PTP IB is a major contributor to the insulin resistant state and that pharmacological inhibition of its activity may go some way towards pharmaceutically alleviating the condition. Indeed, the recent reports ofthe knockout mouse in which PTP IB has been ablated confirm that loss of PTP IB activity leads to enhancement ofthe metabolic effects of insulin (Elechelby, M, Payette, P, Michalszyn, E, Cromlish, W, Collins, S, Loy, AL, Normandin, D, Cheng, A, Himms- Hagen, J, Chan, C-C, Ramachandran, C, Gresser, MJ, Tremblay, M & Kennedy, B P (1999) Science, 283, 1544-1548; Klaman, LD, Ross, O, Peroni, OD, Kim, JK, Martino, JL, Zabolotny, JM, Moghal, N, Lubkin, M, Kim, Y-B, Sharpe, AH, Stricker-Krongrad, A, Shulman, Gl, Neel, BG & Kahn, BB (2000) Mol. Cell. Biol. 20 5479-5489).
Furthermore, inhibition of PTP IB with a specific small molecule has been reported to treat the symptoms of diabetes in the ob/ob mouse (Wrobel, J, Sredy, J, Moxham, C, Dietrich, A, Li, Z, Sawicki, DR, Seestaller, L, Wu. L, Katz, A, Sullivan, D, Tio, C & Zhang, Z-Y (1999) J. Med. Chem. 42 3199-3202). WO 96/40113 discloses heterocyclic nitrogen containing compounds, such as nitropyridine or nitrothiazole, capable of inhibiting protein tyrosine phosphatase activity. Such molecules are disclosed as being useful to modulate or regulate signal transduction by inhibiting protein tyrosine phosphatase activity and to treat various disease states including diabetes mellitus. WO 98/27065 discloses a class of compounds which are stated as being protein tyrosine phosphatase modulating compounds. These prior art compounds are however structurally distinct from the compounds claimed in the present patent application. WO 97/08934 discloses aryl acrylic acid compounds of a certain structure, which compounds are stated as having protein tyrosine protease modulating activity. Also these prior art compounds are however structurally distinct from the compounds claimed in the present patent application.
WO 99/58519 discloses certain phenyl oxo-acetic acid compounds. These compounds are stated as being useful in the treatment of metabolic disorders related to insulin resistance and hyperglycemia. Also these prior art compounds are however structurally distinct from the compounds claimed in the present patent application.
WO 99/58521 discloses the use of ll-aryl-benzo[b]naphtho[2,3-d]furan and 11- aryl-benzo[b]naphtho[2,3-d]thiophene compounds to inhibit protein tyrosine phosphatase activity. Such compounds are disclosed as being useful to modulate or regulate signal transduction by inhibiting protein tyrosine phosphatase activity and to treat various disease states including diabetes mellitus.
The compound 6H-Pyrano[3,4-c]pyridazine-4-carbonitrile, 3-amino-2,8- dihydro-6,6-dimethyl-2-phenyl and structurally related compounds have been disclosed by E.G. Paronikyan et al. in Khim.Geterotsikl. Soedin (1996), 10, pp. 1410-1412. However, E.G. Paronikyan et al. does not disclose or even suggest that these compounds may have therapeutic activity, and particularly not in the diabetes area, such as the area of type 2 diabetes.
The object ofthe present invention was to provide novel compounds for the specific inhibition of PTPs allowing the study og biological processes in which they are active. Furthermore, a second object ofthe current invention was to provide novel compounds having improved advantages over drugs currently used for the treatment of type 2 diabetes. It should be appreciated that the wording "improved advantages" is not necessarily defined as more potent compounds, but as compounds having improved advantages overall, including but not limited to also improved selectivity and less side- effects.
DISCLOSURE OF THE INVENTION The novel compounds according to the present invention are defined by the general formula I
Figure imgf000006_0001
wherein n is an integer of 1 or 2; R1 is
(i) phenyl or naphthyl, each optionally substituted with up to 5 substituents independently selected from the group consisting of:
(a) a straight or branched -ds alkyl;
(b) a C3-C6 cycloalkyl or a C6-C10 aryl; (c) -CO-O-(C1-C6 alkyl) wherein the alkyl group is straight or branched;
(d) a halogen selected from the group consisting of fluoro, chloro, bromo, and iodo;
(e) a straight or branched -C6 alkoxy; (f) nitro;
(g) CF3;
(h) -O-(Cι-C6 alkyl)-phenyl wherein the alkyl group is straight or branched; (i) a heteroaryl having 5 or 6 ring atoms, wherein 1 or 2 ofthe ring atoms are optionally O, N, or S, optionally substituted with one or more methyl or ethyl; (j) a heterocycloalkyl having 5 or 6 ring atoms, wherein 1 or 2 ofthe ring atoms are optionally O, N, or S; (k) -N(R )-CO-R , where R is hydrogen or a straight or branched Ci-Cβ alkyl, and R3 is a straight or branched Ci-C6 alkyl; and (1) -N(R4)(R5), where each of R4 and R5 independently is a straight or branched Ci-Cβ alkyl;
(ii) phenyl that is fused with a cyclohexyl group or naphthyl that is fused with a cyclohexyl group, wherein 1 or 2 ofthe carbon atoms are optionally substituted with O, N, or S, and wherein said cyclohexyl group is optionally substituted with 1 or 2 substituents independently selected from the group consisting of halogen, CF3, a straight or branched d-C6 alkyl, a C3-C6 cycloalkyl, and a C6-C10 aryl; or (iii)
Figure imgf000007_0001
wherein Y is O, S, or N-Rx wherein Rx is a straight or branched Cι-C6 alkyl;
X is
(i) an oxygen atom, a sulfur atom, or a methylene group;
Figure imgf000007_0002
, wherein R° is
(a) benzyl; (b) -CO-(C!-C6 alkyl) wherein the alkyl group is straight or branched;
(c) -CO-A, wherein A is phenyl, naphthyl, or a heteroaryl; said heteroaryl group having 5 to 10 ring atoms wherein at least one of said ring atoms is O, N, or S;
(d) -CO-O-Ry, wherein Ry is hydrogen or a straight or branched C Cό alkyl;
(e) -CO-O-(C1-C6 alkyl)-A wherein A is as defined above; or
(f) -SO2-A, wherein A is as defined above;
(iii)
\ CH— CO— O- -(C C6 alkyl)
/ , wherein the alkyl group is straight or branched; or
Figure imgf000008_0001
of Q1 and Q2 is independently phenyl, naphthyl, or a heteroaryl; said heteroaryl group having 5 to 10 ring atoms wherein at least one of said ring atoms is O, N, or S; each of Ra and Rb is independently hydrogen, CrC6 alkyl, or Ra and Rb together form a carbonyl group; and
Rc is hydrogen, a straight or branched Ci-Cβ alkyl, or carbonyl; with the proviso that the compound 6H-pyrano[3,4-c]pyridazine-4-carbonitrile, 3- amino-2,8-dihydro-6,6-dimethyl-2-phenyl is excluded.
A subset of compounds of formula I is defined by formula la below.
Figure imgf000008_0002
wherein X, R1, Ra, Rb and Rc are as defined in formula I above. A further subset of compounds of formula la is defined by formula la' below.
Figure imgf000009_0001
wherein X, R1, Ra, Rb and Rc are as defined in formula I above.
Another subset of compounds of formula I is defined by formula lb below.
Figure imgf000009_0002
wherein X, R1, Ra, Rb and Rc are as defined above.
Within the scope ofthe invention are also pharmaceutically and pharmacologically acceptable salts ofthe compounds of formula I, la, la', and lb, as well as hydrates thereof. Hydrochloride salts and tosylate salts of a compound ofthe present invention are also within the scope ofthe invention. Hydrochloride salts are preferred.
Some specific examples of R1 in accordance with the present invention are phenyl or naphthyl which is optionally and independently substituted by 1, 2, 3, 4 or 5 substituents selected from the group consisting of methyl; ethyl; straight, branched or cyclic propyl, butyl, pentyl, or hexyl; -CO-O-(CH2)n-CH3 wherein n is an integer 0, 1, 2, 3, 4, or 5; methoxy, ethoxy, propoxy, butyloxy, pentyloxy, or hexyloxy; -O-(CH )„- phenyl where n is an integer 0, 1, 2, 3, 4, 5, or 6. .
A more specific example of R1 is phenyl, optionally substituted with a straight or branched - alkyl (e.g., methyl, ethyl, n-propyl, or isopropyl), a straight or branched -Ce alkoxy (e.g., methoxy, ethoxy, or propoxy), nitro, CF3, halo (e.g., fluoro, chloro, or bromo; especially fluoro and chloro), cycloalkyl (e.g., cyclohexyl), heterocycloalkyl (e.g., heterocyclohexyl such as morpholino), aryl (e.g., phenyl), heteroaryl (e.g., furan),
-CO-O-(Ci-C4 alkyl) (e.g., -CO-O-CH3 or -CO-O-CH2CH3), or -N(R2)-CO-R3, where R2 is hydrogen, methyl, or ethyl, and R3 is a straight or branched - alkyl (e.g., -NH-CO-CH3 or -NH-CO-CH2CH3). Still further specific examples of R1 are shown below:
Figure imgf000010_0001
wherein each of nt and n2 independently is an integer
0, 1, 2, 3, 4, or 5;
, wherein n3 is an integer 0, 1, 2, 3, 4, or 5;
Figure imgf000010_0002
, wherein each of 114 and n5 independently is an integer
0, 1, 2, 3, 4, or 5;
Figure imgf000010_0003
Examples of X include an oxygen atom, a sulfur atom, a methylene group,
\
N— R6 / wherein R6 is benzyl, -CO-(CrC4 alkyl), -CO-O-(CrC4 alkyl), or
\
CH-CO— O— (CrC4 alkyl)
/ . A preferred example of X is oxygen. Another preferred example
\ .
N— R6 of X is /
Some examples of Ra, Rb, and Rc are hydrogen, methyl, or ethyl. Examples of a compound ofthe present invention include compounds of formula la' wherein R1 is phenyl, optionally substituted with a straight or branched Q- C4 alkyl, a straight or branched Ci-Cβ alkoxy, nitro, CF3, fluoro, chloro, bromo, cyclohexyl, heterocyclohexyl, phenyl, -CO-O-(Cι-C4 alkyl), or -N(R2)-CO-R3, where R2 is hydrogen, methyl, or ethyl, and R3 is a straight or branched C C4 alkyl; X is an
oxygen atom; a sulfur atom; a methylene group;
Figure imgf000011_0001
, wherein R6 is benzyl, -CO- (Cι-C4 alkyl), or
\
CH-CO— O — (CrC4 alkyl)
-CO-O-(Ci-C4 alkyl); or / ; and each of Ra, Rb, and Rc independently is hydrogen, methyl, or ethyl. Examples of a compound ofthe present invention also include compounds of formual la' wherein R1 is
Figure imgf000011_0002
wherein each of and n2 independently is an integer 0, 1, 2, 3, 4, or 5;
wherein n is an integer 0, 1, 2, 3, 4, or 5;
Figure imgf000011_0003
wherein each of nt and n5 independently is an integer 0, 1, 2, 3, 4, or 5;
Figure imgf000012_0001
\
N— R6 X is an oxygen atom; a sulfur atom; a methylene group; / , wherein R6 is benzyl, -
\
CH-CO— O — (CrC4 alkyl)
CO-(Cι-C4 alkyl), or -CO-O-(d-C4 alkyl); or / ; and each of Ra, Rb, and Rc independently is hydrogen, methyl, or ethyl. The compounds ofthe present invention are useful in therapy, particular for the treatment of type 2 diabetes mellitus.
Also within the scope ofthe invention is the use of a compound of formula I, for the manufacture of a medicament for the treatment of type 2 diabetes mellitus.
A further aspect ofthe invention is the use of a compound of formula la (e.g., a compound of formula la'), for the manufacture of a medicament for the treatment of type 2 diabetes mellitus.
Still a further aspect ofthe invention is the use of a compound of formula lb, for the manufacture of a medicament for the treatment of type 2 diabetes mellitus.
A further aspect ofthe invention is a method for the treatment of a patient suffering from type 2 diabetes mellitus, whereby an effective amount of a compound according to formula I above, is administered to a patient in need of such treatment.
A further aspect ofthe invention is a method for the treatment of a patient suffering from type 2 diabetes mellitus, whereby an effective amount of a compound according to formula la (e.g., a compound of formula la') above, is administered to a patient in need of such treatment.
A further aspect ofthe invention is a method for the treatment of a patient suffering from type 2 diabetes mellitus, whereby an effective amount of a compound according to formula lb above, is administered to a patient in need of such treatment.
Still a further aspect ofthe invention is a method for the treatment of a patient suffering from type 2 diabetes mellitus, whereby an effective amount of a compound described in each ofthe following Examples above, is administered to a patient in need of such treatment. DEFINITIONS As used herein, an alkyl is a straight or branched hydrocarbon chain containing the indicated number of carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, n- pentyl, 2-methylpentyl, and n-hexyl.
By cycloalkyl is meant a cyclic alkyl group containing the indicated number of carbon atoms. Some examples of cycloalkyl are cyclopropyl, cyclopentyl, cyclohexyl, adamantyl, and norbornyl. Heterocycloalkyl is a cycloalkyl group containing the indicated number of heteroatoms such as nitrogen, oxygen, or sulfur. Examples of heterocycloalkyl include piperidinyl, piperazinyl, tefrahydropyranyl, tefrahydrofuryl, and mo holinyl.
As used herein, aryl is an aromatic group containing the indicated number of ring atoms. Examples of an aryl group include phenyl, naphthyl, phenanthryl, and anthracyl. Heteroaryl is aryl containing the indicated number of heteroatoms such as nitrogen, oxygen, or sulfur. Some examples of heteroaryl are pyridyl, furanyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, and imidazolyl.
Each ofthe cycloalkyl, heterocycloalkyl, aryl, or heteroaryl groups described herein is optionally substituted with C1-4 alkyl, C3.6 cycloalkyl, 3-6 membered heterocycloalkyl, C6-ιo aryl, 6-10 membered heteroaryl, C -14 aralkyl, Cι-4 alkyl- heteroaryl with 6-10 ring atoms, Ci-4 alkoxy, hydroxy, hydroxyl-C1-4 alkyl, carboxyl, halo, halo-CM alkyl, amino, amino-C1.4 alkyl, nitro, cyano, Q-s alkylcarbonyloxy, Ci-5 alkyloxycarbonyl, C1-5 alkylcarbonyl, formyl, oxo, aminocarbonyl, Ci-5 alkylcarbonylamino, Ci-4 alkylsulfonylamino, aminosulfonyl, aminocarbonyloxy, or Cι-4 alkyloxycarbonylamino. Note that an amino group can be unsubstituted, mono-substituted, or di- substituted. It can be substituted with groups such as C1-4 alkyl, C3.6 cycloalkyl, 3-6 membered heterocycloalkyl, C6-10 aryl, or 6-10 membered heteroaryl. Halo refers to fluoro, chloro, bromo, or iodo. METHODS OF PREPARATION The compounds according to the present invention may be prepared by the following methods.
Compounds according to the present invention may be prepared by following the procedure reported by Norayan, Paronikian and Vartanyan, Khim. Geterotsikl.
Soedin. pp. 1464-6 (1983) (see Scheme I and Scheme II below).
Scheme I
Figure imgf000014_0001
Each of X, Ra, R , Rc, R , and n has the same definition as provided above.
Examples of a base that can be used in the reaction of Scheme I include sodium hydride and potassium t-butoxide.
Scheme II
Figure imgf000014_0002
Each of X, Ra, Rb, Rc, and R1 has the same definition as provided above.
Examples of a base that can be used in the reaction of Scheme I include sodium hydride and potassium t-butoxide.
The invention will now be described in more details by the following working examples, which however should not be construed as limiting the invention.
