Title of Invention

"A COMPOUND OF FORMULA IV"

Abstract The invention provides novel 7,8-dihydro-imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4-one compounds and 7,8,9-trihydro-[lH or 2H]-pyrimido [l,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one compounds, substituted at the 1 or 2 position with C2-9 alkyl, 03.9 cycloalkyl, heteroarylalkyl, or substituted arylalkyl, in free, salt or prodrug form, processes for their production, their use as Pharmaceuticals, particularly as PDE1 inhibitors, and pharmaceutical compositions comprising them.
Full Text ORGANIC COMPOUNDS
This application claims priority from U.S. Provisional Application No. 60/687,715, filed June 6, 2005, the contents of which are hereby incorporated by reference.
TECHNICAL FIELD
[0001] The present invention relates to novel 7,8-dihydro-imidazo[l,2-fl]pyrazolo[4,3-e]pyrimidin-4-one compounds and 7,8,9-trihydro-[l Hor 2H]-pyrimido [l,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one compounds, processes for their production, their use as pharmaceuticals and pharmaceutical compositions comprising them. Of particular interest are novel compounds useful as inhibitors of phosphodiesterase 1 (PDE1), e.g., in the treatment of diseases involving disorders of the dopamine Dl receptor intracellular pathway, such as Parkinson's disease, depression and damage to cognitive function, e.g., in schizophrenia.
BACKGROUND OF THE INVENTION
[0002] Eleven families of phosphodiesterases (PDEs) have been identified but only PDEs in Family I, the Ca2+-calmodulin-dependent phosphodiesterases (CaM-PDEs), have been shown to mediate the calcium and cyclic nucleotide (e.g. cAMP and cGMP) signaling pathways. The three known CaM-PDE genes, PDE1A, PDE1B, and PDE1C, are all expressed in central nervous system tissue. PDE1A is expressed throughout the brain with higher levels of expression in the CA1 to CA3 layers of the hippocampus and cerebellum and at a low level in the striatum. PDE1A is also expressed in the lung and heart. PDE1B is predominately expressed in the striatum, dentate gyrus, olfactory tract and cerebellum, and its expression correlates with brain regions having high levels of dopaminergic innervation. Although PDE1B is primarily expressed in the central nervous system, it may be detected in the heart. PDE1C is primarily expressed in olfactory epithelium, cerebellar granule cells, and striatum. PDE1C is also expressed in the heart and vascular smooth muscle.
[0003] Cyclic nucleotide phosphodiesterases decrease intracellular cAMP and cGMP signaling by hydrolyzing these cyclic nucleotides to their respective inactive 5'-monophosphates (5'AMP and 5'GMP). CaM-PDEs play a critical role in mediating signal transduction in brain cells, particularly within an area of the brain known as thebasal ganglia or striatum. For example, NMDA-type glutamate receptor activation and/or dopamine D2 receptor activation result in increased intracellular calcium concentrations, leading to activation of effectors such as calmodulin-dependent kinase II (CaMKII) and calcineurin and to activation of CaM-PDEs, resulting in reduced cAMP and cGMP. Dopamine Dl receptor activation, on the other hand, leads to activation of calcium dependent nucleotide cyclases, resulting in increased cAMP and cGMP. These cyclic nucleotides in turn activate protein kinase A (PKA; cAMP-dependent protein kinase) and/or protein kinase G (PKG; cGMP-dependent protein kinase) that phosphorylate downstream signal transduction pathway elements such as DARPP-32 (dopamine and cAMP-regulated phosphoprotein) and cAMP responsive element binding protein (CREB).
[0004] CaM-PDEs can therefore affect dopamine-regulated and other intracellular signaling pathways in the basal ganglia (striatum), including but not limited to nitric oxide, noradrenergic, neurotensin, CCK, VIP, serotonin, glutamate (e.g., NMDA receptor, AMPA receptor), GABA, acetylcholine, adenosine (e.g., A2A receptor), cannabinoid receptor, natriuretic peptide (e.g., ANP, BNP, CNP) and endorphin intracellular signaling pathways.
[0005] Phosphodiesterase (PDE) activity, in particular, phosphodiesterase 1 (PDE1) activity, functions in brain tissue as a regulator of locomotor activity and learning and memory. PDE1 is a therapeutic target for regulation of intracellular signaling pathways, preferably in the nervous system, including but not limited to a dopamine Dl receptor, dopamine D2 receptor, nitric oxide, noradrenergic, neurotensin, CCK, VIP, serotonin, glutamate (e.g., NMDA receptor, AMPA receptor), GABA, acetylcholine, adenosine (e.g., A2A receptor), cannabinoid receptor, natriuretic peptide (e.g., ANP, BNP, CNP) or endorphin intracellular signaling pathway. For example, inhibition of PDE IB should act to potentiate the effect of a dopamine Dl agonist by protecting cGMP and cAMP from degradation, and should similarly inhibit dopamine D2 receptor signaling pathways, by inhibiting PDE1 activity. Chronic elevation in intracellular calcium is linked to cell death in numerous disorders, particularly in neurodegenerative diseases such as Alzhemer's Parkinson's and Huntington's Diseases, and in disorders of the circulatory system leading to stroke and myocardial infarction. PDE1 inhibitors are therefore potentially useful in diseases characterized by reduced dopamine Dl receptor signaling activity, such as Parkinson's disease, restless
leg syndrome, depression, and cognitive impairment.
[0006] There is thus a need for compounds that selectively inhibit PDE1 activity,
especially PDE1B activity.

SUMMARY OF THE INVENTION
[0007] The invention provides novel, optionally substituted 7,8-dihydro-[lHor 2H]-imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-ones or 7,8,9-trihydro-[lHor 2H]-pyrimido [l,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-ones, substituted at the 1 or 2 position with C2-9 alkyl or €3.9 cycloalkyl, or optionally substituted heteroarylalkyl or substituted arylalkyl, in free, salt or prodrug form (hereinafter "Compounds of the Invention"). The 1- or 2- position substituent is preferably substituted benzyl or pyridylmethyl, e.g. para-substituted relative to the point of attachment, e.g., with aryl, e.g., phenyl, or heteroaryl, e.g., pyridyl or thiadiazolyl. These compounds are surprisingly found to selectively inhibit phosphodiesterase 1 (PDE1) activity, especially PDE1B activity.
[0008] Preferably, the Compounds of the Invention are the 7,8-dihydro-[lHor 2H]-imidazo[l,2-o]pyrazolo[4,3-e]pyrimidin-4(5H)-ones and 7,8,9-trihydro-[lHor 2H]-pyrimido [l,2-aJpyrazolo[4,3-e]pyrimidin-4(5H)-ones, of formula I
Formula
RI is H or C1-4 alkyl (e.g., methyl);
(ii) R4 is H or CM alkyl and R?. and RS are, independently, H or CM alkyl
(e.g., Ra and RS are both methyl, or R2 is H and RS is isopropyl), aryl,
heteroaryl, (optionally hetero)arylalkoxy, or (optionally hetero)arylalkyl;
or
Ra is H and RS and R4 together form a di-, tri- or tetramethylene bridge
(pref. wherein the RS and R4 together have the cis configuration, e.g., where the
carbons carrying Ra and R4 have the R and S configurations, respectively);
(iii) RS is a substituted heteroarylalkyl, e.g., substituted with haloalkyl
or
RS is attached to one of the nitrogens on the pyrazolo portion of Formula 1
and is a moiety of Formula A
wherein X, Y and Z are, independently, N or C, and R8, R9, R11 and R12 are independently H or halogen (e.g., Cl or F), and RIO is halogen, alkyl, cycloalkyl, haloalkyl (e.g., trifluoromethyl), aryl (e.g., phenyl), heteroaryl (e.g., pyridyl (for example pyrid-2-yl), or thiadiazolyl (e.g., l,2,3-thiadiazol-4-yl)), diazolyl, triazolyl, tetrazolyl, arylcarbonyl (e.g., benzoyl), alkylsulfonyl (e.g., methylsulfonyl), heteroarylcarbonyl, or alkoxycarbonyl; provided that when X, Y, or Z is nitrogen, R8, R9, or RIO, respectively, is not present; and
(iv) R(, is H, alkyl, aryl, heteroaryl, arylalkyl (e.g., benzyl), arylamino (e.g.,
phenylamino), heterarylamino, N,N-dialkylamino, N,N-diarylamino, or N-aryl-N-
(arylakyl)amino (e.g., N-phenyl-N-(l,l'-biphen-4-ylmethyl)amino); and
(v) n=0 or 1;
(vi) when n=l, A is -C(Ri3Ri4)-
wherein Rn and RH, are, independently, H or CM alkyl, aryl, heteroaryl, (optionally hetero)arylalkoxy or (optionally hetero)arylalkyl;
in free, salt or prodrug form, including its enantiomers, diastereoisomers and
racemates.
[0009] The invention further provides compounds of Formula I as follows:
1.1 Formula I wherein RI is methyl and n=0;
1.2 Formula I or 1.1 wherein R\ is H or CM alkyl and at least one of R2 and
Ra is lower alkyl, such that when the carbon carrying R3 is chiral, it has the R
configuration, e.g., wherein both R2 and Rj are methyl, or wherein one is
hydrogen and the other isopropyl;
1.3 Formula I or 1.1 wherein R4 is H and at least one of R2 and R3 is
arylalkoxy;
1.4 Formula I wherein RI is methyl, R2, RB, and R4 are H, n=l, and R13 and
RH are, independently, H or CM alkyl (e.g., methyl or isopropyl);
1.5 Formula I or 1.1 wherein R2 is H and RI and R4 together form a tri- or
tetramethylene bridge, having the cis configuration, preferably wherein the
carbons carrying RI and R4 have the R and S configurations respectively;
1.6 Formula I, 1.1 or 1.5 wherein R5 is a substituted heteroarylmethyl, e.g.,
para-substituted with haloalkyl;
1.7 Formula I, 1.1, 1.2, 1.3, 1.4 or 1.5 wherein R5 is a moiety of Formula A
wherein Rg, Rg, RI i, and R12are H and RIO is phenyl;
1.8 Formula I, 1.1, 1.2, 1.3, 1.4 or 1.5 wherein RS is a moiety of Formula A
wherein Rg, RQ, RH, and R12 are H and RIO is pyridyl or thiadiazolyl;
1.9 Formula I, 1.1, 1.2, 1.3, 1.4 or 1.5 wherein R5 is a moiety of Formula A
wherein Rg, Rg, R\\, and R^ are, independently, H or halogen, and RIO is
haloalkyl;
1.10 Formula I, 1.1, 1.2, 1.3, 1.4 or 1.5 wherein R5 is a moiety of Formula A
wherein Rg, Rg, Rn, and R|2 are, independently, H, and RIO is alkyl sulfonyl;
1.11 any of the preceding formulae wherein R5 is attached to the 2-position
nitrogen on the pyrazolo ring;
1.12 any of the preceding formulae wherein R6 is benzyl;
1.13 any of the preceding formulae wherein R6 is phenylamino or
phenylalkylamino (e.g., benzylamino);
1.14 any of the preceding formulae wherein R6 is phenylamino;
1.15 any of the preceding formulae wherein X, Y, and Z are all C;
1.16 any of the preceding formulae wherein X, Y, and Z are all C and RIO is
phenyl or 2-pyridyl; and/or
1.17 any of the preceding formulae wherein the compounds inhibit
phosphodiesterase-mediated (e.g., PDE1-mediated, especially PDEIB-
mediated) hydrolysis of cGMP, e.g., with an IC20 of less than lµM, preferably
less than 25 nM in an immobilized-metal affinity particle reagent PDE assay,
for example, as described in Example 24;
in free or salt form.
[0010] For example, the Compounds of the Invention include 7,8-dihydro-[lH
or 2H]-imidazo[l,2-fl]pyrazolo[4,3-e]pyrimidin-4(5H)-ones of Formula la
(Formula Removed)

