Pyndazinones for the treatment of cancer

Specification

The present invention relates to novel substituted pyrrolo- and pyrazolo- pyridazinones, as well as pharmaceutical compositions containing at least one of these substituted pyrrolo- and pyrazolo- pyridazinones together with at least one pharmaceutically acceptable carrier, excipient and/or diluent. Said novel substituted pyrrolo- and pyrazolo- pyridazinones are binding to the prenyl binding pocket of PDE5 and therefore, are useful for the prophylaxis and treatment of cancer by inhibition of the binding of PDE5 to farnesylated Ras proteins and thereby, inhibition of oncogenic Ras signaling in cells. Background of the invention

In cancer treatment, there is an ongoing need for the development of novel substances, which are effective to induce cell cycle/proliferation arrest or cell death of cancer cells. The fundamental characteristics of these cells are that the control of the cell cycle and proliferation is disturbed and they evade oncogene induced stress response and cell senescence.

Ras proteins, H-Ras, K-Ras and N-Ras are key regulators of diverse cellular processes including proliferation and differentiation. Ras proteins are normally tightly regulated by guanine nucleotide exchange factors (GEFs) promoting GDP dissociation and GTP binding and GTPase-activating proteins (GAPs) that stimulate the intrinsic GTPase activity of Ras to switch off signaling. Ras alternates between an active "on" state with a bound GTP and an inactive "off state with a bound GDP. The active "on" state binds to and activates proteins that control growth and differentiation of cells. Aberrant Ras function is associated with proliferative disorders, cancers and tumors. Mutation at these conserved sites favors GTP binding and produces constitutive activation of Ras. Importantly, all Ras isoforms share sequence identity in all of the regions responsible for GDP/GTP binding, GTPase activity, and effector interactions suggesting functional redundancy (Cancer Res. 2012, 72 (10), 2457).

20 to 30% of the human tumors have activating point mutations in Ras, most frequently in K-Ras, then N-Ras, then H-Ras. These mutations all compromise the GTPase activity of Ras, preventing GAPs from promoting hydrolysis of GTP on Ras and therefore, causing Ras to accumulate in the GTP-bound, active form. Despite substantial efforts to interfere with signaling by oncogenic Ras proteins, in particular by means of Ras-farnesyltransferase inhibitors, up to present no clinically useful drug has been found. Signaling by Ras proteins critically depends on their correct subcellular localization, which in turn is regulated by lipid modifications at their C-terminus. Thus, K-Ras, a major proto-oncogenic isoform of the Ras proteins, is S-farnesylated at its C-terminal CaaX box. Correct localization and signaling by farnesylated Ras is regulated by the prenyl binding protein PDE5 (Phosphodiesterase 6 delta subunit, GDI-like solubilizing factor PDE5) (Nature Cell Biol. 2012, 14, 148 - 158).

PDE5 was originally identified as a regulatory (noncatalytic) subunit of the enzyme phosphodiesterase 6 (PDE6). PDE5 is referred to in the scientific literature using several different designations, including PDE delta, PDEG, PDE65, PDE6 delta, PDE66, PDE6D (Phosphodiesterase 6 delta) and PDED.

PDE5 binds farnesylated Ras via the farnesylated C-terminus and has an essential role in sustaining its spatial organization and proper localization in cells, by facilitating its intracellular diffusion to enhance the kinetics of trapping at the right membrane compartment, i.e. the plasma membrane for Ras (Nature Cell Biol. 2012, 14, 148 - 158). This regulation of Ras localization and signaling by PDE5 suggests that interfering with the PDE5-Ras interaction by means of small molecules might provide a novel opportunity to suppress signaling from oncogenic and normal Ras. Suppressing oncogenic Ras signaling results in halting Ras- dependent tumor proliferation.

It is the objective of the present invention to provide novel pyrrolo- and pyrazolo- pyridazinones that can be used as pharmaceutically active agents, especially for the treatment or prophylaxis of cancers, tumors and proliferative diseases, as well as compositions comprising at least one of these compounds as pharmaceutically active agent.

The objective of the present invention is solved by the teaching of the independent claims. Further advantageous features, aspects and details of the invention are evident from the dependent claims, the description, the figures, and the examples of the present application. Description of the invention

In the present invention, it was surprisingly found that the binding of pyridazinones of the general formula (I) to the prenyl binding pocket of PDE5 induces strong inhibition of the binding of PDE5 to Ras and oncogenic Ras signaling in cells, thereby causing inhibition of tumor cell proliferation and tumor cell death. Hence, these inventive compounds are useful for the treatment or prophylaxis of cancers, tumors and other proliferative diseases. Thus, the present invention is directed to a pyridazinone of general formula (I)

(I)

wherein

X represents N or C(CH ₃ );

R ¹ represents R ³ and R ² represents -CH ₂ CR ¹⁸ R ¹⁹ CH ₂ R ⁴

-CH ₂ CR ¹⁸ R ¹⁹ CH ₂ C(O)NR ¹⁷ R ⁶ , or

R ¹ represents -CH ₂ CR ¹⁸ R ¹⁹ CH ₂ R ⁴ or -CH ₂ CR ¹⁸ R ¹⁹ CH ₂ C(O)NR ²⁰ R ^6* and represents R ³ ;

R ³ represents

R ⁵ is selected from: -H, -F, -CI, -CH ₃ , -CF ₃ , -C ₂ H ₅ , -CH ₂ CH ₂ CH ₃ -CH(CH ₃ ) ₂ , -OCH ₃ , -OCF ₃ , -OC ₂ H ₅ and -CN;

R ⁶ represents -(CR ⁸ R ⁹ ) _n i-(CH ₂ ) _n ^R ⁷ or -(CH ₂ ) _n2 -(CR ⁸ R ⁹ ) _n i-R ⁷ and R ¹⁷ represents -H,

or R ⁶ and R ¹⁷ form together with the nitrogen atom to which they are attached to a residue selected from:

or R ^6* and R ²⁰ form together with the nitrogen atom to which they are attached to a residue selected from:

R ⁷ represents:

-Y- is selected from -O- and -S-;

R ⁸ , R ⁹ are independently of each other selected from: -H, -CH ₃ , -C2H ₅ , and -OH,

or R ⁸ and R ⁹ form together with the carbon to which they are attached to a residue selected from:

R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ are independently of each other selected from: -H,

-CI, -F, -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH ₃ ) ₂ , -OCH3,

-OCH2CH3, -OCH2CH2CH3, -OCH(CH ₃ ) ₂ and -CF ₃ ;

R ¹⁵ represents -H or -CH ₃ ;

R ¹⁶ represents: -H, -CH ₃ or -CI;

R ¹⁸ and R ¹⁹ are independently of each other selected from: -H, -F, and -CH ₃ , or R ¹⁸ and R ¹⁹ form together with the carbon atom to which they are attached to a residue selected fro

n1 and n2 are independently of each other selected from 0 or 1 ;

n3 is an integer selected from 1 , 2 and 3;

and enantiomers, mixtures of enantiomers, diastereomers, mixtures of diastereomers, hydrates, solvates, tautomers, racemates and pharmaceutically acceptable salts thereof. The compound 4-(5,7-dimethyl-1 -oxo-2-phenyl-1 ,2-dihydro-6H-pyrrolo[3,4- d]pyridazin-6-yl)-N-(2-methoxybenzyl)butanamide is excluded from the scope of protection of the present invention.

-dimethyl-1 -oxo-2-phenyl-1 ,2-dihydro-6H-pyrrolo[3,4-d]pyridazin-6-yl)-N-(2- methoxybenzyl)butanamide

Preferred is a pyrrolopyridazinone of general formula (l-A)

(l-A)

wherein

R ¹ represents: -CH ₂ CR ¹⁸ R ¹⁹ CH ₂ R ⁴ or -CH ₂ CR ¹⁸ R ¹⁹ CH ₂ C(O)NR ²⁰ R ^6* ;

R ⁵ is selected from: -H, -F, -CI, -CH ₃ , -CF ₃ , -C ₂ H ₅ , -CH ₂ CH ₂ CH ₃ , -CH(CH ₃ ) ₂ , -OCH _3j -OCF _3j -OC ₂ H ₅ and -CN;

R ⁴ represents: If ^"

17

R ⁶ represents -(CR ⁸ R ⁹ ) _n i-(CH ₂ ) _n2 -R ⁷ or -(CH ₂ ) _n2 -(CR ⁸ R ⁹ )m-R ⁷ and R represents -H,

or R ⁶ and R ¹⁷ form together with the nitrogen atom to which they are attached to a residue selected from:

R represents:

H,

or R ^6* and R ²⁰ form together with the nitrogen atom to which they are attached to a residue selected from:

R ⁷ represents:

-Y- is selected from -O- and -S-;

R ⁸ , R ⁹ are independently of each other selected from: -H, -CH ₃ , -C2H ₅ , and -OH,

or R ⁸ and R ⁹ form together with the carbon to which they are attached to a residue selected from:

R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ are independently of each other selected from: -H, -CI, -F, -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH ₃ ) ₂ , -OCH3, -OCH2CH3, -OCH2CH2CH3, -OCH(CH ₃ ) ₂ and -CF ₃ ;

R ¹⁵ represents -H or -CH ₃ ;

R ¹⁶ represents: -H, -CH ₃ , or -CI;

R ¹⁸ and R ¹⁹ are independently of each other selected from: -H, -F and -CH ₃ , or R ¹⁸ and R ¹⁹ form together with the carbon atom to which they are attached to a residue selected from:

n1 and n2 are independently of each other selected from 0 or 1

and n3 is an integer selected from 1 , 2 and 3.

Also preferred is a pyrazolopyridazinone of general formula (l-B)

(l-B)

wherein

R ² represents: -CH ₂ CR ¹⁸ R ¹⁹ CH ₂ R ⁴ or -CH ₂ CR ¹⁸ R ¹⁹ CH ₂ C(O)NR ¹⁷ R ⁶ ;

R ⁵ is selected from: -H, -F, -CI, -CH ₃ , -CF ₃ , -C ₂ H ₅ , -CH ₂ CH ₂ CH ₃ , -CH(CH ₃ ) ₂ , -OCH _3j -OCF _3j -OC ₂ H ₅ and -CN;

R ⁴ represents:

17

R ⁶ represents -(CR ⁸ R ⁹ ) _n i-(CH ₂ ) _n ^R ⁷ or -(CH ₂ ) _n2 -(CR ⁸ R ⁹ ) _n i-R ⁷ and R represents -H,

or R ⁶ and R ¹⁷ form together with the nitrogen atom to which they are attached to a residue selected from:

R ⁷ represents:

-Y- is selected from: -O- and -S-;

R ⁸ , R ⁹ are independently of each other selected from: -H, -CH ₃ , -C2H ₅ , and -OH,

or R ⁸ and R ⁹ form together with the carbon to which they are attached to a residue selected from:

R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ are independently of each other selected from: -H,

-CI, -F, -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH ₃ ) ₂ , -OCH3,

-OCH2CH3, -OCH2CH2CH3, -OCH(CH ₃ ) ₂ , and -CF ₃ ;

R ¹⁵ represents: -H or -CH ₃ ;

R ¹⁶ represents: -H, -CH ₃ , or -CI;

R ¹⁸ and R ¹⁹ are independently of each other selected from: -H, -F and -CH ₃ , or R ¹⁸ and R ¹⁹ form together with the carbon atom to which they are attached to a residue selected from:

n1 and n2 are independently of each other selected from 0 or 1 ; and

n3 is an integer selected from 1 , 2 and 3.

A preferred embodiment of the present invention is directed to a pyridazinone of the general formula (II)

wherein

X represents N or C(CH ₃ );

R ¹ represents R ³ and R ² represents -CH ₂ CH ₂ CH ₂ R ⁴ or -CH ₂ CH ₂ CH ₂ C(O)NHR ⁶ or

R ¹ represents -CH ₂ CH ₂ H ₂ CH ₂ CH ₂ C(O)NHR ^6* , and R ² represents R ³ ;

R represents:

R ⁵ is selected from: -H, -CH ₃ , -C ₂ H ₅ , -CH ₂ CH ₂ CH ₃ and -CH(CH ₃ ) ₂ ;

R

R ⁶

R ^6*

R ⁷ represents:

-Y- is selected from: -O- and -S-;

R ⁸ , R ⁹ are independently of each other selected from: -H, -CH ₃ and -C2H ₅ ; R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ are independently of each other selected from: -H, -CI, -F, -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH ₃ ) ₂ , -OCH3, -OCH2CH3, -OCH2CH2CH3, -OCH(CH ₃ ) ₂ and -CF ₃ ;

R ¹⁵ represents: -H or -CH ₃ ;

n1 and n2 are independently of each other selected from 0 and 1 ;

and enantiomers, mixtures of enantiomers, diastereomers, mixtures of diastereomers, hydrates, solvates, tautomers, racemates and pharmaceutically acceptable salts thereof, with the proviso that the compound 4-(5,7-dimethyl-1 - oxo-2-phenyl-1 ,2-dihydro-6H-pyrrolo[3,4-d]pyridazin-6-yl)-N-(2-methoxybenz yl) butanamide is excluded from the scope of protection of the present invention.

An embodiment of the present invention relates to a pyrrolopyridazinone of general formula (III)

(III) wherein

R ⁶ represents:

-Y- is selected from: -O- and -S-, and

R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ are independently of each other selected from: -H, -CI, -F, -CH ₃ , -CH2CH3, -CH2CH2CH3, -CH(CH ₃ ) ₂ , -OCH3, -OCH2CH3, -OCH2CH2CH3, -OCH(CH ₃ ) ₂ and -CF ₃ .

Another embodiment of the present invention is directed to a compound of general formula (III)

and R ⁶ has the meaning defined above.

Thus, compounds of the following general formulae (lll-A) and (lll-B) are also preferred. 5

and R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ are independently of each other selected from: -CH ₃ , -OCH ₃ , -CF ₃ , and -F.

A pyrrolopyridazinone of general formula (IV)

wh

and residues R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ are independently of each other selected from: -H, -CI, -F, -CH ₃ , -CH ₂ CH ₃ , -CH2CH ₂ CH _3j -CH(CH ₃ ) ₂ , -OCH ₃ , -OCH ₂ CH ₃ , -OCH ₂ CH ₂ CH ₃ , -OCH(CH ₃ ) ₂ , and -CF ₃ is especially preferred.

A compound of general formula (IV-A), (IV-B), (IV-C) or (IV-D), wherein

R ¹² , R ¹³ and R ¹⁴ have the meanin s defined above is especially preferred.

(IV-B)

(IV-D)

Especially preferred are compounds of general formula (IV-A), wherein at least one of the residues R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ is -CI and compounds of general formula (IV-D), wherein at least one of the residues R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ is -CH ₃ , and preferably two of the residues R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ are -CH ₃ .

Still preferred compounds are pyridazinones of general formulae (I), (II) and (IV), wherein the residue R ^6* is selected from:

7

and the residues R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ are independently of each other selected from: -CH ₃ , -CH ₂ CH ₃ , -CH ₂ CH ₂ CH ₃ , -CH(CH ₃ ) ₂ and -CF ₃ .

Other preferred compounds of the current invention are pyridazinones of general formulae (I), (II) and (IV), wherein R ^6* is selected from:

and R ¹⁰ , R ^{1 1} , R ¹² , R ¹³ and R ¹⁴ are independently of each other selected from: -CH ₃ , -OCHs, -CF ₃ , -CI and -F.

Especially preferred compounds are pyridazinones of general formula (I), (II) and

(IV), wherein R ^6* re resents:

and R ¹⁰ , R ^{1 1} and R ¹² represent independently of each other -CI or -F.

Another preferred embodiment is directed to a pyrazolopyridazinone of general formula (V)

wherein

R ⁵ is selected from: -H, -F, -CI, -CH ₃ , -CF ₃ , -C ₂ H ₅ , -CH2CH2CH3, -CH(CH ₃ ) ₂ , -OCH3, -OCF3, -OC2H5, and -CN;

R ⁶ represents -(CR ⁸ R ⁹ ) _n i-(CH ₂ ) _n ^R ⁷ or -(CH ₂ ) _n HCR ⁸ R ⁹ ) _n i-R ⁷ and R ¹⁷ represents -H, or R ⁶ and R ¹⁷ form together with the nitrogen atom to which they are attached to a residue selected from:

is selected from -O- and

R ⁸ , R ⁹ are independently of each other selected from: -H, -CH ₃ , -C2H ₅ , and -OH,

or R ⁸ and R ⁹ form together with the carbon to which they are attached to a residue selected from:

R ¹⁰ , R ^{1 1} , R ¹² , R ¹³ and R ¹⁴ are independently of each other selected from: -H, -CI, -F, -CH ₃ , -CH ₂ CH ₃ , -CH ₂ CH ₂ CH ₃ , -CH(CH ₃ ) ₂ , -OCH ₃ , -OCH ₂ CH ₃ , -OCH ₂ CH ₂ CH _3j -OCH(CH ₃ ) ₂ , and -CF ₃ ;

R ¹⁵ represents: -H or -CH ₃ ;

R ¹⁶ represents: -H, -CH ₃ , or -CI;

R ¹⁸ and R ¹⁹ are independently of each other selected from: -H, -F and -CH ₃ , or R ¹⁸ and R ¹⁹ form together with the carbon atom to which they are attached to a residue selected from:

n1 and n2 are independently of each other selected from 0 or 1 ; and

n3 is an integer selected from 1 , 2 and 3.

Also preferred is a pyrazolopyridazinone of general formula (V-A)

wherein

R ⁵ is selected from: -H, -F, -CI, -CH ₃ , -CF ₃ , and -OCH ₃ ;

R ⁸ , R ⁹ are independently of each other selected from: -H, -CH ₃ , -C ₂ H ₅ , an

-OH,

or R ⁸ and R ⁹ form together with the carbon to which they are attached to a residu selected from:

R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ are independently of each other selected from: -H, -F, -OCH ₃ , -OCH(CH ₃ ) ₂ , and -CF ₃ ;

R ¹⁵ represents: -H, or -CH ₃ ;

R ¹⁶ represents: -H, -CH ₃ , or -CI;

R ¹⁸ and R ¹⁹ are independently of each other selected from: -H, -F and -CH ₃ , or R ¹⁸ and R ¹⁹ form together with the carbon atom to which they are attached to a residue selected from:

n1 and n2 are independently of each other selected from 0 or 1 ; and at least one of n1 and n2 is 1 .

In general formula (V-A), it is further preferred one of the residues R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ , and particularly residue R ¹² , is selected from: -H, -F, -CI, -CH ₃ , -OCH ₃ , -OCH(CH ₃ ) ₂ , and -CF ₃ , and the remaining residues are -H. Preferred are compounds of general formula (V), wherein R ¹⁵ represents -CH ₃ ;

R ¹⁶ represents -H and R ⁵ is selected from: -H, -F, -CI, -CH ₃ , -CF ₃ , and -OCH ₃ .

Even more preferred is a compound of general formula (V-B)

(V-B)

wherein R ⁵ is selected from: -H, -F, -CI, -CH ₃ , -CF ₃ , and -OCH ₃ ;

R ⁸ , R ⁹ are independently of each other selected from -H, -CH ₃ , -C2H ₅ , and -OH; or R ⁸ and R ⁹ form together with the carbon to which they are attached to a residue selected from:

R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ are independently of each other selected from: -H, -F, -OCH ₃ , -OCH(CH ₃ ) ₂ , and -CF ₃ ;

R ¹⁸ and R ¹⁹ are independently of each other selected from: -H, -F and -CH ₃ , or R ¹⁸ and R ¹⁹ form together with the carbon atom to which they are attached to a residue selected from:

n1 and n2 are independently of each other selected from 0 or 1 ; and at least one of n1 and n2 is 1 .