In the following examples, NMR spectra were recorded on a Narian 400 MHz spectrometer, a Bruker Advance DPX 400 or a Bruker DRX 500 and chemical shifts are given in ppm using tetramethylsilane as an internal standard at 25°C. HPLC analyses were performed using a Waters Xterra MS C18 column (100 x 4.6 mm, 5μ) eluting with a gradient of 5% ACΝ in 95% water to 95% ACΝ in 5% water (0.2% TFA buffer) over 3.5 min. then 95% ACN in 5% water (0.2% TFA buffer) for a further 2.5 min. at a flow rate of 3 ml/min on a Waters 600E system with monitoring at 254 nm and on a on a Hewlett-Packard 1100 instrument with a Nucleosil C-18 column (250x4.6 mm, 3μM) thermostated at 25 °C, eluting with water (0.1% TFA)/acetonitrile at a flow rate of 1 mL/min and gradients with a 5 minute isocratic run followed by a 10 min gradient, with UN detection at 254 nm.. Thin layer chromatography was carried out using pre-coated silica gel F-254 plates (thickness 0.25 mm). IR spectra were recorded on a Perkin Elmer Spectrum 1000 FTIR spectrometer. Electrospray MS spectra were obtained on a Micromass platform LCMS spectrometer. The Biotage Quad 3 system was used for parallel flash purification. Silica gel column chromatography was performed using YMC gel, silica 120 A S-50 μm.
The following methods (i.e., Methods la, lb, and 2) are employed to prepare compounds 2, 3, and 52, which are used as starting materials or intermediates in the following Examples.
Method la (Preparation of compound 2)
Scheme 1
Figure imgf000015_0001
To dry ethanol (60 ml) at -20°C was added sodium hydride (slowly) (3.92 g,
0.098 mol). Tetrahydro-4H-pyran-4-one (1) (9.79g, 0.098mol) was then added, followed by ethyl formate (11.9 ml, 0.147 mol) and the reaction mixture stirred at room temperature overnight. The solvent was then removed under reduced pressure and the residue dissolved in water (40 ml). The resultant solution was added to a solution ofthe diazonium salt of aniline, prepared by the gradual addition of a solution of sodium nitrite (6.76 g, 0.098 mol) in water (20 ml) to a solution of aniline (9.11 g, 0.098 mol) in 2 M HC1 (98 ml, 0.196 mol) at -5 to 0°C. The resultant mixture was stined at this temperature for one hour. The product (2) was then extracted with DCM, washed with water, dried (MgSO4) and the solvent removed under reduced pressure to give a brown solid (14.5 g, 73%), the product was then recrystallized from ethanol (8.17g, 41%); HPLC (92%, Rχ=3.74); 1H NMR (CDC13) δ 7.50-7.45 (m, Ph), 4.58 (s, CH2), 4.01 (t, CH2), 2.65 (t, CH2).
Method lb (Preparation of compound 3)
Scheme 2
Figure imgf000016_0001
To dry ethanol (2 ml) at -20°C was added sodium hydride (slowly) (0.1 g, 0.0025 mol). Tetrahydro-4H-pyran-4-one (0.25 g, 0.0025 mol) was then added, followed by ethyl formate (0.30 ml, 0.0038 mol) and the reaction mixture stirred at room temperature overnight. The solvent was then removed under reduced pressure and the residue dissolved in water (2 ml). The resultant solution was added to a solution of the diazonium salt of 2,6-dichloroaniline, prepared by the gradual addition of a solution of i-pentyl nitrite (0.32 g, 0.0028 mol) to a solution of 2,6-dichloroaniline (0.4 g, 0.0025 mol) in formic acid (4 ml) at 4°C. The resultant mixture was stirred at this temperature for one hour. The product was then extracted with DCM, washed with water, dried (MgSO4) and the solvent removed under reduced pressure. This gave the hydrazone as a brown solid (0.68g, 50%); HPLC (68%). Method lb was carried out according to procedures described in Barbero, M. et al. Synthesis, 1171-1175 (1998).
Method 2 (Preparation of compound 52)
Scheme 3
Figure imgf000016_0002
Diethylaminehydrochloride (50) (HOg, lmol), formaldehyde solution (78 ml, 1 mol), diacetone alcohol (125 ml, 1 mol), concentrated HC1 (4 ml; for pH = 1) and hydroquinone (2 g) were heated at 110°C for three hours. The reaction mixture was then distilled under reduced pressure. The product was a green-black solution that was collected at P = 14 rnmHg, Tva = 40-100°C. The product was then re-distilled to give the divinyl ketone (51) (9.67g, 9% yield). (P = 15 mmHg, Tvap = 70°C; lit: b.p. 60-61°C at 22 mmHg) NMR: 61.95 (s, 3H); 52.2 (s, 3H); 55.9-6.25 (m, 4H). Divinyl ketone (52) (11.17 g, 0.106 mol) was added dropwise to a mixture of
HgSO4 (0.86 g, 0.0029 mol), H2SO4 (0.86 ml) and water (34 ml) at 85°C with stirring. Then HgSO4 (0.251 g, 0.00073 mol) and H2SO (0.22 ml) in water (8.6 ml) were added over a period of one hour. In total the reaction mixture was heated for five hours at 85 to 100°C. This reaction mixture was then steam distilled. The product was then extracted with DCM, washed with water, dried (MgSO4) and the solvent removed under reduced pressure to give a yellow liquid. This crude product was then purified over silica (hexane:EtOAc 2:1), which furnished 2.38g (18%) of 2,2-dimethyl-tetrahydro- pyran-4-one (52) as a colourless oil. NMR: 51.27 (s, 6H); 52.36 (s, 2H); 52.39 (t, 2H); 54.01 (t, 2H). Method 2 was carried out according to the procedures described in J. Gen. Chem. USSR, 33(5), 1476-1480 (1963); Chem. Abs., 2161 (1949); andJ. Org. Chem., 35(3), 589 (1970).
EXAMPLES
Example 1
Preparation of 3-amino-2-phenyl-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4- carbonitrile (compound 4)
Scheme 4
Figure imgf000018_0001
To the hydrazone (2) (1 g, 0.0049 mol) in DMSO (3 ml) was added malononitrile (0.32 g, 0.0049 mol) and mo holine (0.43 ml, 0.0049 mol) and the reaction mixture stirred at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (5:1 to 1:1). This gave the pyridazine as a brown solid (1.12 g, 91%); HPLC (94%); MS (electrospray, [M+H]+) m/z 253.1; 1H NMR (DMSO) δ 7.69-7.25 (m, Ph), 6.14 (s, NH2), 5.05-5.01 (m, CH), 4.27-4.25 (m, CH2), 4.18 (s, CH2). The HCl salt of the pyridazine was then prepared and the compound freeze-dried to give 3-amino-2- ρhenyl-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile; HPLC (95%, Rτ=3.73); MS (electrospray, [M+H]*) m/z 253.1.
Example 2
Preparation of 3-amino-2-phenyl-2,6,7,8-tetrahydro-4-cinnoIinecarbonitrile (compound 5)
Figure imgf000018_0002
Synthetic method la was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.15 g, 0.0007 mol), DMSO (3ml), malononitrile (0.05g, 0.0007mol) and morpholine (0.064 ml, 0.0007 mol). The reaction mixture was stirred at 80°C for 15 minutes. The mixture was cooled and the solution poured into cold water (20 ml). The resultant precipitate was collected by filtration, recrystallized from ethanol, then freeze- dried. This gave 3-amino-2-phenyl-2,6,7,8-tetrahydro-4-cinnolinecarbonitrile as a brown solid (0.107g, 58%); HPLC (91%, Rτ=4.04); MS (electrospray, [M+H]+) m/z 251.2; 1H NMR (CDC13) δ 7.49-7.24 (m, Ph), 5.28 (t, CH), 4.43 (s, NH2), 2.38-2.42 (m, CH2), 2.22-2.25 (m, CH2), 1.77-1.80 (m, CH2).
Example 3
Preparation of 3-amino-2-phenyI-6,7-dihydro-2H-cycIopenta[c]pyridazine-4- carbonitrile (compound 6)
Figure imgf000019_0001
Synthetic method la was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.5g, 0.0027mol), DMSO (1ml), malononitrile (0.18g, 0.0027mol) and morpholine (0.23ml, 0.0027mol). The reaction mixture was stirred at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (5:1 to 1:1), then freeze-dried. This gave 3-amino-2-phenyl-6,7-dihydro-2H-cyclopenta[c]pyridazine-4- carbonitrile as a green solid (0.33g, 53%); HPLC (93%, Rr=3.79); MS (electrospray, [M+H]+) m/z 237.0.
Example 4 Preparation of 3-amino-7-benzyl-2-phenyl-2,6,7,8-tetrahydropyrido[3,4- c]pyridazine-4-carbonitri!e (compound 7)
Figure imgf000020_0001
Synthetic method la was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.2 g, 0.0007 mol), DMSO (1ml), malonomtrile (0.05 g, 0.0007 mol) and moφholine (0.06 ml, 0.0007 mol). The reaction mixture was stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (4:1 to ethyl acetate), then freeze-dried. This gave 3-amino-7-benzyl-2-phenyl-2,6,7,8- tetrahydropyrido[3,4-c]pyridazine-4-carbonitrile as an orange solid (0.07g, 30%); HPLC (82%,
Figure imgf000020_0002
MS (electrospray, [M+H]+) m/z 342.0.
Example 5
Preparation of 7-acetyl-3-amino-2-phenyI-2,6,7,8-tetrahydropyrido[3,4- c]pyridazine-4-carbonitrile (compound 8)
Figure imgf000020_0003
Synthetic method la was used to prepare the hydrazone intermediate. The method described in Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.5 g, 0.0021 mol), DMSO (1ml), malononitrile (0.14 g, 0.0021 mol) and morpholine (0.18 ml, 0.0021 mol). The reaction mixture was stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (4:1 to ethyl acetate), then freeze-dried. This gave 7-acetyl-3-amino-2-phenyl-2,6,7,8- tefrahydropyrido[3,4-c]pyridazine-4-carbonitrile as an orange solid (0.09g, 15%); HPLC (95%, Rτ=3.49); MS (electrospray, [M+H]+) m/z 294.1.
Example 6
Preparation of 3-amino-2-(2-fluorophenyl)-2,8-dihydro-6H-pyrano [3,4- c]pyridazine-4-carbonitrile hydrochloride (compound 9)
Figure imgf000021_0001
9
To the hydrazone (0.405 g, 0.0018 mol) in DMSO (1ml) was added malononitrile (0.12g, 0.0018mol) and morpholine (0.16 ml, 0.0018 mol) and the reaction mixture stirred at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using hexane : ethyl acetate (1:1). This gave the pyridazine as a green solid (0.156g, 32%); HPLC (97%); MS (electrospray, [M+H]+) m/z 271.1. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-(2-fluorophenyl)-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride; HPLC (97%, Rχ=3.81); MS (electrospray, [M+H]+) m/z 271.1.
Example 7
Preparation of 3-amino-2-(4-methylphenyl)-2,8-dihydro-6H-pyrano[3,4- c]pyridazine-4-carbonitrile hydrochloride (compound 10)
Figure imgf000021_0002
To the hydrazone (0.392 g, 0.0018 mol) in DMSO (1ml) was added malononitrile (0.12 g, 0.0018 mol) and moφholine (0.16 ml, 0.0018mol) and the reaction mixture stirred at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using hexane : ethyl acetate (2:1). This gave the pyridazine as a green solid (0.346g, 72%); HPLC (96%); MS (electrospray, [M+H]+) m/z 267.1. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-(4-methylphenyl)-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride; HPLC (96%, Rχ=3.96); MS (electrospray, [M+H]+) m/z 267.1.
Example 8
Preparation of 3-amino-2-[3-(trifluoromethyl)phenyl]-2,8-dihydro-6H-pyrano[3,4- c]pyridazine-4-carbonitrile hydrochloride (compound 11)
Figure imgf000022_0001
To the hydrazone (0.622 g, 0.0023 mol) in DMSO (1ml) was added malononitrile (0.15 g, 0.0023 mol) and moφholine (0.2 ml, 0.0023 mol) and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using hexane : ethyl acetate (2:1). This gave the pyridazine as a green/brown solid; HPLC (100%); MS
(electrospray, [M+H]+) m/z 321.1. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-[3-(trifluoromethyl)phenyl]-2,8-dihydro-
6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (0.747g, 91%); HPLC (100%,
Rτ=4.26); MS (electrospray, [M+H]+) m/z 321.1.
Example 9
Preparation of 3-amino-2-phenyI-2,8-dihydro-6H-thiopyrano[3,4-c]pyridazine-4- carbonitrile hydrochloride (compound 12)
Figure imgf000022_0002
To the hydrazone (0.1 g, 0.00045 mol) in DMSO (0.3 ml) was added malononitrile (0.03 g, 0.00045 mol) and moφholine (0.04 ml, 0.00045 mol) and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane ethyl acetate (4:1 to 1:1). This gave the pyridazine as a green solid; HPLC (82%); MS (electrospray, [M+H]+) m z 269.0. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-phenyl-2,8-dihydro-6H- thiopyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (0.086g, 63%); HPLC (82%, Rτ=3.75); MS (electrospray, [M+H]+) m/z 269.0.
Example 10
Preparation of 3-amino-2-(3-methoxyphenyl)-2,8-dihydro-6H-pyrano [3,4- c]pyridazine-4-carbonitrile hydrochloride (compound 13)
Figure imgf000023_0001
To the hydrazone (0.474 g, 0.002 mol) in DMSO (2ml) was added malononitrile
(0.132 g, 0.002 mol) and moφholine (0.18ml, 0.002mol) and the reaction mixture stirred at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (2:1 to 1:1). This gave the pyridazine as a green solid (0.15g, 27%); HPLC (93%); MS (electrospray, [M+H]+) m/z 283.1. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-(3-methoxyphenyl)-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride; HPLC (93%, Rr=2.37); MS (electrospray, [M+H]+) m/z 283.1.
Example 11
Preparation of 3-amino-2-(2,6-dimethylphenyl)-2,8-dihydro-6H-pyrano [3,4- c]pyridazine-4-carbonitrile hydrochloride (compound 14)
Figure imgf000024_0001
14
To the hydrazone (0.45 g, 0.0019 mol) in DMSO (2 ml) was added malononitrile (0.13 g, 0.0019 mol) and moφholine (0.17 ml, 0.0019 mol) and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using hexane : ethyl acetate (2:1). This gave the pyridazine as a green solid; HPLC (97%); MS (electrospray, [M+H]+) m/z 281.1. The HCl salt of the pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-(2,6-dimethylphenyl)-2,8-dihydro-6H- pyrano[3,4-c]ρyridazine-4-carbonitrile hydrochloride (0.563g, 94%); HPLC (97%, Rτ=3.42); MS (electrospray, [M+H]"1") m/z 281.1.
Example 12
Preparation of Ethyl 3-amino-4-cyano-2-phenyl-2,6,7,8-tetrahydro-7- cinnolinecarboxylate hydrochloride (compound 15)
Figure imgf000024_0002
To the hydrazone (0.2 g, 0.00073 mol) in DMSO (0.6 ml) was added malononitrile (0.048 g, 0.00073 mol) and moφholine (0.064 ml, 0.00073 mol) and the reaction mixture stirred at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (5 : 1 to 1 : 1). This gave the pyridazine as a brown solid; HPLC (92%); MS
(electrospray, [M+H]+) m/z 323.1. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give ethyl 3-amino-4-cyano-2-phenyl-2,6,7,8- tetrahydro-7-cinnolinecarboxylate hydrochloride (0.052g, 22%); HPLC (92%,
Rτ=3.86); MS (electrospray, [M+H]"1") m/z 323.1. Example 13
Preparation of 3-amino-2-(3,4-dichlorophenyl)-2,8-dihydro-6H-pyrano [3,4- c]pyridazine-4-carbonitrile hydrochloride (compound 16)
Figure imgf000025_0001
To the hydrazone (0.3 g, 0.0011 mol) in DMSO (2ml) was added malononitrile
(0.073 g, 0.0011 mol) and moφholine (0.1 ml, 0.0011 mol) and the reaction mixture stirred at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (4:1 to 2:1). This gave the pyridazine as a green solid; HPLC (94%); MS (electrospray,
[M+H]4} m/z 321.1. The HCl salt of the pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-(3,4-dichlorophenyl)-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (0.127g, 32%); HPLC (94%,
Rτ=3.47); MS (electrospray, [M+H]+) m/z 321.1.