Formula la
wherein
(i) RI is H or CM alkyl [e.g., methyl];
(ii) R* is H and R2 and Ra are, independently, H or CM alkyl [e.g., R2 and R3 are both methyl, or R2 is H and Ra is isopropyl], aryl, or arylalkyl;
or R2 is H and RS and R4 together form a di-, tri- or tetramethylene bridge [pref. wherein the Ra and R4 have the cis configuration, e.g., where the carbons carrying RS and R4 have the R and S configurations respectively];
(iii) RS is attached to one of the nitrogens on the pyrazolo portion of formula I and is a substituted benzyl of formula B
(Formula B Removed)
Formula B
wherein R8, R9, R11 and R12 are independently H or halogen (e.g., Cl or F); and RIO is halogen, alkyl, cycloalkyl, haloalkyl (e.g., trifluorornethyl) aryl (e.g., phenyl), heteroaryl (e.g., pyridyl (for example pyrid-2-yl), or thiadiazolyl (e.g., l,2,3-thiadiazol-4-yl)), arylcarbonyl (e.g., benzoyl), alkyl sulfonyl or heteroarylcarbonyl; and
(iv) Re is H, alkyl, aryl, heteroaryl, arylalkyl [e.g., benzyl], arylamino [e.g., phenylamino], heteroarylamino, arylalkylamino, N,N-dialkylamino, N,N-
diarylamino, or N-aryl-N-(arylalkyl)amino [e.g. N-phenyl-N-(l,l'-biphen-4-ylmethyl)amino];
in free, salt or prodrug form.
[0011] The invention further provides compounds of Formula la as follows:
1.1: Formula la wherein RI is methyl;
1.2: Formula la or 1.1 wherein R4 is H and at least one of R2 and R3 is lower alkyl, such that when the carbon carrying R3 is chiral, it has the R configuration, e.g., wherein both R2 and R3 are methyl, or wherein one is hydrogen and the other isopropyl;
1.3: Formula la or 1.1 wherein R2 is H and R3 and R4 together form a tri- or tetramethylene bridge, having the cis configuration, preferably wherein the carbons carrying RS and R4 have the R and S configurations respectively; 1.4: Formula la, 1.1, 1.2 or 1.3 wherein R5 is a moiety of formula A wherein RS, Rg, RI i, and Ri2 are H and RIO is phenyl; 1.5: Formula la, 1.1, 1.2, or 1.3 wherein R5 is a moiety of formula A wherein R8, R 9 RH, and Ri2 are H and RIO is pyridyl or thiadiazolyl; 1.6: Formula la, 1.1, 1.2, 1.3, 1.4, or 1.5 wherein RS is attached to the 2-position nitrogen on the pyrazolo ring;
1.7: Formula la, 1.1, 1.2, 1.3, 1.4, 1.5 or 1.6 wherein Re is benzyl; 1.8: Formula la, 1.1, 1.2, 1.3, 1.4, 1.5 or 1.6 wherein R6 is phenylamino or phenylalkylamino (e.g., benzylamino); and/or 1.9: Formula la, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, or 1.8 wherein the compounds inhibit phosphodiesterase-mediated (e.g., PDE1-mediated, especially PDEIB-mediated) hydrolysis of cGMP, e.g., with an IC50 of less than 1 (µM, preferably less than 25 nM in an immobilized-metal affinity particle reagent PDE assay, for example, as described in Example 15; in free or salt form.
[0012] In an another embodiment, the Compounds of the Invention are
compounds of Formula I wherein
(i) RI is methyl;
(ii) R2, R3 and R4 are H;
(iii) n=l and Raand Rb are, independently, H or methyl;
(iv) RS is a moiety of Formula A wherein Rg, R9, RH and Ri2 are H and RIO
is phenyl, pyridyl (for example pyrid-2-yl), or thiadiazolyl (e.g., 1,2,3-
thiadiazol-4-yl);
(v) R6 is benzyl, phenylamino or benzylamino;
in free or salt form.
[0013] In another embodiment, the Compounds of the Invention are
compounds of Formula I wherein
(i) RI is methyl;
(ii) n=0;
(iii) R2 is H and R3 and Rt together form a tri-or tetra-methylene bridge
[pref. with the carbons carrying R3 and R4 having the R and S configuration
respectively]; or at least one of R2 and R3 is methyl, isopropyl or arylalkoxy
and RI is H; or R2 and R3 are H and RI is a CM alkyl;
(iv) RS is a substituted heteroarylmethyl, e.g., para-substituted with
haloalkyl; or
RS is a moiety of Formula A wherein Rg, Rg, Rn and Ri2 are H or halogen
and RIO is haloalkyl, phenyl, pyridyl (for example pyrid-2-yl), or thiadiazolyl
(e.g., l,2,3-thiadiazol-4-yl); and
(v) Re is benzyl, phenylamino or benzylamino;
in free or salt form.
[0014] In another embodiment, the Compounds of the Invention are compounds
of Formula la wherein
(i) RI is methyl;
(ii) R2 is H and R3 and R» together form a tri- or tetra-methylene bridge
[pref. with the carbons carrying R3 and R4 having the R and S configuration
respectively]; or R2 and R3 are each methyl and RA is H; or R2 and R4 are H
and R3 is isopropyl [pref. the carbon carrying R3 having the R
configuration];
(iii) R5 is a moiety of Formula A wherein R8, R9, R11, and Ri2 are H and RIO
is haloalkyl, phenyl, pyridyl (for example pyrid-2-yl), or thiadiazolyl (e.g.,
l,2,3-thiadiazol-4-yl); and
(iv) Re is benzyl, phenylamino or benzylamino;
in free or salt form.
[0015] For example, Compounds of the Invention include compounds
according to Formulae II, III and IV.
(Formula Removed)

Formula II
wherein
Ra and Rb are, independently, H or C1-4 alkyl;
R6 is phenylamino or benzylamino;
R10 is phenyl, pyridyl (for example pyrid-2-yl), or thiadiazolyl (e.g., 1,2,3-
thiadiazol-4-yl);
in free or salt form. (Formula III Removed)

Formula III
wherein
R2 is H and R3 and R4 together form a tri- or tetra-methylene bridge [pref. with
the carbons carrying R3 and R4 having the R and S configuration respectively];
or at least one of R2 and RS is methyl, isopropyl or arylalkoxy and R4 is H; or R2
and R3 are H and R4 is a CM alkyl;
R6 is phenylamino or benzylamino;
RIO is haloalkyl, phenyl, pyridyl (for example pyrid-2-yl), or thiadiazolyl (e.g.,
l,2,3-thiadiazol-4-yl);

Formula IV
in free or salt form.
(Formula Removed)R 2 is H and R3 and R4 together form a tri- or tetra-methylene bridge [pref. with
the carbons carrying RS and R4 having the R and S configuration respectively];
or at least one of R.2 and RS is methyl, isopropyl or afylalkoxy and R4 is H; or R2
and RS are H and R4 is a CM alkyl;
R6 is phenylamino or benzylamino;
RIO is phenyl, pyridyl (for example pyrid-2-yl), or thiadiazolyl (e.g., 1,2,3-
thiadiazol-4-yl);
in free or salt form.
[0016] Compounds of the Invention include, for example, the title compounds
of Examples 1-23 below.
[0017] If not otherwise specified or clear from context, the following terms as
used herein have the following meetings:
a. "Alkyl" as used herein is a saturated or unsaturated hydrocarbon moiety,
preferably saturated, preferably one to four carbon atoms in length,
which may be linear or branched, and may be optionally substituted,
e.g., mono-, di-, or tri-substitutcd, e.g., with halogen (e.g., chloro or
fluoro), hydroxy, or carboxy.
b. "Cycloalkyl" as used herein is a saturated or unsaturated nonaromatic
hydrocarbon moiety, preferably saturated, preferably comprising three
to nine carbon atoms, at least some of which form a nonaromatic mono-
or bicyclic, or bridged cyclic structure, and which may be optionally
substituted, e.g., with halogen (e.g., chloro or fluoro), hydroxy, or
carboxy.
c. "Aryl" as used herein is a mono or bicyclic aromatic hydrocarbon,
preferably phenyl, optionally substituted, e.g., with alkyl (e.g., methyl),
halogen (e.g., chloro or fluoro), haloalkyl (e.g., trifluoromethyl),
hydroxy, carboxy, or an additional aryl or heteroaryl (e.g., biphenyl or
pyridylphenyl).
d. "Heteroaryl" as used herein is an aromatic moiety wherein one or more
of the atoms making up the aromatic ring is sulfur or nitrogen rather
than carbon, e.g., pyridyl or thiadiazolyl, which may be optionally
substituted, e.g., with alkyl, halogen, haloalkyl, hydroxy or carboxy.
[0018] Compounds of the Invention may exist in free or salt form, e.g., as acid
addition salts. In this specification unless otherwise indicated language such as
Compounds of the Invention is to be understood as embracing the compounds in any
form, for example free or acid addition salt form, or where the compounds contain
acidic substituents, in base addition salt form. The Compounds of the Invention are
intended for use as Pharmaceuticals, therefore pharmaceutically acceptable salts are
preferred. Salts which are unsuitable for pharmaceutical uses may be useful, for
example, for the isolation or purification of free Compounds of the Invention or their
pharmaceutically acceptable salts, are therefore also included.
[0019] Compounds of the Invention may in some cases also exist in prodrug
form. For example when the compounds contain hydroxy or carboxy substituents, these substituents may form physiologically hydrolysable and acceptable esters. As used herein, "physiologically hydrolysable and acceptable ester" means esters of Compounds of the Invention which are hydrolysable under physiological conditions to yield acids (in the case of Compounds of the Invention which have hydroxy substituents) or alcohols (in the case of Compounds of the Invention which have carboxy substituents) which are themselves physiologically tolerable at doses to be administered. As will be appreciated the term thus embraces conventional pharmaceutical prodrug forms.
[0020] The invention also provides methods of making the Compounds of the
Invention, novel intermediates useful for making Compounds of the Invention, and methods of using the Compounds of the Invention for treatment of diseases and disorders as set forth below (especially treatment of diseases characterized by reduced dopamine Dl receptor signaling activity, such as Parkinson's disease, restless leg syndrome, depression, and cognitive impairment of schizophrenia).
DETAILED DESCRIPTION OF THE INVENTION
Methods of Making Compounds of the Invention
[0021] The compounds of the formula I and their pharmaceutically acceptable
salts may be made using the methods as described and exemplified herein and by
methods similar thereto and by methods known in the chemical art. Such methods
include, but not limited to, those described below. If not commercially available,
starting materials for these processes may be made by procedures, which are selectedfrom the chemical art using techniques which are similar or analogous to the synthesis of known compounds. All references cited herein are hereby incorporated in their entirety by reference.
[0022] Some individual compounds within the scope of this invention may
contain double bonds. Representations of double bonds in this invention are meant to include both the E and the Z isomer of the double bond. In addition, some compounds within the scope of this invention may contain one or more asymmetric centers. This invention includes the use of any of the optically pure stereoisomers as well as any combination of stereoisomers.
[0023] Melting points are uncorrected and (dec) indicated decomposition.
Temperature are given in degrees Celsius (°C); unless otherwise stated, operations are
carried out at room or ambient temperature, that is, at a temperature in the range of 18-
25 °C. Chromatography means flash chromatography on silica gel; thin layer
chromatography (TLC) is carried out on silica gel plates. NMR data is in the delta
values of major diagnostic protons, given in parts per million (ppm) relative to
tetramethylsilane (TMS) as an internal standard. Conventional abbreviations for signal
shape are used. Coupling constants (J) are given in Hz. For mass spectra (MS), the
lowest mass major ion is reported for molecules where isotope splitting results in
multiple mass spectral peaks Solvent mixture compositions are given as volume
percentages or volume ratios. In cases where the NMR spectra are complex, only
diagnostic signals are reported.
[0024] Terms and abbreviations:
Bu'OH = tert-butyl alcohol,
CAN = ammonium cerium (IV) nitrate,
DIPEA = diisopropylethylamine,
DMF = N,N-dimethylforamide,
DMSO = dimethyl sulfoxide,
Et2O = diethyl ether,
EtOAc = ethyl acetate,
equiv. = equivalent(s),
h = hour(s),
HPLC =high performance liquid chromatography,
K2CO3 = potassium carbonate, MeOH = methanol, NaHCCb = sodium bicarbonate, NHUOH = ammonium hydroxide, PMB = p-methoxybenzyl, POCls = phosphorous oxychloride, SOC12 = thionyl chloride, TFA = trifluoroacetic acid, THF = tetrahedrofuran.
[0025] The synthetic methods in this invention are illustrated below. The
significances for the R groups are as set forth above for formula I unless otherwise indicated.
[0026] In an aspect of the invention, intermediate compounds of formula IIb
can be synthesized by reacting a compound of formula Ila with a dicarboxylic acid, acetic anhydride and acetic acid mixing with heat for about 3 hours and then cooled:



(Formula Removed)
wherein R1 is H or Chalky! [e.g., methyl].
[0027] Intermediate IIe can be prepared by for example reacting a compound of
IIb with for example a chlorinating compound such as POCb, sometimes with small amounts of water and heated for about 4 hours and then cooled
: (Formula Removed)
)

[0028] Intermediate IId may be formed by reacting a compound of IIe with for
example a P'-X in a solvent such as DMF and a base such as K^COs at room temperature or with heating:
(Formula Removed)

wherein P1 is a protective group [e.g.P-methoxybenzyl group (PMB)]; X is a leaving group such as a halogen, mesylate, or tosylate.
[0029] Intermediate IIe may be prepared by reacting a compound of IId with
hydrazine or hydrazine hydrate in a solvent such as methanol and refluxed for about 4 hours and then cooled:
(Formula Removed)
[ 0030] Intermediate IIf can be synthesized by reacting a compound of IIe with
hydrazine or hydrazine hydrate in a solvent such as methoxymethanol and refluxed for about 30 min and then cooled:
(Formula Removed)
[ 0031] Intermediate Hg (wherein R13 is alkyl, aryl [e.g., phenyl], heteroaryl,
arylalkyl, or heteroarylalkyl), can be synthesized by reacting a compound of He with for example an aryl isothiocyanate or isocyanate in a solvent such as DMF and heated at 110 °C for about 2 days and then cooled:
(Formula Removed)
[ 0032] Intermediate Ilh may be synthesized from a compound of Hg by
removing the protective group P' with an appropriate method. For example, if P1 is ap-methoxybenzyl group, then it can be removed with Aids at room temperature or with TFA under heated conditions. Intermediate Ilh may also be prepared directly from a compound of Hf using the similar methods, but the yields are relatively low.
(Formula Removed)
[0033] Intermediate II-I can be prepared by for example reacting a compound
of Ilh with for example a chlorinating compound such as POCI3. The reaction may be carried out at atmospheric pressure and refluxed for about 2 days, or heated at 150-200 °C for about 10 min in a sealed vial with a microwave instrument.
(Formula Removed)
[ 0034] Intermediate IIJ can be prepared by reacting a compound of II-I with an
amino alcohol under basic condition in a solvent such as DMF. The reaction may be heated overnight and then
(Formula Removed)
[0035] Unless otherwise specified or defined, R2, R3 and R4 are the same as
those defined previously, e.g., with respect to Formula 1.
[0036] Intermediate UK can be formed by reacting a compound of IIJ with for
example a dehydrating agent such as SOCb in a solvent such as CHaCh at room temperature overnight or heated at 35 °C for about 4 hours, and then cooled:
(Table Removed)
Compound la and Ib may be formed by reacting a compound of Ilk
with for example a R5-X in a solvent such as DMF and a base such as K2CO3 at room temperature or with heating:
(Formula Removed)
wherein R5 is as defined previously [e.g. an optionally substituted benzyl group]; X is a leaving group such as a halogen, mesylate, or tosylate,
[0038] R5 may also be introduced earlier by for example reacting Hg with R5X
and then perform similar procedure as described above to form compound la and Ib, as long as R5 will not be cleaved off in the P1 deprotection step.
[0039] The second synthetic route is designated for the preparation of
compound la and Ib wherein R6 is an alkyl, aryl or heteroaryl group.
[0040] Intermediate Ilia (wherein R7 is aryl, preferably phenyl substituted with
Rg.12 corresponding to the substituted benzyl of Formula A supra) may be formed by reacting a compound of lie with an aldehyde R7CHO in a solvent such as EtOAc at 0°C or room temperature: 0
(Formula Removed)
[0041] Intermediate IHb can be prepared by for example reacting a compound
of IIIa with for example an aldehyde in a solvent such as DMF and heated overnight and then cooled:
(Formula Removed)
[0042] Intermediate IIIc may be synthesized from a compound of Illb by
removing the protective group P1 with an appropriate method. For example, if P1 is &p-methoxybenzyl group, then it can be removed with CAN at room temperature. Intermediate IIIc may also be prepared directly from a compound of Ilf using similar methods, but the yields are relatively low.
(Formula Removed)
[ 0043] Intermediate Ille can be prepared by reacting a compound of IIIc with
for example a chlorinating compound such as POCb. The reaction may be carried out at atmospheric pressure and refluxed for about 2 days, or heated at 150-200 °C for about 10 min in a sealed vial with a microwave instrument.

(Formula Removed)
[0044] Intermediate IIlf can be formed by reacting a compound of with an
amino alcohol under basic condition in a solvent such as DMF and heated overnight and then cooled:
(Formula Removed)
[ 0045] Compound la can be formed by reacting a compound of IIIf with for
example a dehydrating agent such as SO2CL3 in a solvent such as CH2Cl3 at room temperature overnight or heated at 35 °C for about 4 hours, and then cooled:
(Formula Removed)
[ 0046] There is an alternative approach for the synthesis of compound la and
Ib wherein R6 is an alkyl or aryl group. If a harsher condition is employed for the deprotection of Illb, then the R7CH2 group can be cleaved off too. For instance, if P1 is a p-methoxybenzyl group and R7 is a substituted phenyl group, then both P1 and R7CH2 can be cleaved with A1C13 at room temperature. Thus, Intermediate Hlg may be formed with this approach:
(Formula Removed)
[0047] Intermediate IIIh can be prepared by reacting a compound of III with
for example a chlorinating compound such as PO2CI2. The reaction may be carried out
at atmospheric pressure and refluxed for about 2 days, or heated at 150-200 °C for about 10 min in a sealed vial with a microwave instrument.
(Formula Removed)
0048] Intermediate III-I can be formed by reacting a compound of Illh with
an amino alcohol under basic condition in a solvent such as DMF and heated overnight and then cooled:

(Formula Removed)
[0049] Intermediate IHJ may be formed by reacting a compound of III-I with
for example a dehydrating agent such as SOCL2 in a solvent such as CH2CI2 or methanol at room temperature overnight or heated at 35 °C for about 4 hours, and then (Formula Removed)
[0050] Intermediate IIIJ may also be formed by reacting a compound of la
with for example a strong acid or Lewis acid such as
(Formula Removed)
[0051] Compound la and Ib may be formed by reacting a compound of IIIJ
with for example a R5-X in a solvent such as DMF and a base such as K2CO3 at room temperature or with heating:
(Formula Removed)
[ 0052] The third synthetic route is designated for the preparation of compound
la and Ib wherein R6 is hydrogen.