In general formula (V-B), it is further preferred that one of the residues R ¹⁰ , R ^{1 1} , R ¹² , R ¹³ and R ¹⁴ , and particularly residue R ¹² , is selected from -H, -F, -OCH ₃ , -OCH(CH ₃ ) ₂ , and -CF ₃ , and the remaining residues are -H.

Also preferred is a pyrazolopyridazinone of general formula (V-C)

(V-C)

wherein

R ⁵ is selected from: -H, -F, -CI, -CH ₃ , -CF ₃ , and -OCH ₃ ;

R ⁸ , R ⁹ are independently of each other selected from -H, -CH ₃ , -C2H ₅ , and -OH; or R ⁸ and R ⁹ form together with the carbon to which they are attached to a residue selected from:

R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ are independently of each other selected from: -H, -F, -OCH ₃ , -OCH(CH ₃ ) ₂ , and -CF ₃ ;

R ¹⁸ and R ¹⁹ are independently of each other selected from: -H, -F and -CH ₃ , or R ¹⁸ and R ¹⁹ form together with the carbon atom to which they are attached to a residue selected from:

n1 and n2 are independently of each other selected from 0 or 1 ; and at least one of n1 and n2 is 1 .

In general formula (V-C), it is preferred that n1 and n2 represent 1 . In same general formula (V-C), it is further preferred that R ¹⁸ and R ¹⁹ represent -H. Also preferred are compounds of general formula (V-C), wherein R ⁵ is selected from -H and -CF ₃ . Hence, a compound of general formula (V-C), wherein n1 and n2 represent 1 , R ¹⁸ and R ¹⁹ represent -H and R ⁵ is selected from -H and -CF ₃ is especially preferred.

Especially preferred is a pyrazolopyridazinone of general formula (V-D)

(V-D)

wherein

R ⁵ is selected from: -H, -F, -CI, -CH ₃ , -CF ₃ , and -OCH ₃ ;

R ⁶ represents -(CR ⁸ R ⁹ ) _n i-(CH ₂ ) _n 2-R ⁷ or -(CH ₂ )nHCR ⁸ R ⁹ )m-R ⁷ and R ¹⁷ represents -H,

or R ⁶ and R ¹⁷ form together with the nitrogen atom to which they are attached to a residue selected from:

R ⁷ represents:

-Y- is selected from -O- and -S-;

R ⁸ , R ⁹ are independently of each other selected from: -H, -CH ₃ , -C2H ₅ , and -OH,

or R ⁸ and R ⁹ form together with the carbon to which they are attached to a residue selected from:

R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ are independently of each other selected from: -H, -CI, -F, -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH ₃ ) ₂ , -OCH3, -OCH2CH3, -OCH2CH2CH3, -OCH(CH ₃ ) ₂ , and -CF ₃ ;

R ¹⁸ and R ¹⁹ are independently of each other selected from: -H, -F and -CH ₃ , or R ¹⁸ and R ¹⁹ form together with the carbon atom to which they are attached to a residue selected from:

n1 and n2 are independently of each other selected from 0 or 1 ; and

n3 is an integer selected from 1 , 2 and 3. 5

Preferably, the residues R ¹⁸ and R ¹⁹ represent -H. Hence, a compound of general formula (I), (l-A), (l-B), (II), (V), (V-A), (V-B), (V-C) or (V-D), wherein the residues R ¹⁸ and R ¹⁹ represent -H, is especially preferred.

Another preferred embodiment of the present invention relates to a pyrazolopyridazinone of general formula (VI)

wherein

R ⁵ is selected from: -H and -CH ₃ ;

R ⁶ represents -(CR ⁸ R ⁹ ) _n i-(CH ₂ ) _n ^R ⁷ or -(CH ₂ )nHCR ⁸ R ⁹ )m-R ⁷ ;

R ⁷ represents:

-Y- is selected from -O- and -S-;

R ⁸ , R ⁹ are independently of each other selected from: -H, -CH ₃ , and -C2H ₅ ; R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ are independently of each other selected from: -H, -CI, — F, — CH3, — CH2CH3, — CH2CH2CH3, — CH(CH3)2, — OCH3, — OCH2CH3, -OCH2CH2CH3, -OCH(CH ₃ ) ₂ , and -CF ₃ ;

and n1 and n2 are independently of each other selected from 0 or 1 .

Preferred compounds are pyrazolopyridazinones of general formula (VI)

and the residues R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ are independently of each other selected from: -H, -CI, -F, -CH ₃ , -CH ₂ CH ₃ , -CH ₂ CH ₂ CH _3j -CH(CH ₃ ) ₂ , -OCH ₃ , -OCH ₂ CH _3j -OCH ₂ CH ₂ CH _3j -OCH(CH ₃ ) ₂ , and -CF ₃ .

Even more preferred compounds are pyrazolopyridazinones of general formula (VI)

wherein the residue R ⁶ is selected from:

7

residues R ⁸ , R ⁹ are independently of each other selected from: -H, -CH ₃ and -C ₂ H ₅ ; and residues R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ are independently of each other selected from: -CI, -F, -CH ₃ , -OCH ₃ , -OCH(CH ₃ ) ₂ and -CF ₃ .

Especially preferred compounds are pyrazolopyridazinones of general formula (VI)

(VI)

wherein the residue R is selected from:

residues R ⁸ , R ⁹ are independently of each other selected from -H, -CH ₃ and -C ₂ H ₅ ; and residues R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ are independently of each other selected from: -CI, -F, -CH ₃ , -OCH ₃ , -OCH(CH ₃ ) ₂ and -CF ₃ . In ge

and the residue R ¹² is defined as above and most preferably is selected from: -CH ₃ , -OCH ₃ , -OCH(CH ₃ ) ₂ and -CF ₃ .

Preferably, the compounds of the present invention are selected from:

Compound Structure, lUPAC name

Number

A/-benzyl-4-(5,7-dimethyl-1 -oxo-2-phenyl-1 /-/-pyrrolo[3,4-c ]pyhdazin- 6(2H)-yl)butanamide

4-(5,7-dimethyl-1 -oxo-2-phenyl-1 H-pyrrolo[3,4-c ]pyhdazin-6(2/-/)-yl)- A/-(3-methoxybenzyl)butanamide

A/-(3-chlorobenzyl)-4-(5,7-dinnethyl-1-oxo-2-phenyl-1/-/-pyr rolo[3,4-

A/-(2-chlorobenzyl)-4-(5,7-dimethyl-1-oxo-2-phenyl-1/-/-pyrr olo[3,4- c)

A/-(4-chlorobenzyl)-4-(5,7-dinnethyl-1-oxo-2-phenyl-1/-/-pyr rolo[3,4-

4-(5,7-dimethyl-1-oxo-2-phenyl-1/-/-pyrrolo[3,4-c]pyndazin-6 (2/-/)-yl)-

4-(5 -dimethyl-1-oxo-2-phenyl-1/-/-pyrrolo[3,4-c]pyndazin-6(2/-/) -yl)- /V-(furan-2-ylmethyl)butanamide

^' -dimethyl-1 -oxo-2-phenyl-1 H-pyrrolo[3,4-c ]pyndazin-6(2/-/)-yl)

/V-(pyridin-2-ylmethyl)butanamide

A/-cyclopropyl-4-(5,7-dinnethyl-1 -oxo-2-phenyl-1 H-pyrrolo[3,4- c ]pyridazin-6(2H)-yl)butanamide

A/-cyclopentyl-4-(5,7-dinnethyl-1 -oxo-2-phenyl-1 H-pyrrolo[3,4- c ]pyridazin-6(2H)-yl)butanamide

A/-cyclohexyl-4-(5,7-dinnethyl-1 -oxo-2-phenyl-1 H-pyrrolo[3,4- c ]pyridazin-6(2H)-yl)butanamide

4-(5,7-dimethyl-1-oxo-2-phenyl-1H-pyrrolo[3,4-c]pyndazin-6(2 H)-yl)- A/-(4-methylcyclohexyl)butanamide

4-(5,7-dimethyl-1-oxo-2-phenyl-1H-pyrrolo[3,4-c]pyndazin-6(2 H)-yl)- /V-(2-methylcyclohexyl)butanannide

4-(5,7-dimethyl-1-oxo-2-phenyl-1H-pyrrolo[3,4-c]pyndazin-6(2 H)-yl)- A/-(2,3-dinnethylcyclohexyl)butanamide

6-(3-(1-benzyl-1r7-benzo[d]imidazol-2-yl)propyl)-5,7-dime thyl-2- phenyl-2,6-dihydro-1 H-pyrrolo[3,4-c]pyndazin-1 -one

5,7-dimethyl-2-phenyl-6-(3-(3-phenyl-1 ,2,4-oxadiazol-5-yl)propyl)-2,6- dihydro-1 H-pyrrolo[3,4-c ]pyndazin-1 -one

S -dimethyl^-phenyl-e-iS-iS-ip-toly -l ^^-oxadiazol-S-y propyl)- 2,6-dihydro-1 /-/-pyrrolo[3,4-c/]pyhdazin-1 -one

6-(3-(3-(4-methoxyphenyl)-1 ,2,4-oxadiazol-5-yl)propyl)-5,7-dimethyl- 2-phenyl-2,6-dihydro-1 H-pyrrolo[3,4-c/]pyhdazin-1 -one

6-(3-(3-(2,5-dinnethoxyphenyl)-1 ,2,4-oxadiazol-5-yl)propyl)-5,7- dimethyl-2-phenyl-2,6-dihydro-1 /-/-pyrrolo[3,4-c/]pyridazin-1 -one

5,7-dimethyl-2-phenyl-6-(3-(3-(4-(thfluoromethyl)phenyl)-1 ,2,4- oxadiazol-5-yl)propyl)-2,6-dihydro-1 H-pyrrolo[3,4-c ]pyhdazin-1 -one

6-(3-(3-(4-fluorophenyl)-1 ,2,4-oxadiazol-5-yl)propyl)-5,7-dimethyl-2- phenyl-2,6-dihydro-1 H-pyrrolo[3,4-c ]pyhdazin-1 -one 5,7-dimethyl-2-phenyl-6-(3-(5-phenyl-1 ,3,4-oxadiazol-2-yl)propyl)-2,6- dihydro-1 H-pyrrolo[3,4-c ]pyndazin-1 -one

5,7-dimethyl-2-phenyl-6-(3-(5-(p-tolyl)-1 ,3,4-oxadiazol-2-yl)propyl)- 2,6-dihydro-1 /-/-pyrrolo[3,4-c/]pyridazin-1 -one

6-(3-(5-(4-methoxyphenyl)-1 ,3,4-oxadiazol-2-yl)propyl)-5,7-dinnethyl- 2-phenyl-2,6-dihydro-1 /-/-pyrrolo[3,4-c/]pyhdazin-1 -one

6-(3-(5-(2-methoxyphenyl)-1 ,3,4-oxadiazol-2-yl)propyl)-5,7-dinnethyl- 2-pheny

5,7-dimethyl-2-phenyl-6-(3-(5-(4-(thfluoromethyl)phenyl)-1 ,3,4- oxadiazol-2-yl)propyl)-2,6-dihydro-1 H-pyrrolo[3,4-c ]pyhdazin-1 -one

6-(3-(5-(4-fluorophenyl)-1 ,3,4-oxadiazol-2-yl)propyl)-5,7-dimethyl-2- phenyl-2,6-dihydro-1 H-pyrrolo[3,4-c ]pyhdazin-1 -one

5,7-dimethyl-2-phenyl-6-(3-(5-(pyhdin-4-yl)-1 ,3,4-oxadiazol-2- yl)propyl)-2,6-dihydro-1 H-pyrrolo[3,4-c ]pyhdazin-1 -one 5

A/-(3-chloro-2-methylphenyl)-4-(3,4-dimethyl-7-oxo-2-phenyl- 2/-/- pyrazolo[3,4-c ]pyridazin-6(7H)-yl)butanamide

4-(3,4-dimethyl-7-oxo-2-phenyl-2H-pyrazolo[3,4-c ]pyndazin-6(7/-/)-yl)- A/-(furan-2-ylnnethyl)butanannide

/V-benzyl-4-(3,4-dimethyl-7-oxo-2-phenyl-2/-/-pyrazolo[3,4-c ]pyndazin- 6(7/-/)-yl)butanamide

4-(3,4-dimethyl-7-oxo-2-phenyl-2H-pyrazolo[3,4-c ]pyndazin-6(7/-/)-yl)- /V-(4-methoxybenzyl)butanannide

4-(3,4-dimethyl-7-oxo-2-phenyl-2H-pyrazolo[3,4-c ]pyndazin-6(7/-/)-yl)- /V-(3-methoxybenzyl)butanannide

4-(3,4-dimethyl-7-oxo-2-phenyl-2H-pyrazolo[3,4-c ]pyndazin-6(7/-/)-yl)- /V-(2-methoxybenzyl)butanamide

4-(3,4-dimethyl-7-oxo-2-phenyl-2H-pyrazolo[3,4-c ]pyndazin-6(7/-/)-yl)- /V-(4-methylbenzyl)butanamide

4-(3,4-dimethyl-7-oxo-2-phenyl-2H-pyrazolo[3,4-c ]pyndazin-6(7/-/)-yl)- /V-phenethylbutanamide

4-(3,4-dimethyl-7-oxo-2-phenyl-2H-pyrazolo[3,4-c ]pyndazin-6(7/-/)-yl)-

/V-(4-methylphenethyl)butanamide

4-(3,4-dimethyl-7-oxo-2-(p-tolyl)-2H-pyrazolo[3,4-c ]pyndazin-6(7H)- yl)-/V-(furan-2-ylnnethyl)butanannide

4-(3,4-dimethyl-7-oxo-2-(p-tolyl)-2H-pyrazolo[3,4-c ]pyndazin-6(7H)- yl)-/V-((tetrahydrofuran-2-yl)nnethyl)butanannide

4-(3,4-dimethyl-7-oxo-2-(p-tolyl)-2H-pyrazolo[3,4-c ]pyndazin-6(7H)- yl)-/V-(pyridin-2-ylnnethyl)butanannide

4-(3,4-dinnethyl-7-oxo-2-(p-tolyl)-2H-pyrazolo[3,4-c ]pynclazin-6(7H)- yl)-/V-(pyridin-3-ylmethyl)butanamide

A/-benzyl-4-(3,4-dimethyl-7-oxo-2-(p-tolyl)-2H-pyrazolo[3,4- c/]pyridazin-6(7H)-yl)butanamide

4-(3,4-dimethyl-7-oxo-2-(p-tolyl)-2H-pyrazolo[3,4-c ]pyndazin-6(7H)- yl )-A/-(4-methoxybenzyl )butanam ide

4-(3,4-dimethyl-7-oxo-2-(p-tolyl)-2H-pyrazolo[3,4-c ]pyndazin-6(7H)- yl )-A/-(4-nnethylbenzyl )butanam ide

4-(3,4-dimethyl-7-oxo-2-(p-tolyl)-2H-pyrazolo[3,4-c ]pyndazin-6(7H)- yl)-A/-(4-isopropoxybenzyl)butanamide

4-(3,4-dimethyl-7-oxo-2-(p-tolyl)-2H-pyrazolo[3,4-c ]pyndazin-6(7H)- yl)-/V-phenethylbutanamide

4-(3,4-dimethyl-7-oxo-2-(p-tolyl)-2H-pyrazolo[3,4-c ]pyndazin-6(7H)- yl)-/V-(4-methylphenethyl)butanannide

A/-(4-chlorophenethyl)-4-(3,4-dimethyl-7-oxo-2-(p-tolyl)-2/- /- pyrazolo[3,4-c ]pyridazin-6(7H)-yl)butanamide

A/-(3-chlorophenethyl)-4-(3,4-dimethyl-7-oxo-2-(p-tolyl)-2/- /- pyrazolo[3,4-c ]pyridazin-6(7H)-yl)butanamide

4-(3,4-dimethyl-7-oxo-2-(p-tolyl)-2H-pyrazolo[3,4-c ]pyndazin-6(7H)- yl)-/V-(2-phenylpropyl)butanamide

A/-cyclopropyl-4-(3,4-dinnethyl-7-oxo-2-(p-tolyl)-2/-/-pyraz olo[3,4- c )pyridazin-6(7/-/)-yl)butanannide

A/-cyclopentyl-4-(3,4-dinnethyl-7-oxo-2-(p-tolyl)-2/-/-pyraz olo[3,4- c )pyridazin-6(7/-/)-yl)butanannide

4-(3,4-dimethyl-7-oxo-2-(p-tolyl)-2H-pyrazolo[3,4-Q]pynclazi n-6(7H)- yl)-/V-(4-methylcyclohexyl)butanamide

N-(4-fluorobenzyl)-4-(4-methyl-7-oxo-2-phenyl-2H-pyrazolo[3, 4- d]pyridazin-6(7H)-yl)butanamide

6-(4-(3,4-dihydroisoquinolin-2(1 H)-yl)-4-oxobutyl)-2-ph

pyrazolo[3,4-d]pyridazin-7(6H)-one

N-(1 -(4-fluorophenyl)ethyl)-4-(7-oxo-2-phenyl-2H-pyrazolo[3,4- d]pyridazin-6(7H)-yl)butanannide

4-(7-oxo-2-phenyl-2H-pyrazolo[3,4-d]pyridazin-6(7H)-yl)-N- phenethylbutanamide

N-(4-isopropoxybenzyl)-4-(7-oxo-2-phenyl-2H-pyrazolo[3,4-d]p yridazin- 6(7H)-yl)butanamide

4-(3-chloro-7-oxo-2-phenyl-2H-pyrazolo[3,4-d]pyridazin-6(7H) -yl)-N-(4- fluorobenzyl)butanannide

4-(3-chloro-7-oxo-2-phenyl-2H-pyrazolo[3,4-d]pyridazin-6(7H) -yl)-N-(4- isopropoxybenzyl)butanamide

3-chloro-6-(4-oxo-4-(pyrrolidin-1 -yl)butyl)-2-phenyl-2H-pyrazolo[3,4- d]pyridazin-7(6H)-one

4-(3-chloro-7-oxo-2-phenyl-2H-pyrazolo[3,4-d]pyridazin-6(7H) -yl)-N-(1 - (4-fluorophenyl)ethyl)butanamide

4-(3-chloro-7-oxo-2-phenyl-2H-pyrazolo[3,4-d]pyridazin-6(7H) -yl)-N- phenethylbutanamide

3-chloro-6-(4-(3,4-dihydroisoquinolin-2(1 H)-yl)-4-oxobutyl)-2-phenyl- 2H-pyrazolo[3,4-d]pyridazin-7(6H)-one

4-(3-chloro-7-oxo-2-phenyl-2H-pyrazolo[3,4-d]pyridazin-6(7H) -yl)-N-(4 (trifluoromethyl)benzyl)butanannide

4-(3-chloro-7-oxo-2-phenyl-2H-pyrazolo[3,4-d]pyridazin-6(7H) -yl)-N-(4 fluorophenethyl)butanamide