Example 14
Preparation of 7-benzoyI-3-amino-2-phenyl-2,6,7,8-tetrahydropyrido [3,4- c]pyridazine-4-carbonitrile (compound 17)
Figure imgf000025_0002
17 To the hydrazone (0.37 g, 0.00121 mol) in DMSO (2 ml) was added malononitrile (0.08 g, 0.00121 mol) and moφholine (0.11 ml, 0.00121 mol) and the reaction mixture stirred at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (1 :1 to ethyl acetate). This gave the pyridazine as a green solid (0.1 lg, 26%); HPLC (91%); MS (electrospray, [M+H]+) m/z 356.4. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 7-benzoyl-3- amino-2-phenyl-2,6,7,8-tetrahydropyrido[3,4-c]pyridazine-4-carbonitrile; HPLC (91 %, Rτ=4.45); MS (electrospray, [M+H]4) m/z 356.4.
Example 15
Preparation of 3-amino-2-(4-methoxyphenyl)-2,8-dihydro-6H-pyrano [3,4- c]pyridazine-4-carbonitrile hydrochloride (compound 18)
Figure imgf000026_0001
To the hydrazone (0.131 g, 0.00055 mol) in DMSO (1 ml) was added malononitrile (0.037 g, 0.00055 mol) and moφholine (0.049 ml, 0.00055 mol) and the reaction mixture stirred at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane ethyl acetate (4:1 to ethyl acetate). This gave the pyridazine as a green solid (0.095 g, 61%); HPLC (98%); MS (electrospray, [M+H]4") m/z 283.3. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-(4- methoxyphenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride; HPLC (98%, Rχ-4.21); MS (electrospray, [M+H]+) m/z 283.3.
Example 16 Preparation of 3-amino-2-(4-isopropyIphenyl)-2,8-dihydro-6H-pyrano[3,4- c]pyridazine-4-carbonitrile hydrochloride (compund 19)
Figure imgf000026_0002
To the hydrazone (0.25 g, 0.001 mol) in DMSO (1 ml) was added malononitrile (0.067 g, 0.001 mol) and moφholine (0.089 ml, 0.001 mol) and the reaction mixture stirred at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using hexane : ethyl acetate (3:1). This gave the pyridazine as a green solid (0.103g, 35%); HPLC (94%); MS (electrospray, [M+H]4) m/z 295.3. The HCl salt ofthe pyridazine was then prepared and the compound freeze- dried to give 3-amino-2-(4-isopropylphenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine- 4-carbonitrile hydrochloride; HPLC (94%, Rχ=4.72); MS (electrospray, [M+H]4) m/z 295.3.
Example 17
Preparation of 3-amino-2-[4-(4-morpholinyl)phenyl]-2,8-dihydro-6H-pyrano[3,4- c]pyridazine-4-carbonitrile hydrochloride (compound 20)
Figure imgf000027_0001
To the hydrazone (0.1 g, 0.00035 mol) in DMSO (1 ml) was added malononitrile (0.023 g, 0.00035 mol) and moφholine (0.03 ml, 0.00035 mol) and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane ethyl acetate (2:1 to ethyl acetate). This gave the pyridazine as a brown solid (0.026g, 19%); HPLC (95%); MS (electrospray, [M+H]4) m/z 338.5. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-[4-(4- moφholinyl)phenyl]-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride; HPLC (95%, Rχ=4.32); MS (electrospray, [M+H]4) m/z 338.5.
Example 18
Preparation of 7-acetyl-3-amino-2-(2,6-dimethyIphenyl)-2,6,7,8- tetrahydropyrido[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 21)
Figure imgf000028_0001
21 To the hydrazone (0.33 g, 0.0012 mol) in DMSO (2ml) was added malononitrile
(0.08 g, 0.0012 mol) and moφholine (0.11 ml, 0.0012 mol) and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (1:1 to ethyl acetate). This gave the pyridazine as a brown solid (0.1 lg, 28%); HPLC (84%); MS (electrospray, [M+H]4") m/z 322.4. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 7-acetyl-3-amino-2-(2,6- dimethylphenyl)-2,6,7,8-tefrahydropyrido[3,4-c]pyridazine-4-carbonitrile hydrochloride; HPLC (84%, Rr=4.58); MS (electrospray, [M+H]4") m/z 322.4.
Example 19
Preparation of ethyl 3-(3-amino-4-cyano-6H-pyrano[3,4-c]pyridazin-2(8H)- yl)benzoate hydrochloride (compound 22)
Figure imgf000028_0002
To the hydrazone (0.125 g, 0.00045 mol) in DMSO (0.5 ml) was added malononitrile (0.03 g, 0.00045 mol) and moφholine (0.04 ml, 0.00045 mol) and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (4:1 to 1:1). This gave the pyridazine as a green solid (0.133g, 91%); HPLC (97%); MS (electrospray, [M+H]4) m/z 325.3. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give ethyl 3-(3-amino-4-cyano-6H- ρyrano[3,4-c] pyridazin-2(8H)-yl)benzoate hydrochloride; HPLC (97%, Rχ=4.68); MS (electrospray, [M+H]4") m/z 325.3.
Example 20
Preparation of ethyl 3-amino-4-cyano-2-phenyI-2,8-dihydropyrido[3,4- c]pyridazine-7(6H)-carboxylate hydrochloride (compound 23)
Figure imgf000029_0001
To the hydrazone (0.305 g, 0.0011 mol) in DMSO (2 ml) was added malononitrile (0.073 g, 0.0011 mol) and moφholine (0.09 ml, 0.0011 mol) and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (4:1 to 1:1). This gave the pyridazine as a green solid (0.14g, 40%); HPLC (94%); MS (electrospray, [M+H]4") m/z 324.4. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give ethyl 3-amino-4-cyano-2-phenyl-2,8- dihydropyrido[3,4-c]pyridazine-7(6H)-carboxylate hydrochloride; HPLC (94%, Rτ=4.52); MS (electrospray, [M+H]4") m/z 324.4.
Example 21
Preparation of 3-amino-2-(2-chloro-6-fluorophenyl)-2,8-dihydro-6H-pyrano[3,4- c]pyridazine-4-carbonitriIe hydrochloride (compound 24)
Figure imgf000029_0002
24
To the hydrazone (0.46 g, 0.0018 mol) in DMSO (0.5 ml) was added malononitrile (0.12 g, 0.0018 mol) and moφholine (0.16 ml, 0.0018 mol) and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (4:1 to 1:1). This gave the pyridazine as a green solid (0.224g, 41%); HPLC (98%); MS (electrospray, [M+H]4") m/z 305.2. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-(2-chloro-6- fluorophenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride; HPLC (98%, Rχ=4.45); MS (electrospray, [M+H]4) m/z 305.2.
Example 22
Preparation of 3-amino-2-(2-bromo-5-trifluoromethylphenyl)-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 25)
Figure imgf000030_0001
25
To the hydrazone (0.1 g, 0.00027 mol) in DMSO (0.5 ml) was added malononitrile (0.02 g, 0.00027 mol) and moφholine (0.024 ml, 0.00027 mol) and the reaction mixture stirred at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane ethyl acetate (2:1 to 1:1). This gave the pyridazine as a green solid; HPLC (95%); MS (electrospray, [M+H]4") m/z 401.2. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-(2-bromo-5-trifluoromethylphenyl)- 2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (0.084g, 70%); HPLC (95%, Rχ=4.31); MS (electrospray, [M+H]4) m/z 401.2.
Example 23
Preparation of 3-amino-2-(l ,3-benzothiazol-6-yl)-2,8-dihydro-6H-pyrano [3,4- c]pyridazine-4-carbonitriIe hydrochloride (compound 26)
Figure imgf000030_0002
To the hydrazone (0.072 g, 0.00028 mol) in DMSO (0.5 ml) was added malononitrile (0.018 g, 0.00028 mol) and moφholine (0.024 ml, 0.00028 mol) and the reaction mixture stirred at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (2:1 to ethyl acetate). This gave the pyridazine as a beige solid; HPLC (91%); MS (electrospray, [M+H]4") m/z 310.3. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-(l,3-benzothiazol-6-yl)-2,8- dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (0.06g, 63%); HPLC (91%, Rr=3.66); MS (electrospray, [M+H]4) m/z 310.3.
Example 24
Preparation of N-[4-(3-amino-4-cyano-6H-pyrano[3,4-c]pyridazin~2(8H)- yl)phenyl]-N-methylacetamide hydrochloride (compound 27)
Figure imgf000031_0001
27 To the hydrazone (0.37 g, 0.0014 mol) in DMSO (0.5 ml) was added malononitrile (0.089 g, 0.0014 mol) and moφholine (0.12 ml, 0.0014 mol) and the reaction mixture stirred at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (2:1 to ethyl acetate). This gave the pyridazine as a brown solid; HPLC (100%); MS (electrospray, [M+H]4") m/z 324.4. The HCl salt of the pyridazine was then prepared and the compound freeze-dried to give N-[4-(3-amino-4-cyano-6H- pyrano[3,4-c]pyridazin-2(8H)-yl)phenyl]-N-methylacetamide hydrochloride (0.097g, 20%); HPLC (100%, Rχ=3.58); MS (electrospray, [M+H]4) m/z 324.4. Example 25
Preparation of 3-amino-2-[4-(dimethylamino)phenyl]-2,8-dihydro-6H-pyrano[3,4- c]pyridazine-4-carbonitrile hydrochloride (compound 28)
Figure imgf000032_0001
To the hydrazone (0.26 g, 0.0011 mol) in DMSO (0.5 ml) was added malononitrile (0.07 g, 0.0011 mol) and moφholine (0.09 ml, 0.0011 mol) and the reaction mixture stirred at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (2:1 to 1:1). This gave the pyridazine as a green solid; HPLC (91%); MS (electrospray, [M+H]4") m/z 296.4. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-[4-(dimethylamino)phenyl]-2,8- dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (0.039g, 11%); HPLC (91%, Rχ=3.87); MS (electrospray, [M+H]4") m/z 296.4.
Example 26
Preparation of 3-amino-2-(4-chloro-2-methylphenyl)-2,8-dihydro-6H-pyrano [3,4- c]pyridazine-4-carbonitrile hydrochloride (compound 29)
Figure imgf000032_0002
29 To the hydrazone (0.061 g, 0.00024 mol) in DMSO (0.5ml) was added malononitrile (0.016 g, 0.00024 mol) and moφholine (0.021 ml, 0.00024 mol) and the reaction mixture stirred at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (2:1 to 1:1), then repeated over alumina. This gave 3-amino-2-(4-chloro-2- methylphenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride as a green solid (O.Olδg, 22%); HPLC (91%, Rχ=4.08); MS (electrospray, [M+H]4) m/z 301.3.
Example 27
Preparation of 3-amino-2-(2-chloro-6-methylphenyl)-2,8-dihydro-6H-pyrano[3,4- c]pyridazine-4-carbonitrile hydrochloride (compound 30)
Figure imgf000033_0001
30
To the hydrazone (0.87 g, 0.0034 mol) in DMSO (0.5 ml) was added malononitrile (0.23 g, 0.0034 mol) and moφholine (0.3 ml, 0.0034 mol) and the reaction mixture stirred at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using hexane : ethyl acetate (1:1), then repeated over alumina. This gave the pyridazine as a green solid (0.5g, 49%); HPLC (96%); MS (electrospray, [M+H]4") m/z 301.3. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-(2- chloro-6-methylphenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride; HPLC (96%, 3.12); MS (electrospray, [M+H]4) m/z 301.3.
Example 28 Preparation of 3-amino-2-(2-methoxyphenyl)-2,8-dihydro-6H-pyrano[3,4- c]pyridazine-4-carbonitrile hydrochloride (compound 31)
Figure imgf000033_0002
31
To the hydrazone (0.546 g, 0.0023 mol) in DMSO (0.5 ml) was added malononitrile (0.154 g, 0.0023 mol) and moφholine (0.2 ml, 0.0023 mol) and the reaction mixture stirred at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane ethyl acetate (2:1 to ethyl acetate). This gave the pyridazine as a pale green solid (0.265g, 41%); HPLC (99%); MS (electrospray, [M+H]4") m/z 283.3. The HCl salt of the pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-(2- methoxyphenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (0.084g, 70%); HPLC (99%, Rf=4.35); MS (electrospray, [M+H]4") m/z 283.3.
Example 29 Preparation of 3-amino-2-mesityI-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4- carbonitrile hydrochloride (compound 32)
Figure imgf000034_0001
32
To the hydrazone (1.82 g, 0.0074 mol) in DMSO (0.5 ml) was added malononitrile (0.49 g, 0.0074 mol) and moφholine (0.65 ml, 0.0074 mol) and the reaction mixture stirred at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane ethyl acetate (1:1 to ethyl acetate). This gave the pyridazine as a green solid (0.262g, 12%); HPLC (91%); MS (electrospray, [M+H]4) m z 295.3. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3-amino-2- mesityl-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride; HPLC (91%, MS (electrospray, [M+H]4) m/z 295.3. Example 30
Preparation of 3-amino-2-(2-isopropylphenyl)-2,8-dihydro-6H-pyrano[3,4- c]pyridazine-4-carbonitrile hydrochloride (compound 33)
Figure imgf000035_0001
To the hydrazone (4.6 g, 0.0187 mol) in DMSO (1 ml) was added malononitrile
(1.24 g, 0.0187 mol) and moφholine (1.63 ml, 0.0187 mol) and the reaction mixture stirred at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (1:1 to ethyl acetate). This gave the pyridazine as a brown solid; HPLC (93%); MS (electrospray, [M+H]4") m/z 295.3. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-(2-isopropylphenyl)-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (1.5g, 27%); HPLC (91%, Rτ=3.13); MS (electrospray, [M+H]4") m z 295.3.
Example 31
Preparation of 3-amino-2-(2,6-dichlorophenyl)-2,8-dihydro-6H-pyrano[3,4- c]pyridazine-4-carbonitrile hydrochloride (compound 34)
Figure imgf000035_0002
To the hydrazone (0.68 g, 0.0025 mol) in DMSO (0.5 ml) was added malonomtrile (0.16 g, 0.0025 mol) and moφholine (0.22 ml, 0.0025 mol) and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane ethyl acetate (2: 1 to 1 : 1), then repeated over alumina. This gave the pyridazine as a green solid (0.074g, 9%); HPLC (93%); MS (electrospray, [M+H]4) m/z 321.3. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3- amino-2-(2,6-dichlorophenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride; HPLC (93%, Rχ=2.96); MS (electrospray, [M+H]4") m/z 321.3.
Example 32
Preparation of 3~amino-2-(2-methyI~4-nitrophenyl)-2,8-dihydro-6H-pyrano[3,4- c]pyridazine-4-carbonitrile tosylate (compound 35)
Figure imgf000036_0001
To the hydrazone (0.9 g, 0.0034 mol) in DMSO (0.5 ml) was added malononitrile (0.23 g, 0.0034 mol) and moφholine (0.3 ml, 0.0034 mol) and the reaction mixture stirred at room temperature for 15 minutes. The product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (2:1 to 1:1), then repeated over alumina. This gave the pyridazine as a green solid (0.105g, 10%); HPLC (87%); MS (electrospray, [M+H]4) m/z 312.4. The tosylate salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-(2- methyl-4-m^ophenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carboriitrile tosylate; HPLC (77%, Rτ=2.96); MS (electrospray, [M+H]4") m/z 312.4.
Example 33 Preparation of benzyl 3-amino-4-cyano-2-phenyl-2,8-dihydropyrido[3,4- c]pyridazine-7(6H)-carboxylate hydrochloride (compound 36)
Figure imgf000036_0002
To the hydrazone (0.48 g, 0.0014 mol) in DMSO (0.5 ml) was added malononitrile (0.09 g, 0.0014 mol) and moφholine (0.12 ml, 0.0012 mol) and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (2:1 to ethyl acetate). This gave the pyridazine as a green solid; HPLC (94%); MS (electrospray, [M+H]4") m/z 386.5. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give benzyl 3-amino-4-cyano-2-phenyl-258- dihydropyrido[3,4-c]pyridazine-7(6H)-carboxylate hydrochloride (0.32g, 58%); HPLC (90%, Rτ=3.38); MS (electrospray, [M+H]4") m/z 386.5.