[0053] Intermediate IVa may be formed by for example reacting a compound
of He with POC13 and DMF:
(Formula Removed)
[ 0054] Intermediate IVb may be formed by reacting a compound of IVa with
for example a R5-X in a solvent such as DMF and a base such as K2CO3 at room temperature or with heating:
(Formula Removed)
[ 0055] Intermediate IVc may be synthesized from a compound of IVb by
removing the protective group P1 with an appropriate method. For example, if P1 is a PMB group, then it can be removed with CAN at room temperature:
(Formula Removed)
[ 0056] Intermediate IVd can be prepared by reacting a compound of IVc with
for example a chlorinating compound such as POCb and refluxed for about 2 days, or heated at 150-200 °C for about 10 min in a sealed vial with a microwave instrument and then cooled:
(Formula Removed)
[ 0057] Intermediate IVe can be formed by reacting a compound of IVd with an
amino alcohol under basic condition in a solvent such as DMF and heated overnight then cooled:
(Formula Removed)
[ 0058] Compound la may be formed by reacting a compound of IVe with for
example a dehydrating agent such as SOCl2 in a solvent such as CH2Cl2 at room temperature overnight or heated at 35 °C for about 4 hours, and then cooled. Similar to the methods described above, the R5 group in a compound of la can be cleaved off using an appropriate method, and then the obtained intermediate can react with another R5X to give compound la and Ib.
(Formula Removed)
[ 0059] The invention thus provides methods of making Compounds of the
Invention as described above, for example, comprising
(i) reacting a 7,8-dihydro-imidazo[l,2-o]pyrazolo[4,3-e]pyrimidin-4(5H)-one or a 7,8,9-trihydro-[lHor 2H]-pyrimido [l,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one with a compound of formula X-R5 wherein X is a leaving group, e.g., halogen, mesylate, or tosylate, and R5 is C2-9 alkyl, C cycloalkyl, heteroarylalkyl, or substituted arylalkyl, for example wherein R is a substituted benzyl of formula A as defined above, e.g., under basic conditions, for example wherein the 7,8-dihydro-imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one is a compound of Formula IIIJ:
(ii) (Formula Removed)
(iii) w herein Ri-e are as defined above, e.g., with reference to Formula I; and /or (ii) dehydrating a compound of Formula V
(Formula Removed)
wherein R1-6 and [A]n are as defined above, e.g., with reference to Formula I, e.g., using a dehydrating agent, for example thionyl chloride; and isolating the Compound of the Invention thus obtained.
Methods of using Compounds of the Invention
[0060] The Compounds of the Invention are useful in the treatment of diseases
characterized by disruption of or damage to cAMP and cGMP mediated pathways, e.g., as a result of increased expression of PDE1 or decreased expression of cAMP and cGMP due to inhibition or reduced levels of inducers of cyclic nucleotide synthesis, such as dopamine and nitric oxide (NO). By preventing the degradation of cAMP and cGMP by PDE1B, thereby increasing intracellular levels of cAMP and cGMP, the Compounds of the Invention potentiate the activity of cyclic nucleotide synthesis inducers.
[0061] The invention provides methods of treatment of any one or more of the
following conditions:
(i) Neurodegenerative diseases, including Parkinson's disease, restless
leg, tremors, dyskinesias, Huntington's disease, Alzheimer's disease,
and drug-induced movement disorders; (ii) Mental disorders, including depression, attention deficit disorder,
attention deficit hyperactivity disorder, bipolar illness, anxiety, sleep
disorders, cognitive impairment, dementia, psychostimulant
withdrawal, and drug addiction; (iii) Circulatory and cardiovascular disorders, including cerebrovascular
disease, stroke, congestive heart disease, hypertension, pulmonary
hypertension, and sexual dysfunction; (iv) Respiratory and inflammatory disorders, including asthma, chronic
obstructive pulmonary disease, and allergic rhinitis, as well as
autoimmune and inflammatory diseases; and/or (v) Any disease or condition characterized by low levels of cAMP
and/or cGMP (or inhibition of cAMP and/or cGMP signaling
pathways) in cells expressing PDE1.
comprising administering an effective amount of a Compound of the Invention to a human or animal patient in need thereof.
[0062] The invention also provides a method for enhancing or potentiating dopamine Dl intracellular signaling activity in a cell or tissue comprising contacting said cell or tissue with an amount of a Compound of the Invention sufficient to inhibit PDEIB activity.
[0063] The invention also provides a method for treating a PDE 1-related, especially PDE IB-related disorder, or a dopamine Dl receptor intracellular signaling pathway disorder, in a patient in need thereof comprising administering to the patient an effective amount of a Compound of the Invention that inhibits PDE IB, wherein PDE1B activity modulates phosphorylation of DARPP-32 and/or the GluRl AMPA receptor.
[0064] The present invention also provides
(i) a Compound of the Invention for use as a pharmaceutical, for
example for use in any method or in the treatment of any disease or
condition as hereinbefore set forth, (ii) the use of a Compound of the Invention in the manufacture of a
medicament for treating any disease or condition as hereinbefore set
forth, and (iii) a pharmaceutical composition comprising a Compound of the
Invention in combination or association with a pharmaceutically
acceptable diluent or carrier.
[0065] The words "treatment" and "treating" are to be understood accordingly
as embracing prophylaxis and treatment or amelioration of symptoms of disease as well as treatment of the cause of the disease
[0066] Compounds of the Invention are in particular useful for the treatment of
Parkinson's disease.
[0067] Compounds of the Invention may be used as a sole therapeutic agent,
but may also be used in combination or for co-administration with other active agents. For example, as Compounds of the Invention potentiate the activity of Dl agonists, such as dopamine, they may be simultaneously, sequentially, or contemporaneously administered with conventional dopaminergic medications, such as levodopa and levodopa adjuncts (carbidopa, COMT inhibitors, MAO-B inhibitors), dopamine agonists, and anticholinergics, e.g., in the treatment of a patient having Parkinson's disease.
[0068] Dosages employed in practicing the present invention will of course
vary depending, e.g. on the particular disease or condition to be treated, the particular Compound of the Invention used, the mode of administration, and the therapy desired. Compounds of the Invention may be administered by any suitable route, including orally, parenterally, transdermally, or by inhalation, but are preferably administered orally. In general, satisfactory results, e.g. for the treatment of diseases as hereinbefore set forth are indicated to be obtained on oral administration at dosages of the order from about 0.01 to 2.0 mg/kg. In larger mammals, for example humans, an indicated daily dosage for oral administration will accordingly be in the range of from about 0.75 to 150 mg, conveniently administered once, or in divided doses 2 to 4 times, daily or in sustained release form. Unit dosage forms for oral administration thus for example may comprise from about 0.2 to 75 or 150 mg, e.g. from about 0.2 or 2.0 to 50, 75 or 100 mg of a Compound of the Invention, together with a pharmaceutically acceptable diluent or carrier therefor.
[0069] Pharmaceutical compositions comprising Compounds of the Invention
may be prepared using conventional diluents or excipients and techniques known in the galenic art. Thus oral dosage forms may include tablets, capsules, solutions, suspensions and the like.
EXAMPLES
Example 1:
2-(Biphenyl-4-ylmethyl)-7,8-dihydro-5,7,7-trimethyl-[2H]-imidazo-[l,2-a] pyrazolo [4,3-e] pyrimidin-4(5H)-one
(Formula Removed)
[0070] (a) l-Methylpyrimidine-2,4,6(lH3H,5H)-trione
To a solution of malonic acid (80 g, 0.79 mol) and methylurea (50 g, 0.68 mol) in 180 ml of acetic acid at 70 °C, acetic anhydride (130 ml, 1.37 mol) is added slowly. After the completion of the addition, the reaction mixture is stirred at 90 °C for 3 hours, and then cooled to room temperature. The solvent is removed under reduced pressure, and the residue is treated with 350 mL of ethanol to precipitate out yellowish solid. The solid is recrystallized from ethanol to give 63.1 g product as crystalline solids (Yield: 65.8%). m.p. = 131.2-133.1 °C [Lit.1: m.p. = 130-131.5 °C].
[0071] (b) 6-Chloro-3-methylpyrimidine-2,4(lH,3H)-dione
Water (2.7 mL) is added dropwise to a suspension of 1 -methylpyrimidine-2,4,6(1 H,3H,5H)-trione (14.2 g, 100 mol) in POC13 (95 mL) at 0 °C. The reaction mixture is then heated at 80 °C for 5 hours. The resulting brownish solution is cooled, and POCls is evaporated under reduced pressure. The residue is treated with MeOH, and the obtained solid is recrystallized from ethanol to give 1 1.5 g product (Yield: 71.6%). m.p. = 279-282 °C (dec) [Lit.2: 280-282 °C]. !H NMR (400 MHz, DMSO-d6) 53.10 (S, 3H), 5.90 (S, 1H), 12.4 (br, 1H).
[0072] (c)6-Chloro-l-(4-methoxybenzyl)-3-methylpyrimidine-2,4(lH,3H)
dione
A mixture of 6-chloro-3-methylpyrimidine-2,4(lH,3H)-dione (16.2 g, 101 mmol),P
methoxybenzyl chloride (16.5 mL, 122 mmol) and potassium carbonate (7.0 g, 50.7
mmol) in anhydrous DMF (200 mL) is heated at 60 °C for 3 hours. Additionalpotassium carbonate (3.0 g, 21.7 mmol) is added, and the reaction mixture heated at 60 °C for another 3 hours. After hot filtration, the filtrate is evaporated to dryness under reduced pressure. The obtained oil is directly used for the synthesis in the next step. A small amount of product is further purified by silica-gel flash chromatography to give pure product as crystals. !H NMR (400 MHz, MeOH-d4) δ3.37 (s, 3H), 3.83 (s, 3H), 5.24 (s, 2H), 5.96 (s, 1H), 6.91 and 7.32 (AB, 4H, J= 6.8 Hz). MS (FAB) m/z 281.23 [M+Hf.
[0073] (d) 6-Hydrazinyl-l-(4-methoxybenzyl)-3-methylpyrimidine-
2,4(lH,3H)-dione
To a solution of 6-chloro-l-(4-methoxybenzyl)-3-methylpyrimidine-2,4(lH,3H)-dione (2.4 g 8.6 mmol) in EtOH (25 mL) and MeOH (50 mL), anhydrous hydrazine (1.2 mL) is added slowly. The reaction mixture is refluxed for three hours, and then cooled. A large amount of ether is added into the reaction mixture, and then filtered to give 2.0 g of product as crystalline solids (Yield: 84%). 'H NMR (DMSO-d6) δ3.13 (s, 3H), 3.73 (s, 3H), 4.42 (br, 1H), 5.03 (s, 2H), 5.15 (s, 1H), 6.88 and 7.15 (AB, 4H, J= 6.4 Hz), 8.08 (br, 1H). MS (FAB) m/z 277.28 [M+H]+.
[0074] (e) 7-(4-Methoxybenzyl)-5-methyl-lH-pyrazolo[3,4-d]pyrimidine-
4,6(5H,7H)-dione
To a solution of 6-hydrazinyl-l-(4-methoxybenzyl)-3-methylpyrimidine-2,4(lH,3H)-dione (0.45 g, 1.6 mmol) in DMF (2 mL), POC13 (0.3 mL, 3.3 mmol) is added dropwise at 0 °C. After the reaction mixture is stirred at 0 °C for 1 hour, the mixture is treated with methanol carefully to give white solid. The solid is further purified by chromatography to give 0.4 g product (Yield: 85%). !H NMR (DMSO-d6) £3.23 (s, 3H), 3.71 (s, 3H), 5.05 (s, 2H), 6.85 and 7,31 (AB, 4H, J= 11.6 Hz), 8.47 (s, 1H), 13.5 (br, 1H). MS (FAB) m/z 287.21 [M+H]+.
[0075] (f)2-(Biphenyl-4-ylmethyl)-7-(4-methoxybenzyl)-5-methyl-2H-
pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione
A mixture of 7-(4-methoxybenzyl)-5-methyl-lH-pyrazolo[3,4-d]pyrimidine-
4,6(5H,7H)-dione (0.312 g, 1.09 mmol), p-biphenylmethyl bromide (0.296g, 1.20
mmol) and potassium carbonate (0.151 g, 1.09 mmol) in acetone (20 mL) is stirred at room temperature overnight. The solvent is evaporated under reduced pressure. The residue is directly purified by chromatography to give 0.382 g product as white solids (Yield: 77.5). 'H NMR (400 MHz, CDC13) £3.37 (s, 3H), 3.75 (s, 3H), 5.15 (s, 2H), 5.34 (s, 2H), 6.81 (m, 2H), 7.27 (m, 3H), 7.47 (m, 4H), 7.60 (m, 4H), 7.87 (s, 1H). MS (FAB)m/z453.3[M+H]+.
[0076] (g)2-(Biphenyl-4-ylmethyl)-5-methyl-2H-pyrazolo[3,4-d]pyrimidine-
4,6(5H,7H)-dione
To a solution of 2-(biphenyl-4-ylmethyl)-7-(4-methoxybenzyl)-5-methyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (300 mg, 0.663 mmol) in THF (9 mL), a solution of ammonium cerium (IV) nitrate (1.82 g, 3.32 mmol) in water (3 mL) is added. The resulting orange solution is stirred at room temperature overnight. Another batch of CAN (1.82 g, 3.32 mmol) is added and the mixture is stirred for 6 hours, and then the third batch of CAN (1.82 g) is added, and the mixture is stirred at r.t. overnight. The reaction mixture is evaporated to dryness. The residue is treated with brine, and extracted with methylene chloride five times. The organic phase is combined and concentrated. The residue is purified by chromatography to give product as white solids with a high yield. ]H NMR (400 MHz, DMDO-d6) £3.16 (s, 3H), 5.37 (s, 2H), 7.38 (m, 3H), 7.46 (m, 2H), 7.65 (m, 4H), 8.59 (s, 1H), 11.6 (s, 1H). MS (FAB) m/z 333.3 [M+H]+.
[0077] (h)2-(Biphenyl-4-ylmethyl)-6-chloro-5-methyl-2H-pyrazolo[3,4-
d]pyrimidin-4(5H)-one
2-(biphenyl-4-ylmethyl)-5-methyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (25 mg, 0.075 mmol) is refluxed in POC13 (10 mL) for 60 hours, and the mixture is evaporated to dryness. The residue is purified by silica gel flash chromatography to give 26 mg product as white solids (Yield: 98.5%). !H NMR (400 MHz, CDC13) £3.68 (s, 3H), 5.45 (s, 2H), 7.39 (m, 3H), 7.43 (m, 2H), 7.59 (m, 4H), 8.01 (s, 1H). MS (FAB) m/z351.2[M+H]+.
[0078] (i) 2-(Biphenyl-4-ylmethyl)-6-( 1 -hydroxy-2-methylpropan-2-ylamino)-
5-methyl-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-one
A solution of 2-(biphenyl-4-ylmethyl)-6-chloro-5-methyl-2H-pyrazolo[3,4-
d]pyrimidin-4(5H)-one (26 mg, 0.074 mmol) and 2-amino-2-methyl-l-propanol (71
µ,L, 0.74 mmol) in DMF (1 mL) is heated at 110 °C overnight. The reaction mixture is
then purified by chromatography to give 21.1 mg product (Yield: 71%). MS (FAB) m/z
404.2 [M+H]+.
[0079] G)2-(Biphenyl-4-ylmethyl)-7,8-dihydro-5,7,7-trimethyl-[2H|-imidazo-
[l,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one
To a solution of 2-(biphenyl-4-ylmethyl)-6-(l-hydroxy-2-methylpropan-2-ylamino)-5-methyl-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (17 mg, 0.042 mmol) in methylene chloride (1 mL), is added 2.0 M CH2C12 solution of thionyl chloride (63 µL, 0.126 mmol) under argon. The reaction mixture is stirred at r.t. overnight. The reaction is quenched with 5% NaHCCh, and the resulting mixture is purified by chromatography to give 11 mg of the final product as white solids (Yield: 68%). !H NMR (400 MHz, DMSO-de + CDC13) 61.36 (s, 6H), 3.30 (s, 3H), 3.69 (s, 2H), 5.30 (s, 2H), 7.36 (m, 3H), 7.43 (m, 2H), 7.58 (m, 4H), 8.10 (s, 1H). MS (FAB) m/z 386.1 [M+H]+.
Example 2 C/5-(6aR*,10aS*)-l-(4-BenzoylbenzyI)-5,6a,7,8,9,10,10a-heptahydro-5-
methyI-3-(phenylamino)cyclohex[4,5]imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(lH)-one
(Formula Removed)
[0080] (a) 7-(4-Methoxybenzyl)-5-methyl-3-(phenylamino)-lH-pyrazolo[3,4-
d]pyrimidine-4,6(5H,7H)-dione
Phenyl isothiocyanate (3.9 mL, 32.7 mmol) is added to a suspension of 6-hydrazinyl-l-(4-methoxybenzyl)-3-methylpyrimidine-2,4(lH,3H)-dione (0.45 g, 1.6 mmol) in DMF (12 mL). The reaction mixture is heated at 120 °C for 40 hours, and then evaporated to remove solvent under reduced pressure. The residue is washed with hexanes, and then treated with MeOH (125 mL), and stored at -15 °C for 2 days to give a crystalline solid. The solid is recrystallized from CH3OH-EtOAc to afford 2.5 g product (Yield: 61%). 'H NMR (400 MHz, DMSO-d6) £3.21 (s, 3H), 3.73 (s, 3H), 5.01 (s, 2H), 6.88-7.36 (m, 9H). MS (FAB) m/z 378.3 [M+H]+.
[0081] (b) 5-Methyl-3-(phenylamino)-lH-pyrazolo[3,4-d]pyrimidine-
4,6(5H,7H)-dione
AlCb (0.733 g, 5.50 mmol) is added to a solution of 7-(4-methoxybenzyl)-5-methyl-3-(phenylamino)-lH-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (0.692g, 1.83 mmol) and anisole (40 µL, 0.367 mmol) in 1,2-dichloroethane (10 mL) under argon. The reaction mixture is stirred at room temperature for 30 min, and then quenched with water with cooling. The resulting suspension is filtered through a layer of celite and the celite is washed with MeOH (20 mL). The product is eluted from the celite with a large amount of THF. The THF eluent is evaporated to afford 0.47 g of product (Yield: 99%). MS (FAB) m/z 258.2 [M+H]+.
[0082] (c) 6-Chloro-5-methyl-3-(phenylamino)-lH-pyrazolo[3,4-d]pyrimidin-
4(5fl)-one
5-methyl-3-(phenylamino)-lH-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione(450mg, 1.75 mmol) is refluxed in POCb (20 mL) for 60 hours, and the mixture is evaporated to dryness. The residue is purified by silica gel flash chromatography to give 122 mg product as white solids and 207 mg starting material is recovered (Yield: 47%). MS (FAB) m/z 276.1 [M+H]+.
[0083] (d) 6-((lR,2R)-2-Hydroxycyclohexylamino)-5-methyl-3-
(phenylamino)-lH-pyrazolo[3,4-d]pyrimidin-4(5H)-one
A solution of 6-chloro-5-methyl-3-(pheny!amino)-lH-pyrazolo[3,4-d]pyrimidin-4(5H)-one (75.8 mg, 0.275 mmol), rrara-2-amino-cyclohexanol hydrochloride (83.4 mg, 0.55 mmol) and DIPEA (144 uL, 0.825 mmol) in DMF (3 mL) is heated at 110 °C overnight. The reaction mixture is evaporated to remove DMF under reduced pressure. The residue is then purified by chromatography to give 63.1 mg product (Yield: 64.7%). MS (ESI) m/z 355.0 [M+H]+.
[0084] (e) Cis-(6aR*, 1 OaS*)-5,6a,7,8,9,10,1 0a-heptahydro-5-methyl-3-
(phenylamino)cyclohex[4,5]imidazo[l,2-c]pyrazolo[4,3-e]pyrimidin-4(lH)-one
[0085] 2.0 M solution of thionyl chloride in CH2C12 (267 µL, 0.534 mmol) is
added to a solution of 6-((l/?*,2^*)-2-hydroxycyclohexylamino)-5-methyl-3-(phenylamino)-lH-pyrazolo[3,4-d]pyrimidin-4(5H)-one (63.1 mg, 0.178 mmol) in ClHbCla (6 mL) and THF (4 mL). The reaction mixture is stirred at r.t. overnight, and then quenched with 100 uL of 28% NI-fyOH. The resulting mixture is concentrated and purified by chromatography to give 25 mg product as white solids (Yield: 42%).MS (ESI) m/z 337.1 [M+H]+.
[0086] (f) Cis-(6&R* 1 OaS*)-1 -(4-Benzoylbenzyl)-5,6a,7,8,9,10,1 Oa-
heptahydro-5-methyl-3-(phenylamino)cyclohex[4,5]imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4( 1 H)-one
A mixture of Cis-(6aR *, 1 OaS*)-5,6a,7,8,9,10,1 Oa-heptahydro-5-methyl-3-(phenylamino)cyclohex[4,5]imidazo[l,2-a]pyrazolo[4,3-^]pyrimidin-4(lH)-one (7.1 mg, 0.021 mmol), 4-benzoylbenzyl bromide (5.8 mg, 0.021 mmol), and K2C03 (2.92 mg, 0.021 mmol) in DMF (1 mL) is stirred at room temperature overnight under argon. The reaction mixture is purified by a semi-preparative HPLC to give 3.5 mg of the final product (Yield: 31%). MS (ESI) m/z 531.1 [M+H]+.
Example 3
3-Benzyl-2-(biphenyl-4-yImethyl)-7,8-dihydro-5,7,7-trimethyl-[2^]-imidazo- [ 1,2-fl] py razolo [4,3-e] pyrimidin-4(5H)-one
(Formula Removed)
[0087] (a) 6-(2-(Biphenyl-4-ylmethylene)hydrazinyl)-1 -(4-methoxybenzyl)-3-
methylpyrimidine-2,4(l H,3H)-dione
A solution of 4-phenylbenzaldehyde (395 mg, 2.17 mmol) in EtOAc is slowly added into a dry ice cooled slurry of 6-hydrazinyl-l-(4-methoxybenzyl)-3-methylpyrimidine-2,4(1 H,3H)-dione (200 mg, 0.724 mmol) in EtOAc. After the addition, the reaction mixture is stirred at room temperature for 2 hours. The solvent is evaporated under reduced pressure, and the residue is triturated with MeOH, followed by filtration to give 256 mg product as pale yellow solids (Yield: 80.3%). !H NMR (400 MHz, DMSO-d6) δ3.17 (s, 3H), 3.71 (s, 3H), 5.22 (s, 2H), 5.59 (s, 1H), 6.91 and 7.21 (AB, J = 7.2 Hz, 4H), 7.37-7.81 (m, 9H), 8.36 (s, 1H), 10.67 (s, 1H). MS (FAB) m/z 441.4
[0088] (b)3-Benzyl-2-(biphenyl-4-ylmethyl)-7-(4-methoxybenzyl)-5-methyl-
2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione Acetic acid (4.4 mL) is added in to a solution of 6-(2-(Biphenyl-4-ylmethylene)hydrazinyl)-l-(4-methoxybenzyl)-3-methylpyrimidine-2,4(l//,3//)-dione (3.2 g, 7.26 mmol) in DMF (50 mL) and Bu'OH (25 mL) at 50 °C. Piperidine (8.7 mL) is mixed with a solution of 2-phenylacetaldehyde (8.5 mL, 72.6 mmol) in DMF (20 mL), and the resulting greenish solution is added in to the above solution. The reaction mixture is stirred at 40-45 °C for 36 hours under argon, and the solvent is evaporated under high vacuum. The residue is treated with MeOH (200 mL) to precipitate out 1.23 g product as sandy solids (Yield: 31.4%). MS (FAB) m/z 543.4 [M+H]+.
[0089] (c)3-Benzyl-2-(biphenyl-4-ylmethyl)-5-methyl-2H-pyrazolo[3,4-
d]pyrimidine-4,6(5H,7H)-dione
A solution of ammonium cerium (IV) nitrate (204 mg, 0.371 mmol) in water (0.6 mL) is added to a solution of 3-benzyl-2-(biphenyl-4-ylmethyl)-7-(4-methoxybenzyl)-5-methyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (40.3 mg, 0.0743 mmol) in THF (2 mL). The resulting orange solution is stirred at room temperature overnight. Another batch of CAN (204 mg, 0.371 mmol) is added and the mixture is stirred for 3 hours, and then the third batch of CAN (204 mg) is added, and the mixture is stirred at r.t. overnight. The reaction mixture is evaporated to dryness. The residue is treated with brine, and extracted with methylene chloride five times. The organic phase is combined and concentrated. The residue is purified by chromatography to give 11.6 mg product as white solids (Yield: 36.9%). !H NMR (400 MHz, acetone-d6) £3.27 (s, 3H), 4.51 (s, 2H), 5.33 (s, 2H), 7.13-7.62 (m, 14H), 10.26 (s, 1H). MS (FAB) m/z 423.2 [M+H]+.
[0090] (d)3-Benzyl-2-(biphenyl-4-ylmethyl)-6-chloro-5-methyl-2H-
pyrazolo[3,4-d]pyrimidin-4(5H)-one
3-benzyl-2-(biphenyl-4-ylmethyl)-5-methyl-2H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-dione (10 mg, 0.024 mmol) is refluxed in POC13 (10 mL) for 4 days, and then the mixture is evaporated to dryness. The residue is purified by silica gel flashchromatography to give 10.4 mg product as white solids (Yield: 100%). MS (FAB) m/z 441.2[M+H]+.
[0091] (e) 3-Benzyl-2-(biphenyl-4-ylmethyl)-6-(l -hydroxy-2-methylpropan-2-
ylimino)-5-methyl-6,7-dihydro-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-one
A solution of 3-Benzyl-2-(biphenyl-4-ylmethyl)-6-chloro-5-methyl-2H-pyrazolo[3,4-
d]pyrimidin-4(5H)-one (9.5 mg, 0.022 mmol) and 2-amino-2-methyl-l-propanol (21
µL, 0.22 mmol) in DMF (2 mL) is heated at 1 10 °C overnight. The reaction mixture is
then purified by chromatography to give 5.5 mg product (Yield: 52%). MS (FAB) m/z
494.4 [M+H]+.
[0092] (f)3-Benzyl-2-(biphenyl-4-ylmethyl)-7,8-dihydro-5,7,7-trimethyl-[2H]-
imidazo-[l,2-fl]pyrazolo[4,3-e]pyrimidin-4(5H)-one
A 2.0 M solution of thionyl chloride (25 µL, 0.050 mmol) in CH2Cl2 is added into a solution of 3-benzyl-2-(biphenyl-4-ylmethyl)-6-(l-hydroxy-2-methylpropan-2-ylimino)-5-methyl-6,7-dihydro-2H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (5.0 mg, 0.010 mmol) in methylene chloride (1 mL). The reaction mixture is stirred at r.t. overnight, and then quenched with 5% NaHCOs. The resulting mixture is purified by chromatography to give 3.2 mg of the final product (Yield: 67%).MS (FAB) m/z 476.4
Example 4
l-(Biphenyl-4-ylmethyl)-7,8-dihydro-5,7,7-trimethyl-3-(phenylamino)-[lH|-imidazo-[l,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one
(Formula Removed)
[0093] The synthesis method is analogous to example 2 wherein 2-amino-2-
methyl-1-propanol is added in step (d) instead of trans-2-amino-cyclohexanol hydrochloride; and p-biphenylmethyl bromide is added in step (f) instead of benzoylbenzyl bromide.
Example 5
l-(4-(l,2,3-thiadiazol-4-yl)benzyl)-7,8-dihydro-5,7,7-trimethyl-3-(phenylamino)-[lH]-imidazo- [1,2-a] pyrazolo [4,3-e] pyrimidin-4(5H)-one
(Formula Removed)
[0094] The synthesis method is analogous to example 2 wherein 2-amino-2-
methyl-1-propanol is added in step (d) instead of trans-2-amino-cyclohexanol hydrochloride; and 4-(l,2,3-thiadiazol-4yl)benzyl bromide is added in step (f) instead of benzoylbenzyl bromide.
Example 6
l-(Biphenyl-4-ylmethyl)-3-((biphenyl-4-yImethyl)(phenyl)amino)-7,8-dihydro-5,7,7-trimethyl-[lH]-imidazo-[l,2-a]pyrazolo[4,3-^]pyrimidin-4(5H)-one
(Formula Removed)
[0095] The synthesis method is analogous to example 2 wherein 2-amino-2-
methyl-1-propanol is added in step (d) instead of trans-2-amino-cyclohexanol hydrochloride; andp-biphenylmethyl bromide is added in step (0 instead of benzoylbenzyl bromide.
Example 7 Cw-(6aR*,10aS*)-5,6a,7,8,9,10,10a-heptahydro-5-methyl-3-(phenylamino)-l-(4-
(pyridin-2yI)benzyl)-cyclohex[4,5]imidazo[l,2-a]pyrazolo[4,3-e]pyimidin-4-(lH)-one
(Formula Removed)
[ 0096] The synthesis method is analogous to example 2 wherein 2-(4-
(bromomethyl)phenyl)pyridine is added in step (f) instead of benzoylbenzyl bromide.
Example 8
Cw-(6aR*,10aS*)-2-(4-(Pyridin-2yl)benzyl)-5,6a,7,8,9,10,10a-heptahydro-5-methyI-3-(phenylamino)cyclohex[4,5]imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one
(Formula Removed)
[0097] The synthesis method is analogous to example 2 wherein 4-pyrid-2-
ylbenzyl bromide is added in step (f) instead of benzoylbenzyl bromide.
Example 9
41
C/5-(6sRMOaS)-3-(Benzyl)-5,6a,7,8,9,10,10a-heptahydro-5-methyl-2-(4-(l,2,3-thiadiazol-4-yl)benzyl)- cyclohex[4,5]imidazo[l,2-a]pyrazolo[4,3-e ]pyi'iniidin-4(2H)-one
(Formula Removed)
[0098] The synthesis method is analogous to example 3 wherein 4-(l,2,3-
thiadiazol-4yl)benzaldehyde and DMF is added in step (a) instead of 4-phenylbenzaldehyde and heated overnight; and ^ow-2-amino-cyclohexanol hydrochloride is added in step (e) instead of 2-amino-2-methyl-l-propanol.
Example 10
Cw-(6aRMOaS*)-3-(Benzyl)-2-(4-Biphenyl-4-ylmethyl)-5,6a,7,8,9,10,10a-heptahydro-5-methyl-cyclohex[4,5]imidazo[l,2-a]pyrazolo[4,3-e ]pyrimidin-4(2H)-one
(Formula Removed)
[0099] The synthesis method is analogous to example 3 wherein trans-2-amino-
cyclohexanol hydrochloride is added in step (e) instead of 2-amino-2-methyl-l-propanol.
Example 11
(R)-3-(Benzyl)-2-(biphenyl-4-ylmethyI)-7,8-dihydro-7-isopropyl-5-methyl-[2H]-imidazo- [ 1,2-a]pyrazolo[4,3-e] pyrimidin-4(5H)-one
(Formula Removed)
[0100] The synthesis method is analogous to example 3 wherein (R)-2-amino-
3-methylbutan-l-ol is added in step (e) instead of 2-amino-2-methyl-l-propanol.
Example 12
(6a/f,9aiS)-5,6a,7,8,9,9a-hexahydro-5-methyI-3-(phenylamino)-2-(4-Pyridin-2yl)-
benzyl)-cyclopent[4,5]imidazo[l,2-fl]pyrazolo[43-£]pynmidin-4(2H)-one
(Formula Removed)
[ 0101] The synthesis method is analogous to example 2 wherein (lR,2R)-2-
amino-cyclopentanol is added in step (d) instead of trans-2-amino-cyclohexanol hydrochloride; and 2-(4-(bromomethyl)phenyl)pyridine is added in step (f) instead of benzoylbenzyl bromide.
Example 13
(6ar,9aS)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-l-(4-Pyridin-2yl)-
benzyl)-cyclopent[4,5]imidazo[l,2-a]pyrazolo[4,3- e ]pyrimidin-4(lH-one

