# Patent

## Title
Substituted tetrahydro-1H-pyrazolo [3,4-c] pyridines, compositions comprising them, and use

## Abstract

Substituted tetrahydro-1H-pyrazolo[3,4-c]pyridines, compositions comprising them and use. The present invention relates in particular to novel substituted tetrahydro-1H-pyrazolo[3,4-c]pyridines having therapeutic activity, which can be used in particular in oncology.

## Background
This application claims the benefit of priority of French Patent Application No. 03/08,441, filed Jul. 10, 2003 and French Patent Application No. 03/08,442, filed Jul. 10, 2003, both of which are incorporated herein by reference in their entirety.
The present invention relates to novel chemical compounds, particularly novel tetrahydro-1H-pyrazolo[3,4-c]pyridines, compositions comprising them, and to their use as medicinal products.
More particularly, the invention relates to novel tetrahydro-1H-pyrazolo-[3,4-c]pyridines exhibiting anticancer activity, and in particular kinase-inhibiting activity, especially Tie2-inhibiting activity.
Only a few tetrahydro-1H-pyrazolo[3,4-c]pyridines are known.
Thus, WO 02/012442 discloses tetrahydro-1H-pyrazolo[3,4-c]pyridines substituted in the 5-position with an optionally substituted amino group. These products are useful in the treatment of cancer and of other diseases related to cell proliferation.
P. Krogsgaard-Larsen et al. in Eur. J. Med. Chemical—Chim. Ther. (1979), 14(2), p. 157–164, discloses two tetrahydro-1H-pyrazolo[3,4-c]pyridines substituted in the 3-position with a hydroxyl group.
WO 96/12720 claims tetrahydro-1H-pyrazolo[3,4-c]pyridines substituted in the 3-position with substituents chosen from H, alkyl, alkylene, cycloalkyl and methylenecycloalkyl, and in the 1- and 6-positions with varied substituents. These products are described as inhibitors (i) of phosphodiesterase type IV (PDE-IV), and (ii) of tumour necrosis factor (TNF), and are, as a result, considered to be useful in the treatment of inflammatory diseases. No example of a compound according to the invention is disclosed.
Attempts to obtain effective inhibitors of Tie2 have already been successful in the past (in this respect, see, for example, WO 98/02434; WO 98/41525; WO 99/10325; WO 99/17770; WO 99/54286; WO 99/21859; WO 99/55335; WO 00/17202; WO 00/17203; WO 00/27822; WO 00/75139; WO 01/37835; WO 01/57008; WO 01/72751; WO 02/060382; WO 02/076396; WO 02/076463; WO 02/076954; WO 02/076984; WO 02/076985; WO 02/080926; WO 03/004488).
However, none of those documents discloses 4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine derivatives as defined below, exhibiting activity against kinases, in particular Tie2.
To this effect, the products in accordance with the invention, according to its first aspect, satisfy formula (I) below:




and its tautomers, in which:

L is chosen from a bond, CH2, CO, SO2, CONH, COO, NHCO, NH, NHSO2, SO2NH, NHCONH, CH2NH and NHCH2,
X is chosen from a bond, CH2, CO, SO2, CONH and COO;
R1 is chosen from OH, H, alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, which is optionally substituted, and, when X is a bond, then R1 may also be halogen;
R2 is H or is chosen from alkyl, alkylene, cycloalkyl, heterocyclyl, aryl, heteroaryl, which is optionally substituted;
the substituents being chosen independently from R3, O—R3, halogen, NO2, SO2—R3, CO—R3, SO2NH—R3, CONH—R3, N—(R3)2, NHCO—R3, NHSO2—R3, NHCONH—R3, NHSO2NH—R3, OCO—R3, COO—R3, OSO2—R3, SO2O—R3, OCONH—R3 and OSO2NH—R3, where each R3 is chosen independently from H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, heterocyclyl, which is optionally substituted with halogen, aryl, heteroaryl, R4, OR4 or N(R4)2, each R4 being chosen independently from H, C1–C4 alkyl and halogenated C1–C4 alkyl.
Products in accordance with the invention, according to its first aspect, are more particularly chosen from products of formula (II) below:




and its tautomers, in which:

X is chosen from a bond, CH2, CO, SO2, CONH and COO;
R1 is chosen from alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, which is optionally substituted;
R2 is H or is chosen from alkyl, alkylene, cycloalkyl, heterocyclyl, aryl, heteroaryl, which is optionally substituted;
the substituents being chosen independently from R3, O—R3, halogen, NO2, SO2—R3, CO—R3, SO2NH—R3, CONH—R3, N—(R3)2, NHCO—R3, NHSO2—R3, NHCONH—R3, NHSO2NH—R3, OCO—R3, COO—R3, OSO2—R3, SO2O—R3, OCONH—R3 and OSO2NH—R3, where each R3 is chosen independently from H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, heterocyclyl, which is optionally substituted with halogen, aryl, heteroaryl, OR4 or N(R4)2, where each R4 is chosen independently from H and C1–C4 alkyl.
Products in accordance with the invention, according to its first aspect, are more particularly chosen from products of formula (III) below:




and its tautomers, in which:

X is chosen from a bond, CH2, CO, SO2, CONH and COO;
R1 is chosen from alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, which is optionally substituted;
R2 is H or is chosen from alkyl, alkylene, cycloalkyl, heterocyclyl, aryl, heteroaryl, which is optionally substituted;
in which the substituents are chosen independently from R3, O—R3, halogen, NO2, SO2—R3, CO—R3, SO2NH—R3, CONH—R3, N—(R3)2, NHCO—R3, NHSO2—R3, NHCONH—R3, NHSO2NH—R3, OCO —R3, COO—R3, OSO2—R3, SO2O—R3, OCONH—R3 and OSO2NH—R3, in which each R3 is chosen independently from H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, heterocyclyl, which is optionally substituted with halogen, aryl, heteroaryl, OR4 or N(R4)2, and in which each R4 is chosen independently from H and C1–C4 alkyl.
A product in accordance with the invention is advantageously chosen from the products according to its first aspect, in which R1 is heteroaryl, which is optionally substituted, in which a preferred heteroaryl is chosen from benzimidazolyl, indolyl, pyrrolyl, optionally substituted with halogen, R4 or O—R4.
More particularly, a preferred heteroaryl is chosen from benzimidazol-2-yl, indol-2-yl, pyrrol-2-yl, optionally substituted with halogen, R4 or O—R4.
A product in accordance with the invention according to its first aspect advantageously has a substituent R2 chosen from phenyl, pyridyl, thienyl, C1–C4 alkyl, and C3–C7 cycloalkyl, which is optionally substituted.
X may advantageously be chosen from CO and SO2.
A product in accordance with the invention according to its first aspect is advantageously chosen from the products of formula (I) in which R1 is H.
A preferred product is advantageously chosen from the products of formula (I) in which R1 is substituted aryl.
According to a first preferred embodiment, a preferred product is advantageously chosen from the products of formula (I) in which R1-L is R1-NH—CO, and more preferably when R1 is H.
Very preferably, and according to a second preferred embodiment, a preferred product is advantageously chosen from (i) the products of formula (I), or (ii) preferably the products according to the first preferred embodiment, in which X is a bond, and in which R2 is chosen from substituted aryl and substituted heteroaryl.
According to a third preferred embodiment, a more preferred product is chosen from the products in accordance with the invention according to its second embodiment, in which R2 is chosen from:

    
    
        aryl substituted with NHSO2—R3 or NHCONH—R3, and
        heteroaryl substituted with NHSO2—R3 or NHCONH—R3.
    
    


Products according to the third preferred embodiment are advantageously chosen from:

    
    
        aryl substituted with NHSO2—R3 or NHCONH—R3, and
        heteroaryl substituted with NHSO2—R3 or NHCONH—R3, in which aryl is phenyl and in which heteroaryl is chosen from pyridyl and pyrimidyl.
    
    


According to a fourth embodiment, products according to the third preferred embodiment are advantageously chosen from:

    
    
        aryl substituted with NHSO2—R3 or NHCONH—R3, and
        heteroaryl substituted with NHSO2—R3 or NHCONH—R3, in which R3 is chosen from substituted aryl and substituted heteroaryl, in which R3 is advantageously substituted with a substituent selected from the group consisting of halogen, R4, OR4 and N(R4)2, in which each R4 is chosen independently from H, C1–C4 alkyl and halogenated C1–C4 alkyl.
    