Example 34 Preparation of 3-amino-2-(2-methoxy-6-methylphenyl)-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 37)
Figure imgf000037_0001
37
To the hydrazone (0.44 g, 0.0018 mol) in DMSO (1 ml) was added malononitrile (0.117 g, 0.0018 mol) and moφholine (0.16 ml, 0.0018 mol) and the reaction mixture stirred at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (5:1 to 2:1). This gave the pyridazine as a green solid; HPLC (91%); MS (electrospray, [M+H]4") m/z 297.0. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-(2-methoxy-6-methylphenyι)-2,8- dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (0.29g, 55%); HPLC (88%, Rτ=2.98); MS (electrospray, [M+H]4") m/z 297.0.
Example 35
Preparation of 3-amino-2-(3,5-dimethoxyphenyl)-2,8-dihydro-6H-pyrano[3,4- c]pyridazine-4-carbonitrile hydrochloride (compound 38)
Figure imgf000038_0001
38
To the hydrazone (0.5 g, 0.0019 mol) in DMSO (0.5 ml) was added malononitrile (0.125 g, 0.0019 mol) and moφholine (0.17 ml, 0.0019 mol) and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane ethyl acetate (2:1 to ethyl acetate). This gave the pyridazine as a brown solid (0.154g, 26%); HPLC (93%); MS (electrospray, [M+H]4") m/z 313.2. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-(3,5- dimethoxyphenyl)-2, 8 -dihydro-6H-pyrano [3 ,4-c]pyridazine-4-carbonitrile hydrochloride; HPLC (95%, Rτ=3.08); MS (electrospray, [M+H]4) m/z 313.2.
Example 36
Preparation of 3-amino-2-(2-(l-methylpropyl)phenyl)-2,8-dihydro-6H-pyrano[3,4- c]pyridazine-4-carbonitrile hydrochloride (compound 39)
Figure imgf000038_0002
To the hydrazone (0.15 g, 0.00057 mol) in DMSO (1 ml) was added malononitrile (0.038 g, 0.00057 mol) and moφholine (0.05 ml, 0.00057 mol) and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane ethyl acetate (5:1 to 1:1). This gave the pyridazine as a green solid (0.086g, 49%); HPLC (89%); MS (electrospray, [M+H]4") m/z 309.2. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-(2-(l- methylpropyl)phenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride; HPLC (85%, Rχ=3.22); MS (electrospray, [M+H]4") m/z 309.2.
Example 37
Preparation of 3-amino~2-(2-chloro-5-methoxyphenyl)~2,8-dihydro-6H-pyrano [3,4- c]pyridazine-4-carbonitrile hydrochloride (compound 40)
Figure imgf000039_0001
To the hydrazone (0.413 g, 0.00154 mol) in DMSO (1 ml) was added malononitrile (0.102 g, 0.00154 mol) and moφholine (0.13 ml, 0.00154 mol) and the reaction mixture stirred at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane ethyl acetate (1:1 to ethyl acetate). This gave the pyridazine as a green solid (0.291g, 60%); HPLC (96%); MS (electrospray, [M+H]4) m/z 319.2. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-(2- chloro-5-methoxyphenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride; HPLC (93%, Rχ=2.96); MS (electrospray, [M+H]4") m/z 319.2.
Example 38
Preparation of 3-amino-2-(2-tert-butylphenyl)-2,8-dihydro-6H-pyrano[3,4- c]pyridazine-4-carbonitrile hydrochloride (compound 41)
Figure imgf000039_0002
To the hydrazone (1.09 g, 0.0042 mol) in DMSO (1 ml) was added malononitrile (0.28 g, 0.0042 mol) and moφholine (0.37 ml, 0.0042 mol) and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane ethyl acetate (2:1 to 1:1). This gave the pyridazine as a green solid (0.327g, 25%); HPLC (96%); MS (electrospray, [M+H]4") m/z 309.4. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-(2-tert- butylphenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride; HPLC (91%,
Figure imgf000040_0001
MS (electrospray, [M+H]4") m/z 309.4.
Example 39
Preparation of 3-amino-2-(2-propylphenyl)-2,8-dihydro-6H-pyrano [3,4- c]pyridazine-4-carbonitrile hydrochloride (compound 42)
Figure imgf000040_0002
To the hydrazone (0.988 g, 0.00402 mol) in DMSO (1 ml) was added malononitrile (0.27 g, 0.00402 mol) and moφholine (0.35 ml, 0.00402 mol) and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (2:1 to 1:1). This gave the pyridazine as a green solid (0.102g, 9%); HPLC (91%); MS (electrospray, [M+H]4") m/z 295.2. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-(2-propylphenyl)-2,8- dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride; HPLC (91%, Rχ=3.17); MS (electrospray, [M+H]4") m/z 295.2.
Example 40
Preparation of 3-amino-2-[2-(4-morpholinyl)phenyl]-2,8-dihydro-6H-pyrano[3,4- c]pyridazine-4-carbonitrile hydrochloride (compound 43)
Figure imgf000041_0001
To the hydrazone (0.15 g, 0.00052 mol) in DMSO (1 ml) was added malononitrile (0.035 g, 0.00052 mol) and moφholine (0.045 ml, 0.00052 mol) and the reaction mixture stined at 80oC for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (5:1 to 1:1). This gave the pyridazine as a brown oil (0.08g, 46%); HPLC (98%); MS (electrospray, [M+H]+) m/z 338.2. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give BF7550; HPLC (98%, RT=1.41); MS (electrospray, [M+H]+) m/z 338.2.
Example 41
Preparation of 3-amino-2-(5,6,7,8-tetrahydro-l-naphthalenyl)-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile (compound 44)
Figure imgf000041_0002
44 To the hydrazone (0.989 g, 0.00383 mol) in DMSO (1 ml) was added malononitrile (0.253 g, 0.00383 mol) and moφholine (0.33 ml, 0.00383 mol) and the reaction mixture stined at 80oC for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane ethyl acetate (5:1 to 2:1). This gave the pyridazine as a brown solid (0.472g, 40%); HPLC (98%); MS (electrospray, [M+H]+) m/z 307.4. The HCl salt of the pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-(5,6,7,8- tefrahydro-l-naphthalenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile; HPLC (98%, RT=1.68); MS (electrospray, [M+H]+) m/z 307.4. Example 42
Preparation of 3-amino-2-[l,l '-bipheny!]-2-yl-2,8-dihydro-6H-pyrano[3,4- c]pyridazine-4-carbonitrile (compound 45)
Figure imgf000042_0001
To the hydrazone (0.351 g, 0.00125 mol) in DMSO (0.5 ml) was added malononitrile (0.083 g, 0.00125 mol) and moφholine (0.11 ml, 0.00125 mol) and the reaction mixture stirred at 80oC for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane ethyl acetate (2:1 to ethyl acetate). This gave the pyridazine as a green solid (0.407 g, 91%); HPLC (96%); MS (electrospray, [M+H]+) m/z 329.3. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-[l,r- biphenyl]-2-yl-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile; HPLC (98%, RT=1.75); MS (electrospray, [M+H]+) m/z 329.3.
Example 43
Preparation of 3-amino-2-[2-(5-methyl-2-furyl)phenyl]-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile (compound 46)
Figure imgf000042_0002
To the hydrazone (0.26 g, 0.000915 mol) in DMSO (0.5 ml) was added malononitrile (0.06 g, 0.000915 mol) and moφholine (0.08 ml, 0.000915 mol) and the reaction mixture stined at 80oC for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane ethyl acetate (2:1 to 1:1). This gave the pyridazine as a brown solid (0.224g, 74%); HPLC (99%); MS (electrospray, [M+H]+) m/z 333.3. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-[2-(5-methyl-2- furyl)phenyl]-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile; HPLC (99%, RT=1.74); MS (electrospray, [M+H]+) m/z 333.3.
Example 44
Preparation of 3-amino-2-(2,2,3,3-tetrafluoro-2,3-dihydro-l ,4-benzodioxin-5-yl)-
2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile (compound 47)
Figure imgf000043_0001
To the hydrazone (0.229 g, 0.000686 mol) in DMSO (0.5 ml) was added malononitrile (0.045 g, 0.000686 mol) and moφholine (0.06 ml, 0.00402 mol) and the reaction mixture stined at 80oC for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane ethyl acetate (2:1 to ethyl acetate). This gave the pyridazine as a green solid (0.215g, 82%); HPLC (97%); MS (electrospray, [M+H]+) m/z 383.2. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3-amino-2- (2,2,3,3-tetrafluoro-2,3-dihydro-l,4-benzodioxin-5-yl)-2,8-dihydro-6H-pyrano[3,4- c]pyridazine-4-carbonitrile; HPLC (97%, RT=1.83); MS (electrospray, [M+H]+) m/z 383.2.
Example 45
Preparation of 3-amino-2-[3-(benzyIoxy)phenyI]-2,8-dihydro-6H-pyrano[3,4- c]pyridazine-4-carbonitrile (compound 48)
Figure imgf000044_0001
48
To the hydrazone (0.42 g, 0.00135 mol) in DMSO (1 ml) was added malononitrile (0.09 g, 0.00135 mol) and moφholine (0.12 ml, 0.00135 mol) and the reaction mixture stirred at 80oC for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane ethyl acetate (5:1 to 1:1). This gave the pyridazine as a brown solid (0.27 g, 56%); HPLC (98%); MS (electrospray, [M+H]+) m/z 359.1. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-[3- (benzyloxy)phenyl]-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile; HPLC (98%, RT=1.93); MS (electrospray, [M+H]+) m z 359.1.
Example 46
Preparation of 3-amino-2-phenyl-2,5,6,8-tetrahydro -3H-pyrano [3,4-c]pyridazine-
4-carbonitrile (compound 49)
Scheme 5
Figure imgf000044_0002
To the pyridazine (4) (0.2 g, 0.0008 mol) in THF / EtOH (2 ml / 0.5 ml) was added sodium borohydride (0.06 g, 0.0016 mol) and the reaction mixture stined at room temperature for 12 hours. The solvent was then removed under reduced pressure, the residue dissolved in DCM, washed with water, dried (MgSO ) and the solvent removed under reduced pressure, then freeze-dried to give 3-amino-2-phenyl-2,5,6,8-tetrahydro- 3H-pyrano[3,4-c]pyridazine-4-carbonitrile (49) as a yellow solid (0.15g, 74%); HPLC (87%, Rχ=4.20); MS (electrospray, [M+H]+) m/z 255.3.
Example 47
Preparation of 3-amino-2-(2-methoxyphenyI)-2,5,6,8-tetrahydro~4aH-pyrano[3,4- c]pyridazine-4-carbonitrile acetate (compound 53)
Figure imgf000045_0001
To the pyridazine (31) (0.2 g, 0.0007 mol) in dry dioxan / EtOH (8 ml / 2 ml) was added sodium triacetoxyborohydride (0.751 g, 0.00355 mol) and the reaction mixture stirred at 0°C to room temperature for 12 hours according to the method described in Scheme 5. The reaction mixture was quenched by the gradual addition of water then the solvent was removed under reduced pressure. The residue was dissolved in DCM, washed with saturated sodium bicarbonate solution, water, dried (MgSO4) and the solvent removed under reduced pressure, then freeze-dried to give 3-amino-2-(2- memoxyphenyl)-2,5,6,8-tefrahy(fro-4aH-pyrano[3,4-c]pyridazine-4-carbonitrile acetate (53) as a green solid (0.13g, 65%); HPLC (91%,
Figure imgf000045_0002
MS (electrospray, [M+H]4") m/z 285.1.
Example 48 Preparation of 3-amino-2-[l,l'-biphenyl]-2-yI-2,5,6,8-tetrahydro-4aH-pyrano[3,4- c]pyridazine-4-carbonitri!e acetate (compound 54)
Figure imgf000045_0003
To the pyridazine (45) (0.150 g, 0.00046 mol) in dry dioxan / EtOH (4 ml / 0.5 ml) was added sodium triacetoxyborohydride (0.485 g, 0.00229 mol) and the reaction mixture stirred at 0°C to room temperature for 12 hours according to the method described in Scheme 5. The reaction mixture was quenched by the gradual addition of water then the solvent was removed under reduced pressure. The residue was dissolved in DCM, washed with saturated sodium bicarbonate solution, water, dried (MgSO4) and the solvent removed under reduced pressure, then freeze-dried to give 3-amino-2-[l,l '- biphenyl]-2-yl-2,5,6,8-tefrahydro-4aH-pyrano[3,4-c]pyridazine-4-carbonitrile acetate (54) as a green solid (0.147g, 97%); HPLC (89%, Rτ=3.72); MS (electrospray, [M+H]4") m/z 330.6.
Example 49
Preparation of 3-amino-2-(2,6-dimethylphenyl)-2,5,6,8-tetrahydro-4aH- pyrano[3,4-c]pyridazine-4-carbonitrile acetate (compound 55)
Figure imgf000046_0001
55
To the pyridazine (14) (0.100 g, 0.00032 mol) in dry dioxan / EtOH (4 ml / 0.5 ml) was added sodium triacetoxyborohydride (0.344 g, 0.0016 mol) and the reaction mixture stirred at 0°C to room temperature for 12 hours according to the method described in Scheme 5. The reaction mixture was quenched by the gradual addition of water then the solvent was removed under reduced pressure. The residue was dissolved in DCM, washed with saturated sodium bicarbonate solution, water, dried (MgSO4) and the solvent removed under reduced pressure, then freeze-dried to give 3- amino-2-(2,6-dimethylphenyl)-2,5,6,8-tetrahydro-4aH-pyrano[3,4-c]pyridazine-4- carbonitrile acetate ( 55) as a yellow solid (0.07g, 71%); HPLC (87%, Rχ=3.66); MS (electrospray, [M+H]4) m/z 310.64.
Example 50
Preparation of 3-amino-2-[4-(trifluoromethyl)phenyl]-2,5,6,8-tetrahydro-4aH- pyrano[3,4-c]pyridazine-4-carbonitrile acetate (compound 56)
Figure imgf000047_0001
To the coπesponding pyridazine (0.02 g, 0.000065 mol) in dry dioxan / EtOH (4 ml / 0.5 ml) was added sodium triacetoxyborohydride (0.066 g, 0.00031 mol) and the reaction mixture stirred at 0°C to room temperature for 12 hours according to the method described in Scheme 5. The reaction mixture was quenched by the gradual addition of water then the solvent was removed under reduced pressure. The residue was dissolved in DCM, washed with saturated sodium bicarbonate solution, water, dried (MgSO4) and the solvent removed under reduced pressure, then freeze-dried to give 3- amino-2-[4-(trifluoromethyl)phenyl]-2,5,6,8-tetrahydro-4aH-pyrano[3,4-c]pyridazine-4- carbonitrile acetate (56) as a green powder (0.017g, 81%); HPLC (75%, Rτ=4.09); MS (electrospray, [M+H]4") no m/z .
Example 51
Preparation of 3-amino-2-(2-methoxyphenyl)-6,6-dimethyl-2,5,6,8-tetrahydro-4aH- pyrano[3,4-c]pyridazine-4-carbonitrile acetate (compound 57)
Figure imgf000047_0002
57
To the corresponding pyridazine (0.100 g, 0.00032 mol) in dry dioxan / EtOH (4 ml / 0.5 ml) was added sodium triacetoxyborohydride (0.342 g, 0.0016 mol) and the reaction mixture stined at 0°C to room temperature for 12 hours according to the method described in Scheme 5. The reaction mixture was quenched by the gradual addition of water then the solvent was removed under reduced pressure. The residue was dissolved in DCM, washed with saturated sodium bicarbonate solution, water, dried (MgSO4) and the solvent removed under reduced pressure, then freeze-dried to give 3- amino-2-(2-methoxyphenyl)-6,6-dimethyl-2,5,6,8-tetrahydro-4aH-pyrano[3,4- c]pyridazine-4-carbonifrile acetate (57) as a beige solid (0.029g, 29%); HPLC (92%, Rτ=3.31); MS (electrospray, [M+H]+) m/z 312.62.