(Formula Removed)
[0102] The synthesis method is analogous to example 2 wherein (lR,2R)-2-
amino-cyclopentanol is added in step (d) instead of trans-2-amino-cyclohexanol hydrochloride; and 4-pyrid-2-ylbenzyl bromide is added in step (f) instead of benzoylbenzyl bromide.
Example 14
(6aR,9aS)-3-(benzyIamino)-5,6a,7,8,9,9a-hexahydro-5-methyI-2-(4-Pyridin-2yl)-
benzyl)-cyclopent[4,5]imidazo[l,2-fl]pyrazolo[4,3-e ]pyrimidin-4(2H)-one
(Formula Removed)
[0103] The synthesis method is analogous to example 2 wherein benzyl
isothiocyanate is added in step (a) instead of phenyl isothiocyanate; (1R,2R)-2-amino-cyclopentanol is added in step (d) instead of frvmy-2-amino-cyclohexanol hydrochloride; and 4-pyrid-2-ylbenzyl bromide is added in step (f) instead of benzoylbenzyl bromide.
Example 15
(6aR,9aS)-3-(phenylamino)-5,6a,7,8,9,9a-hexahydro-5-methyl-2-(biphenyl-4-
ylmethyl)-cyclopent[4,5]imidazo[l,2-a]pyrazolo[4,3-^]pyrimidin-4(2H)-one
(Formula Removed)
[0104] The synthesis method is analogous to example 2 wherein (lR,2R)-2-
amino-cyclopentanol is added in step (d) instead of fraw-2-amino-cyclohexanol hydrochloride; and 4-(bromomethyl)biphenyl is added in step (f) instead of benzoylbenzyl bromide.
Example 16
2-(Biphenyl-4-ylmethyl)-7,8,9-trihydro-5,8,8-trimethyl-3-(phenylamino)-[2H]-
pyrimido-[l,2-a]pyrazolo[4,3-e]pyrimidm-4(5H)-one(
Formula Removed)
[0105] The synthesis method is analogous to example 2 wherein 3-amino-2,2-
dimethyl- 1-propanol is added in step (d) instead of trans-2-amino-cyclohexanol hydrochloride; and 4-(bromomethyl)biphenyl is added in step (f) instead of benzoylbenzyl bromide. Example 17
imidazo-[l,2-fl]pyrazolo[4,3-e]pyrimidin-4(5H)-one[0106] The synthesis method is analogous to example 2 wherein (R)-2-
aminoprop-1-ol is added in step (d) instead of trans-2-amino-cyclohexanol hydrochloride; and 4-(bromomethyl)biphenyl is added in step (f) instead of benzoylbenzyl bromide.
Example 18
(8R)-2-(Biphenyl-4-ylmethyl)-7,8-dihydro-5,8-dimethyl-3-(phenylamino)-[2H-imidazo-[l ,2-e] pyrazolo [4,3-e ] pyrimidin-4(5H)-one
(Formula Removed)
[0107] The synthesis method is analogous to example 2 wherein (R)-l-
aminopropan-2-ol is added in step (d) instead of trans-amino-cyclohexanol hydrochloride; and 4-(bromomethyl)biphenyl is added in step (f) instead of benzoylbenzyl bromide.
Example 19
(7R)-2-(Biphenyl-4-ylmethyl)-7,8-dihydro-3-(pheaylamino)-5-methyl-7-(l-
methylethyl)-[2H]-imidazo-[l,2-α]pyrazolo[4,3-e]pynmidin-4(5H)-one
(Formula Removed)
[0108] The synthesis method is analogous to example 2 wherein (R)-2-amino-
3-methylbutan-l-ol is added in step (d) instead of trans-2-amino-cyclohexanolhydrochloride; and 4-(bromomethyl)biphenyl is added in step (f) instead of benzoylbenzyl bromide.
Example 20
(6aR,9aS)-3-(phenylamino)-5,6a,7,8,9,9a-hexahydro-5-methyI-2-(4-(trifluoromethyl)-benzyl)-cyclopent[4,5]imidazo[l,2-fl]pyrazolo[4,3-e]pyrimidin-4(2H)-one
(Formula Removed)
[0109] The synthesis method is analogous to example 2 wherein (lR,2R)-2-
amino-cyclopentanol is added in step (d) instead of trans-2-amino-cyclohexanol hydrochloride; an p-trifluoromethyl benzyl bromide is added in step (f) instead of benzoylbenzyl bromide.
Example 21
(6aR,9aS)-3-(phenylamino)-5,6a,7,8,9,9a-hexahydro-5-methyl-2-((6-trifluoromethyl)-pyridin-3-yl)methyl)-cyclopent[4,5]imidazo[l,2-fl]pyrazolo[4,3-e]pyr\midin-4(2H)-one
(Formula Removed)
[0110] The synthesis method is analogous to example 2 wherein (]R,2R)-2-
amino-cyclopentanol is added in step (d) instead of frara'-2-amino-cyclohexanol hydrochloride; and 5-(bromomethyl)-2-(trifluoromethyl)pyridine is added in step (f) instead of benzoylbenzyl bromide.
Example 22
(6aR,9aS)-3-(phenylamino)-5,6a,7,8,9,9a-hexahydro-5-methyl-2-(3-fluoro-4-(trifluoromethyl)-benzyl)-cyclopent[4,5]imidazo[l,2-a]pyrazolo[4,3-e]pynmidin-4(2H)-one
(Formula Removed)
[0111] The synthesis method is analogous to example 2 wherein (lR,2R)-2-
amino-cyclopentanol is added in step (d) instead of trans-2-amino-cyclohexanol
hydrochloride; and 3-fluoro-4-trifluoromethyl-benzyl bromide is added in step (f) instead of benzoylbenzyl bromide.
Example 23
(6aR,9aS)-3-(phenyIamino)-5,6a,7,8,9,9a-hexahydro-5-methyl-2-(4-methylsulfonyl-benzyl)-cyclopent[4,5]imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-one(Formula Removed)
[0112] The synthesis method is analogous to example 2 wherein (lR,2R)-2-
amino-cyclopentanol is added in step (d) instead of trans-2-amino-cyclohexanol hydrochloride; and p-methylsulfonyl-benzyl bromide is added in step (f) instead of benzoylbenzyl bromide.
Example 24
Measurement of PDEIB inhibition in vitro using IMAP Phosphodiesterase Assay
Kit
[0113] Phosphodiesterase IB (PDEIB) is a calcium/calmodulin dependent
phosphodiesterase enzyme that converts cyclic guanosine monophosphate (cGMP) to
5'-guanosine monophosphate (5'-GMP). PDEIB can also convert a modified cGMP
substrate, such as the fluorescent molecule cGMP-fluorescein, to the corresponding GMP-fluorescein. The generation of GMP-fluorescein from cGMP-fluorescein can be quantitated, using, for example, the IMAP (Molecular Devices, Sunnyvale, CA) immobilized-metal affinity particle reagent.
[0114] Briefly, the IMAP reagent binds with high affinity to the free 5'-
phosphate that is found in GMP-fluorescein and not in cGMP-fluorescein. The
resulting GMP-fluorescein - IMAP complex is large relative to cGMP-fluorescein.
Small fluorophores that are bound up in a large, slowly tumbling, complex can be
distinguished from unbound fluorophores, because the photons emitted as they
fluoresce retain the same polarity as the photons used to excite the fluorescence.
[0115] In the phosphodiesterase assay, cGMP-fluorescein, which cannot be
bound to IMAP, and therefore retains little fluorescence polarization, is converted to
GMP-fluorescein, which, when bound to IMAP, yields a large increase in fluorescence
polarization (Amp). Inhibition of phosphodiesterase, therefore, is detected as a
decrease in Amp.
[0116] Enzyme assay
Materials: All chemicals are available from Sigma-Aldrich (St. Louis, MO) except for IMAP reagents (reaction buffer, binding buffer, FL-GMP and IMAP beads), which are available from Molecular Devices (Sunnyvale, CA).
Assay: 3',5'-cyclic-nucleotide-specific bovine brain phosphodiesterase (Sigma, St.
Louis, MO) is reconstituted with 50% glycerol to 2.5 U/ml. One unit of enzyme will
hydrolyze 1.0 umole of 3',5'-cAMP to 5'-AMP per min at pH 7.5 at 30°C. One part
enzyme is added to 1999 parts reaction buffer (30 µM CaCl2, 10 U/ml of calmodulin
(Sigma P2277), l0mM Tris-HCl pH 7.2, l0mM MgCl2, 0.1% BSA, 0.05% NaN3) to
yield a final concentration of 1.25mU/ml. 99 ul of diluted enzyme solution is added
into each well in a flat bottom 96-well polystyrene plate to which 1 ul of test
compound dissolved in 100% DMSO is added. The compounds are mixed and pre-
incubated with the enzyme for 10 min at room temperature.
[0117] The FL-GMP conversion reaction is initiated by combining 4 parts
enzyme and inhibitor mix with 1 part substrate solution (0.225 µM) in a 384-well microtiter plate. The reaction is incubated in dark at room temperature for 15 min. The reaction is halted by addition of 60 µl of binding reagent (1:400 dilution of IMAP beads in binding buffer supplemented with 1:1800 dilution of antifoam) to each well of the 384-well plate. The plate is incubated at room temperature for 1 hour to allow IMAP binding to proceed to completion, and then placed in an Envision multimode
microplate reader (PerkinElmer, Shelton, CT) to measure the fluorescence polarization
(Amp).
[0118] A decrease in GMP concentration, measured as decreased Amp, is
indicative of inhibition of PDE activity. ICso values are determined by measuring
enzyme activity in the presence of 8 to 16 concentrations of compound ranging from
0.0037 nM to 80,000 µM and then plotting drug concentration versus AmP, which
allows ICso values to be estimated using nonlinear regression software (XLFit; IDBS,
Cambridge, MA).
[0119] The compounds of Examples 1-14 have ICso values of less than lµM in
this assay, generally less than 10 µM.