N-(4-fluorobenzyl)-4-(7-oxo-2-phenyl-2H-pyrazolo[3,4-d]pyrid azin- 6(7H)-yl)butanamide

N-(1 -(4-fluorophenyl)ethyl)-4-(4-methyl-7-oxo-2

pyrazolo[3,4-d]pyridazin-6(7H)-yl)butanamide

4-(4-methyl-7-oxo-2-phenyl-2H-pyrazolo[3,4-d]pyridazin-6(7H) -yl)-N-(2- phenylpropyl)butanamide

N-(4-fluorophenethyl)-4-(4-methyl-7-oxo-2-phenyl-2H-pyrazolo [3,4- d]pyridazin-6(7H)-yl)butanannide

N-(4-isopropoxybenzyl)-4-(4-methyl-7-oxo-2-phenyl-2H-pyrazol o[3,4- d]pyridazin-6(7H)-yl)butanannide

4-(3,4-dimethyl-7-oxo-2-phenyl-2H-pyrazolo[3,4-d]pyridazin-6 (7H)-yl)- N-(4-fluorobenzyl)butanamide

4-(3,4-dimethyl-7-oxo-2-phenyl-2H-pyrazolo[3,4-d]pyridazin-6 (7H)-yl)- N-(1 -(4-fluorophenyl)ethyl)butanamide

4-(3,4-dimethyl-7-oxo-2-phenyl-2H-pyrazolo[3,4-d]pyridazin-6 (7H)-yl)- N-(2-phenylpropyl)butanamide

4-(3,4-dimethyl-7-oxo-2-phenyl-2H-pyrazolo[3,4-d]pyridazin-6 (7H)-yl)- N-(4-fluorophenethyl)butanamide

4-(3,4-dimethyl-7-oxo-2-phenyl-2H-pyrazolo[3,4-d]pyridazin-6 (7H)-yl)- N-(4-isopropoxybenzyl)butanamide

4-(3,4-dimethyl-7-oxo-2-phenyl-2H-pyrazolo[3,4-d]pyridazin-6 (7H)-yl)- N-(4-(trifluoronnethyl)benzyl)butanannide

CI

4-(3-chloro-7-oxo-2-(4-(trifluoromethyl)phenyl)-2H-pyrazolo[ 3,4- d]pyridazin-6(7H)-yl)-N-(4-fluorobenzyl)butanannide

4-(3-chloro-7-oxo-2-(4-(trifluoronnethyl)phenyl)-2H-pyrazolo [3,4- d]pyridazin-6(7H)-yl)-N-(4-isopropoxybenzyl)butanannide

N-(4-fluorophenethyl)-4-(2-(4-fluorophenyl)-4-nnethyl-7- pyrazolo[3,4-d]pyridazin-6(7H)-yl)butanannide

4-(2-(4-fluorophenyl)-4-methyl-7-oxo-2H-pyrazolo[3,4-d]pynda zin- 6(7H)-yl)-N-(4-isopropoxybenzyl)butanamide

4-(2-(4-fluorophenyl)-4-nnethyl-7-oxo-2H-pyrazolo[3,4-d]pynd azin- 6(7H)-yl)-N-(4-(trifluoronnethyl)benzyl)butanannide

4-(2-(4-chlorophenyl)-4-methyl-7-oxo-2H-pyrazolo[3,4-d]py ridazin- 6(7H)-yl)-N-(4-fluorobenzyl)butanamide

4-(2-(4-chlorophenyl)-4-methyl-7-oxo-2H-pyrazolo[3,4-d]pyrid azin- 6(7H)-yl)-N-(1 -(4-fluorophenyl)ethyl)butanamide

4-(3-chloro-7-oxo-2-(4-(trifluoromethyl)phenyl)-2H-pyrazolo[ 3,4- d]pyridazin-6(7H)-yl)-N-(1 -(4-fluorophenyl)ethyl)butanamide

4-(3-chloro-7-oxo-2-(4-(trifluoromethyl)phenyl)-2H-pyrazolo[ 3,4- d]pyridazin-6(7H)-yl)-N-phenethylbutanannide

3-chloro-6-(4-(3,4-dihydroisoquinolin-2(1 H)-yl)-4-oxobutyl)-2-(4- (trifluoromethyl)phenyl)-2H-pyrazolo[3,4-d]pyridazin-7(6H)-o ne

88

4-(3-chloro-7-oxo-2-(4-(trifluoronnethyl)phenyl)-2H-pyrazolo [3,4- d]pyridazin-6(7H)-yl)-N-(4-(tnfluoromethyl)benzyl)butanannid e

89

4-(3-chloro-7-oxo-2-(4-(trifluoronnethyl)phenyl)-2H-pyrazolo [3,4- d]pyridazin-6(7H)-yl)-N-(4-fluorophenethyl)butanannide

90

4-(2-(4-chlorophenyl)-4-methyl-7-oxo-2H-pyrazolo[3,4-d]pyrid azin- 6(7H)-yl)-N-(2-phenylpropyl)butanamide

91

4-(2-(4-chlorophenyl)-4-methyl-7-oxo-2H-pyrazolo[3,4-d]pyrid azin- 6(7H)-yl)-N-(4-fluorophenethyl)butanamide

92 4-(2-(4-chlorophenyl)-4-methyl-7-oxo-2H-pyrazolo[3,4-d]pyrid azin- 6(7H)-yl)-N-(4-isopropoxybenzyl)butanamide

4-(2-(4-chlorophenyl)-4-methyl-7-oxo-2H-pyrazolo[3,4-d]pyrid azin- 6(7H)-yl)-N-(4-(trifluoronnethyl)benzyl)butanannide

N-(4-fluorobenzyl)-4-(4-methyl-7-oxo-2-(p-tolyl)-2H-pyrazolo [3,4- d]pyridazin-6(7H)-yl)butanamide

N-(1 -(4-fluorophenyl)ethyl)-4-(4-methyl-7-oxo-2

pyrazolo[3,4-d]pyridazin-6(7H)-yl)butanamide

4-(4-methyl-7-oxo-2-(p-tolyl)-2H-pyrazolo[3,4-d]pyridazin-6( 7H)-yl)-N- (2-phenylpropyl)butanamide

N-(4-fluorophenethyl)-4-(4-methyl-7-oxo-2-(p-tolyl)-2H-pyraz olo[3,4- d]pyridazin-6(7H)-yl)butanamide

N-(4-isopropoxybenzyl)-4-(4-nnethyl-7-oxo-2-(p-tolyl)-2H-pyr azolo[3,4- d]pyridazin-6(7H)-yl)butanannide

4-(4-methyl-7-oxo-2-(p-tolyl)-2H-pyrazolo[3,4-d]pyridazin-6( 7H)-yl)-N- (4-(trifluoromethyl)benzyl)butanamide

N-(4-fluorobenzyl)-4-(2-(4-fluorophenyl)-4-methyl

pyrazolo[3,4-d]pyridazin-6(7H)-yl)butanamide

4-(2-(4-fluorophenyl)-4-nnethyl-7-oxo-2H-pyrazolo[3,4-d]pyri dazin- 6(7H)-yl)-N-(1 -(4-fluorophenyl)ethyl)butanamide

4-(2-(4-fluorophenyl)-4-nnethyl-7-oxo-2H-pyrazolo[3,4-d]pynd azin- 6(7H)-yl)-N-(2-phenylpropyl)butanamide

2-(1 -((3-chloro-7-oxo-2-(4-(trifluoromethyl)phenyl)-2H-pyrazolo[ 3,4- d]pyridazin-6(7H)-yl)nnethyl)cyclopropyl)-N-(4-fluorobenzyl) acetannide

2-(1 -((3-chloro-7-oxo-2-(4-(trifluoromethyl)phenyl)-2H-pyrazolo[ 3,4- d]pyridazin-6(7H)-yl)nnethyl)cyclopropyl)-N-(1 -(4- fluorophenyl)ethyl)acetamide

2-(1 -((3-chloro-7-oxo-2-(4-(trifluoromethyl)phenyl)-2H-pyrazolo[ 3,4- d]pyridazin-6(7H)-yl)nnethyl)cyclopropyl)-N-phenethylacetann ide

2-(1 -((3-chloro-7-oxo-2-(4-(trifluoromethyl)phenyl)-2H-pyrazolo[ 3,4- d]pyridazin-6(7H)-yl)nnethyl)cyclopropyl)-N-(4-

(trifluoromethyl)benzyl)acetannide 5

2-(1 -((3-chloro-7-oxo-2-(4-(trifluoromethyl)phenyl)-2H-pyrazolo[ 3,4- d]pyridazin-6(7H)-yl)methyl)cyclopropyl)-N-(4-fluorophenethy l)acetamide

4-(4-methyl-7-oxo-2-phenyl-2H-pyrazolo[3,4-d]pyridazin-6(7H) -yl)-N-(4- (trifluoromethyl)benzyl)butanannide

4-(4-methyl-7-oxo-2-phenyl-2H-pyrazolo[3,4-d]pyridazin-6(7H) -yl)-N- phenethylbutanamide

4-(2-(4-chlorophenyl)-4-methyl-7-oxo-2H-pyrazolo[3,4-d]pyrid azin-6(7H)- yl)-N-phenethylbutanamide

4-(2-(4-fluorophenyl)-4-methyl-7-oxo-2H-pyrazolo[3,4-d]pynda zin-6(7H)- yl)-N-phenethylbutanamide

4-(2-(4-chlorophenyl)-4-methyl-7-oxo-2H-pyrazolo[3,4-d]py ndazin-6(7H)- yl)-N-(2-hydroxy-2-phenylethyl)butanamide

4-(2-(4-chlorophenyl)-4-methyl-7-oxo-2H-pyrazolo[3,4-d]pyrid azin- 6(7H)-yl)-N-(3-methoxybenzyl)butanannide

N-(4-fluorobenzyl)-4-(2-(4-methoxyphenyl)-4-nnethyl

pyrazolo[3,4-d]pyridazin-6(7H)-yl)butanannide

N-(3-(2-(2-methoxyethoxy)ethoxy)propyl)-4-(2-(4-nnethoxyphen yl)-4- methyl-

N-(4-isopropoxybenzyl)-4-(2-(4-methoxyphenyl)-4-methyl-7-oxo -2H- pyrazolo[3,4-d]pyridazin-6(7H)-yl)butanamide

N-(1 -(4-fluorophenyl)ethyl)-4-(2-(4-methoxyphenyl)-4-methyl-7-ox o-2H- pyrazolo[3,4-d]pyridazin-6(7H)-yl)butanamide

4-(2-(4-methoxyphenyl)-4-nnethyl-7-oxo-2H-pyrazolo[3,4-d]pyn dazin- 6(7H)-yl)-N-phenethylbutanamide

4-(2-(4-chlorophenyl)-4-methyl-7-oxo-2H-pyrazolo[3,4-d]pyrid azin-6(7H)- yl)-N-(3,4-dimethoxyphenethyl)butanannide

4-(2-(4-chlorophenyl)-4-methyl-7-oxo-2H-pyrazolo[3,4-d]pyrid azin-6(7H)- yl)-N-(3-(trifluoronnethyl)benzyl)butanannide

4-(2-(4-chlorophenyl)-4-methyl-7-oxo-2H-pyrazolo[3,4-d]pyrid azin-6(7H)- yl)-N-(2-(trifluoronnethyl)benzyl)butanannide 5

4-(3,4-dimethyl-7-oxo-2-(p-tolyl)-2H-pyrazolo[3,4-d]pyridazi n-6(7H)-yl)- N-(4-fluorobenzyl)butanamide

N-(4-fluorophenethyl)-4-(2-(4-methoxyphenyl)-4-nnethyl-7-oxo -2H- pyrazolo[3,4-d]pyridazin-6(7H)-yl)butanamide

4-(2-(4-methoxyphenyl)-4-nnethyl-7-oxo-2H-pyrazolo[3,4-d]pyn dazin- 6(7H)-yl)-N-(4-(trifluoronnethyl)benzyl)butanannide

6-(4-(3,4-dihydroisoquinolin-2(1 H)-yl)-4-oxobutyl)-2-(4- meth -7(6H)-one

I

4-(2-(4-chlorophenyl)-4-methyl-7-oxo-2H-pyrazolo[3,4-d]pyrid azin- 6(7H)-yl)-N-(2-fluorobenzyl)butanamide

4-(2-(4-chlorophenyl)-4-nnethyl-7-oxo-2H-pyrazolo[3,4-cl]pyn dazin- 6(7H)-yl)-N-((1 -phenylcyclohexyl)methyl)butanamide

4-(3,4-dimethyl-7-oxo-2-(p-tolyl)-2H-pyrazolo[3,4-d]pyridazi n-6(7H)-yl)- N-(3-(2-(2-methoxyethoxy)ethoxy)propyl)butanamide

4-(2-(4-chlorophenyl)-4-methyl-7-oxo-2H-pyrazolo[3,4-d]pyrid azin-6(7H)- yl)-N-(2,3-dihydro-1 H-inden-1 -yl)butanamide

4-(2-(4-chlorophenyl)-4-methyl-7-oxo-2H-pyrazolo[3,4-d]pyrid azin-6(7H)- yl)-N-(1 -phenylcyclopropyl)butanamide

4-(3,4-dimethyl-7-oxo-2-(p-tolyl)-2H-pyrazolo[3,4-d]pyridazi n-6(7H)-yl)- N-(4-fluorobenzyl)butanamide As suggested by the docking experiment (see Figure 2) and confirmed by the crystal structure of the complex compound 22 / PDE5 (see Figure 1 ) and 29 / PDE5 (see Figure 3), the inventive compounds of general formula (I) are able to bind deeply in the hydrophobic tunnel of PDE5 by establishing hydrophobic interactions and two H-bondings to Tyr-149 and Arg-61 .

Chemical synthesis

The inventive pyridazinones of general formula (I) can be prepared by methods known to one skilled in the art.

For example, pyrrolopyridazinones of general formula (l-A) with R ¹⁵ being -H and R ¹⁶ being -CH ₃ can be obtained from carboxylic acid 2* that can be further disconnected to commercially available phenylhydrazine 3* and ester 4*, which can be easily accessed from commercially available ethylacetoacetate, chloroacetone and 4-aminobutanoic acid derivative (see Scheme 1 ).

Scheme 1 : Retrosynthesis of pyrrolopyridazinones of general formula (l-A), wherein R ¹⁵ represents -H and R ¹⁶ represents -CH ₃ .

The assembly of carboxylic acid 2* commences with the treatment of commercially available ethylacetoacetate with chloroacetone in the presence of sodium hydride to provide intermediate 5* that is subsequently reacted with commercially available 5

4-aminobutanoic acid derivative in presence of acetic acid to furnish /V-substituted pyrrole 6* (see Scheme 2).

After masking the carboxylic acid as a methyl ester, intermediate 7* is reacted with Vilsmeier reagent to introduce the aldehyde function at the fourth position of the pyrrole moiety. Condensation of compound 8* with commercially available phenylhydrazine 9* afforded pyrrolopyridazinone 10*. Cleavage of the methyl ester on compound 10* with potassium hydroxide provided target carboxylic acid 2*.

10*

Scheme 2: Synthesis of carboxylic acid 2*

With carboxylic acid 2* in hands, pyrrolopyridazinones of general formula (l-A) with R ¹ being -CH ₂ CR ¹⁸ R ¹⁹ CH ₂ C(O)NR ²⁰ R ^6* , R ¹⁵ representing -H and R ¹⁶ representing -CH ₃ , can be easily accessed by simple amide coupling (see Scheme 3). Thus, treatment of carboxylic acid 2* with the suitable amine 1* in the presence of 1 - ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC ^' HCI) and 4- dimethylaminopyridine (DMAP) provides target pyrrolopyridazinone of general formula (l-A). Conveniently, amines of general formula R ^6* R ²⁰ NH are commercially available or can be easily synthesized using methods known to the person skilled in the art.

Scheme 3: Synthesis of pyrrolopyridazinone of general formula (I-A), wherein R ¹ represents -CH ₂ CR ¹⁸ R ¹⁹ CH ₂ C(O)NR ²⁰ R ^6* , R ¹⁵ is -H and R ¹⁶ represents -CH ₃ . Pyrrolopyridazinones of general formula (I-A), wherein R ¹⁵ represents -H, R ¹⁶ represents -CH ₃ , R ¹ represents -CH ₂ CR ¹⁸ R ¹⁹ CH ₂ R ⁴ and R ⁴ represents a 1 ,2,4- oxadiazole ring substituted at the third position with the substituent R ⁶ having the meaning defined above, can be synthesized by treatment of the common carboxylic acid intermediate 2* with the suitable amidoxime 11* in the presence of 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC ^' HCI) and triethylamine (see Scheme 4). Amidoxime 11* can be easily prepared by treating the corresponding commercially available nitrile 12* with hydroxylamine and ethanol at reflux.

R ⁶ -CN

12*

Scheme 4: Synthesis of pyrrolopyridazinone of general formula (I-A), wherein R ¹ represents -CH ₂ CR ¹⁸ R ¹⁹ CH ₂ R ⁴ , R ⁴ represents a 1 ,2,4-oxadiazole ring substituted at the third position with the substituent R ⁶ , R ¹⁵ is -H and R ¹⁶ represents -CH ₃ .

Pyrrolopyridazinones of general formula (I-A), wherein R ¹⁵ represents -H, R ¹⁶ represents -CH ₃ , R ¹ represents -CH ₂ CR ¹⁸ R ¹⁹ CH ₂ R ⁴ and R ⁴ represents a 1 ,3,4- oxadiazole ring substituted at the second position with the substituent R ⁶ having the meaning defined above, can be accessed via a two-step synthetic pathway starting from carboxylic acid 2* (see Scheme 5). Hence, treatment of carboxylic 5 acid 2* with acylhydrazine derivatives 13* in the presence of Λ/,Λ/,Λ/',Λ/'-tetrannethyl- 0-(1 H-benzotriazol-1 -yl)uronium hexafluorophosphate (HBTU) and diisopropylethylamine (DIPEA) furnishes an intermediate that via cyclocondensation in the presence of tosyl chloride and DIPEA furnishes target pyrrolopyridazinones.

R ¹⁵ = -H;

R ¹⁶ = -CH ₃ ;

Scheme 5: Synthesis of pyrrolopyridazinone of general formula (l-A), wherein R ¹ represents -CH ₂ CR ¹⁸ R ¹⁹ CH ₂ R ⁴ , R ⁴ represents a 1 ,3,4-oxadiazole ring substituted at the second position with the substituent R ⁶ , R ¹⁵ represents -H and R ¹⁶ represents -CH ₃ .

The pyrazolopyridazinones of general formula (l-B) can be disconnected to carboxylic acid 19* that can be easily prepared from 4-bromo-butanoic acid derivative 20* and pyrazolopyridazinone 21* (see Scheme 6). Conveniently, pyrazolopyridazinone 21* can be purchased (providers: Enamine, Innovapharm) or can be prepared following procedures known to the skilled person in the art (J. Heterocyclic Chem. 2014, 51 , 635 or Scheme 8).