Example 52 Preparation of 3-amino-2-(3-methoxyphenyl)-2,5,6,8-tetrahydro-4aH-pyrano[3,4- c]pyridazine-4-carbonitrile acetate (compound 58)
Figure imgf000048_0001
To the pyridazine (13) (0.110 g, 0.00037 mol) in dry dioxan / EtOH (4 ml / 0.5 ml) was added sodium triacetoxyborohydride (0.393 g, 0.0018 mol) and the reaction mixture stined at 0°C to room temperature for 12 hours according to the method described in Scheme 5. The reaction mixture was quenched by the gradual addition of water then the solvent was removed under reduced pressure. The residue was dissolved in DCM, washed with saturated sodium bicarbonate solution, water, dried (MgSO4) and the solvent removed under reduced pressure, then freeze-dried to give 3- amino-2-(3-methoxyphenyl)-2,5,6,8-tetrahydro-4aH-pyrano[3,4-c]pyridazine-4- carbonitrile acetate (58) as a yellow powder (0.109g, 99%); HPLC (84%, Rχ=3.54); MS (electrospray, [M+H]4") m/z 298.59.
Example 53 Preparation of 3-amino-2-(3-chlorophenyl)-2,5,6,8-tetrahydro-4aH-pyrano[3,4- c]pyridazine-4-carbonitrile acetate (compound 59)
Figure imgf000048_0002
To the corresponding pyridazine (0.100 g, 0.00035 mol) in dry dioxan / EtOH (4 ml / 0.5 ml) was added sodium triacetoxyborohydride (0.371 g, 0.0018 mol) and the reaction mixture stined at 0°C to room temperature for 12 hours according to the method describe in Scheme 5. The reaction mixture was quenched by the gradual addition of water then the solvent was removed under reduced pressure. The residue was dissolved in DCM, washed with saturated sodium bicarbonate solution, water, dried (MgSO4) and the solvent removed under reduced pressure, then freeze-dried to give 3- amino-2-(3-chlorophenyl)-2,5,6,8-tetrahydro-4aH-pyrano[3,4-c]pyridazine-4- carbonitrile acetate (59) as a beige solid (0.037g, 32%); HPLC (100%, Rι=3.75); MS (electrospray, [M+H]4") m/z 288.5.
Example 54
Preparation of 3-amino-2-(2-isopropyIphenyl)-6,6-dimethyl-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 60)
Figure imgf000049_0001
The starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.256g, 0.000934mol) in DMSO(0.5ml). Malononitrile (0.062g, 0.000934mol) and moφholine (0.08ml, 0.000934mol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (3:1 to 2:1). This gave the pyridazine as a yellow oil(0.270g, 89%); HPLC (98%); MS (electrospray, [M+H]4") m/z323.05. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 3-amino-2-(2- isopropylphenyl)-6,6-dimethyl-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (60); HPLC (100%, Rf=3.34); MS (electrospray, [M+H]4) m/z323.05. Example 55
Preparation of 3-amino-2-(2-methoxyphenyl)-6,6-dimethyl-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 61)
Figure imgf000050_0001
61 The starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone(0.360 g, 0.00137 mol) in DMSO(0.5 ml). Malononitrile (0.091 g, 0.00137 mol) and moφholine (0.12 ml, 0.00137 mol) was added and the reaction mixture stirred at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (5:1 to ethyl acetate). This gave the pyridazine as a yellow solid(0.384 g, 90%); HPLC (90%); MS (electrospray, [M+H]4) m/z311.18. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 61. HPLC (100%, Rχ=3.01); MS (electrospray, [M+H]4) m/z311.18.
Example 56
Preparation of 3-amino-2-(2,6-dimethyIphenyl)-6,6-dimethyl-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 62)
Figure imgf000050_0002
62
The starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone(0.350g, 0.00135mol) in DMSO(0.5ml). Malononitrile (0.089g, 0.00135mol) and moφholine (0.12ml, 0.00135mol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane ethyl acetate (5:1 to 1:1). This gave the pyridazine as a yellow solid(0.235g, 58%); HPLC (96%); MS (electrospray, [M+H]4") m/z309.19. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 62. HPLC (100%, Rχ=3.14); MS (electrospray, [M+H]4") m/z309.19.
Example 57
Preparation of 3-amino-2-(3-chlorophenyI)-6,6-dimethyl-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitriIe hydrochloride (compound 63)
Figure imgf000051_0001
The starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.344g, 0.0013mol) in DMSO (1ml). Malononitrile (0.085g, 0.0013mol) and moφholine (0.11ml, 0.0013mol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using hexane : ethyl acetate 4:1 as eluant. This gave the pyridazine as a yellow solid (0.373g, 91%); HPLC (97%); MS (electrospray, [M+H]4") m/z 314.56. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 63. HPLC (99%, R =3.14); MS (electrospray, [M+H]4) m/z 314.56.
Example 58
Preparation of 3-amino-2-(3-methoxyphenyl)-6,6-dimethyl-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitriIe hydrochloride (compound 64)
Figure imgf000052_0001
The starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.47 g, 0.0018 mol) in DMSO (0.5ml). Malononitrile (0.119 g, 0.0018 mol) and moφholine (0.16ml, 0.0018mol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (4:1 to 1:1) as eluant. This gave the pyridazine as a yellow solid (0.481g, 86%); HPLC (96%); MS (electrospray, [M+H]4) m/z 310.61. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 64. HPLC (97%, Rτ=3.02); MS (electrospray, [M+H]4) m/z 310.61.
Example 59 Preparation of 3-amino-2-(2-methoxy-6-methyϊphenyl)-6,6-dimethyl-2,8-dihydro-
6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 65)
Figure imgf000052_0002
65
The starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.291g, 0.001 lmol) in DMSO (1ml). Malononitrile (0.070g, 0.001 lmol) and moφholine (0.09ml, 0.001 lmol) was added and the reaction mixture stirred at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using using a gradient of hexane : ethyl acetate (4:1 to 1:1) as eluant. This gave the pyridazine as a yellow solid (0.379g, 100%); HPLC (98%); MS (electrospray, [M+H]4") m/z 325.20. The HCl salt of the pyridazine was then prepared and the compound freeze-dried to give 65. HPLC (96%, Rχ=3.03); MS (electrospray, [M+H]4) m/z 325.20.
Example 60
Preparation of 3-amino-6,6-dimethyl-2-[2-(methylsuIfanyl)phenyl]-2,8-dihydro-
6H~pyrano[3,4-c]pyridazine-4-carbonitriIe hydrochloride (compound 66)
Figure imgf000053_0001
66
The starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.291g, 0.001 lmol) in DMSO (1ml). Malononitrile (0.084g, 0.0013mol) and moφholine (0.11ml, 0.0013mol) was added and the reaction mixture stirred at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using using a gradient of hexane : ethyl acetate (2:1 to 1:1) as eluant. This gave the pyridazine as a yellow solid (0.361g, 85%); HPLC (98%); MS (electrospray, [M+H]4) m/z 327.16. The HCl salt of the pyridazine was then prepared and the compound freeze-dried to give 66. HPLC (100%, Rτ=3.13); MS (electrospray, [M+H]4") m/z 327.16.
Example 61
Preparation of 3-amino-6,6-dimethyl-2-[3-isopropylphenyl]-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 67)
Figure imgf000053_0002
The starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.39g, 0.0014mol) in DMSO (1ml). Malononitrile (0.094g, 0.0014mol) and moφholine (0.12ml, 0.0014mol) was added and the reaction mixture stirred at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using using a gradient of hexane : ethyl acetate (4:1 to 2:1) as eluant. This gave the pyridazine as a yellow solid (0.226g, 50%); HPLC (97%); MS (electrospray, [M+H]4") m/z 323.21. The HCl salt of the pyridazine was then prepared and the compound freeze-dried to give 67. HPLC (97%, Rχ=3.39); MS (electrospray, [M+H]4) m/z 323.21.
Example 62
Preparation of 3-amino-6,6-dimethyl-2-[2-phenoxyphenyl]-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 68)
Figure imgf000054_0001
The starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.197g, O.OOOόlmol) in DMSO (0.5ml). Malononitrile (0.040g, 0.00061mol) and moφholine (0.05ml, O.OOOόlmol) was added and the reaction mixture stirred at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using using a gradient of hexane : ethyl acetate (3:1 to 1:1) as eluant. This gave the pyridazine as a yellow solid (0.141g, 62%); HPLC (98%); MS (electrospray, [M+H]4) m/z 373.17. The HCl salt of the pyridazine was then prepared and the compound freeze-dried to give 68. HPLC (98%, Rr=3.41); MS (electrospray, [M+H]4") m/z 373.17. Example 63
Preparation of 3-amino-6,6-dimethyl-2-[2-chlorophenyl]-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 69)
Figure imgf000055_0001
The starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.320g, 0.0012mol) in DMSO (1ml). Malononitrile (0.080g, 0.0012mol) and moφholine (0.11ml, 0.0012mol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane : ethyl acetate (5:1 to 2:1) as eluant. This gave the pyridazine as a yellow solid (0.22 g, 58%); HPLC (91%); MS (electrospray, [M+H]4) m/z 315.1. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 69. HPLC (91%, Rχ=1.70); MS (electrospray, [M+H]4") m/z 315.1.
Example 64
Preparation of 3-amino-6,6-dimethyl-2-[4-fluorophenyl]-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 70)
Figure imgf000055_0002
The starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.190g, 0.00076mol) in DMSO (1ml). Malononitrile (0.050g, 0.00076mol) and moφholine (0.07ml, 0.00076mol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using a gradient of hexane ethyl acetate (5:1 to 2:1) as eluant. This gave the pyridazine as a yellow solid (0.18g, 79%); HPLC (96%); MS (electrospray, [M+H]4") m/z 299.1. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 70. HPLC (96%, Rf=1.61); MS (elecfrospray, [M+H]4") m/z 299.1.
Example 65
Preparation of 3-amino-6,6-dimethyl-2-[4-chIorophenyl]-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 71)
Figure imgf000056_0001
The starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.381g, 0.0014mol) in DMSO (1ml). Malononitrile (0.094g, 0.0014mol) and moφholine (0.12ml, 0.0014mol) was added and the reaction mixture was stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using hexane : ethyl acetate (2:1) as eluant. This gave the pyridazine as a yellow solid (0.164g, 37%); HPLC (100%); MS (electrospray, [M+H]4) m/z 315.09. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 71. HPLC (98%, Rχ=1.80); MS (electrospray, [M+H]4) m/z 315.09.
Example 66
Preparation of 3-amino-6,6-dimethyl-2-[3-methylphenyl]-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 72)
Figure imgf000056_0002
The starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.253g, O.OOlOmol) in DMSO (0.5ml). Malononitrile (0.068g, O.OOlOmol) and moφholine (0.09ml, O.OOlOmol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using hexane : ethyl acetate (1:1) as eluant. This gave the pyridazine as a yellow solid (0.132g, 45%); HPLC (98%); MS (electrospray, [M+H]4) m/z 295.12. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 72. HPLC (98%, Rf=l .80); MS (electrospray, [M+H]4) m/z 295.12.
Example 67
Preparation of 3-amino-6,6-dimethyl-2-[3-(dimethylamino)phenyl]-2,8-dihydro- 6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 73)
Figure imgf000057_0001
The starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.138g, 0.00050mol) in DMSO (0.5ml). Malononitrile (0.033g, 0.00050mol) and moφholine (0.044ml, 0.00050mol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using hexane : ethyl acetate (1:1) as eluant. This gave the pyridazine as a yellow solid (0.18g, 100%); HPLC (99%); MS (electrospray, [M+H]4") m/z 324.17. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 73. HPLC (98%, Rχ=1.64); MS (electrospray, [M+H]4") m/z 324.17. Example 68
Preparation of 3-amino-6,6-dimethyl-2-[2-methylphenyl]-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitriIe hydrochloride (compound 74)
Figure imgf000058_0001
74 The starting material 2,2-dimethyl-tetτalιydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.259g, 0.001 lmol) in DMSO (1ml). Malononitrile (0.070g, 0.001 lmol) and moφholine (0.092ml, 0.001 lmol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by column chromatography over silica using hexane : ethyl acetate (1:1) as eluant. This gave the pyridazine as a yellow solid (0.576g, 100%); HPLC (100%); MS (electrospray, [M+H]4) m/z 295.14. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 74. HPLC (100%, Rχ=l .65); MS (electrospray, [M+H]4") m/z 295.14.
Example 69
Preparation of 3-amino-6,6-dimethyI-2-[3,5-dichlorophenyI]-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 75)
Figure imgf000058_0002
The starting material 2,2-dimethyl-tetraliydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.349g, 0.0012mol) in DMSO (1ml). Malononitrile (0.077g, 0.0012mol) and moφholine (0.101ml, 0.0012mol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified using the Biotage Quad 3 system using a gradient of hexane : ethyl acetate (4:1 to ethyl acetate) as eluant. This gave the pyridazine as a yellow solid (0.108g, 26%); HPLC (97%); MS (electrospray, [M+H]4") m/z 348.95. The HCl salt of the pyridazine was then prepared and the compound freeze-dried to give 75. HPLC (100%, Rτ=1.93); MS (electrospray, [M+H]4") m/z 348.95.
Example 70
Preparation of 3-amino-6,6-dimethyl-2-[5-chloro-2-methoxyphenyl]-2,8-dihydro-
6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 76)
Figure imgf000059_0001
The starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.489g, 0.0017mol) in DMSO (1ml). Malononitrile (0.109g, 0.0017mol) and moφholine (0.144ml, 0.0017mol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified using the Biotage Quad 3 system using a gradient of hexane : ethyl acetate (4:1 to ethyl acetate) as eluant. This gave the pyridazine as a yellow solid (0.406g, 69%); HPLC (100%); MS (electrospray, [M+H]4) m/z 345.01. The HCl salt of the pyridazine was then prepared and the compound freeze-dried to give 76. HPLC (99%, Rχ=1.87); MS (electrospray, [M+H]4") m/z 345.01.
Example 71
Preparation of 3-amino-6,6-dimethyl-2-[3,5-(dimethoxy)phenyl]-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 77)
Figure imgf000060_0001
The starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the the hydrazone (0.304g, O.OOlOmol) in DMSO (1ml). Malonomtrile (0.069g, O.OOlOmol) and moφholine (0.09ml, O.OOlOmol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified using the Biotage Quad 3 system using a gradient of hexane : ethyl acetate (4:1 to ethyl acetate) as eluant. This gave the pyridazine as a yellow solid (0.234g, 69%); HPLC (100%); MS (electrospray, [M+H]4) m/z 341.04. The HCl salt of the pyridazine was then prepared and the compound freeze-dried to give 77. HPLC (99%, Rχ=1.83); MS (electrospray, [M+H]4) m/z 341.04.
Example 72 Preparation of 3-amino-6,6-dimethyl-2-[ 4-chloro-3-(trifluoromethyl)phenyl]-2,8- dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitriIe hydrochloride (compound 78)
Figure imgf000060_0002
The starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.290g, 0.00087mol) in DMSO (1ml). Malononitrile (0.057g, 0.00087mol) and moφholine (0.08ml, 0.00087mol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified using the Biotage Quad 3 system using a gradient of hexane : ethyl acetate (2:1 to ethyl acetate) as eluant. This gave the pyridazine as a yellow oil (0.17g, 51%); HPLC (90%); MS (electrospray, [M+H]4") m/z 382.9. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 78. HPLC (90%, Rτ=2.09); MS (electrospray, [M+H]4") m/z 382.9.
Example 73
Preparation of 3-amino-6,6-dimethyl-2- [2-(methylsulf ony!)phenyI]-2,8-dihydro- 6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 79)
Figure imgf000061_0001
The starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.220 g, 0.00071 mol) in DMSO (1 ml). Malononitrile (0.047 g, 0.00071 mol) and moφholine (0.06 ml, 0.00071 mol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified using the Biotage Quad 3 system using a gradient of hexane : ethyl acetate (2:1 to ethyl acetate) as eluant. This gave the pyridazine as a yellow oil (0.15 g, 59%); HPLC (97%); MS (electrospray, [M+H]4) m/z 358.9. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 79. HPLC (97%, Rτ=1.50); MS (electrospray, [M+H]4) m/z 358.9.