CLAIMS
1. A 7,8-dihydro-[lHor 2H]-imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one or
7,8,9-trihydro-[lHor2H]-pyrimido[l,2-(7]pyrazolo[4)3-e]pyrimidin-4(5H)-one,
substituted at the 1 or 2 position with C2-9 alkyl, C3.9 cycloalkyl, heteroarylalkyl, or
substituted arylalkyl, in free, salt or prodrug form.
2. The compound according to claim 1 of Formula I
(Formula Removed)
wherein
(i) RI is H or CM alkyl [e.g., methyl];
(ii) R4 is H or C 1.4 alkyl and R2 and R3 are, independently, H or CM alkyl, aryl,
heteroaryl, heteroarylalkoxy, arylalkoxy, heteroarylaklyl, or arylalkyl;
or RI is H and R3 and R4 together form a di-, tri-, or tetra-methylene bridge;
(iii) RS is a substituted heteroarylalkyl, e.g., substituted with haloalkyl or R5
is attached to one of the nitrogens on the pyrazolo portion of Formula A and is a
moiety of Formula A
formula A
wherein X, Y and Z are, independently, N or C; R8, R9, R11 and R12are independently
H or halogen; and RIO is halogen, alkyl, cycloalkyl, haloalkyl, aryl, heteroaryl, or
thiadiazolyl, diazolyl, triazolyl, tetrazolyl, arylcarbonyl, alkylsulfonyl,
heteroarylcarbonyl, or alkoxycarbonyl; provided that when X, Y, or Z is nitrogen, R8,
R9 or R10 respectively, is not present;
(iv) R6 is H, alkyl, aryl, heteroaryl, arylalkyl, arylamino, heterarylamino, N,N-
dialkylamino, N,N-diarylamino, or N-aryl-N-(arylakyl)amino; and
(v) n=0 or 1;
(vi) when n=l, A is -C(R13R14)-
wherein R13 and R14are, independently, H or CM alkyl, aryl, heteroaryl, heteroarylalkoxy, arylalkoxy, heteroarylalkyl or arylalkyl; in free, salt or prodrug form-.
3. The compound according to claim 2 which is a compound of Formula II
(Formula ll Removed)
Formula II
wherein
Ra and Rb are, independently, H or CM alkyl; Re is phenylamino or benzylamino; RIO is phenyl, pyridyl, or thiadiazolyl; in free or salt form.
4. The compound according to claim 2 which is a compound of Formula III
(Formula III Removed)