20* 21*

Scheme 6: Retrosynthesis of pyrazolopyridazinones of general formula (l-B). 5

For example, carboxylic acid 19* with R ¹⁵ and R ¹⁶ being -CH ₃ and R ¹⁸ and R ¹⁹ being -H i.e. carboxylic acid 14* can be disconnected to methyl-4- bromobutanoate, hydrazine and ester 15*, which can be easily accessed from commercially available aniline derivatives, 2-chloroethylacetoacetate and acetylacetone (see Scheme 7).

15*

Scheme 7: Retrosynthesis of carboxylic acid 14*. The assembly of carboxylic acid 14* commences with the treatment of commercially available aniline derivative with sodium nitrite under acidic conditions to afford intermediate diazonium salt that is subsequently treated with 2- chloroethylacetoacetate to provide benzenediazonium chloride 16* (see Scheme 7). The sodium enolate of acetylacetone was then reacted with 16* to furnish pyrazole 15* that is without further purification subjected to condensation reaction with hydrazine to give pyrazolopyridazinone 17*. Finally, treatment of 17* with methyl-4-bromobutanoate in the presence of sodium hydride delivered ester 18*. Cleavage of the methyl ester with potassium hydroxide provided target carboxylic acid 14*.

18* 14*

Scheme 8: Synthesis of carboxylic acid 14*.

With carboxylic acid 14* in hands, pyrazolopyndazinones of general fornnula (VI) can be easily accessed by simple amide coupling (see Scheme 9). Thus, treatment of carboxylic acid 14* with the suitable amine in the presence of 1 -ethyl- 3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC ^' HCI) and 4- dimethylaminopyridine (DMAP) provides target pyrazolopyridazinone of general formula (VI). Conveniently, amines of general formula R ⁶ NH ₂ are commercially available or can be easily synthesized using methods known to the person skilled in the art.

R ⁶ NH ₂

Scheme 9: Synthesis of pyrazolopyridazinone of general formula (IV).

Some of the compounds of the present invention may be crystallized or recrystallized from solvents such as aqueous and organic solvents. In such cases solvates may be formed. This invention includes within its scope stoichiometric solvates including hydrates as well as compounds containing variable amounts of water that may be produced by processes such as lyophilization. The compounds of the general formulas (I) may exist in the form of optical isomers, i.e. enantiomers and mixtures of said isomers in all ratios, e.g. racemic mixtures. The invention includes all such forms, in particular the pure isomeric forms or enantiomeric forms. The different isomeric forms may be separated or resolved one from the other by conventional methods, or any given isomer may be obtained by conventional synthetic methods or by stereospecific or asymmetric syntheses.

The compounds of the general formula (I) may form salts with organic or inorganic acids. Examples of suitable acids for such acid addition salt formation are trifluoroacetic acid, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, oxalic acid, malonic acid, salicylic acid, p- aminosalicylic acid, malic acid, fumaric acid, succinic acid, ascorbic acid, maleic acid, sulfonic acid, phosphonic acid, perchloric acid, nitric acid, formic acid, propionic acid, gluconic acid, lactic acid, tartaric acid, hydroxymaleic acid, pyruvic acid, phenylacetic acid, benzoic acid, p-aminobenzoic acid, p-hydroxybenzoic acid, methanesulfonic acid, ethanesulfonic acid, nitrous acid, hydroxyethanesulfonic acid, ethylenesulfonic acid, p-toluenesulfonic acid, naphthylsulfonic acid, sulfanilic acid, camphorsulfonic acid, china acid, mandelic acid, o-methylmandelic acid, hydrogen-benzenesulfonic acid, picric acid, adipic acid, d-o-tolyltartaric acid, tartronic acid, (o, m, p)-toluic acid, naphthylamine sulfonic acid, and other mineral or carboxylic acids well known to those skilled in the art. The salts are prepared by contacting the free base form with a sufficient amount of the desired acid to produce a salt in the conventional manner. Indications and Pharmaceutical compositions

Another aspect of the present invention relates to the use of the inventive substituted pyrrolo- and pyrazolo-pyridazinones as drugs, i.e. as pharmaceutically active agents applicable in medicine.

Surprisingly, it was found that the above-mentioned pyridazinones, as well as the pharmaceutical compositions comprising said pyridazinones are useful for treatment or prophylaxis of cancer, tumors and proliferative diseases, preferably cancer, tumors and proliferative diseases caused by and/or associated with activating Ras mutations, and more preferably cancer, tumors and proliferative diseases caused by and/or associated with activating K-Ras mutations.

The term "mutation", as used herein, means a difference in the amino acid or nucleic acid sequence of a particular protein or nucleic acid (gene, RNA) relative to the wild-type protein or nucleic acid, respectively. A mutated protein or nucleic acid can be expressed from or found on one allele (heterozygous) or both alleles (homozygous) of a gene, and may be somatic or germ line. In the instant invention, mutations are generally somatic. Mutations include sequence rearrangements such as insertions, deletions, and point mutations.

The term "Ras mutation(s)" as used herein refers to a constitutive active form of the Ras protein caused by mutations mainly in the codons 12, 13, and 61 . Most common ones are the following point mutations: G12D, G12V, G12C, G12A, G12S, G12R, G13D, G13C, Q61 H.

Thus, the pyridazinone compounds of the present invention can be used for prophylaxis and/or treatment of cancers, tumors and proliferative diseases or for the preparation of a pharmaceutical formulation for prophylaxis and/or treatment of cancers, tumors and proliferative diseases, preferably cancer, tumors and proliferative diseases caused by and/or associated with activating Ras mutations.

As already mentioned, K-Ras is the most frequently mutated oncogene is tumors. Cancer cell lines harboring K-Ras mutations have been classified based on K-Ras dependency for cell viability into K-Ras dependent and K-Ras independent groups (Cancer Cell 2009, 15, 489). Examples of K-Ras dependent cell lines include, but are not restricted to: Capan-1 , Mia PaCa-2, Panc-Tu-I, NCI-H358, NCI-H441 . The compounds of general formula (I) are able to inhibit the proliferation of Ras dependent cells, leading to cell death. More specifically, the cancers, tumors and proliferative diseases that can be treated and/or prevented by the inventive compounds are selected from the group comprising or consisting of: adenocarcinoma, choroidal melanoma, acute leukemia, acoustic neurinoma, ampullary carcinoma, anal carcinoma, astrocytoma, basal cell carcinoma, pancreatic cancer, desmoid tumor, bladder cancer, bronchial carcinoma, non-small cell lung cancer (NSCLC), breast cancer, Burkitt's lymphoma, corpus cancer, CUP-syndrome (carcinoma of unknown primary), colorectal cancer, small intestine cancer, small intestinal tumors, ovarian cancer, endometrial carcinoma, ependymoma, epithelial cancer types, Ewing's tumors, gastrointestinal tumors, gastric cancer, gallbladder cancer, gall bladder carcinomas, uterine cancer, cervical cancer, glioblastomas, gynecologic tumors, ear, nose and throat tumors, hematologic neoplasias, hairy cell leukemia, urethral cancer, skin cancer, skin testis cancer, brain tumors (gliomas), brain metastases, testicle cancer, hypophysis tumor, carcinoids, Kaposi's sarcoma, laryngeal cancer, germ cell tumor, bone cancer, colorectal carcinoma, head and neck tumors (tumors of the ear, nose and throat area), colon carcinoma, craniopharyngiomas, oral cancer (cancer in the mouth area and on lips), cancer of the central nervous system, liver cancer, liver metastases, leukemia, eyelid tumor, lung cancer, lymph node cancer (Hodgkin's/Non-Hodgkin's), lymphomas, stomach cancer, malignant melanoma, malignant neoplasia, malignant tumors gastrointestinal tract, breast carcinoma, rectal cancer, medulloblastomas, melanoma, meningiomas, Hodgkin's disease, mycosis fungoides, nasal cancer, neurinoma, neuroblastoma, kidney cancer, renal cell carcinomas, non-Hodgkin's lymphomas, oligodendroglioma, esophageal carcinoma, osteolytic carcinomas and osteoplastic carcinomas, osteosarcomas, ovarial carcinoma, pancreatic carcinoma, penile cancer, plasmocytoma, squamous cell carcinoma of the head and neck (SCCHN), prostate cancer, pharyngeal cancer, rectal carcinoma, retinoblastoma, vaginal cancer, thyroid carcinoma, Schneeberger disease, esophageal cancer, spinalioma, T-cell lymphoma (mycosis fungoides), thymoma, tube carcinoma, eye tumors, urethral cancer, urologic tumors, urothelial carcinoma, vulva cancer, wart appearance, soft tissue tumors, soft tissue sarcoma, Wilm's tumor, cervical carcinoma and tongue cancer.

Cancer cells often depend on the survival signaling emanating from oncogene products, particularly from oncogenic K-Ras, for their survival. The induction of programmed cell death by the loss of such survival signaling, as disclosed for the compounds of the present invention, is especially useful in the treatment of cancer by inducing the death of oncogenic Ras dependent malignant cells. Since all kinds of cancer cells are destroyable through the induction of programmed cell death, all different kinds of cancer and abnormal proliferating cells can be treated with the compounds of the present invention.

Another aspect of the present invention is directed to a pharmaceutical composition comprising at least one compound of the present invention as active ingredient, together with at least one pharmaceutically acceptable carrier, excipient and/or diluent. The pharmaceutical compositions of the present invention can be prepared in a conventional solid or liquid carrier or diluent and a conventional pharmaceutically-made adjuvant at suitable dosage level in a known way. The preferred preparations are adapted for oral application. These administration forms include, for example, pills, tablets, film tablets, coated tablets, capsules, powders and deposits.

Furthermore, the present invention also includes pharmaceutical preparations for parenteral application, including dermal, intradermal, intragastral, intracutan, intravasal, intravenous, intramuscular, intraperitoneal, intranasal, intravaginal, intrabuccal, percutan, rectal, subcutaneous, sublingual, topical, or transdermal application, which preparations in addition to typical vehicles and/or diluents contain at least one compound according to the present invention. The pharmaceutical compositions according to the present invention containing at least one compound according to the present invention as active ingredient will typically be administered together with suitable carrier materials selected with respect to the intended form of administration, i.e. for oral administration in the form of tablets, capsules (either solid filled, semi-solid filled or liquid filled), powders for constitution, extrudates, deposits, gels, elixirs, dispersable granules, syrups, suspensions, and the like, and consistent with conventional pharmaceutical practices. For example, for oral administration in the form of tablets or capsules, the active drug component may be combined with any oral non-toxic pharmaceutically acceptable carrier, preferably with an inert carrier like lactose, starch, sucrose, cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, talc, mannitol, ethyl alcohol (liquid filled capsules) and the like. Moreover, suitable binders, lubricants, disintegrating agents and coloring agents may also be incorporated into the tablet or capsule. Powders and tablets may contain about 5 to about 95 weight % of the inventive pyridazone of general formula (I) as active ingredient.

Suitable binders include starch, gelatin, natural sugars, corn sweeteners, natural and synthetic gums such as acacia, sodium alginate, carboxymethylcellulose, 5 polyethylene glycol and waxes. Among suitable lubricants there may be mentioned boric acid, sodium benzoate, sodium acetate, sodium chloride, and the like. Suitable disintegrants include starch, methylcellulose, guar gum, and the like. Sweetening and flavoring agents as well as preservatives may also be included, where appropriate. The disintegrants, diluents, lubricants, binders etc. are discussed in more detail below.

Moreover, the pharmaceutical compositions of the present invention may be formulated in sustained release form to provide the rate controlled release of any one or more of the components or active ingredients to optimize the therapeutic effect(s), e.g. antihistaminic activity and the like. Suitable dosage forms for sustained release include tablets having layers of varying disintegration rates or controlled release polymeric matrices impregnated with the active components and shaped in tablet form or capsules containing such impregnated or encapsulated porous polymeric matrices.

Liquid form preparations include solutions, suspensions, and emulsions. As an example, there may be mentioned water or water/propylene glycol solutions for parenteral injections or addition of sweeteners and opacifiers for oral solutions, suspensions, and emulsions. Liquid form preparations may also include solutions for intranasal administration. Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be present in combination with a pharmaceutically acceptable carrier such as an inert, compressed gas, e.g. nitrogen. For preparing suppositories, a low melting fat or wax, such as a mixture of fatty acid glycerides like cocoa butter is melted first, and the active ingredient is then dispersed homogeneously therein e.g. by stirring. The molten, homogeneous mixture is then poured into conveniently sized moulds, allowed to cool, and thereby solidified. Also included are solid form preparations, which are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration. Such liquid forms include solutions, suspensions, and emulsions.

The compounds according to the present invention may also be delivered transdermally. The transdermal compositions may have the form of a cream, a lotion, an aerosol and/or an emulsion and may be included in a transdermal patch of the matrix or reservoir type as is known in the art for this purpose. The term capsule as recited herein refers to a specific container or enclosure made e.g. of methyl cellulose, polyvinyl alcohols, or denatured gelatins or starch for holding or containing compositions comprising the active ingredient(s). Capsules with hard shells are typically made of blended of relatively high gel strength gelatins from bones or pork skin. The capsule itself may contain small amounts of dyes, opaquing agents, plasticisers and/or preservatives. Under tablet a compressed or moulded solid dosage form is understood, which comprises the active ingredients with suitable diluents. The tablet may be prepared by compression of mixtures or granulations obtained by wet granulation, dry granulation, or by compaction well known to a person of ordinary skill in the art.

Oral gels refer to the active ingredients dispersed or solubilized in a hydrophilic semi-solid matrix. Powders for constitution refers to powder blends containing the active ingredients and suitable diluents which can be suspended e.g. in water or in juice.

Suitable diluents are substances that usually make up the major portion of the composition or dosage form. Suitable diluents include sugars such as lactose, sucrose, mannitol, and sorbitol, starches derived from wheat, corn rice, and potato, and celluloses, such as microcrystalline cellulose. The amount of diluent in the composition can range from about 5 to about 95 % by weight of the total composition, preferably from about 25 to about 75 weight %, and more preferably from about 30 to about 60 weight %.

The term disintegrants refers to materials added to the composition to support break apart (disintegrate) and release the pharmaceutically active ingredients of a medicament. Suitable disintegrants include starches, "cold water soluble" modified starches such as sodium carboxymethyl starch, natural and synthetic gums such as locust bean, karaya, guar, tragacanth and agar, cellulose derivatives such as methylcellulose and sodium carboxymethylcellulose, microcrystalline celluloses, and cross-linked microcrystalline celluloses such as sodium croscaramellose, alginates such as alginic acid and sodium alginate, clays such as bentonites, and effervescent mixtures. The amount of disintegrant in the composition may range from about 2 to about 20 weight % of the composition, more preferably from about 5 to about 10 weight %.

Binders are substances which bind or "glue" together powder particles and make them cohesive by forming granules, thus serving as the "adhesive" in the 7 formulation. Binders add cohesive strength already available in the diluent or bulking agent. Suitable binders include sugars such as sucrose, starches derived from wheat corn rice and potato, natural gums such as acacia, gelatin and tragacanth, derivatives of seaweed such as alginic acid, sodium alginate and ammonium calcium alginate, cellulose materials such as methylcellulose, sodium carboxymethylcellulose and hydroxypropylmethylcellulose, polyvinylpyrrolidone, and inorganic compounds such as magnesium aluminum silicate. The amount of binder in the composition may range from about 2 to about 20 weight % of the composition, preferably from about 3 to about 10 weight %, and more preferably from about 3 to about 6 weight %.

Lubricants refer to a class of substances, which are added to the dosage form to enable the tablet granules etc. after being compressed to release from the mould or die by reducing friction or wear. Suitable lubricants include metallic stearates such as magnesium stearate, calcium stearate, or potassium stearate, stearic acid, high melting point waxes, and other water soluble lubricants such as sodium chloride, sodium benzoate, sodium acetate, sodium oleate, polyethylene glycols and D,L-leucine. Lubricants are usually added at the very last step before compression, since they must be present at the surface of the granules. The amount of lubricant in the composition may range from about 0.2 to about 5 weight % of the composition, preferably from about 0.5 to about 2 weight %, and more preferably from about 0.3 to about 1 .5 weight % of the composition.

Glidents are materials that prevent caking of the components of the pharmaceutical composition and improve the flow characteristics of granulate so that flow is smooth and uniform. Suitable glidents include silicon dioxide and talc. The amount of glident in the composition may range from about 0.1 to about 5 weight % of the final composition, preferably from about 0.5 to about 2 weight %. Coloring agents are excipients that provide coloration to the composition or the dosage form. Such excipients can include food grade dyes adsorbed onto a suitable adsorbent such as clay or aluminum oxide. The amount of the coloring agent may vary from about 0.1 to about 5 weight % of the composition, preferably from about 0.1 to about 1 weight %.

Another aspect of this invention provides a method of treating a disease or a medical condition in a patient comprising administering to said patient one or more compound(s) of general formula (I) in an amount effective to treat or prevent said disease or condition. In a particular embodiment, the invention provides a method of treating or preventing of a proliferative disease, a tumor and/or a cancer in a patient, which comprises administering to said patient a therapeutically effective amount of a compound of general formula (I).

The term "effective amount" means an amount of compound that, when administered to a patient in need of such treatment, is sufficient to

(i) treat or prevent a particular disease, condition, or disorder;

(ii) attenuate, ameliorate, or eliminate one or more symptoms of the particular disease, condition, or disorder; or

(iii) prevent or delay the onset of one or more symptoms of the particular disease, condition, or disorder described herein. The amount of a compound of general formula (I) that will correspond to such an amount will vary depending upon factors such as the particular compound, disease condition and its severity, the identity (e.g., weight) of the patient in need of treatment, but can nevertheless be routinely determined by one skilled in the art.

Description of the Figures

Figure 1 shows a) the structure of compound 22; b) the crystal structure of inhibitor 22 in complex with PDE5 at 2.0 A resolution showing H-bonding interactions with Tyr149 and Arg61 .

Figure 2 shows a) the structure of compound 1 ; b) the modeling (Schrodinger, Maestro suite) of compound 1 into the prenyl binding site of PDE5, showing H- bonding interactions with Tyr149 and Arg61 .

Figure 3 shows a) the structure of compound 29; b) crystal structure of compound 29 in complex with PDE5 at 2.6 A resolution H-bonding interactions with Tyr149 and Arg61 .

Figure 4 shows a) compound 50 dose dependence of molar fraction (a) of interacting mCitrine-Rheb with mCherry-PDE5. First row shows fluorescence intensity distribution of mCitrine-Rheb, second row shows fluorescence intensity distribution of mCherry-PDE5, third row represents average mCit ne fluorescence lifetime (t _av ) in ns and forth row represents molar fraction (a) of interacting mCitrine-Rheb with mCherry-PDE5. The concentration of compound 50 is indicated at the top of the panel in nM; b) Fit of the dose-response relationship of molar interacting fraction (a) in dependence to increasing concentration of compound 50 to a binding model (n=5).

Figure 5 shows K-Ras-PDE5 interaction and inhibition. Upper row: fluorescence intensity of mCitrine-K-Ras distribution, lower row: molar fraction (a) of interacting mCitrine-Rheb with mCherry-PDE5. To monitor if K-Ras is indeed a client of the Arl-PDE5 system, the non-plasma membrane bound fraction of K-Ras was increased by a PKC induced (Bryostatin) phosphorylation of a Serine (S181 ) located in its polybasic stretch. The administration of Bryostatin relocated K-Ras and increased the interacting fraction (middle column) with PDE5. Subsequent addition of compound 50 (right column) abolished the interaction between K-Ras and PDE5.