Example 74
Preparation of 3-amino-6,6-dimethyl-2-[3-nitrophenyI]-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 80)
Figure imgf000061_0002
The starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.300g, 0.00108mol) in DMSO (1ml). Malononitrile (0.072g, 0.00108mol) and moφholine (0.094ml, 0.00108mol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified using the Biotage Quad 3 system using a gradient of hexane : ethyl acetate (2:1 to ethyl acetate) as eluant. This gave the pyridazine as an orange solid (0.16g, 46%); HPLC (98%); MS (electrospray, [M+H]4") m/z 326.0. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 80. HPLC (98%, Rτ=1.69); MS (electrospray, [M+H]4) m/z 326.0.
Example 75
Preparation of 3-amino-6,6-dimethyl-2-[3-(methylsulfonyl)phenyl]-2,8-dihydro-
6H-pyrano[3,4-c]pyridazine-4-carbonitrile trifluoroacetate (compound 81)
Figure imgf000062_0001
The starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.240g, 0.00077mol) in DMSO (1ml). Malononitrile (0.05 lg, 0.00077mol) and moφholine (0.07ml, 0.00077mol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified using the Biotage Quad 3 system using a gradient of hexane : ethyl acetate (2:1 to ethyl acetate) as eluant. This gave the pyridazine as a yellow oil (0.1 Og, 36%); HPLC (99%); MS (electrospray, [M+H]4") m/z 358.7. The HCl salt ofthe pyridazine was then prepared and the compound freeze-dried to give 81. HPLC (99%, Rτ=l .55); MS (electrospray, [M+H]4") m/z 358.7.
Example 76
Preparation of 3-amino-6,6-dimethyI-2-[3-fluorophenyl]-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile (compound 82)
Figure imgf000063_0001
The starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.57g, 0.00228mol) in DMSO (1ml). Malononitrile (0.151g, 0.00228mol) and moφholine (0.2ml, 0.00228mol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by prep hplc and then freeze-dried to give the TFA salt ofthe pyridazine as a yellow solid. 82 (0.273g, 40%); HPLC (100%, Rχ=1.65); MS (electrospray, [M+H]4") m/z 299.1.
Example 77
Preparation of 3-amino-2-(l,3-benzothiazoI-6-yl)-6,6-dimethyl-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile trifluoroacetate (compound 83)
Figure imgf000063_0002
The starting material 2,2-dimethyl-tetraliydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.30g, 0.00104mol) in DMSO (1ml). Malononitrile (0.069g, 0.00104mol) and moφholine (0.09ml, 0.00104mol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by prep hplc and then freeze-dried to give the TFA salt ofthe pyridazine as a yellow solid. 83 (0.218g, 62%); HPLC (100%, Rχ=1.60); MS (electrospray, [M+H]4") m z 337.9. Example 78
Preparation of 3-amino-2- [4-(lH-imidazol-l-yl)phenyl]-6,6-dimethyl-2,8-dihydro-
6H-pyrano[3,4-c]pyridazine-4-carbonitrile trifluoroacetate (compound 84)
Figure imgf000064_0001
The starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.201g, 0.00067mol) in DMSO (0.5ml). Malononitrile (0.045g, 0.00067mol) and moφholine (0.02ml, 0.00067mol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by prep hplc and then freeze-dried to give the TFA salt ofthe pyridazine as a brown oil. 84 (0.162g, 70%); HPLC (95%, Rτ=1.22); MS (electrospray, [M+H]4") m/z 347.34.
Example 79
Preparation of 3-amino-6,6-dimethyl-2-[4-(4-morpholinyIcarbonyϊ)phenyl]-2,8- dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile trifluoroacetate (compound 85)
Figure imgf000064_0002
The starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.437g, 0.0013mol) in DMSO (1ml). Malononitrile (0.084g, 0.0013mol) and moφholine (0.11ml, 0.0013mol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by prep hplc and then freeze-dried to give the TFA salt ofthe pyridazine as a brown oily-solid. 85 (0.406g, 79%); HPLC (97%, R[=150); MS (electrospray, [M+H]4") m/z 394.07.
Example 80
Preparation of 3-amino-6,6-dimethyl-2-[2-bromophenyl]-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile trifluoroacetate (compound 86)
Figure imgf000065_0001
The starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.306g, 0.00098mol) in DMSO (1ml). Malononitrile (0.065g, 0.00098mol) and moφholine (0.086ml, 0.00098mol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by prep hplc and then freeze-dried to give the TFA salt ofthe pyridazine as a brown solid. 86 (0.251g, 71%); HPLC (100%, Rχ=1.75); MS (electrospray, [M+H]4") m/z 358.74, 361.22.
Example 81
Preparation of 2-(diethylamino)ethyl 4-(3-amino-4-cyano-6,6-dimethyl-6H- pyrano[3,4-c]pyridazin-2(8H)-yl)benzoate trifluoroacetate (compound 87)
Figure imgf000065_0002
The starting material 2,2-dimethyl-tetrahydro-ρyran-4-one (52) was prepared according to method 2, Scheme 3. Synthetic method la, Scheme 1, was used to prepare the hydrazone intermediate. The method according to Scheme 4 was undertaken to synthesize the pyridazine using the hydrazone (0.053g, 0.00014mol) in DMSO (0.5ml). ; Malononitrile (0.0093g, 0.00014mol) and moφholine (0.012ml, 0.00014mol) was added and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by prep hplc and then freeze-dried to give the TFA salt of the pyridazine as a green oil. 87 (0.038g, 64%); HPLC (100%, Rχ=1.45); MS (electrospray, [M+H]4) m/z 424.07.
Example 82
Preparation of 3-amino-6,6-dimethyl-2- [2-(trifluoromethyI)phenyl] -2,8-dihy dro- 6H-pyrano[3,4-c]pyridazine-4-carbonitrile trifluoroacetate (compound 89)
Scheme 5
Figure imgf000066_0001
The starting material 2,2-dimethyl-tetrahydro-pyran-4-one (52) was prepared according to method 2, Scheme 3. A solution of 2,2-dimethyl-tetrahydro-pyran-4-one (3.00g, 23.4 mmol) and ethyl formate (3.0 ml, 37.2 mmol) in dry THF (30 mL) was placed under nitrogen. To the solution was added KlBuO (2.63 g, 23.4 mmol) in small portions during 10 min. A yellow precipitate was formed almost immediately. The mixture was stined at room temperature for 3 hours. The solvent was then evaporated and the crude product 88 was dried in vacuum. The crude material was dissolved in EtOH (18 mL) and divided into 6 portions and stored in freezer until used.
A solution ofthe diazoniumsalt of 2-trifluoromethyl-aniline was prepared by the gradual addition of a solution of sodium nitrite (273 mg, 3.95 mmol) in water (2 mL) to a solution of 2-trifluoromethyl aniline (637 mg, 3.95 mol) in 1.00 M HCl (7.90 mL, 7.90 mmol) at -5°C. The cold ethanol solution (3 mL) ofthe the potassium salt of 5- (hydroxymethylene)-2,2-dimethyltetrahydro-4H-pyran-4-one 88 (3.90 mmol) was added dropwise and the resulting mixture was stined vigorously at -5° C for 1 h. The reaction mixture was diluted with DCM and washed with water. The organic phase was dried with MgSO4 and evaporated and gave 0.9755 g ofthe hydrazone intermediate as an orange solid. (83%) 1H NMR (400 MHz, CDC13) δ 1.36 (s, 6 H), 2.58 (s, 2 H), 4.64 (s, 2 H), 7.08 (t, 1 H), 7.50 (t, 1 H), 7.55 (d, 1 H), 7.86 (d, 1 H), 14.05 (s, 0.7 H). MS (El) m/z 300.0 (M4). A solution of hydrazone (0.97 g, 3.23 mmol), malononitrile (261 mg, 3.95 mmol) and moφholine (345 μl, 3.95 mmol) in DMSO (2 ml) was heated at 80° C 30 min. The reaction mixture was diluted with CHC13, was washed with water, dried (MgSO4) and evaporated. The crude product was purified by column chromatography on silica gel with a gradient of hexane : ethyl acetate (4:1 to 1:1). The purified material was dissolved in EtOAc and HCl in EtOAc was added. The product was filtered, washed with EtOAc and dried in vacuum. Gave 0.667 g ofthe title compound 89. (54%) MS (ESI+) m/z 349.29 (M+H)4". HPLC (30-60%) rt : 12.46 min (99%).
Example 83
Preparation of 3-amino-6,6-dimethyl-2-[3-(trifluoromethyl)phenyl]-2,8-dihydro- 6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 90)
Figure imgf000067_0001
The intermediate 6,6-dimethyldihydro-2H-pyran-3,4-3-{[3-(frifluoromethyl)- phenyl]hydrazone} was synthesized according to the modified method lc described in example 82, Scheme 5. The hydrazone (0.948 g, 3.16 mmol) was dissolved in DMSO (2 mL) and malononitrile (263 mg, 3.98 mmol) and moφholine (345 μL, 3.96 mmol) was added. The reaction conditions and work-up procedure followed Method 2 a and gave 0.514 g ofthe title compound 90. (42%) MS (ESI+) m/z 349.31 (M+H)+. HPLC (30-60%) rt: 13.75 min (99 %)
Example 84
Preparation of 3-amino-2-(4-methoxyphenyl)-6,6-dimethyl-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 91)
Figure imgf000068_0001
The intermediate 6,6-dimethyldihydro-2H-pyran-3,4-dione 3-[(4-methoxy- phenyl)hydrazone] was synthesized according to the modified method lc described in example 82, Scheme 5. The hydrazone (0.832 g, 3.17 mmol) was dissolved in DMSO (2 mL) and malononitrile (265 mg, 4.01 mmol) and moφholine (345 μL, 3.96 mmol) was added. The reaction conditions and work-up procedure followed Method 2a and gave 0.651 g ofthe title compound 91. (59%) MS (ESI+) m/z 311.31 (M+H)+.HPLC (30-60%) rt:9.74 min
Example 85
Preparation of 3-amino-2-(2-fluorophenyl)-6,6-dimethyl-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 92)
Figure imgf000068_0002
The intermediate 6,6-dimethyldihydro-2H-pyran-3,4-dione 3-[(2- fluorophenyl)hydrazone] was synthesized according to the modified method lc described in example 82, Scheme 5. The hydrazone (0.778 g, 3.16 mmol) was dissolved in DMSO (2 mL) and malononitrile (268 mg, 4.06 mmol) and moφholine (345 μL, 3.96 mmol) was added. The reaction conditions and work-up procedure followed Method 2a and gave 0.625 g ofthe title compound 92. (60%) MS (ESI+) m/z 299.30 (M+H)+. HPLC (30-60%) rt:8 45 min (99%)
Example 86
Preparation of 3-amino-2-[4-(2-hydroxyethyl)phenyl]-6,6-dimethyI-2,8-dihydro- 6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 93)
Figure imgf000069_0001
The intermediate 6,6-dimethyldihydro-2H-pyran-3,4-dione 3-{[4-(2- hydroxyethyl)-phenyl]hydrazone} was synthesized according to the modified method lc described in example 82, Scheme 5. The hydrazone (0.832 g, 3.01 mmol) was dissolved in DMSO (2 mL) and malononitrile (266 mg, 4.03 mmol) and moφholine (345 μL, 3.96 mmol) was added. The reaction conditions and work-up procedure followed Method 2a and gave 0.690 g ofthe title compound 93. (64%) MS (ESI+) m/z 325.33 (M+H)+. HPLC (30-60%) rt:5.88 min (97%)
Example 87
Preparation of 3-amino-2-[3-(hydroxymethyl)phenyl]-6,6-dimethyl-2,8-dihydro- 6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 94)
Figure imgf000069_0002
The intermediate 6,6-dimethyldihydro-2H-pyran-3,4-dione 3-{[3- (hydroxymethyl)phenyl]-hydrazone}was synthesized according to the modified method lc described in example 82, Scheme 5. The hydrazone (0.546 g, 2.08 mmol) was dissolved in DMSO (2 mL) and malononitrile (170 mg, 2.57 mmol) and piperidine (255 μL, 2.58 mmol) was added. The reaction conditions and work-up procedure followed Method 2a and gave 0.538 g ofthe title compound 94 (75%) MS (ESI+) m/z 311.26 (M+H)4". HPLC (30-60%) rt:5.35 (94%).
Example 88
Preparation of 3-amino-2-(4-ethylphenyI)-6,6-dimethyl-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 95)
Figure imgf000070_0001
The intermediate 6,6-dimethyldihydro-2H-pyran-3,4-dione 3-[(4- ethylphenyl)hydrazone] was synthesized according to the modified method lc described in example 82, Scheme 5. The hydrazone (0.526 g, 2.02 mmol) was dissolved in DMSO (2 mL) and malononitrile (170 mg, 2.57 mmol) and piperidine (255 μL, 2.58 mmol) was added. The resulting mixture was heated at 80°C for 30 min. The reaction mixture was diluted with CHC13, washed with water, dried (MgSO4) and evaporated. The crude product was dissolved in EtOAc/Et2O and HCl in EtOAc was added dropwise. The product was filtered, washed with EtOAc and Et2O and dried in vacuum and gave 0.488 g ofthe title product 95. (70%) MS (ESI+) m/z 309 (M+H)4". HPLC (30- 60%) rt:13.46 min (99%)
Example 89
Preparation of 3-amino-2-[(3-carboxymethyl)phenyl]-6,6-dimethyl-2,8-dihydro- 6H-pyrano[3,4-c]pyrazidine-4-carbonitrile hydrochloride (compound 96)
Figure imgf000070_0002
The intermediate 6,6-dimethyldihydro-2H-pyran-3,4-dione [3-(3- carboxymethylphenyl)-hydrazone] was synthesized according to the modified method lc described in example 82, Scheme 5. The hydrazone (0.461 g, 1.59 mmol) was dissolved in DMSO (2 mL) and malononitrile (126 mg, 1.91 mmol) and moφholine (170 μL, 1.95 mmol) was added. The reaction mixture was stined at room temperature for 1 h. The reaction mixture was diluted with EtOAc and washed with water, then extracted with 1 M HCl (aq.) x 3. The aqueous phase was neutralized and the free amine was back-extracted to EtOAc. The organic phase was dried (MgSO4) and evaporated. The pure amine was redissolved in EtOAc/Et2O and HCl (Et2O) was added. The product was filtered, washed with Et2O and dried in vacuum and gave 0.281 g ofthe title compound 96. (47%) MS (ESI+) for m/z 339.23 (M+H)+. HPLC (30- 60% in 10 min) rt:8.36 min (96%)
Example 90
Preparation of 3-amino-2-(3-acetamidophenyl)- 6,6-dimethyI-2,8-dihydro-6H- pyrano[3,4-c]pyrazidine-4-carbonitrile hydrochloride (compound 97)
Figure imgf000071_0001
The intermediate 6,6-dimethyldihydro-2H-pyran-3,4-dione [3-(3- acetamidophenyl)-hydrazone] was synthesized according to the modified method lc described in example 82, Scheme 5. The hydrazone (0.481 g, 1.66 mmol) was dissolved in DMSO (2 mL) and malononitrile (133 mg, 2.01 mmol) and moφholine (170 μL, 1.95 mmol) was added. The reaction conditions and work-up procedure followed the method described in Example 89 and gave 0.337 g ofthe title compound 97. (54%) MS (ESI+) m/z 338.26 (M+H)+. HPLC (30-60%) rt:6.13 min (97%)
Example 91
Preparation of 3-amino-6,6-dimethyl-2-(2-nitrophenyl)-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 98)
Figure imgf000071_0002
The intermediate 6,6-dimethyldihydro-2H-pyran-3,4-dione 3-[(2-nitrophenyl)- hydrazone]was synthesized according to the modified method lc described in example 82, Scheme 5. The hydrazone (0.443 g, 1.59 mmol) was dissolved in DMSO (2 mL) and malononitrile (145 mg, 2.19 mmol) and moφholine (170 μL, 1.95 mmol) was added. The reaction conditions and work-up procedure followed the method described in Example 89 and gave 0.348 g ofthe title compound 98. (61%). MS (ESI+) m/z 326.23 (M+H)4". HPLC (30-60%) rt:9.58 min (97%)
Example 92
Preparation of 3~amino-2-(2-butyIphenyl)-6,6-dimethyl-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 99)
Figure imgf000072_0001
99
The intermediate 6,6-dimethyldihydro-2H-pyran-3,4-dione 3-[(2- butylphenyl)hydrazone] was synthesized according to the modified method lc described in example 82, Scheme 5. The hydrazone (0.477 g, 1.65 mmol) was dissolved in DMSO (2 mL) and malononitrile (131 mg, 1.98 mmol) and moφholine (170 μL, 1.95 mmol) was added. The reaction conditions and work-up procedure followed the method described in Example 89 and gave 0.339 g ofthe title compound 99. (55%). MS (ESI+) m/z 337.29 (M+H)4". HPLC (60-90%) rt:5.39 min (94%).