Wherein
R2 is H and R3 and R4 together form a tri- or tetra-methylene bridge [pref. with the carbons carrying R3 and R4 having the R and S configuration respectively]; or at least one of R2 and R3 is methyl, isopropyl or arylalkoxy and R4 is H; or R2 and R3 are H and R4 is a CM alkyl; R6 is phenylamino or benzylamino;
RIO is haloalkyl, phenyl, pyridyl (for example pyrid-2-yl); or thiadiazolyl (e.g., l,2,3-thiadiazol-4-yl); in free or salt form.
5. The compound according to claim 2 which is a compound of Formula IV
(Formula V Removed)Formula IV wherein
R2 is H and R3 and R4 together form a tri- or tetra-methylene bridge [pref. with the carbons carrying R3 and R4 having the R and S configuration respectively]; or at least one of R2 and R3 is methyl, isopropyl or arylalkoxy and R1 is H; or R2 and R3 are H and R4 is a CM alkyl; R6is phenylamino or benzylamino; RIO is phenyl, pyridyl, or thiadiazolyl; in free or salt form.
6. The compound according to claim 1 of formula Ia
(Formula Removed)Formula la
wherein (i) Ri is H or C1-4 alkyl;
(ii) R4 is H and R2 and RI are, independently, H or CM alkyl, aryl, or arylalkyl; or R.2 is H and RS and R4 together form a di-, tri- or tetramethylene bridge;
(iii) RS is attached to one of the nitrogens on the pyrazolo portion of formula I and is a substituted benzyl of formula B
(Formula B Removed)Formula B
wherein R8, R9, R11 and R112 are independently H or halogen; and RIO is halogen, alkyl, cycloalkyl, haloalkyl, aryl, heteroaryl, arylcarbonyl, or heteroarylcarbonyl, and(iv) R6 is H, alkyl, aryl, heteroaryl, arylalkyl, arylamino, heteroarylamino, arylalkylamino, N,N-dialkylamino, N,N-diarylamino, or N-aryl-N-(arylalkyl)amino;
in free, salt or prodrug form.
7. The compound according to claim 6 which is a compound of Formula VFormula V
wherein
R2 is H and R3 and R4 together form a tri- or tetra-methylene bridge [pref. with
the carbons carrying R3 and R4 having the R and S configuration respectively];
or R2 and R3, are each methyl and R4 is H; or R2 and R4 are II and R3 is
isopropyl [pref. the carbon carrying R^ having the R configuration];
R6 is phenylamino or benzylamino,
RIO is phenyl, pyridyl, or thiadiazolyl;
in free or salt form.