Figure 6 shows continuous impedance measurements (RTCA) (monitored every 15 min up to 100 hours) of compound 50 dose-dependent cell proliferation response (for PANC-1 (see Figure 6d), BxPC-3 (see Figure 6e), Panc-Tu-I (see Figure 6f), Capan-1 (see Figure 6g)). Compound 50 was administered 24 hours after seeding at indicated concentrations. Figures 6a, 6b, 6c show the growth rates calculated from the RTCA curves shown in Figures 6d-6g over the indicated time span (Figure 6a: 40 to 60 min; Figure 6b: 50 to 70 min; Figure 6c: 50 to 70 min; time span indicated by a black bar in Figures 6d, 6f and 6g) at given concentrations of compound 50.

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. 7

Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims.

EXAMPLES A. Chemical Synthesis General Information:

All reactions involving air- or moisture-sensitive reagents or intermediates were carried out in flame dried glassware under an argon atmosphere. Dry solvents (THF, toluene, MeOH, DMF) were used as commercially available; CH2CI2 was purified by the Solvent Purification System M-BRAUN Glovebox Technology SPS- 800. Analytical thin-layer chromatography (TLC) was performed on Merck silica gel aluminium plates with F-254 indicator. Compounds were visualized by irradiation with UV light or potassium permanganate staining. Column chromatography was performed using silica gel Merck 60 (particle size 0.040- 0.063 mm). ¹ H-NMR and ¹³ C-NMR were recorded on a Bruker DRX400 (400 MHz), Bruker DRX500 (500 MHz) and INOVA500 (500 MHz) at 300 K using CDCI ₃ or (CD ₃ ) ₂ SO as solvent. All resonances are reported relative to TMS. Spectra were calibrated relative to solvent's residual proton and carbon chemical shift: CDCI3 (δ = 7.26 ppm for ¹ H NMR and δ = 77.16 ppm for ¹³ C NMR); (CD ₃ ) ₂ SO: δ= 2.50 ppm for ¹ H NMR and δ= 39.52 ppm for ¹³ C NMR). Multiplicities are indicated as: br s (broadened singlet), s (singlet), d (doublet), t (triplet), q (quartet), quin (quintet), m (multiplet); and coupling constants (J) are given in Hertz (Hz). High resolution mass spectra were recorded on a LTQ Orbitrap mass spectrometer coupled to an Acceka HPLC-System (HPLC column: Hypersyl GOLD, 50 mm x 1 mm, particle size 1 .9 μιτι, ionization method: electron spray ionization). Fourier transform infrared spectroscopy (FT-IR) spectra were obtained with a Bruker Tensor 27 spectrometer (ATR, neat) and are reported in terms of frequency of absorption 7

(cm ^"1 ). Atorvastatin was purchased from Sequoia Reseach Products. All chemicals and solvents were purchased from Sigma-Aldrich, Fluka, TCI, Acros Organics, ABCR, Alfa Aesar, Enamine, VWR and Innovapharm. Unless otherwise noted, all commercially available compounds were used as received without further purifications.

Example A.1 : Synthesis of ethyl 2-acetyl-4-oxopentanoate (5 ^' )

A solution of ethyl acetoacetate (13.01 g, 100.0 mmol) in THF (150 mL) was cooled to 0 °C and NaH (60 % in mineral oil, 4.40 g, 1 10.0 mmol, 1 .1 equiv) was added to it slowly over 15 min. After complete addition, the mixture was stirred at room temperature for another 1 h. Then chloroacetone (7.96 mL, 100.0 mmoL, 1 .0 equiv.) was added dropwise and the mixture was stirred for an additional 16 h at rt. The volatile components were removed and the residue was re-dissolved in diethyl ether (100 mL). The solution was washed with water (20 ml χ 2) before being dried over Na2SO ₄ and concentrated in vacuo. Crude product was purified by flash column chromatography (FCC) on silica gel using 10% ethyl acetate/Petroleum ether (EA PE) as an eluent to provide analytically pure product 5 ^' (6.0 g, 33% yield) as a colorless liquid. FT-IR (neat): v = 2984, 1739, 171 1 , 1359, 1259, 1 157, 1019, 866, 744 cm ^"1 . ¹ H NMR (500 MHz, CDCI ₃ ) δ 4.17 (q, J = 7.1 Hz, 2H), 3.99 (dd, J = 8.2, 5.7 Hz, 1 H), 3.12 (dd, J = 18.5, 6.9 Hz, 1 H), 2.93 (dd, J = 18.5, 6.9 Hz, 1 H), 2.33 (s, 3H), 2.17 (s, 3H), 1 .26 (t, J = 7.1 Hz, 3H). ¹³ C NMR (126 MHz, CDCIs) δ 205.7, 202.3, 168.9, 61 .8, 53.9, 41 .6, 30.2, 29.8, 14.1 . HRMS (ESI): calc. for [M+Na] ⁺ C ₉ Hi ₄ O ₄ Na: 209.07843, found: 209.07843.

Example A.2: Synthesis of 4-(3-(ethoxycarbonyl)-2,5-dimethyl-1 H-pyrrol-1 - yl)butanoic acid (6 ^' )

6 ^'

Ethyl 2-acetyl-4-oxopentanoate (5 ^' ) (0.56 g, 3.0 mmol, 1 equiv) and 4- aminobutanoic acid (0.31 g, 1 .0 equiv.) were dissolved in acetic acid (2.62 mL) and stirred at ambient temperature for 30 min. After all solid had dissolved, the 7 reaction mixture was heated in a CEM microwave reactor (close vessel, 150 W, 120 °C) for 30 min. The reaction was completed after 30 min as monitored by TLC (40% EA/PE). After completion, the reaction mixture was diluted with ethyl acetate (10 mL) and acetic acid was neutralized using saturated NaHCO3 (If excess NaHCOs was used, then the desired compound was transferred to the H ₂ O layer, which could be recovered by neutralization with acid to pH 4). The aqueous layer was extracted with ethyl acetate (2 x 20 mL); the combined organic layers were sequentially washed with H ₂ O (10 ml x 2), brine (10 mL), dried over Na ₂ SO ₄ , filtered and concentrated in vacuo to give the desired product 6 ^' (0.66 g, 87%) as a brown solid in good purity. The compound was used in the next step without further purification. FT-IR (neat): v = 3545, 2942, 1942, 1743, 1708, 1559, 1475, 1371 , 1248, 1222, 1095, 815, 775 cm ^"1 . ¹ H NMR (400 MHz, CDCI ₃ ) δ 10.75 (s, 1 H), 6.25 (s, 1 H), 4.23 (q, J = 7.1 Hz, 2H), 3.82 (t, J = 7.6 Hz, 2H), 2.51 (s, 3H), 2.40 (t, J = 7.0 Hz, 2H), 2.19 (s, 3H), 2.02 - 1 .81 (m, 2H), 1 .31 (t, J = 7.1 Hz, 3H). ¹³ C NMR (101 MHz, CDCI3) δ 178.1 , 166.0, 135.1 , 127.4, 1 10.9, 107.8, 59.4, 42.6, 30.7, 25.2, 14.6, 12.20, 1 1 .40. HRMS (ESI): calc. for [M+H] ⁺ Ci ₃ H ₂₀ O ₄ N: 254.13868, found: 254.13817.

Example A.3: Synthesis of ethyl 1 -(4-methoxy-4-oxobutyl)-2,5-dimethyl-1 H- pyrrole-3-carboxylate (7 ^' )

T

To a solution of 6 ^' (3,36 g, 13.26 mmol, 1 equiv) and DIPEA (4.62 mL, 26. 53 mmol, 2.0 equiv) in anhydrous methanol (40 mL) at 0 °C was added drop wise thionyl chloride (1 .15 mL, 15.92 mmol, 1 .2 equiv) and the resulting reaction mixture was left to stir for 16 h. After completion, volatile components were removed and the reaction mixture was diluted with dichloromethane (25 mL) and water (20 mL). The aqueous layer was extracted with dichloromethane (2 x 10 mL); the combined organic layers were sequentially washed with H ₂ O (10 ml χ 2), brine (20 mL), dried over Na ₂ SO ₄ , filtered and concentrated under vacuum to give the desired product T (3.47 g, 98%) in good purity. FT-IR (neat): v = 2951 , 1735, 1689, 1578, 1532, 1420, 1371 , 1218, 1 184, 1 171 , 1093, 1062, 867, 772 cm ^"1 . ¹ H NMR (400 MHz, CDCI ₃ ) δ 6.24 (s, 1 H), 4.22 (q, J = 7.1 Hz, 2H), 3.80 (t, J = 7.6 Hz, 2H), 3.68 (s, 3H), 2.51 (s, 3H), 2.34 (t, J = 7.0 Hz, 2H), 2.19 (s, 3H), 1 .92 (quin, J = 7.4 Hz, 2H), 1 .31 (t, J = 7.1 Hz, 3H). ¹³ C NMR (101 MHz, CDCI ₃ ) δ 173.1 , 165.8, 7

135.0, 127.4, 1 1 1 .0, 107.8, 59.2, 51 .9, 42.7, 30.7, 25.5, 14.6, 12.2, 1 1 .4. HRMS (ESI): calc. for [M+H] ⁺ Ci ₄ H ₂₂ O ₄ N: 268.15433, found: 268.15384.

Example A.4: Synthesis of ethyl 4-formyl-1 -(4-methoxy-4-oxobutyl)-2,5-dimethyl- 1 H-pyrrole-3-carboxylate (

8 ^'

Phosphorus oxychloride (1 .91 mL, 20.5 mmol, 1 .6 equiv) was added to anhydrous DMF (1 .99 mL, 25.7 mmol, 2.0 equiv) at 0 °C during 8 min, and the resulting mixture was stirred at the same temperature for 30 min. Then, the reaction mixture was diluted by 1 ,2-dichloroethane (12 mL), followed by addition of a solution of pyrrole derivative 7 ^' (3.43 g, 12.83 mmol, 1 .0 equiv) in 1 ,2-dichloroethane (20 mL). The mixture was then heated at reflux for 45 min. After slight cooling, a solution of sodium acetate (4.21 g, 51 .32 mmol, 4.0 equiv) in H ₂ O (20.15 mL) was added and the mixture was reheated at reflux for an additional 30 min. After completion, the reaction mixture was allowed to come to room temperature and was diluted with dichloromethane (30 mL) and water (20 mL). The aqueous layer was extracted with dichloromethane (2 x 10 mL); the combined organic layers were sequentially washed with H ₂ O (10 ml χ 2), brine (25 mL), dried over Na ₂ SO ₄ , filtered and concentrated under vacuum to give the desired product 8 ^' (3.79 g, 99%) in good purity. FT-IR (neat): v = 2986, 2925, 1732, 1702, 1648, 1555, 1525, 1438, 1361 , 1267, 1 197, 1 178, 1 141 , 1096, 971 , 890, 869, 777 cm ^"1 . ¹ H NMR (400 MHz, CDCIs) δ 10.37 (s, 1 H), 4.29 (q, J = 7.1 Hz, 2H), 3.86 (t, J = 8.0 Hz, 2H), 3.67 (s, 3H), 2.53 (s, 3H), 2.50 (s, 3H), 2.37 (t, J = 6.8 Hz, 2H), 1 .90 (quin, J = 7.4 Hz, 2H), 1 .33 (t, J = 7.1 Hz, 3H). ¹³ C NMR (101 MHz, CDCI ₃ ) δ 190.1 , 172.8, 165.1 , 136.2, 136.1 , 120.0, 1 12.3, 60.1 , 52.0, 42.5, 30.5, 25.0, 14.5, 1 1 .6, 1 1 .4. HRMS (ESI): calc. for [M+H] ⁺ Ci ₅ H ₂₂ O ₅ N: 296.14925, found: 296.14893.

Example A.5: Synthesis of methyl 4-(5,7-dimethyl-1 -oxo-2-phenyl-1 H-pyrrolo[3,4- d]pyridazin-6(2H)-yl)butanoate (10 ^' )

10 ^' 7

Pyrrolo precursor 8 ^' (3.74 g, 12.7 mmol, 1 equiv) and phenyl hydrazine 9 ^' (1 .31 ml_, 13.31 mmol, 1 .05 equiv) were dissolved in acetic acid (52 ml_) and the mixture was heated to 90 °C for 24 h. After completion, the reaction was diluted with DCM and aqueous layer was extracted with dichloromethane (3 x 20 ml_). The combined organic layers were sequentially washed with saturated NaHCO3 solution (20 ml_ x 2), H ₂ O (20 ml 2) and brine (25 ml_), dried over Na ₂ SO ₄ , filtered and concentrated under vacuum. The crude residue was further purified by FCC on silica-gel using 2% MeOH/CH ₂ CI ₂ as an eluent to afford desired 10 ^' (3.42 g, 79%). FT-IR (neat): v = 2941 , 1726, 1658, 1561 , 1313, 1255, 1089, 978, 956, 764, 697 cm ^"1 . ¹ H NMR (400 MHz, CDCI ₃ ) δ 8.03 (s, 1 H), 7.58 (d, J = 7.7 Hz, 2H), 7.44 (t, J = 7.7 Hz, 2H), 7.30 (t, J = 7.7 Hz, 2H), 4.04 (t, J = 7.8 Hz, 2H), 3.72 (s, 3H), 2.75 (s, 3H), 2.50 (s, 3H), 2.42 (t, J = 6.8 Hz, 2H), 2.09 - 1 .97 (m, 2H). ¹³ C NMR (101 MHz, CDCI ₃ ) δ 172.8, 159.3, 142.5, 134.0, 129.8, 128.5, 126.9, 126.4, 122.6, 1 15.6, 1 1 1 .3, 52.0, 43.4, 30.5, 25.2, 1 1 .1 , 10.3. HRMS (ESI): calc. for [M+H] ⁺ Ci9H ₂₂ O ₃ N ₃ : 340.16557, found: 340.16539.

Example A.6: Synthesis of 4-(5,7-Dimethyl-1 -oxo-2-phenyl-1 H-pyrrolo[3,4- d]pyridazin-6(2H)-yl)butanoic acid (2 ^' )

2 ^'

A heterogeneous mixture of pyrrolopyridazone derivative 10 ^' (3.37 g, 9.93 mmol, 1 .0 equiv) and KOH (0.67 g, 1 1 .92 mmol, 1 .2 equiv) in H ₂ O (43 ml_) was heated to reflux until all the solid had dissolved (it takes around 30 min). Then the reaction mixture was allowed to come to room temperature, cooled to 0 °C and was acidified with 10% HCI solution to a pH of 5-6. The resultant precipitation was then filtered, dried under vacuum to yield analytically pure 2 ^' as a light brown solid (3.0 g, 92%). FT-IR (neat): ? = 3304, 1687, 1645, 1542, 1505, 1455, 1391 , 1316, 1207, 1024, 1091 , 962, 832, 767, 698 cm ^"1 . ¹ H NMR (400 MHz, DMSO) δ 12.28 (s, 1 H), 8.23 (s, 1 H), 7.48 (d, J = 7.5 Hz, 2H), 7.42 (t, J = 7.7 Hz, 2H), 7.29 (t, J = 7.2 Hz, 1 H), 4.05 (t, J = 7.8 Hz, 2H), 2.64 (s, 3H), 2.49 (s, 3H), 2.34 (t, J = 6.9 Hz, 2H), 1 .85 (quin, J = 7.6 Hz, 2H). ¹³ C NMR (101 MHz, DMSO) δ 173.7, 158.2, 142.5, 134.4, 129.3, 128.1 , 126.3, 126.3, 123.7, 1 14.6, 109.9, 43.1 , 30.4, 24.9, 10.7, 9.7. HRMS (ESI): calc. for [M+H] ⁺ Ci ₈ H ₂₀ O ₃ N ₃ : 326.14992, found: 326.14948. Example A.7: General procedure I for amide coupling

To a solution of acid derivative 2 ^' (0.049 g, 0.15 mmol, 1 .0 equiv) and DMAP (0.024 g, 0.195 mmol, 1 .3 equiv) in THF/DCM (1 :1 , 0.6 ml_ and 0.6 ml_) was added EDC ^' HCI (0.037 g, 0.195 mmol, 1 .3 equiv), followed by the corresponding amine derivative 1* (0.158 mmol, 1 .05 equiv) and the resulting solution was stirred for 16 h at ambient temperature. Once completed, as observed by TLC, the reaction mixture was diluted with CH2CI2 (5 ml_) and quenched by adding saturated NaHCO3 solution (5 ml_). The aqueous layer was extracted with dichloromethane (2 x 5 ml_) and combined organic layers were sequentially washed with saturated NH ₄ CI solution (5 ml_ x 2), H ₂ O (5 ml 2) and brine (5 ml_); dried over Na ₂ SO ₄ , filtered and concentrated under vacuum. The crude residue was further purified by FCC on silica-gel using 2% MeOH/CH ₂ Cl2 as an eluent to afford desired compounds. Example A.7.1 : Synthesis of A/-cyclohexyl-4-(5,7-dimethyl-1 -oxo-2-phenyl-1 /-/- pyrrolo[3,4-c/]pyridazin-6(2H)-yl)butanamide (11 )

(11 )

The title compound (0.05 g, 89%) was prepared according to general procedure I. FT-IR (neat): v = 3287, 2929, 2359, 2341 , 1634, 1543, 1446, 1321 , 1 124, 963, 757, 725, 693 cm ^"1 . ¹ H NMR (400 MHz, DMSO) δ 8.23 (s, 1 H), 7.74 (d, J = 7.7 Hz, 1 H), 7.47 (d, J = 7.4 Hz, 2H), 7.42 (t, J = 7.6 Hz, 2H), 7.34 - 7.26 (m, 1 H), 4.02 (t, J = 7.5 Hz, 2H), 3.58 - 3.46 (m, 1 H), 3.35 (s, 3H), 2.63 (s, 3H), 2.13 (t, J = 6.9 Hz, 2H), 1 .94 - 1 .79 (m, 2H), 1 .77 - 1 .61 (m, 4H), 1 .60 - 1 .49 (m, 1 H), 1 .32 - 1 .18 (m, 2H), 1 .17 - 1 .03 (m, 3H). ¹³ C NMR (101 MHz, DMSO) δ 169.9, 158.2, 142.5, 134.4, 129.3, 128.1 , 126.3, 126.2, 123.8, 1 14.6, 109.9, 47.4, 43.3, 32.5, 31 .8, 25.4, 25.3, 24.6, 10.7, 9.8. HRMS (ESI): calc. for [M+H] ⁺ C ₂ H ₃ iO ₂ N ₄ : 407.24415, found: 407.24318.