Example 93
Preparation of 3-amino-6,6-dimethyl-2-(3-phenoxyphenyl)-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 100)
Figure imgf000072_0002
The intermediate 6,6-dimethyldihydro-2H-pyran-3,4-dione 3-[(3- phenoxyphenyl)-hydrazone] was synthesized according to the modified method lc described in example 82, Scheme 5. The hydrazone (0.579 g, 1.78 mmol) was dissolved in DMSO (2 mL) and malononitrile (132 mg, 2.00 mmol) and moφholine (170 μL, 1.95 mmol) was added. The reaction conditions and work-up procedure followed the method described in Example 89 and gave 0.367 g ofthe title compound 100. (50%) MS (ESI+) m/z 373.23 (M+H)4". HPLC (60-90%) rt:4.99 min (94%).
Example 94 Preparation of 3-amino-6,6-dimethyl-2-[2-(4-morpholinyl)phenyl]-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 101)
Figure imgf000073_0001
The intermediate 6,6-dimethyldihydro-2H-pyran-3,4-dione 3-{[2-(4- moφholinyl)phenyl] -hydrazone} was synthesized according to the modified method lc described in example 82, Scheme 5, with the addition of an extra equivalent of acid when forming the diazonium salt. The hydrazone (0.428 g, 1.35 mmol) was dissolved in DMSO (10 mL) and malononitrile (187 mg, 2.83 mmol) and piperazinomethyl polystyrene resin (1.30g, 1.08 mmol/g, 1 eq.) was added. The reaction mixture was heated at 80°C for 8 h, then diluted with CH2C12 and the solid phase reagent was filtered and washed with CH2C12. The organic phase was washed with water, dried (MgSO4) and evaporated. The crude product was purified by column chromatography on silica gel with petroleum ether:EtOAc 4:1. The purified material was dissolved in EtOAc/Et2O and HCl in Et O was added dropwise. The product was filtered, washed with Et2O and dried in vacuum and gave 0.302 g ofthe title compound 101. (51%) MS (ESI+) m/z 366.28 (M+H)4". HPLC (30-60%) rt: 10.58 min (98%).
Example 95
Preparation of3-amino-2-(2-ethoxyphenyl)-6,6-dimethyI-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 102)
Figure imgf000073_0002
The intermediate 6,6-dimethyldihydro-2H-pyran-3,4-dione 3-[(2- ethoxyphenyl)hydrazone] was synthesized according to the modified method lc described in example 82, Scheme 5. The hydrazone (0.491 g, 1.78 mmol) was dissolved in DMSO (10 mL) and malononitrile (289mg, 4.37 mmol) and piperazinomethyl polystyrene resin (1.67 g, 1.08 mmol/g, 1 eq.) was added. The reaction mixture was heated at 80°C for 12h. The reaction mixture was diluted with CH2C12 and the resin was filtered and washed with CH2C1 . The organic phase was washed with water and extracted with 1 M HCl (aq.) x 3. The aqueous phase was neutralized and the free amine was back-extracted with EtOAc, dried (MgSO4) and evaporated. The pure product was dissolved in Et2O and HCl in Et2O was added dropwise. The product was filtered, washed with Et2O and dried in vacuum and gave 0.349 g ofthe title compound 102. (54%) MS (ESI+) m/z 325.29 (M+H)4". HPLC (30- 60%) rt:12.23 min (98%).
Preparation of the intermediate monomethyl pyranone according to method 2:
Figure imgf000074_0001
Diethylaminehydrochloride(24.87 g, 0.24 mol), formaldehyde solution (19.51 g, 0.24mol), 3-penten-2-one (23.2 ml, 0.24 mol), concenfrated HCl(lml; for pH = 1) and hydroquinone (0.53 g) were heated at 80°C for two hours. The reaction mixture was then distilled under reduced pressure. The divinyl ketone was collected at P = 16mmHg, Tvap = 45-70°C. (8.08 g, 35% yield).
Divinyl ketone (8.0 g, 0.083 mol) was added dropwise to a mixture of HgSO4(0.616 g, 0.0027 mol), H2SO4(0.616 ml) and water (24 ml) at 85°C with stirring. Then HgSO4 (0.152 g, 0.00052 mol) and H2SO4 (0.152 ml) in water (6 ml) were added over a period of one hour. In total the reaction mixture was heated for five hours at 85 to 100°C. This reaction mixture was then steam distilled. The product was then extracted with DCM, washed with water, dried (MgSO4) and the solvent removed under reduced pressure to give a yellow liquid. This crude product 103 (80%) was then used to synthesize the monomethyl analogues. The pyranone (colourless liquid) had a yield of 2.38g, 18%. NMR: δl.29 (d, 3H), 62.34 (d, 2H), 52.36 (t, 2H), 53.77 (t, 2H), 54.22 (q, IH).
Example 96 Preparation of 3-amino-6-methyl-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4- carbonitrile trifluoroacetate (compound 104)
Figure imgf000075_0001
To the monomethyl hydrazone 103 (0.166 g, 0.00076 mol) in DMSO (0.5 ml) was added malononitrile (0.050 g, 0.00076 mol) and moφholine (0.07 ml, 0.00076 mol) and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by prep HPLC and then freeze-dried to give the TFA salt ofthe pyridazine as a green-brown solid, 104 (0.07g, 35%); HPLC (99%, Rχ=1.42); MS (electrospray, [M+H]4") m/z266.99.
Example 97
Preparation of 3-amino-2-(2-ethoxyphenyl)-6-methyl-2,8-dihydro-6H-pyrano [3,4- c]pyridazine-4- carbonitrile trifluoroacetate (compound 105)
Figure imgf000075_0002
105
To the monomethyl hydrazone 103 (0.248 g, 0.001 mol) in DMSO (1.0 ml) was added malononitrile (0.05 g, 0.001 mol) and moφholine (0.06 ml, 0.001 mol) and the reaction mixture stined at 80°C for 15 minutes. The mixture was cooled and the product was purified by prep HPLC and then freeze-dried to give the TFA salt ofthe pyridazine as a green oil, 105 (0.252g, 85%); HPLC (99%, Rτ=1.54); MS (electrospray, [M+H]+) m/z297.12 PHARMACEUTICAL COMPOSITIONS The novel compounds according to the present invention may be administered orally, intranasally, transdermally, subcutaneously, parenterally, intramusculary, as well as intravenously. Oral administration is the prefened route.
The dosage will depend on the route of administration, the severity ofthe disease, age and weight ofthe patient, and other factors normally considered by the attending physician when determining the individual regimen and dosage level as the most appropriate for a particular patient. Either solid or fluid dosage forms can be prepared for oral administration. Solid compositions, such as compressed tablets, are prepared by mixing the compounds ofthe invention with conventional ingredients such as talc, magnesium stearate, dicalcium phosphate, magnesium aluminum silicate, calcium sulfate, starch, lactose, acacia, methyl cellulose, or functionally similar pharmaceutical diluents and carriers. Capsules are prepared by mixing the compounds of this invention with an inert pharmaceutical diluent and placing the mixture into an appropriately sized hard gelatin capsule. Soft gelatin capsules are prepared by machine encapsulation of a sluny ofthe compounds of this invention with an acceptable inert oil such as vegetable oil or light liquid petrolatum. Syrups are prepared by dissolving compounds ofthe invention in an aqueous vehicle and adding sugar, aromatic flavoring agents and preservatives. Elixirs are prepared using a hydroalcoholic vehicle such as ethanol, suitable sweeteners such as sugar or saccharin and an aromatic flavoring agent. Suspensions are prepared with an aqueous vehicle and a suspending agent such as acacia, tragacanth, or methyl cellulose. When the compounds ofthe invention are administered parenterally, they can be given by injection or by intravenous infusion. Parenteral solutions are prepared by dissolving the compounds ofthe invention in aqueous vehicle and filter sterilizing the solution before placing in a suitable sealable vial or ampule. Parenteral suspensions are prepared in substantially the same way except a sterile suspension vehicle is used and the compounds ofthe present invention are sterilized with ethylene oxide or suitable gas before it is suspended in the vehicle.
Thus, a further aspect ofthe present invention is a pharmaceutical composition comprising a compound of formula I, la, la', and lb (e.g., a compound specified in the following Examples) above respectively together with a pharmacologically and pharmaceutically acceptable carrier. It is prefened to use pharmaceutically inert carriers which may be solid or liquid. Solid form preparations include but is not limited to powders, tablets, dispersible granules, capsules etc. The skilled person within the formulation field will readily know which carrier to use for the specific circumstance when formulating a composition in accordance with the present invention.
Pharmaceutically acceptable salts ofthe compounds of formula I above, which salts are useful in accordance with the present invention, may be formed from organic and inorganic acids. Examples of such salts are hydrochloride salts, tosylate salts, citrate salts, maleate salts, acetate salts, hydrobromide salts, malate salts, stearate salts, aluminium salts, lithium salts, calcium salts, and magnesium salts among others. This list should however not in any way be regarded as exhaustive. The hydrochloride salts are the prefened salts ofthe invention.
BIOLOGICAL EVALUATION The compound 6H-Pyrano[3,4-c]pyridazine-4-carbonitrile, 3-amino-2,8- dihydro-6,6-dimethyl-2-phenyl which is commercially available from the company Labotest in Germany, was tested for biological activity in accordance with the present invention, together with other compounds within the scope ofthe present invention.
Expression and purification of recombinant human PTPs
Human PTP1B (amino acid residues 1-298, cloned from a human placental library), without the GST tag and thrombin cleavage site, was inserted into a pMB replicon and transformed into E. coli BL21(DE3), a strain containing a chromosomal copy ofthe gene for T7 RNA polymerase under control of a lacUN5 promoter. Expression of PTP IB was induced with isopropyl thiogalactose and cells were lysed in lysis buffer comprising 50mM Tris-HCl pH 7.5, 10% glycerol, lmM EDTA, 3mM DTT, 3mM MgCl2, and 0.2mg/ml lysozyme with lmg/ml DΝAse I. The soluble protein was purified by ion exchange, hydrophobic interaction and gel exclusion chromatography for use in assays to identify PTP IB inhibitors. The plasmid pGEX2K- SHP2 which encoded the catalytic domain of human SHP-2 (residues 252-529) was used to transform E. coli cells. After induction of protein expression, cells were lysed in PBS containing 1% Triton X100 and lysozyme (2mg/ml). Recombinant protein was purified by glutathione sepharose 4B chromatography followed by Superdex 200 size exclusion chromatography. Recombinant proteins were stored at -70°C until used. Recombinant T cell PTP (TCPTP) and LAR were purchased from New England Biolabs.
Measurement of PTP activity
Human PTP IB activity was measured using p-nitrophenol phosphate (pNPP) as substrate in a 96- well microtiter plate format. An assay pH of 7.2 is used for standard assays (measured extinction coefficient =9800 at pH 7.2).
Standard assays were conducted at room temperature in a total volume of 0.2 ml that contains Hepes buffer (50 mM, pH 7.2), NaCl (50 mM), EDTA (1 mM), DTT (1 mM), bovine serum albumin (1 mg/ml), pNPP (1.25mM) and PTP1B (500ng/ml, 13.5nM). A master plate was set up for each compound in which a stock solution of compound in DMSO (19mM or ImM) was diluted 1 to 10 with assay buffer in column 1 (giving a ImM or 100 μM concentration). Substances were subsequently diluted serially by two thirds in all columns across the plate. For enzyme assays, 20 μl of each diluted compound was removed to a new plate and diluted to 200 μl (final volume) with 160 μl pNPP solution and 20 μl PTP IB solution. Reactions were thus started immediately and were stopped after 60 minutes by addition of 100 μl 0. IN NaOH. The OD405 was subsequently measured. Two wells on each plate contained DMSO controls and two wells contained sodium orthovanadate (2mM) which inhibits PTP IB-catalyzed hydrolysis of pNPP completely. Data were conected for background absorbance by the subtraction ofthe optical densities from a no-enzyme control plate and were expressed as percent inhibition relative to the average ofthe vanadate controls measured on the same microtiter plate. The activity ofthe other PTPs was determined in a similar fashion except that the concentration of pNPP was varied according to the Km values for individual enzymes (0.6mM for TCPTP and 6.25mM for each of SHP-2 and LAR) and the buffer used for TCPTP was 25mM Tris-HCl pH 7.2.
Cell-based analysis of compound activity
The effects of compounds on the phosphorylation status ofthe insulin receptor was measured using L6 muscle cells expressing the receptor endogenously. L6 myocytes were cultured in α-MEM with 10% foetal bovine serum and antibiotics. Cells were differentiated into myotubes in 24-well plates by culturing for 10 days in medium containing 2% serum. The medium was refreshed on alternate days and 0.24mg/ml cytidine was included from day 7 to stop any remaining cycling cells. Cells were starved of serum overnight prior to use. Cells were pretreated with compound at approximately five times the IC50 for inhibition of PTP IB for 30 minutes, prior to being stimulated with insulin (25nM) for five minutes. Cells were lysed in buffer comprising 25mM Tris-HCl pH 7.4, 150mM NaCl, ImM EDTA, ImM EGTA, 1% Nonidet-40, 0.25% sodium deoxycholate, ImM sodium orthovanadate, lOmM β-glycerophosphate, 5mM sodium pyrophosphate and protease inhibitors. Cleared lysates were stored at - 70°C until used.
Insulin receptor phosphorylation was assessed using a lanthanide-based fluorescent assay (DELFIA). An anti-insulin receptor antibody was captured on the wells of a 96-well plate using an anti-rabbit IgG antibody. After incubation with lysates containing between 100-250μg protein which was consistent for all wells in a single experiment, phosphate on the receptor was detected with a biotinylated anti- phosphotyrosine antibody (PY99B from Santa Cruz) and europium-labelled streptavidin.
Results
IC50 values were determined for all compounds against each of four PTPs (PTP1B, SHP-2, LAR and TCPTP). Compounds were active across a wide range of concentrations from 100 nM to 50 μM.
Representative compounds from across the series were analysed in cell-based assays in which effects on insulin-stimulated auto-phosphorylation ofthe insulin receptor was determined. Compounds caused statistically-significant increases in receptor phosphorylation by between 20% to 70%.