8. A compound selected from the following:
2-(Biphenyl-4-ylmethyl)-7,8-dihydro-5,7,7-trimethyl-[2H]-imidazo-[l,2-
fl]pyrazolo[4,3-e]pyrimidin-4(5H)-one;
Cis-(6αR *, 1 0aS*)-1 -(4-Benzoylbenzyl)-5,6a,7,8,9,10,1 Oa-heptahydro-5-
methyl-3-(phenylammo)cyclohex[4,5]imidazo[l,2-fl]pyrazolo[4,3-
e]pyrimidin-4( 17/)-one;3-Benzyl-2-(biphenyl-4-ylmethyl)-7,8-dihydro-5,7,7-trimethyl-[2H]-
imidazo-[l,2-fl]pyrazolo[4,3-e]pyrimidin-4(5H-one;
l-(Biphenyl-4-ylmethyl)-7,8-dihydro-5,7,7-trimethyl-3-(phenylamino)-[lH]-
imidazo-[l,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-°ne;
l-(4-(l,2,3-thiadiazol-4-yl)benzyl)-7,8-dihydro-5,7,7-trimethyl-3-
(phenylamino)-[lHl-imidazo-[],2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one;
l-(Biphenyl-4-ylmethyl)-3-((biphenyl-4-ylmethyl)(phenyl)amino)-7,8-
dihydro-5,7,7-trimethyl-[lH]-imidazo-[l,2-a]pyrazolo[4,3-e]pyrimidin-
4(5H)-one;
Cis-(6aR *, 1 OaS*)-5,6a, 7,8,9,10,1 Oa-heptahydro-5-methyl-3-(phenylamino)-
l-(4.(pyridin-2yl)benzyl)benzyl)-cyclohex[4,5]imidazo[l,2-a]pyrazolo[4,3-
e]pyrimidin-4-(lH)-one;
Cis-(6aR * 1 Oa5*)-2-(4-(Pyridin-2yl)benzyl)-5,6a,7,8,9,10,1 Oa-heptahydro-5-
methyl-3-(phenylamino)cyclohex[4,5]imidazo[l,2-(7]pyrazolo[4,3-
e]pyrimidin-4(2 H)-one;
Cw-(6a7R*10a5'*)-3-(Benzyl)-5)6a,7,8,9,10,10a-heptahydro-5-methyl-2-(4-
(l,2,3-thiadiazol-4-yl)benzyl)- cyclohex[4,5]imidazo[l,2-a]pyrazolo[4,3-
e]pyrimidin-4(2//)-°ne;
Cis-(6aR * 1 Oa6'*)-3-(Benzyl)-2-(4-Biphenyl-4-ylmethyl)-5,6a,7,8,9,10,1 Oa-
heptahydro-5-methyl-cyclohex[4,5]imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-
4(2/0-one;
(R)-3-(Benzyl)-2-(biphenyl-4-ylmethyl)-7,8-dihydro-7-isopropyl-5-methyl-
[2H]-imidazo-[l,2-£/]pyrazolo[4,3-e]pyrimidin-4(5/H)-one;
(6aR,9a5)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-2-(4-Pyridin-
2yl)-benzyI)-cyclopent[4,5]imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(2H)-
one;
(6a/?,9alS)-5,6a,7,8,9,9a-hexahydro-5-methyl-3-(phenylamino)-l-(4-Pyridin-
2yl)-benzyl)-cyclopent[4,5]imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(l//)-
one;
(6a^,9a5)-3-(benzylamino)-5,6a,7,8,9,9a-hexahydro-5-methyl-2-(4-Pyridin-
2yl)-benzyl)-cyclopent[4,5]imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(2//)-
one;
(6aR,9aS-(phenylamino)-5,6a,7,8,9,9a-hexahydro-5-inethyl-2-(biphenyl-
4-ylmethyl)-cyclopent[4,5]imidazo[l,2-(3]pyrazolo[4,3-e]pyrimidin-4(2/H)-
one;
2-(Biphenyl-4-ylmethyl)-7,8,9-trihydro-5,8,8-trimethyl-3-(phenylamino)-
[2H]-pyrimido-[ 1,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one;
(7R)-2-(Biphenyl-4-ylmethyl)-7,8-dihydro-5,7-dimethyl-3-(phenylamino)-
[2/H-imidazo-[l,2-a]pyrazolo[4,3-e]pyrimidin-4(5/f)-one;
(8R)-2-(Biphenyl-4-ylmethyl)-7,8-dihydro-5,8-dimethyl-3-(phenylamino)-
[2H]-imidazo-[l,2-α]pyrazolo[4)3-e]pyrimidin-4(5H)-one;
(7R)-2-(Biphenyl-4-ylmethyl)-7,8-dihydro-3-(phenylamino)-5-methyl-7-(l-
methylethyl)-[2H]-imidazo-[l,2-(7]pyrazolo[4,3-e]pyrimidin-4(5H)-one;
(6aR,9aS)-3-(phenylamino)-5)6a,7)8,9,9a-hexahydro-5-methyl-2-(4-
(trifluoromethyl)-benzyl)-cyclopent[4,5]imidazo[l,2-(3]pyrazolo[4,3-
e]pyrimidin-4(2H)-one;
(6aR,9aS)-3-(pheny\ammo)-5,6'd,7,8,9,9a-he\ahydro-5-melhy\-2-((6-
trifluoromethyl)-pyridin-3-yl)methyl)-cyclopent[4,5]imidazo[l,2-
a]pyrazolo[4,3-e]pyrimidin-4(2H)-one;
(6aR,9aS)- 3-(phenylamino)-5,6a,7,8,9,9a-hexahydro-5-methyl-2-(3-fluoro-
4-(trifluoromethyl)-benzyl)-cyclopent[4,5]imidazo[l,2-a]pyrazolo[4,3-
e]pyrimidin-4(2H)-one; and
(6aR,9a S)-3-(phenylamino)-5,6a,7,8,9,9a-hexahydro-5-methyl-2-(4-
methylsulfonyl-benzyl)-cyclopent[4,5]imidazo[l,2-a]pyrazolo[4,3-
e]pyrimidin-4(2H)-one;
in free or salt form, or in pure enantiomeric form.
9. A pharmaceutical composition comprising a compound according to any of the preceding claims in admixture with a pharmaceutically acceptable diluent or carrier.
10. A method of making a compound according to any one of claims 1, 2, 3, 4, 5, 6, 7 or 8 comprising reacting a 7,8-dmydro-imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one or a 7,8,9-trihydro-[lH or 2H]-pyrimidq [l,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one with a compound of formula X-Rs wherein X is a leaving group and RS is C2-9 alkyl, C3-9 cycloalkyl, heteroarylalkyl, or substituted arylalkyl, and isolating the , compound according to claim 1, 2, 3, 4, 5, 6, 7 or 8 thus obtained.
11. The method of claim 10 wherein the 7,8-dihydro-imidazo[l,2-a]pyrazolo[4,3-e]pyrimidin-4(5H)-one is a compound of Formula IIIJ:
(Formula IIIJ Removed)
wherein R1-4 and R6 are as defined in any of claim 2, 3, 4 or 5 for Formula I or claim 6 and 7 for Formula la.
12. A method of making a compound according to any of claims 1, 2, 3, 4, 5, 6,, or 8 comprising dehydrating a compound of Formula VI
Docket No. IT-01-PCT Li, et al. (Formula VI Removed)
wherein R1-6 and [A]n are as defined in any of claim 2, 3, 4 or 5 for Formula I or claim 6 and 7 for Formula la., and recovering the compound according to the claims 1, 2, 3,4, 5, 6, 7 or 8.
13. A method of making a compound according to claim 6 or 7, comprising
dehydrating a compound of Formula V of claim 12, wherein n is 0 and wherein R1-6 are as defined for Formula la of claim 6 or 7, and recovering the compound according to the claim 6 or 7.




Documents:

http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=JVrcbGxA6tcUmeLeyfwrTA==&loc=+mN2fYxnTC4l0fUd8W4CAA==


Patent Number 278409
Indian Patent Application Number 9296/DELNP/2007
PG Journal Number 53/2016
Publication Date 23-Dec-2016
Grant Date 22-Dec-2016
Date of Filing 03-Dec-2007
Name of Patentee TAKEDA PHARMACEUTICAL COMPANY LIMITED
Applicant Address 1-1,DOSHOMACHI 4-CHOME,CHUO-KU,OSAKA 541-0045,JAPAN
Inventors:
# Inventor's Name Inventor's Address
1 PENG LI 3960 BROADWAY, NEW YORK, NY 10032, USA.
2 HAIYAN WU 3960 BROADWAY, NEW YORK, NY 10032, USA.
PCT International Classification Number A61K 31/519
PCT International Application Number PCT/US2006/022066
PCT International Filing date 2006-06-06
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/687,715 2005-06-06 U.S.A.