Example A.8

Synthesis of pyrrolopyridazinones of general formula l-A, wherein R ¹ represents -CH ₂ CR ¹⁸ R ¹⁹ CH ₂ R ⁴ , R ⁴ represents a 1 ,2,4-oxadiazole ring substituted at the third position with the substituent R ⁶ , R ¹⁵ represents -H and R ¹⁶ represents -CH ₃ . 7

aq. NH ₂ OH (4.0 equiv)

EtOH, reflux, 6 h 39-56% yield quantitative

CN

General procedure II for the synthesis pyrrolopyridazinones of general formula l-A presenting a 1 ,2,4-oxadiazole ring:

Hydroxylamine (50% by weight in H ₂ O, 1 .32 mL, 20 mmol, 4 equiv) and the corresponding nitrile derivatives 12 ^' (5 mmol, 1 equiv) were combined in EtOH (20 mL) and heated to reflux for 2 h. Then the reaction mixture was cooled to 23 °C and was concentrated in vacuo to give the corresponding amidoxime derivatives 11 ^' as white solids in quantitative yields. ( A. R. Gangloff, J. Litvak, E. J. Shelton, D. Sperandio, V. R. Wang, K. D. Rice, Tetrahedron Lett. 2001 , 42, 1441 .) The analytical data of synthesized amidoxime derivatives 11 ^' were in good correlation with literature reported data. (X. Yang, G. Liu, H. Li, Y. Zhang, D. Song, C. Li, R. Wang, B. Liu, W. Liang, Y. Jing, G. Zhao, J. Med. Chem. 2010, 53, 1015; C. Quan, M. Kurth, J. Org. Chem. 2004, 69, 1470.)

Synthesized amidoxime derivatives 11 ^' (0.101 mmol, 1 .01 equiv) were treated with acid precursor 2 ^' (0.033 g, 0.1 mmol, 1 equiv) in the presence of EDC HCI (0.038 g, 0.2 mmol, 2 equiv) and triethylamine (0.028 mL, 0.2 mmol, 2 equiv) in diglyme (2-3 mL) and heated to 50 °C for 12 h. Then the reaction mixture was further heated to 1 10 °C for 5 h for the cyclodehydration step to take place. After completion, the reaction mixture was diluted with dichloromethane (5 mL) and water (5 mL). The aqueous layer was extracted with dichloromethane (2 x 5 mL); the combined organic layers were sequentially washed with H ₂ O (10 ml x 2), saturated brine (5 mL), dried over Na ₂ SO ₄ , filtered and concentrated in vacuo to give crude l-A. The resulting crude material was purified by FCC on silica gel using 3% methanol/dichloromethane as an eluent to give the desired 1 ,2,4- oxadiazole derivatives l-A in 39-56% yields.

Example A.8.1 : Synthesis of 5,7-dimethyl-2-phenyl-6-(3-(3-phenyl-1 ,2,4- oxadiazol-5-yl)propyl)-2,6-dihydro-1 H-pyrrolo[3,4-d]pyridazin-1 -one (16)

Ph

(16)

The title compound was prepared according to general procedure II (0.023 g, 53%). FT-IR (neat): v= 2922, 1652, 1594, 1444, 1351, 1306, 1116, 1090, 961, 903, 720, 692 cm ^"1 . ¹ H NMR (500 MHz, CDCI ₃ ) δ 8.07 (dd, J = 8.1, 1.6 Hz, 2H), 8.01 (s, 1H), 7.55 (dd, J = 8.4, 1.1 Hz, 2H), 7.53 - 7.48 (m, 3H), 7.45 - 7.40 (m, 2H), 7.30 (t, J= 7.4 Hz, 1H), 4.19 (t, J = 7.8 Hz, 2H), 3.05 (t, J = 7.0 Hz, 2H), 2.78 (s, 3H), 2.52 (s, 3H), 2.33 (quin, J = 7.5 Hz, 2H). ¹³ C NMR (126 MHz, CDCI ₃ ) δ 178.1, 168.5, 142.5, 133.9, 131.5, 129.8, 129.1, 128.8, 128.6, 127.5, 127.0, 126.6, 126.5, 122.5, 115.9, 111.6, 43.3, 26.7, 23.8, 11.3, 10.5. HRMS (ESI): calc. for [M+H] ⁺ C25H ₂ O2N ₅ : 426.19245, found: 426.19208.

Example A.8.2: Synthesis of 5,7-dimethyl-2-phenyl-6-(3-(3-(p-tolyl)-1 ,2,4- oxadiazol-5-yl)propyl)-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazi n-1-one (17)

(17)

The title compound was prepared according to general procedure II (0.018 g, 41%). FT-IR (neat): v= 2918, 1653, 1590, 1560, 1305, 1180, 1115, 1090, 960, 797, 693 cm ^"1 . ¹ H NMR (500 MHz, CDCI ₃ ) δ 8.01 (s, 1 H), 7.95 (d, J = 8.2 Hz, 2H), 7.55 (d, J = 7.4 Hz, 2H), 7.42 (t, J = 7.9 Hz, 2H), 7.30 (dd, J = 7.6, 3.4 Hz, 3H), 4.18 (t, J = 7.8 Hz, 2H), 3.04 (t, J = 7.0 Hz, 2H), 2.78 (s, 3H), 2.51 (s, 3H), 2.42 (s, 3H), 2.32 (quin, J = 7.5 Hz, 2H). ¹³ C NMR (126 MHz, CDCI ₃ ) δ 177.9, 168.6, 159.3, 142.6, 141.9, 133.8, 129.9, 129.8, 128.6, 127.4, 126.9, 126.5, 123.8, 122.5, 7

116.0, 111.7, 43.3, 26.8, 23.8, 21.7, 11.3, 10.5. HRMS (ESI): calc. for [M+H] ⁺ C26H26O2N5: 440.20810, found: 440.20793.

Example A.8.3: Synthesis of 6-(3-(3-(4-methoxyphenyl)-1 ,2,4-oxadiazol-5- yl)propyl)-5,7-dimethyl-2-phenyl-2,6-dihydro-1H-pyrrolo[3,4- d]pyridazin-1-one (18)

(18)

The title compound was prepared according to general procedure II (0.020 g, 44%). FT-IR (neat): v = 2937, 1652, 1612, 1590, 1303, 1252, 1172, 1116, 1090, 1025, 960, 901, 839, 754, 693 cm ^"1 . ¹ H NMR (500 MHz, CDCI ₃ ) δ 8.05 - 7.97 (m, 3H), 7.55 (d, J = 7.7 Hz, 2H), 7.42 (t, J = 7.8 Hz, 2H), 7.29 (t, J = 7.4 Hz, 1 H), 6.99 (d, J = 8.8 Hz, 2H), 4.17 (t, J = 7.8 Hz, 2H), 3.86 (s, 3H), 3.03 (t, J = 6.9 Hz, 2H), 2.78 (s, 3H), 2.51 (s, 3H), 2.31 (quin, J = 7.5 Hz, 2H). ¹³ C NMR (126 MHz, CDCI ₃ ) δ 178.1, 168.5, 162.5, 159.6, 142.8, 134.1, 130.1, 129.4, 128.8, 127.2, 126.7, 122.8, 119.3, 116.3, 114.8, 111.9, 55.8, 43.6, 27.0, 24.1, 11.5, 10.7. HRMS (ESI): calc. for [M+HJ^e^eOsNs: 456.20302, found: 456.20280.

Example A.8.4: Synthesis of 6-(3-(3-(2,5-dimethoxyphenyl)-1 ,2,4-oxadiazol-5- yl)propyl)-5,7-dimeth l-2-phenyl-2,6-dihydro-1H-pyrrolo[3,4-d]pyridazin-1-one (19)

7

The title compound was prepared according to general procedure II (0.027 g, 56%). FT-IR (neat): v = 2925, 1653, 1575, 1500, 1460, 1272, 1217, 1 179, 1042, 959, 810, 767, 694 cm ^"1 . ¹ H NMR (400 MHz, CDCI ₃ ) δ 8.03 (s, 1 H), 7.60 - 7.53 (m, 3H), 7.45 (t, J = 7.7 Hz, 2H), 7.31 (t, J = 7.4 Hz, 1 H), 7.10 - 6.99 (m, 2H), 4.19 (t, J = 7.8 Hz, 2H), 3.95 (s, 3H), 3.85 (s, 3H), 3.07 (t, J = 6.9 Hz, 2H), 2.79 (s, 3H), 2.52 (s, 3H), 2.32 (quin, J = 7.6 Hz, 2H). ¹³ C NMR (101 MHz, CDCI ₃ ) δ 176.9, 166.9, 159.3, 153.6, 152.5, 142.4, 133.9, 129.8, 128.6, 127.0, 126.4, 122.5, 1 18.4, 1 16.0, 1 15.8, 1 15.7, 1 13.3, 1 1 1 .5, 56.7, 56.0, 43.3, 26.7, 23.6, 1 1 .2, 10.5. HRMS (ESI): calc. for [M+H] ⁺ C27H ₂ 8O ₄ N ₅ : 486.21358, found: 486.21360.

Example A.8.5: Synthesis of 5,7-dimethyl-2-phenyl-6-(3-(3-(4- (trifluoromethyl)phenyl)-1 ,2,4-oxadiazol-5-yl)propyl)-2,6-dihydro-1 H-pyrrolo[3,4- d]pyridazin-1 -one (20)

(20)

The title compound was prepared according to general procedure II (0.024 g, 47%). FT-IR: v = 1654, 1620, 1416, 1345, 1321 , 1 171 , 1 1 12, 1065, 853, 770, 717, 702 cm ^"1 . ¹ H NMR (600 MHz, CDCI ₃ ) δ 8.19 (d, J = 8.2 Hz, 2H), 8.04 (s, 1 H), 7.76 (d, J = 8.2 Hz, 2H), 7.51 (dd, J = 8.2, 1 .0 Hz, 2H), 7.43 (t, J = 7.9 Hz, 2H), 7.31 (t, J = 7.4 Hz, 1 H), 4.20 (t, J = 7.5 Hz, 2H), 3.08 (t, J = 7.0 Hz, 2H), 2.78 (s, 3H), 2.53 (s, 3H), 2.35 (quin, J = 7.3 Hz, 2H). ¹³ C NMR (151 MHz, CDCI ₃ ) δ 178.6, 167.6, 159.4, 142.2, 134.1 , 133.27 (q, J = 32.8 Hz), 130.0, 129.98 (q, J = 1 .1 Hz), 128.7, 127.9, 127.3, 126.5, 126.1 (q, J = 3.7 Hz), 125.6 (q, J = 272.5 Hz), 122.7, 1 15.9, 1 1 1 .6, 43.4, 26.7, 23.9, 1 1 .3, 10.5.HRMS (ESI): calc. for [M+H] ⁺ C26H ₂₃ O2N ₅ F ₃ : 494.17984, found: 494.17988.

Example A.8.6: Synthesis of 6-(3-(3-(4-fluorophenyl)-1 ,2,4-oxadiazol-5-yl)propyl)- 5,7-dimethyl-2-phenyl-2,6-dihydro-1 H-pyrrolo[3,4-d]pyridazin-1 -one (21)

(21 )

The title compound was prepared according to general procedure II (0.017 g, 39%). FT-IR (neat): v = 2918, 1654, 1606, 1483, 1417, 1308, 1224, 1 155, 1 1 16, 1091 , 846, 752, 694 cm ^"1 . ¹ H NMR (500 MHz, CDCI ₃ ) δ 8.09 - 8.04 (m, 2H), 8.01 (s, 1 H), 7.55 (d, J = 7.7 Hz, 2H), 7.43 (t, J = 7.8 Hz, 2H), 7.30 (t, J = 7.4 Hz, 1 H), 7.18 (t, J = 8.7 Hz, 2H), 4.18 (t, J = 7.8 Hz, 2H), 3.04 (t, J = 7.0 Hz, 2H), 2.78 (s, 3H), 2.51 (s, 3H), 2.33 (quin, J = 7.3 Hz, 2H). ¹³ C NMR (126 MHz, CDCI ₃ ) δ 178.3, 166.8 (d, J = 243.3 Hz), 163.8, 159.3, 142.5, 133.8, 129.8, 129.6 (d, J = 8.8 Hz), 128.6, 127.0, 126.4, 122.9 (d, J = 3.3 Hz), 122.5, 1 16.3 (d, J = 22.1 Hz), 1 1 1 .7, 1 10.1 , 43.3, 26.7, 23.8, 1 1 .3, 10.5. HRMS (ESI): calc. for [M+H] ⁺ C2 ₅ H23O2N ₅ F: 444.18303, found: 444.18276.

Example A.9: Synthesis of pyrrolopyridazinone of general formula (l-A) wherein R ¹ represents -CH ₂ CR ¹⁸ R ¹⁹ CH ₂ R ⁴ , R ⁴ represents a 1 ,3,4-oxadiazole ring substituted at the second position with the substituent R ⁶ , R ¹⁵ represents -H and R ¹⁶ represents -CH ₃ .

1. HBTU (1.1 equiv)

DIPEA (3 equiv), 40 °C, 5 h

2. DIPEA (2 equiv)

TsCI (3 equiv), 40 °C, 6 h

58-80% yield General procedure III for the synthesis of 1 ,3,4-oxadiazole derivatives:

To a mixture of carboxylic acid 2 ^' (0.033 g, 0.1 mmol, 1 .0 equiv), respective hydrazide 13 ^' (0.1 mmol, 1 equiv) and diisopropylethylamine (0.052 mL, 0.3 mmol, 3 equiv) in acetonitrile (1 .2 mL) at room temperature was added HBTU (0.042 g, 0.1 1 mmol, 1 .1 equiv) and the resulting mixture was heated to 40 °C for 6 h. Then the reaction mixture was allowed to come to room temperature and diisopropylethylamine (0.035 mL, 2 equiv), followed by 4-methylbenzenesulfonyl chloride (0.057 g, 3 equiv) were added. The resulting reaction mixture was stirred at 40 °C for another 6 h. After completion, the reaction mixture was diluted with dichloromethane (5 mL) and water (5 mL). The aqueous layer was extracted with dichloromethane (2 x 5 mL); the combined organic layers were sequentially washed with H ₂ O (5 ml ^χ 2), saturated brine (5 mL), dried over Na2SO ₄ , filtered and concentrated in vacuo to give crude l-A. The resulting crude material was purified by FCC on silica gel using 3% methanol/dichloromethane as an eluent to give the desired 1 ,3,4-oxadiazole derivatives l-A in 58-80% yields.

Example A.9.1 : Synthesis of 5,7-dimethyl-2-phenyl-6-(3-(5-phenyl-1 ,3,4- oxadiazol-2-yl)propyl)-2,6-dihydro-1 H-pyrrolo[3,4-d]pyridazin-1 -one (22)

(22)

The title compound was prepared according to general procedure III (0.033 g, 78%). FT-IR (neat): v = 2360, 2341 , 1660, 1549, 1489, 1313, 1 121 , 1086, 1014, 991 , 842, 768, 689 cm ^"1 . ¹ H NMR (600 MHz, CDCI ₃ ) δ 8.03 - 7.98 (m, 3H), 7.56 - 7.52 (m, 3H), 7.52 - 7.48 (m, 2H), 7.42 (t, J = 7.9 Hz, 2H), 7.31 - 7.26 (m, 1 H), 4.19 (t, J = 7.8 Hz, 2H), 3.00 (t, J = 7.0 Hz, 2H), 2.75 (s, 3H), 2.49 (s, 3H), 2.31 (quin, J = 7.4 Hz, 2H). ¹³ C NMR (151 MHz, CDCI ₃ ) δ 165.3, 159.3, 142.4, 133.9, 132.0, 129.8, 129.2, 129.0, 127.0, 126.9 (x 2), 126.4, 123.7, 122.8, 1 15.8, 1 1 1 .5, 43.3, 26.51 , 22.7, 1 1 .3, 10.5. HRMS (ESI): calc. for [M+H] ⁺ C2 ₅ H ₂ O2N ₅ : 426.19245, found: 426.19209.

Example A.9.2: Synthesis of 5,7-dimethyl-2-phenyl-6-(3-(5-(p-tolyl)-1 ,3,4- oxadiazol-2-yl)propyl)-2,6-dihydro-1 H-pyrrolo[3,4-d]pyridazin-1 -one (23) (23)

The title compound was prepared according to general procedure III (0.035, 80%). FT-IR (neat): v = 2260, 2341 , 1657, 1560, 1496, 1450, 1389, 1313, 1 120, 1085, 988, 958, 823, 758, 726, 692 cm ^"1 . ¹ H NMR (500 MHz, CDCI ₃ ) δ 7.99 (s, 1 H), 7.89 (d, J = 8.2 Hz, 2H), 7.54 (dd, J = 8.4, 1 .0 Hz, 2H), 7.41 (t, J = 7.9 Hz, 2H), 7.32 - 7.27 (m, 3H), 4.18 (t, J = 7.8 Hz, 2H), 2.99 (t, J = 7.0 Hz, 2H), 2.74 (s, 3H), 2.48 (s, 3H), 2.41 (s, 3H), 2.29 (quin, J = 7.5 Hz, 2H). ¹³ C NMR (126 MHz, CDCI ₃ ) δ 165.3, 164.9, 159.2, 142.5, 142.5, 133.9, 129.9, 129.8, 128.5, 126.9, 126.8, 126.4, 122.7, 120.9, 1 15.8, 1 1 1 .5, 43.3, 26.5, 22.7, 21 .7, 1 1 .2, 10.5. HRMS (ESI): calc. for [M+H] ⁺ C26H26O2N5: 440.20810, found: 440.20794.

Example A.9.3: Synthesis of 6-(3-(5-(4-Methoxyphenyl)-1 ,3,4-oxadiazol-2- yl)propyl)-5,7-dimethyl-2-phenyl-2,6-dihydro-1 H-pyrrolo[3,4-d]pyridazin-1 -one (24)

The title compound was prepared according to general procedure III (0.032, 70%). FT-IR (neat): v = 2360, 2341 , 1655, 1612, 1498, 1306, 1256, 1 173, 1017, 958, 839, 761 , 693 cm ^"1 . ¹ H NMR (500 MHz, CDCI ₃ ) δ 7.99 (s, 1 H), 7.94 (d, J = 8.8 Hz, 2H), 7.58 - 7.51 (m, 2H), 7.41 (t, J = 7.9 Hz, 2H), 7.28 (t, J = 7.4 Hz, 1 H), 6.99 (d, J = 8.8 Hz, 2H), 4.20 (t, J = 7.8 Hz, 2H), 3.86 (s, 3H), 2.98 (t, J = 7.0 Hz, 2H), 2.75 (s, 3H), 2.49 (s, 3H), 2.30 (quin, J = 7.5 Hz, 2H). ¹³ C NMR (126 MHz, CDCI ₃ ) δ

165.1 , 164.7, 162.5, 159.2, 142.5, 133.9, 129.8, 128.6, 128.6, 126.9, 126.4, 122.7,

1 16.2, 1 15.8, 1 14.7, 1 1 1 .6, 55.6, 43.3, 26.6, 22.7, 1 1 .3, 10.5. HRMS (ESI): calc. for [M+H] ⁺ C26H26O3N ₅ : 456.20302, found: 456.20281 .