Claims

1. A compound ofthe formula I
Figure imgf000080_0001
wherein n is an integer of 1 or 2; R* is (i) phenyl or naphthyl, each optionally substituted with up to 5 substituents independently selected from the group consisting of: (a) a straight or branched Cι-C6 alkyl;
(b) a C3-C6 cycloalkyl or a C6-C10 aryl;
(c) -CO-O-(Cι-C6 alkyl) wherein the alkyl group is straight or branched;
(d) a halogen selected from the group consisting of fluoro, chloro, bromo, and iodo; (e) a straight or branched Ci-Cβ alkoxy;
(f) nitro;
(g) CF3;
(h) -O-(d-C6 alkyl)-phenyl wherein the alkyl group is straight or branched; (i) a heteroaryl having 5 or 6 ring atoms, wherein 1 or 2 ofthe ring atoms are optionally O, N, or S, optionally substituted with one or more methyl or ethyl;
(j) a heterocycloalkyl having 5 or 6 ring atoms, wherein 1 or 2 ofthe ring atoms are optionally O, N, or S; (k) -N(R )-CO-R3, where R2 is hydrogen or a straight or branched Ci-Cβ alkyl, and R3 is a straight or branched C C6 alkyl; and (1) -N(R4)(R5), where each of R4 and R5 independently is a straight or branched
Ci-Cβ alkyl; (ii) phenyl that is fused with a cyclohexyl group or naphthyl that is fused with a cyclohexyl group, wherein 1 or 2 ofthe carbon atoms are optionally substituted with O, N, or S, and wherein said cyclohexyl group is optionally substituted with 1 or 2 substituents independently selected from the group consisting of halogen, CF , a straight or branched C Cβ alkyl, a C3-C6 cycloalkyl, and a C6-C10 aryl; or
Figure imgf000081_0001
wherein Y is O, S, or N-Rx wherein Rx is a straight or branched -C6 alkyl;
is (i) an oxygen atom, a sulfur atom, or a methylene group;
Figure imgf000081_0002
, wherein R6 is
(a) benzyl;
(b) -CO-(d-C6 alkyl) wherein the alkyl group is straight or branched;
(c) -CO-A, wherein A is phenyl, naphthyl, or a heteroaryl; said heteroaryl group having 5 to 10 ring atoms wherein at least one of said ring atoms is O, N, or S;
(d) -CO-O-R , wherein Ry is hydrogen or a straight or branched C Cδ alkyl;
(e) -CO-O-(C1-C6 alkyl)-A wherein A is as defined above; or
(f) -SO -A, wherein A is as defined above;
(«i)
\
CH— CO— O — (C C6 alkyl)
/ , wherein the alkyl group is straight or branched; or
Figure imgf000081_0003
, wherein each of Q 1 and Q 2 independently is phenyl, naphthyl, or a heteroaryl; said heteroaryl group having 5 to 10 ring atoms wherein at least one of said ring atoms is O, N, or S; each of Ra and Rb is independently hydrogen or Ci-Cβ alkyl, or Ra and Rb together form a carbonyl group; and
Rc is hydrogen, a straight or branched CrC6 alkyl, or carbonyl; with the proviso that the compound 6H-pyrano[3,4-c]pyridazine-4-carbonitrile, 3- amino-2,8-dihydro-6,6-dimethyl-2-phenyl is excluded; or a pharmaceutically and pharmacologically acceptable salt or hydrate thereof.
2. The compound of claim 1, said compound having formula la:
Figure imgf000082_0001
wherein X, R p i , r R>a , τ R>b , and R are as defined in claim 1.
3. The compound of claim 2, said compound having formula la':
Figure imgf000082_0002
wherein X, R1, Ra, R , and Rc are as defined in claim 1.
4. The compound of claim 1, said compound having formula lb:
Figure imgf000082_0003
wherein X, R1, Ra, RD, and Rc are as defined in claim 1.
5. The compound of claim 1, wherein R1 is phenyl or naphthyl, each optionally and independently substituted with 1, 2, 3, 4, or 5 substituents selected from the group consisting of methyl; ethyl; straight, branched or cyclic propyl, butyl, pentyl, or hexyl; -CO-O-(CH2)n-CH3 wherein n is an integer 0, 1, 2, 3, 4, or 5; methoxy, ethoxy, propoxy, butyloxy, pentyloxy, or hexyloxy; and -O-(CH2)n-phenyl where n is an integer 0, 1, 2, 3, 4, 5, or 6.
6. The compound of claim 1, wherein R1 is phenyl, optionally substituted with a straight or branched C C4 alkyl, a straight or branched Cι-C6 alkoxy, nitro, CF3, fluoro, chloro, bromo, cyclohexyl, heterocyclohexyl, phenyl, -CO-O^ - alkyl), or -N(R2)-CO-R3, where R2 is hydrogen, methyl, or ethyl, and R3 is a straight or branched Cι-C4 alkyl.
7. The compound of claim 1, wherein R1 is phenyl, optionally substituted with methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, propoxy, CF3, moφholino, fluoro, chloro, phenyl, -CO-O-CH3, -CO-O-CH2CH3, -NH-CO-CH3, or -NH-CO-CH2CH3.
8. The compound of claim 1, wherein R1 is
Figure imgf000083_0001
wherein each of nt and n2 independently is an integer 0, 1, 2, 3, 4, or 5;
Figure imgf000083_0002
Figure imgf000084_0001
C 3 . wherein each of x\4 and n5 independently is an integer
0, 1, 2, 3, 4, or 5.
9. The compound of claim 1, wherein R1 is
Figure imgf000084_0002
10. The compound of claim 1, wherein X is an oxygen atom; a sulfur atom; a
alkyl);
Figure imgf000084_0003
11. The compound of claim 1 , wherein X is an oxygen atom.
12. The compound of claim 1, wherein each of Ra and Rb is independently hydrogen, methyl, or ethyl.
13. The compound of claim 1, wherein Rc is hydrogen, methyl, or ethyl.
14. The compound of claim 3, wherein
R1 is phenyl, optionally substituted with a straight or branched C1-C4 alkyl, a straight or branched Ci-C6 alkoxy, nitro, CF3, fluoro, chloro, bromo, cyclohexyl, heterocyclohexyl, phenyl, -CO-O-(C1-C4 alkyl), or -N(R2)-CO-R3, where R2 is hydrogen, methyl, or ethyl, and R3 is a sfraight or branched C1-C4 alkyl; X is an oxygen atom; a sulfur atom; a methylene group;
Figure imgf000085_0001
, wherein R6 is
\ CH— CO— O — (CrC4 alkyl) benzyl, -CO-(d-C4 alkyl), or -CO-O-(d-C4 alkyl); or / ; and each of R , Rb, and Rc independently is hydrogen, methyl, or ethyl.
15. The compound of claim 3, wherein RJ is
Figure imgf000085_0002
and n independently is an integer 0, 1, 2, 3, 4, or 5;
Figure imgf000085_0003
wherein n3 is an integer 0, 1, 2, 3, 4, or 5;
Figure imgf000085_0004
and n5 independently is an integer 0, 1, 2, 3, 4, or 5;
Figure imgf000085_0005
\ ;N— . R.B
X is an oxygen atom; a sulfur atom; a methylene group; / , wherein R6 is
\ CH-CO— O — (CrC4 alkyl) benzyl, -CO-(d-C4 alkyl), or -CO-O-(d-C4 alkyl); or / ; and each of Ra, Rb, and Rc independently is hydrogen, methyl, or ethyl.
16. The compound of claim 1, said compound being
3-amino-2-phenyl-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile;
3-amino-2-phenyl-2,6,7,8-tetrahydro-4-cinnolinecarbonitrile;
3-amino-2-phenyl-6,7-dihydro-2H-cyclopenta[c]pyridazine-4-carbonitrile;
3-amino-7-benzyl-2-phenyl-2,6,7,8-tetrahydropyrido[3,4-c]pyridazine-4-carboriitrile; 7-acetyl-3-amino-2-phenyl-2,6,7,8-tefrahycfropyrido[3,4-c]pyridazine-4-carbonitrile;
3-amino-2-(2-fluorophenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonifrile;
3-amino-2-(4-methylphenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile;
3 -amino-2- [3 -(trifluoromethyl)phenyl] -2, 8-dihydro-6H-pyrano [3 ,4-c]pyridazine-4- carbonitrile; 3-amino-2-phenyl-2,8-dihydro-6H-tMopyrano[3,4-c]pyridazine-4-carbonitrile;
3 -amino-2-(3 -methoxyphenyl)-2, 8 -dihydro-6H-pyrano [3 ,4-c]pyridazine-4-carbonitrile;
3-ammo-2-(3-methoxyphenyl)-2,8-dmy(fro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile; ethyl 3-amino-4-cyano-2-phenyl-2,6,7,8-tetrahydro-7-cinnolinecarboxylate;
3-amino-2-(3,4-dichlorophenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4- carbonitrile;
7-benzoyl-3-amino-2-phenyl-2,6,7,8-tefrahydropyrido[3,4-c]pyridazine-4-carbonitrile;
3 -amino-2-(4-methoxyphenyl)-2, 8 -dihydro-6H-pyrano [3 ,4-c]pyridazine-4-carbonitrile;
3 -amino-2-(4-isopropylphenyl)-2, 8-dihydro-6H-pyrano [3 ,4-c]pyridazine-4-carbonitrile;
3-amino-2-[4-(4-moφholinyl)phenyl]-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4- carbonitrile;
7-acetyl-3-amino-2-(2,6-dimethylphenyl)-2,6,7,8-tefrahydropyrido[3,4-c]pyridazine-4- carbonitrile; ethyl 3 -(3 -amino-4-cyano-6H-pyrano [3 ,4-c]pyridazin-2(8H)-yl)b enzoate; ethyl 3-amino-4-cyano-2-phenyl-2,8-dihydropyrido[3,4-c]pyridazine-7(6H)- carboxylate; 3-amino-2-(2-chloro-6-fluorophenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4- carbonitrile;
3-amino-2-(2-bromo-5-trifluoromethylphenyl)-2,8-dihydro-6H-pyrano[3,4- c]pyridazine-4-carbonitrile; 3 -amino-2-( 1 ,3 -benzothiazol-6-yl)-2, 8 -dihydro-6H-pyrano [3 ,4-c]pyridazine-4- carbonitrile;
N-[4-(3-amino-4-cyano-6H-pyrano[3,4-c]pyridazin-2(8H)-yl)phenyl]-N- methylacetamide;
3-amino-2-[4-(dimethylamino)phenyl]-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4- carbonitrile;
3-amino-2-(4-chloro-2-methylphenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4- carbonitrile;
3-amino-2-(2-chloro-6-methylphenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4- carbonitrile; 3 -amino-2-(2-methoxyphenyl)-2, 8 -dihydro-6H-pyrano [3 ,4-c]pyridazine-4-carbonitrile;
3-amino-2-mesityl-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile;
3-amino-2-(2-isopropylphenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile;
3-amino-2-(2,6-dichlorophenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4- carbonitrile; 3 -amino-4-cyano-2-phenyl-2, 8 -dihydropyrido [3 ,4-c]pyridazine-7(6H)-carboxylate;
3-amino-2-(2-methoxy-6-methylphenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4- carbonitrile;
3-amino-2-(2-methyl-4-nitrophenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4- carbonitrile; 3-amino-2-(3,5-dimethoxyphenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4- carbonitrile;
3-amino-2-(2-(l-methylpropyl)phenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4- carbonitrile;
3-amino-2-(2-chloro-5-methoxyphenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4- carbonitrile;
3-amino-2-(2-ter-butylphenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile;
3 -amino-2-(2-propylphenyl)-2, 8 -dihydro-6H-pyrano [3 ,4-c]pyridazine-4-carbonitrile; 3-amino-2-[2-(4-moφholinyl)phenyl]-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4- carbonitrile;
3-amino-2-(5,6,7,8-tetrahydro-l-naphthalenyl)-2,8-dihydro-6H-pyrano[3,4- c]pyridazine-4-carbonitrile; 3 -amino-2- [1,1' -biphenyl] -2-yl-2,8-dihydro-6H-pyrano [3 ,4-c]pyridazine-4-carbonitrile;
3 -amino-2- [2-(5 -methyl-2-furyl)phenyl] -2,8-dihydro-6H-pyrano [3 ,4-c]pyridazine-4- carbonitrile;
3-amino-2-(2,2,3,3-tetrafluoro-2,3-dihydro-l,4-benzodioxin-5-yl)-2,8-dihydro-6H- pyrano [3 ,4-c]pyridazine-4-carbonitrile; 3-amino-2-(2,2,3,3-tetrafluoro-2,3-dihydro-l,4-benzodioxin-5-yl)-2,8-dihydro-6H- pyrano[3,4-c]pyridazine-4-carbonitrile; or
3-amino-2-phenyl-2,5,6,8-tetrahydro-3H-pyrano[3,4-c]pyridazine-4-carbonitrile.
17. A hydrochloride salt or tosylate salt of a compound of claim 1.
18. A hydrochloride salt of a compound of claim 1.
19. A compound of claim 18, said compound being
3-ainino-2-(2-fluorophenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 9);
3-armno-2-(4-methylphenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 10);
3-amino-2-[3-(trifluoromethyl)phenyl]-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4- carbonitrile hydrochloride (compound 11); 3-amino-2-phenyl-2,8-dihydro-6H-thiopyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 12);
3-amino-2-(3-methoxyphenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 13);
3 -amino-2-(3 -methoxyphenyl)-2, 8-dihydro-6H-pyrano [3 ,4-c]pyridazine-4-carbonitrile hydrochloride (compound 14); ethyl 3-amino-4-cyano-2-phenyl-2,6,7,8-tetrahydro-7-cinnolinecarboxylate hydrochloride (compound 15); 3-amino-2-(3,4-dichlorophenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 16);
3 -amino-2-(4-methoxyphenyl)-2, 8 -dihydro-6H-pyrano [3 ,4-c]pyridazine-4-carbonitrile hydrochloride (compound 18); 3-amino-2-(4-isopropylphenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 19);
3-amino-2-[4-(4-moφholinyl)phenyl]-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4- carbonitrile hydrochloride (compound 20);
7-acetyl-3 -amino-2-(2,6-dimethylphenyl)-2,6,7, 8 -tetrahydropyrido [3 ,4-c]pyridazine-4- carbonitrile hydrochloride (compound 21); ethyl 3-(3-amino-4-cyano-6H-pyrano[3,4-c]pyridazin-2(8H)-yl)benzoate hydrochloride
(compound 22); ethyl 3-amino-4-cyano-2-phenyl-2,8-dihydropyrido[3,4-c]pyridazine-7(6H)-carboxylate hydrochloride (compound 23); 3-amino-2-(2-chloro-6-fluorophenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4- carbonitrile hydrochloride (compound 24);
3-amino-2-(2-bromo-5-trifluoromethylphenyl)-2,8-dihydro-6H-pyrano[3,4- c]pyridazine-4-carbonitrile hydrochloride (compound 25);
3 -amino-2-( 1 ,3 -benzothiazol-6-yl)-2, 8-dihydro-6H-pyrano [3 ,4-c]pyridazine-4- carbonitrile hydrochloride (compound 26);
N-[4-(3-amino-4-cyano-6H-pyrano[3,4-c]pyridazin-2(8H)-yl)phenyl]-N- methylacetamide hydrochloride (compound 27);
3-amino-2-[4-(dimethylamino)phenyl]-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4- carbonitrile hydrochloride (compound 28); 3-amino-2-(4-chloro-2-methylphenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4- carbonitrile hydrochloride (compound 29);
3-amino-2-(2-chloro-6-methylphenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4- carbonitrile hydrochloride (compound 30);
3-amino-2-(2-me oxyphenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 31);
3-amino-2-mesityl-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 32); 3-amino-2-(2-isopropylphenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 33);
3-amino-2-(2,6-dichlorophenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 34); 3 -amino-2-(2-methyl-4-mtrophenyl)-2, 8-dihydro-6H-pyrano [3 ,4-c]pyridazine-4- carbonitrile hydrochloride (HCl salt of compound 35);
3-amino-4-cyano-2-phenyl-2,8-dihydropyrido[3,4-c]pyridazine-7(6H)-carboxylate hydrochloride (compound 36);
3-amino-2-(2-methoxy-6-methylphenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4- carbonitrile hydrochloride (compound 37);
3-amino-2-(3,5-dimethoxyphenyl)-2,8-dmydro-6H-pyrano[3,4-c]pyridazine-4- carbonitrile hydrochloride (compound 38);
3-amino-2-(2-(l-methylpropyl)phenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4- carbonitrile hydrochloride (compound 39); 3-amino-2-(2-chloro-5-methoxyphenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4- carbonitrile hydrochloride (compound 40);
3-amino-2-(2-ter-butylphenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 41);
3-amino-2-(2-propylphenyl)-2,8-dihydro-6H-pyrano[3,4-c]pyridazine-4-carbonitrile hydrochloride (compound 42); or
3 -amino-2- [2-(4-moφholinyl)phenyl] -2,8-dihydro-6H-pyrano [3 ,4-c]pyridazine-4- carbonitrile hydrochloride (compound 43).
20. A pharmaceutical composition comprising a compound according to any one of claims 1 to 19, and a pharmaceutically acceptable carrier.
21. Use of a compound according to any one of claims 1 to 19 in the manufacture of a medicament for the treatment of type 2 diabetes mellitus.
PCT/SE2001/002097 2000-09-26 2001-09-26 Novel pyridazine compounds for the treatment of diabetes WO2002026743A1 (en)

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