Example A.9.4: Synthesis of 6-(3-(5-(2-methoxyphenyl)-1 ,3,4-oxadiazol-2- yl)propyl)-5,7-dimethyl-2-phenyl-2,6-dihydro-1 H-pyrrolo[3,4-d]pyridazin-1 -one (25)

(25)

The title compound was prepared according to general procedure III (0.032, 70%). FT-IR (neat): v = 2360, 2341 , 1655, 1494, 1451 , 1308, 1257, 1 165, 1 120, 1016, 958, 758, 692 cm ^"1 . ¹ H NMR (400 MHz, CDCI ₃ ) δ 8.01 (s, 1 H), 7.89 (d, J = 7.5 Hz, 1 H), 7.59 - 7.47 (m, 3H), 7.42 (t, J = 7.8 Hz, 2H), 7.29 (t, J = 7.4 Hz, 1 H), 7.07 (t, J = 8.0 Hz, 2H), 4.20 (t, J = 7.6 Hz, 2H), 3.96 (s, 3H), 3.02 (t, J = 6.9 Hz, 2H), 2.75 (s, 3H), 2.50 (s, 3H), 2.30 (quin, J = 7.2 Hz, 2H). ¹³ C NMR (101 MHz, CDCI ₃ ) δ 164.9, 164.0, 159.3, 157.9, 142.4, 133.9, 133.3, 130.4, 129.8, 128.6, 127.0, 126.4, 122.8, 120.9, 1 15.7, 1 12.7, 1 12.0, 1 1 1 .5, 56.1 , 43.3, 26.6, 22.7, 1 1 .2, 10.5. HRMS (ESI): calc. for [M+H] ⁺ C26H26O3N ₅ : 456.20302, found: 456.20278.

Example A.9.5: Synthesis of 5,7-dimethyl-2-phenyl-6-(3-(5-(4- (thfluoromethyl)phenyl)-1 ,3,4-oxadiazol-2-yl)propyl)-2,6-dihydro-1 H-pyrrolo[3,4- d]pyridazin-1 -one (26)

(26)

The title compound was prepared according to general procedure III (0.039, 79%). FT-IR (neat): v = 1654, 1558, 1321 , 1 167, 1 1 19, 1089, 1063, 961 , 849, 759, 694 cm ^"1 . ¹ H NMR (500 MHz, CDCI ₃ ) δ 8.13 (d, J = 8.2 Hz, 2H), 7.99 (s, 1 H), 7.77 (d, J = 8.3 Hz, 2H), 7.53 (d, J = 7.7 Hz, 2H), 7.41 (t, J = 7.8 Hz, 2H), 7.29 (t, J = 7.4 Hz, 1 H), 4.20 (t, J = 7.8 Hz, 2H), 3.01 (t, J = 7.1 Hz, 2H), 2.75 (s, 3H), 2.49 (s, 3H), 2.32 (quin, J = 7.5 Hz, 2H). ¹³ C NMR (126 MHz, CDCI ₃ ) δ 165.9, 164.0, 159.2, 142.5, 133.8, 133.6 (q, J = 32.9 Hz), 129.7, 128.5, 127.2, 126.8, 126.4, 126.3 (q, J = 3.8 Hz), 125.8 (q, J = 272.6 Hz), 122.6, 1 15.9, 1 1 1 .6, 43.2, 26.4, 22.8, 1 1 .3, 10.5. HRMS (ESI): calc. for [M+H] ⁺ C26H23O2N5F3: 494.17984, found: 494.17985.

Example A.9.6: Synthesis of 6-(3-(5-(4-fluorophenyl)-1 ,3,4-oxadiazol-2-yl)propyl)- 5,7-dimethyl-2-phenyl-2,6-dihydro-1 H-pyrrolo[3,4-d]pyridazin-1 -one (27)

(27)

The title compound was prepared according to general procedure III (0.036, 81 %). FT-IR (neat): v = 2360, 2341 , 1660, 1496, 1312, 1242, 1 158, 1 120, 957, 839, 759, 735, 692 cm ^"1 . ¹ H NMR (400 MHz, CDCI ₃ ) δ 8.08 - 7.98 (m, 3H), 7.56 (d, J = 7.6 Hz, 2H), 7.44 (t, J = 7.7 Hz, 2H), 7.35 - 7.27 (m, 1 H), 7.21 (t, J = 8.6 Hz, 2H), 4.22 (t, J = 7.0 Hz, 2H), 3.01 (t, J = 7.0 Hz, 2H), 2.77 (s, 3H), 2.52 (s, 3H), 2.33 (t, J = 7.0 Hz, 2H). ¹³ C NMR (101 MHz, CDCI ₃ ) δ 165.2, 164.9 (d, J = 253.4 Hz), 164.4, 159.2, 142.4, 133.9, 129.8, 129.2 (d, J = 9.0 Hz), 128.6, 126.9, 126.4, 122.7, 120.0 (d, J = 3.4 Hz), 1 16.6 (d, J = 22.4 Hz), 1 15.8, 1 1 1 .5, 43.2, 26.5, 22.7, 1 1 .3, 10.5. HRMS (ESI): calc. for [M+H] ⁺ C2 ₅ H ₂ 3O2N ₅ F: 444.18303, found: 444.18283.

Example A.9.7: Synthesis of 5,7-dimethyl-2-phenyl-6-(3-(5-(pyridin-4-yl)-1 ,3,4- oxadiazol-2-yl)propyl)-2,6-dihydro-1 H-pyrrolo[3,4-d]pyridazin-1 -one (28)

(28)

The title compound was prepared according to general procedure III (0.025, 59%). FT-IR (neat): v = 1651 , 1558, 1492, 1305, 1 1 18, 1090, 962, 830, 768, 723, 693 cm ^"1 . ¹ H NMR (400 MHz, CDCI ₃ ) δ 8.81 (d, J = 5.5 Hz, 2H), 7.99 (s, 1 H), 7.87 (d, J = 5.8 Hz, 2H), 7.53 (d, J = 7.7 Hz, 2H), 7.41 (t, J = 7.7 Hz, 2H), 7.29 (t, J = 7.2 Hz, 1 H), 4.20 (t, J = 7.4 Hz, 2H), 3.03 (t, J = 7.1 Hz, 2H), 2.75 (s, 3H), 2.50 (s, 3H), 2.33 (quin, J = 7.2 Hz, 2H). ¹³ C NMR (101 MHz, CDCI ₃ ) δ 166.3, 163.4, 159.2, 150.9, 142.4, 133.9, 130.8, 129.7, 128.6, 127.0, 126.4, 122.6, 120.3, 1 15.8, 1 1 1 .5, 43.2, 26.4, 22.8, 1 1 .3, 10.5. HRMS (ESI): calc. for [M+H] ⁺ C ₂ H23O2N ₆ : 427.18770, found: 427.18713.

Example A.10: Synthesis of 6-(3-(1 H-benzo[d]imidazol-2-yl)propyl)-5,7-dimethyl- 2-phenyl-2,6-dihydro-1 H-pyrrolo[3,4-d]pyridazin-1 -one (S1 )

(S1 )

The acid derivative 2 ^' (0.195 g, 0.6 mmol, 1 equiv) and o-phenylenediamine (0.065 g, 0.6 mmol, 1 equiv) in polyphosphoric acid (1 .2 g) were heated at 175 °C for 24 h. The reaction mixture was then allowed to come to room temperature and poured on ice. The resulting suspension was brought to pH « 8 using ammonium hydroxide. The resulting light brown solid was filtered, washed with water, dried under vaccum to yield the desired benzimidazole derivative S1 (0.20 g, 84%) as a brown solid, which was used in the next step without further purification. FT-IR (neat): v = 2915, 1640, 1557, 1453, 1307, 1270, 1089, 960, 725, 693 cm ^"1 . ¹ H NMR (500 MHz, DMSO) δ 8.23 (s, 1 H), 7.52 - 7.46 (m, 4H), 7.42 (t, J = 7.9 Hz, 5

2H), 7.29 (t, J = 7.3 Hz, 1 H), 7.15 - 7.10 (m, 2H), 4.20 (t, J = 7.8 Hz, 2H), 2.91 (t, J = 7.3 Hz, 2H), 2.65 (s, 3H), 2.49 (s, 3H), 2.20 (quin, J = 7.5 Hz, 2H). ¹³ C NMR (126 MHz, DMSO) δ 158.2, 153.8, 142.5, 141 .7, 134.3, 129.3, 128.3, 128.0, 126.2, 126.2, 126.1 , 123.71 , 121 .2, 1 14.6, 109.9, 43.2, 27.4, 25.4, 10.7, 9.7. HRMS (ESI): calc. for [M+H] ⁺ C ₂ H ₂₄ ON ₅ : 398.19754, found: 398.19571 .

Example A.11 : Synthesis of 6-(3-(1 -Benzyl-1 H-benzo[d]imidazol-2-yl)propyl)-5,7- dimethyl-2-phenyl-2,6-dihydro-1 H-pyrrolo[3,4-d]pyridazin-1 -one (15)

(15)

To a suspension of benzimidazole derivative S1 (0.016 g, 0.04 mmol, 1 equiv) in acetonitrile (0.5 ml_) was added CS2CO3 (0.020 g, 0.06 mmol, 1 .5 equiv) and benzyl bromide (5.0 μΙ_, 0.044 mmol, 1 .1 equiv). The reaction mixture was stirred at room temperature for 12 h. After completion, the reaction mixture was concentrated in vacuo and the residue was re-dissolved in CH2CI2 (5 ml_) and sat. NaHCO3 (5 ml_) was added. The aqueous layer was extracted with CH2CI2 (2 x 5 ml_). The combined organic layers were dried over Na2SO ₄ , filtered and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography using 4% MeOH/CH ₂ Cl2 as an eluent to give the desired product 15 (0.014 g, 72%) as a brown foam like solid. FT-IR (neat): v = 2923, 1650, 1494, 1453, 1306, 1 1 17, 1089, 960, 725, 693 cm ^"1 . ¹ H NMR (500 MHz, CDCI3) δ 7.98 (s, 1 H), 7.82 - 7.77 (m, 1 H), 7.57 (d, J = 7.5 Hz, 2H), 7.43 (t, J = 7.9 Hz, 2H), 7.34 - 7.27 (m, 7H), 7.04 - 6.97 (m, 2H), 5.29 (s, 2H), 4.14 (t, J = 7.5 Hz, 2H), 2.82 (t, J = 7.2 Hz, 2H), 2.64 (s, 3H), 2.38 (s, 3H), 2.30 (t, J = 7.3 Hz, 2H). ¹³ C NMR (126 MHz, CDCI ₃ ) δ 159.4, 153.1 , 142.6, 135.7, 135.6, 134.0, 129.9, 129.3, 128.6, 128.4, 126.9, 126.5, 126.3, 123.2, 122.8, 122.7, 1 19.4, 1 15.7, 1 1 1 .5, 109.7, 109.6, 47.1 , 43.6, 27.3, 24.2, 1 1 .3, 10.4. HRMS (ESI): calc. for [M+H] ⁺ C31 H30ON ₅ : 488.24449, found: 488.24406. Example A.12: Ethyl 2-chloro-2-(2-(p-tolyl)hydrazono)acetate (16 ^' )

(16 ^' )

A solution of aniline derivative (20 mmol) in dilute HCI (1 :1 , 14 ml_) was cooled to 0 °C, and a cold solution of NaNO ₂ (1 .1 equiv.) in H ₂ O (19 ml_) was added drop wise over 15 min while maintaining internal solution temperature below 5 °C. After addition, the reaction mixture was stirred for another 10 min keeping the internal temperature below 0 °C. The resulting ice-cold solution of diazonium derivative was then added drop wise via cannula to a precooled (0 °C) solution of ethyl-2- chloroacetoacetate (1 .0 equiv.) and NaOAc (1 .5 equiv.) in H ₂ O/EtOH (1 :7, 80 ml_). After complete addition, the reaction mixture was stirred for 4 h at the same temperature and then was quenched by addition of 200 ml_ of cold water. The resultant precipitate was filtered and dried under vacuum to furnish the desired product 16 ^' (3.50 g, 72%) as an off-white solid. ¹ H NMR (400 MHz, CDCI ₃ ) δ 8.34 (s, 1 H), 7.13 (s, 4H), 4.38 (q, J = 7.1 Hz, 2H), 2.31 (s, 3H), 1 .40 (t, J = 7.1 Hz, 3H). ¹³ C NMR (101 MHz, CDCI ₃ ) δ 159.9, 139.4, 132.8, 130.0, 1 15.4, 1 14.2, 62.8, 20.8, 14.4. HRMS: calc. for [M+H] ⁺ Ci iHi ₄ O ₂ N ₂ CI: 241 .07383, found: 241 .07342.

Example A.13: 3,4-Dimethyl-2-(p-tolyl)-2H-pyrazolo[3,4-d]pyridazin-7(6H)-o ne (17 ^' )

(17 ^' )

2,4-Pentanedione (1 .34 ml_, 13.0 mmol, 1 equiv) was added drop wise to a solution of NaOEt (21 % wt in EtOH, 4.85 ml_, 13 mmol, 1 .0 equiv.) in anhydrous MeOH (10 ml_) at ambient temperature and the reaction mixture was stirred for 4 h. Then the corresponding solid hydrazonyl chloride 16 ^' (3.13 g, 13 mmol, 1 .0 equiv) was added in portions and the reaction was left to stir for 16 h. After completion, the volatile components were removed and the crude material was re- dissolved in dichloromethane (30 ml_). The organic layer was then sequentially washed with H ₂ O (10 ml x 2), saturated brine (15 ml_), dried over Na ₂ SO ₄ , filtered 7 and concentrated in vacuo to give crude 15 ^' (15 ^' was isolated as a mixture of ethyl and methyl ester, which was confirmed by GC-MS. Transesterification was observed as MeOH was used as solvent. Reaction works equally well in EtOH. So trans-esterification can be avoided by using NaOEt as a base in combination with anhydrous EtOH as solvent). Crude 15 ^' was subjected to the next step without further purification.

15 ^' (3.75 g, 13.0 mmol) and hydrazine monohydrate (1 .91 mL, 39.3 mmol, 3.0 equiv.) were dissolved in EtOH (20 mL) and the mixture was heated in a sealed tube at 1 10 °C for 6 h. After completion, the reaction mixture was allowed to come to room temperature and then was cooled in an ice-bath. The resulting precipitation was filtered, washed with water and dried under vacuum to yield the desired product 17 ^' (2.4 g, 9.44 mmol, 72% over two step) as grey solid. ¹ H NMR (500 MHz, DMSO) δ 1 1 .98 (s, 1 H), 7.49 (d, J = 8.2 Hz, 2H), 7.43 (d, J = 8.2 Hz, 2H), 2.59 (s, 3H), 2.49 (s, 3H), 2.43 (s, 3H). ¹³ C NMR (126 MHz, DMSO) δ 156.4, 141 .2, 141 .0, 139.2, 137.1 , 135.9, 129.8, 125.7, 1 17.4, 20.7, 19.2, 1 1 .8. HRMS: calc. for [M+H] ⁺ Ci ₄ Hi ₅ ON ₄ : 255.12404, found: 255.12345.

Example A.14: Methyl 4-(3,4-dimethyl-7-oxo-2-(p-tolyl)-2H-pyrazolo[3,4- d]pyridazin-6(7H)-yl)butanoate (18 ^' )

(18 ^' )

NaH (60 % in mineral oil, 0.236 g, 5.89 mmol, 1 .50 equiv.) was added in small portions to a solution of 17 ^' (1 .0 g, 3.93 mmol, 1 .0 equiv) in DMF (3 mL) at 0 °C and the reaction mixture was stirred for an hour at this temperature. Then a solution of methyl 4-bromobutanoate (0.99 mL, 7.86 mmol, 2.0 equiv) in DMF (0.5 mL) was added drop wise and the resulting reaction mixture was stirred for an additional 4 h at room temperature. After completion, the reaction was quenched by adding H ₂ O (20 mL) and diluted with EtOAc (20 mL). The aqueous layer was extracted with EtOAc (2 x 10 mL); combined organic layers were sequentially washed with H ₂ O (2 x 10 mL), brine (2 x 10 mL), dried over Na ₂ SO ₄ , filtered, and concentrated under vacuum. The crude residue was purified by flash column chromatography using 4% MeOH/CH ₂ Cl ₂ as an eluent to give the desired product 18 ^' (1 .2 g, 3.39 mmol, 86%) as a white solid. ¹ H NMR (400 MHz, CDCI ₃ ) δ 7.34 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H), 4.22 (t, J = 6.9 Hz, 2H), 3.65 (s, 3H), 2.61 (s, 3H), 2.53 (s, 3H), 2.43 (s, 3H), 2.40 (t, J = 8.0 Hz , 2H), 2.15 (quin, J = 7.1 Hz, 2H). ^{l d} C NMR (101 MHz, CDCI ₃ ) δ 173.6, 156.2, 142.0, 140.9, 139.7, 136.3, 136.0, 129.9, 125.9, 125.8, 1 17.7, 51 .6, 48.8, 31 .3, 24.1 , 21 .3, 19.9, 12.3. HRMS: calc. for [M+H] ⁺ Ci ₉ H23O ₃ N ₄ : 355.17647, found: 355.17677. Example A.15: 4-(3,4-dimethyl-7-oxo-2-(p-tolyl)-2H-pyrazolo[3,4-d]pyridazi n- 6(7H)-yl)butanoic acid (14 ^' )

(14 ^' )

A heterogeneous mixture of pyrazolopyridazone derivative 18 ^' (1 .1 g, 3.10 mmol, 1 .0 equiv) and KOH (0.21 g, 3.72 mmol, 1 .2 equiv) in H ₂ O (15 ml_) was heated to reflux until the entire solid had dissolved (30 min). Then the reaction mixture was allowed to come to room temperature, cooled to 0 °C and was acidified with 10% aqueous HCI solution to a pH of 5-6. The resultant precipitation was then filtered, dried under vacuum to yield analytically pure 14 ^' as a white solid (1 .03 g, 3.03 mmol, 98%). ¹ H NMR (400 MHz, DMSO) δ 12.09 (s, 1 H), 7.47 (d, J = 8.2 Hz, 2H), 7.41 (d, J = 8.2 Hz, 2H), 4.06 (t, J = 6.9 Hz, 2H), 2.58 (s, 3H), 2.50 (s, 3H), 2.41 (s, 3H), 2.25 (t, J = 7.3 Hz, 2H), 1 .92 (quin, J = 7.1 Hz, 2H). ¹³ C NMR (101 MHz, DMSO) δ 174.0, 155.2, 141 .1 , 141 .1 , 139.3, 137.4, 135.9, 129.9, 125.7, 1 17.1 , 48.2, 30.8, 23.8, 20.8, 19.4, 1 1 .9. HRMS: calc. for [M+H] ⁺ Ci ₈ H ₂ iO ₃ N ₄ : 341 .16082, found: 341 .16096.

Example A.16: General procedure IV for amide coupling

To a solution of acid derivative 14 ^' (0.049 g, 0.15 mmol, 1 .0 equiv) and DMAP (0.024 g, 0.195 mmol, 1 .3 equiv) in THF/DCM (1 :1 , 0.6 ml_ and 0.6 ml_) was added EDC HCI (0.037 g, 0.195 mmol, 1 .3 equiv), followed by the corresponding amine derivative (0.158 mmol, 1 .05 equiv) and the resulting solution was stirred for 16 h at ambient temperature. Once completed, as observed by TLC, the reaction mixture was diluted with CH2CI2 (5 ml_) and quenched by adding saturated NaHCO ₃ solution (5 ml_). The aqueous layer was extracted with dichloromethane (2 x 5 ml_) and combined organic layers were sequentially washed with saturated NH ₄ CI solution (5 ml_ x 2), H ₂ O (5 ml 2) and brine (5 ml_); dried over Na ₂ SO ₄ , filtered and concentrated under vacuum. The crude residue was further purified by FCC on silica-gel using 3.5% MeOH/CH ₂ Cl2 as an eluent to afford desired amide compounds. Example A.16.1 : 4-(3,4-dimethyl-7-oxo-2-(p-tolyl)-2H-pyrazolo[3,4-d]pyridazi n- 6(7H)-yl)-N-(2-phenylpropyl)butanamide (50)

(50)

The title compound (1 .20 g, 2.62 mmol, 94%) was prepared according to general procedure IV by treating acid derivative 14 ^' (0.95 g, 2.79 mmol) with β- methylphenethylamine (0.426 ml_, 2.93 mmol), EDC HCI (0.696 g, 3.63 mmol) and DMAP (0.44 g, 3.628 mmol) in a DCM/THF (9/9 ml_) solvent mixture. ¹ H NMR (400 MHz, CDCIs) δ 7.40 - 7.31 (m, 4H), 7.30 - 7.24 (m, 4H), 7.23 - 7.16 (m, 1 H), 4.1 1 - 3.97 (m, 2H), 3.58 - 3.49 (m, 1 H), 3.48 - 3.39 (m, 1 H), 3.09 (sex, J = 7.1 Hz, 1 H), 2.64 (s, 3H), 2.54 (s, 3H), 2.46 (s, 3H), 2.17 (t, J = 6.5 Hz, 2H), 2.12 - 2.02 (m, 2H), 1 .31 (d, J = 7.0 Hz, 3H). ¹³ C NMR (101 MHz, CDCI ₃ ) δ 173.2, 157.3, 144.9, 142.2, 142.1 , 140.3, 136.8, 136.3, 130.3, 128.9, 127.7, 126.8, 126.2, 1 18.1 , 48.8, 46.7, 40.0, 33.6, 26.1 , 21 .7, 20.3, 20.1 , 12.7. HRMS: calc. for [M+H] ⁺ C27H32O2N5: 458.25505, found: 458.25514.

Example A.17: 4-(4-methyl-7-oxo-2-phenyl-2H-pyrazolo[3,4-d]pyridazin-6(7H) - yl)butanoic acid (19 ^' )

(19 ^' )

Methyl 4-bromobutanoate (1 .885g, 10.41 mmol, 1 .314ml_, 1 .2eq.) was added to a stirred suspension of commercial 4-methyl-2-phenyl-2H-pyrazolo[3,4-d]pyridazin- 7(6H)-one (1 .963g, 8.68mmol, 1 .0eq.) and cesium carbonate (7.07g, 21 .69mmol, 2.5eq.) in dry Ν,Ν-dimethylformamide (40ml_) at RT under nitrogen atmosphere. The reaction mixture was heated at 50°C for 15h. The mixture was cooled, filtered through Celite® and the filter cake was rinsed with EtOAc. The filtrate was diluted with water and extracted three times with EtOAc. The combined organic phase was washed with brine and dried over Na ₂ SO ₄ . Solvents were removed in vacuo. The crude was stirred with heptane. The solid was filtered off, rinsed with a small amount of heptane and dried in vacuo yielding methyl 4-(4-methyl-7-oxo-2-phenyl- 2H-pyrazolo[3,4-d]pyridazin-6(7H)-yl)butanoate (1 .83g, 5.61 mmol, 63%) as a white solid. For hydrolysis of the ester, the methyl 4-(4-methyl-7-oxo-2-phenyl-2H- pyrazolo[3,4-d]pyridazin-6(7H)-yl)butanoate (1 .80g, 5.52mmol, 1 .0eq.) was solved in THF (40ml_) and a solution of lithium hydroxide monohydrate (0.289g, 6.89mmol, 1 .25 eq.) in water (20ml_) was added. After stirring 3 h at RT, the solvent THF was removed in vacuo. The aqueous solution was acidified to pH~4 by addition of aq. 1 N HCI solution. The white solid was filtered off, rinsed with water and dried in vacuo yielding the acid 19 ^' (1 .65g, 5.30mmol, 96%) as a white solid. ¹ H NMR (400 MHz, DMSO) δ 9.28 (s, 1 H), 8.00 (d, J = 7.4 Hz, 2H), 7.60 (t, J = 7.4 Hz, 2H), 7.48 ( t, J = 7.4 Hz, 1 H), 4.07 (t, J = 7.1 Hz, 2H), 2.44 (s, 3H), 2.26 (t, J = 7.1 Hz, 2H), 1 .93 (quin., J = 7.1 Hz, 2H). Ci ₆ Hi ₆ N ₄ O ₃ requires: 312, found: 313 (M+H) ⁺ .

Example A.18: N-(4-fluorobenzyl)-4-(4-methyl-7-oxo-2-phenyl-2H-pyrazolo[3, 4- d]pyridazin-6(7H)-yl)butanamide (54)

To a solution of 4-(4-methyl-7-oxo-2-phenyl-2H-pyrazolo[3,4-d]pyridazin-6(7H) - yl)butanoic acid (19 ^' ) (50mg, 0.16mmol, LOOeq.) and DIPEA (0.03ml_, 0.19mmol, 1 .20eq.) in dry DMF (2.5ml_) was added HATU (64mg, 0.17mmol, 1 .05eq.). After stirring for 5 min at RT 4-fluorobenzylamine (25mg, 0.20mmol, 1 .25eq.) was added and the solution was stirred for 15h at RT. Then the mixture was diluted with sat. aq. NaHCO ₃ solution (3ml_) and water (3ml_). After stirring for 5 min the solid was filtered off, rinsed with water and dried in vacuo yielding the desired product 54 (54mg, 0.13mmol, 80%) as a white solid. ¹ H NMR (400 MHz, DMSO) δ 9.34 (s, 1 H), 8.35 (t, J = 5.9 Hz, 1 H), 8.04 (d, J = 7.3 Hz, 2H), 7.63 (t, J = 8.3 Hz, 2H), 7.51 (t, J = 7.3 Hz, 1 H), 7.28 ( dd, J = 5.7 Hz, J = 8.6 Hz, 2H), 7.12 (t, J = 8.9 Hz, 2H), 4.22 (d, J = 5.8 Hz, 2H), 4.08 (t, J = 7.4 Hz, 2H), 2.47 (s, 3H), 2.19 (t, J = 7.4 Hz, 2H), 1 .96 (quint., J = 7.4 Hz, 2H). C23H22FN ₅ O2 requires: 419, found: 420 (M+H) ⁺ .

The compounds in the following table were prepared similar to the procedure described in the previous Example A.17 and A.18. The purification of the crude product was performed by flash silica gel column chromatography with MeOH and CH2CI2 as an eluents, by reverse phase RP-HPLC (column: C18), using H ₂ O (0.1 %TFA) and ACN (0.1 %TFA) as eluents or by precipitation as described in Example A.18. 5



B. Crystallography Example B.1. Crystallization of connpound 22 (see Figure 1 )

Connpound 22 was co-crystallized with PDE5 by mixing a solution of 500 μΜ of compound 22 with an equimolar solution of PDE5 resulting in 1 % DMSO as a final concentration. Crystals were obtained from a Qiagen PEGs suite (0.2 M Calcium chloride, 20% (w/v) PEG 3350). For flash freezing the crystals in liquid nitrogen, we used cryoprotectant solution containing the mother liquor components in addition to glycerol. X-rays data were collected at the X10SA beamline of the Suisse Light Source, Villigen. XDS program was used for processing the data. For solving the structures, molecular replacement was applied using the program MolRep from the CCP4 software and PDE5 without bound farnesyl group, from the complex PDE5-farnesylated Rheb complex (PDB 3T5G) as a search model. Further refinement of the model was performed using a combination of manual refinement (COOT program) and the maximum likelihood restrained refinement (REFMAC5 program). Final validation of the model using Ramachandran plot statistics showed none of the residues to be outliers (see data collection and refinement statistics in Table 1 ). Table 1. Data collection and refinement statistics.

22

Data collection

Space group P 32 21

Cell dimensions

a, b, c (A) 55.71 ,55.71 , 1 14.67

A, β, Y (°) 90, 90, 120

Resolution (A) 2.10

/¾ym ΟΓ Rmerge 6.4 (46.4)

Ι / σΙ 20.17 (4.39)

Completeness (%) 99.9 (100)

Redundancy 7.91 (8.23)

Refinement

Resolution (A) 2.10

No. reflections 1 1932

/¾vork / Rfree 19.53 / 24.80

No. atoms

Protein 1237

Ligand/ion 32

Water 23

S-factors

Protein 39.3

Ligand/ion 40.7

Water 39.8

R.m.s. deviations

Bond lengths (A) 0.020

Bond angles (°) 1 .945

Values in parentheses correspond to the high resolution shell

Example B.2: Crystallization of compound 29 (see Figure 3)

Compound 29 was co-crystallized with PDE5 by mixing a solution of 500 μΜ of compound 29 with an equimolar solution of PDE5 resulting in 1 % DMSO as a final concentration.

Crystals were obtained from a Qiagen PEGs suite (0.2 M Calcium acetate, 20% (w/v) PEG 3350). For flash freezing the crystals in liquid nitrogen, we used cryoprotectant solution containing the mother liquor components in addition to glycerol. X-rays data were collected at the X10SA beamline of the Suisse Light Source, Villigen. XDS program was used for processing the data. For solving the structures, molecular replacement was applied using the program MolRep from the CCP4 software and PDE5 without bound farnesyl group, from the complex PDE5-farnesylated Rheb complex (PDB 3T5G) as a search model. Further refinement of the model was performed using a combination of manual refinement (COOT program) and the maximum likelihood restrained refinement (REFMAC5 program). Final validation of the model using Ramachandran plot statistics showed none of the residues to be outliers (see data collection and refinement statistic in Table 2).

Table 2. Data collection and refinement statistics.

Data collection

Space group P 1

Cell dimensions

a, b, c (A) 31 .76, 40.90, 68.80

A, β, Y (°) 97.70,102.38, 89.31

Resolution (A) 2.60

/¾ym ΟΓ Rmerge 1 1 .3 (46.6)

l / ol 9.45 (3.26)

Completeness (%) 97.7 (96.7)

Redundancy 3.45 (3.40)

Refinement

Resolution (A) 2.60

No. reflections 9570

Rwork / Rfree 18.13 / 25.57

No. atoms

Protein 2432

Ligand/ion 64

Water 16

S-factors

Protein 37.7

Ligand/ion 40.2

Water 37.3

R.m.s. deviations

Bond lengths (A) 0.013

Bond angles (°) 1 .732

Values in parentheses correspond to the high resolution shell. C. Biochemical experiments

Example C.1 : Alpha-Screen (Nature 2013, 497, 638)

Screening based on Alpha-technology was conducted in white, non-binding 1536- well plates (Corning) in a final volume of 6 μΙ_. For the screen a mixture of His6- PDE5, and biotinylated K-Ras-peptide (final concentrations 100 nM and 250 nM in HEPES 20 mM, 100 mM NaCI, 0,005% Chaps, pH 7.5) were added to the 1536- well plates. Compound solutions were directly added from 10 mM DMSO stock solutions to a final concentration of 10 μΜ and the resulting mixture was incubated for 30 min (For dose-response curves, compounds were tested at concentrations betwen 10 μΜ and 5 nM). Premixed Nickel Chelate Acceptor Beads and Streptavidin Donor Beads were added to a final concentration of 10 g/mL. The resulting mixture was incubated at 4 °C overnight. Plates were read on a Paradigm reader (Molecular devices, Alphascreen 1536 HTS detection cartridge, temperature 29°C-33°C).

Example C.2: Displacement titrations of labeled Atorvastatin-probe for the determination of K _D values:

Binding to PDE5 was validated and quantified by means of a displacement assay employing a fluorescent-tagged analog of the HMG-CoA reductase inhibitor Atorvastatin (Lipitor®) which has previously been shown to also bind to PDE5. (Nat. Chem. Biol. 2011 , 7, 375-383) The K _d values were determined by the Fluorescence polarization competition binding assay previously developed by the inventors (Nature 2013, 497, 638).

Table 3. K _D values for compounds of general formula (I) as determined by means of fluorescence polarization assay

Compound K _D [nM] Compound K _D [nM]

1 +++ 29 ++++

2 +++ 30 +++

3 +++ 31 +++

4 ++++ 32 +++

5 +++ 33 +++ 5

6 ++ 34 +++

7 ++ 35 +++

8 ++ 36 ++++

9 ++ 37 ++++

10 +++ 38 +++

11 ++++ 39 +++

12 ++++ 40 ++

13 ++++ 41 ++

14 ++++ 42 ++++

15 ++++ 43 ++++

16 ++ 44 ++++

17 +

45 ++++

+

18 46 ++++

+

19 47 ++++

+

20 48 ++++

21 ++ 49 ++++

22 ++

50 ++++

+

23 51 ++

24 + 52 ++++

++

25 53 ++++

++

26

27 ++

+

28

K _D ≤30 nM

30 nM < K _D < 100 nM

100 nM < K _D < 1000 nM

K _D > 1000 nM

Example C.3: Inhibitory effect of compound 50 at different concentrations on interaction of Rheb (Ras homolog enriched in brain) with PDE5 and localization in live MDCK cells (See Figure 4).

To address whether compound 50 inhibits the interaction between PDE5 and Ras family proteins in live cells, fluorescence lifetime imaging microscopy (FLIM)- based quantitative fluorescence resonance energy transfer (FRET) measurements were carried out. These experiments allow the quantitative determination of the fraction (a) of interacting mCitrine (a yellow fluorescent protein) tagged Ras proteins with mCherry (a red fluorescent protein) tagged PDE5 in living cells by the change in the fluorescence lifetime (x _av ) of the mCitrine fused to Ras. mCitrine- Rheb was selected as the farnesylated Ras family protein due to its more clearly measurable interaction with mCherry-PDE5 in live cells.

Hence, MDCK cells were co-transfected with mCherry-PDE5 and mCitrine-Rheb. The fluorescence patterns of both mCitrine-Rheb (first row) and mCherry-PDE5 (second row) were homogeneous in untreated cells (Pre) showing a clear solubilization of mCitrine-Rheb by mCherry-PDE5. Under these conditions a substantial drop in the mCitrine fluorescence lifetime (third row) corresponds to a substantial molar fraction (a) of mCitrine-Rheb (fourth row) that was in complex with mCherry-PDE5. Compound 50 was administered to the final indicated concentrations and 5 min after the administration, fluorescence lifetime images were acquired by using a confocal laser-scanning microscope (FV1000, Olympus) equipped with a time-correlated single-photon counting module (LSM Upgrade Kit, Picoquant). Intensity thresholds were applied to segment the cells from the background fluorescence. Data were further analyzed to obtain images of the molar fraction (a) of interacting mCherry-PDE5/mCitrine-Rheb. Thus, it can be observed that the interaction mCherry-PDE5/mCitrine-Rheb is lost upon incubation with different concentrations of compound 50 leading to an increased average fluorescence lifetime of mCitrine and reduced computed fraction (a) of interacting PDE5-Rheb with the increase in the concentration of compound 50.

Example C.4: Study of the interaction K-Ras-PDE5 and inhibition of said interaction by the compounds of the present invention (see Figure 5)

MDCK cells were co-transfected with mCherry-PDE5 and mCitrine-K-Ras. Fluorescence lifetime images were acquired using a confocal laser-scanning microscope (FV1000, Olympus) equipped with a time-correlated single-photon counting module (LSM Upgrade Kit, Picoquant). 5 min after the administration of Bryostatin, fluorescence lifetime images were acquired and 5 μΜ of Compound 50 was administered. After 10 minutes in total, another fluorescence lifetime image was taken. Intensity thresholds were applied to segment the cells from the background fluorescence. Data were further analyzed to obtain images of the molar fraction (a) of interacting mCherry-PDE5/mCitrine-Rheb. 7

Example C.5: Inhibition of the K-Ras dependent cell (Cancer Cell 2009, 15, 489) proliferation by the inventive compounds (See Figure 6) Real time cell analysis (RTCA) measurements were performed in order to determine the dose-dependent effect of the compounds of the present invention on the proliferation of K-Ras independent cells (PANC-1 (see Figures 6a, 6d), BxPC-3 (see Figures 6e)) and K-Ras dependent cells (Panc-Tu-I (see Figures 6b, 6f), Capan-1 (see Figures 6c, 6g)). RTCA measurements were performed using 16-well E-plates on the Dual Plate xCELLigence instrument (Roche Applied Science, Indianapolis IN). E-Plates were blanked with 100 μΙ of DMEM medium containing 10% FCS. After blanking, trypsinized cells were counted with a Beckman Coulter Vi-cell™ counter and 7.500 to 15.000 cells (cell line dependent) were plated in each well of the 16-well E-plates (ACEA) in a final volume of 200 μΙ. After seeding, cells were allowed to settle for 5 to 10 minutes at room temperature before being reinserted into the xCELLigence instrument. The RTCA is localized in a humidified incubator at 37°C with 10% CO2. Continuous impedance measurements were monitored every 15 min up to 100 hours. 24 hours after seeding, compound 50 was administered. The derivatives were calculated for the time frame indicated by the black bar.

As shown by Figures 6b, 6f, 6c and 6g, addition of compound 50 inhibits the proliferation of K-Ras dependent cells Panc-Tu-I and BxPC-3, leading after a certain time to cell death (see Figure 6f).

Example C.6: Displacement titrations of fluorescein-labeled Atorvastatin labeled probe for the determination of IC ₅ 0 values 10μΙ of Hise-tagged PDE5 in PBS-buffer (containing 0.05% Chaps, 0,9% DMSO) was transfered in a black, non-binding 384-well plate (Corning Life Sciences). 15nl of a DMSO stock solution of tested compound was added using a Labcyte Echo 520 accoustic dispenser. Reaction was started by adding 5μΙ of fluorescein- labeled Atorvastatin. Final concentrations of PDE5 and fluorescein-labeled Atorvastatin are 40 nM and 24 nM, repectively. The compounds were tested in a concentration range from 10μΜ to 5nM. The plates were incubated at room temperature over night and the fluorescence polarization values (Ex: 470 nm, Em: 525 nm) were read on a EnVision plate reader (PerkinElmer). IC ₅ 0 values were determinded using Quattro Workflow software (Quattro Research GmbH). The activity of the compounds was classified according to their IC ₅ o values into the following ranges: IC ₅ o≤100 nM +++

100 nM < IC ₅₀ ≤ 1000 nM ++

IC ₅₀ > 1000 nM +

Table 4. Evaluation of the IC ₅ o values of the compounds of the present invention

Compound ICso [nM] Compound ICso [nM]

54 + 100 ++

55 ++ 101 ++

56 ++ 102 +++

57 ++ 103 ++

58 + 104 ++

59 ++ 105 ++

60 ++ 106 +

61 + 107 ++

62 ++ 109 +++

63 +++ 110 +++

64 ++ 111 +++

65 ++ 112 +++

66 ++ 113 +++

67 + 114 +++

68 +++ 115 ++

69 +++ 116 +++

70 +++ 117 +++

71 ++ 118 +++

72 ++ 119 +++

73 ++ 120 +++

74 ++ 121 +++

75 ++ 122 +++

76 + 123 +++

77 ++ 124 +++

78 ++ 125 +++

79 + 126 +++ 80 ++ 127 +++

81 + 128 ++

82 +++ 129 +++

83 +++ 130 +++

84 +++ 131 +++

85 ++ 109 +++

86 +++ 110 +++

87 ++

88 +

89 +++

90 +++

91 +++

92 ++

93 +++

94 +++

95 +++

96 +++

97 +++

98 ++

99 +++