    


According to a fifth embodiment, products according to the fourth preferred embodiment are advantageously chosen from:




A product in accordance with the invention according to its first aspect may be in:

    
    
        1) racemic form, or
        2) a form enriched in a stereoisomer, or
        3) a form enriched in an enantiomer;

and may optionally be salified.

    
    


According to a second aspect, the invention relates to pharmaceutical compositions comprising a product as defined above, in combination with a pharmaceutically acceptable excipient.
According to a third aspect, the invention relates to the use of a product as defined above, as an agent for modulating the activity of a kinase. A preferred kinase will advantageously be chosen from Tie2 and KDR. Tie2 is more preferred.
According to its third aspect, the invention relates to the use of a product as defined above, for producing a medicinal product that is useful for treating a pathological condition, in particular cancer.
Products in accordance with the invention can be obtained by methods well known to those skilled in the art, in particular as regards the techniques of coupling between an acid and an amine; see, for example, J. March, Advanced organic chemistry, (J. Wiley & Sons, ed.), fourth edition, 1992.
The products of the invention are useful as agents which inhibit a reaction catalysed by a kinase. Tie2 is a kinase for which the products of the invention will be particularly useful as inhibitors. These products can also be used as inhibitors of other kinases, such as KDR.
Reasons for which the kinases are chosen are given below:
Tie2
Tie-2 (TEK) is a member of a family of tyrosine kinase receptors specific for endothelial cells. Tie2 is the first receptor with tyrosine kinase activity for which both the agonist (angiopoietin 1 or Ang1), which stimulates autophosphorylation of the receptor and cell signalling [S. Davis et al (1996) Cell 87, 1161–1169] and the antagonist (angiopoietin 2 or Ang2) [P.C. Maisonpierre et al. (1997) Science 277, 55–60] are known. Angiopoietin 1 can synergize with VEGF in the final stages of neoangiogenesis [AsaharaT. Circ. Res.(1998) 233–240]. Knockout experiments and transgenic manipulations of the expression of Tie2 or of Ang1 result in animals which exhibit vascularization defects [D. J. Dumont et al (1994) Genes Dev. 8, 1897–1909 and C. Suri (1996) Cell 87, 1171–1180]. The binding of Ang1 to its receptor results in autophosphorylation of the kinase domain of Tie2, which is essential for neovascularization and for the recruitment and the interaction of the vessels with pericytes and smooth muscle cells; these phenomena contribute to the maturing and the stability of the newly formed vessels [P. C. Maisonpierre et al (1997) Science 277, 55–60]. Lin et al (1997), J. Clin. Invest. 100, 8: 2072–2078 and Lin P. (1998) PNAS 95, 8829–8834, have shown inhibition of tumour growth and vascularization and a decrease in lung metastases during adenoviral infections or during injections of the Tie-2 (Tek) extracellular domain in breast tumour and melanoma xenograph models.
Tie2 inhibitors can be used in situations where neovascularization takes place inappropriately (i.e. in diabetic retinopathy, chronic inflammation, psoriasis, Kaposi's sarcoma, chronic neovascularization due to macular degeneration, rheumatoid arthritis, infantile haemoangioma and cancers).
KDR
KDR (Kinase insert Domain Receptor), also known as VEGF-R2 (Vascular Endothelial Growth Factor Receptor 2), is expressed only in endothelial cells. This receptor binds to the angiogenic growth factor VEGF and thus acts as a mediator to a transduction signal via the activation of its intracellular kinase domain. Direct inhibition of kinase activity of VEGF-R2 makes it possible to reduce the phenomenon of angiogenesis in the presence of exogenous VEGF (Vascular Endothelial Growth Factor) (Strawn et al., Cancer Research, 1996, vol. 56, p. 3540–3545). This process has been demonstrated in particular using VEGF-R2 mutants (Millauer et al., Cancer Research, 1996, vol. 56, p. 1615–1620). The VEGF-R2 receptor does not appear to have any function in adults other than that related to the angiogenic activity of VEGF. Consequently, a selective inhibitor of the kinase activity of VEGF-R2 should only show slight toxicity.
In addition to this central role in the dynamic angiogenic process, recent results suggest that VEGF expression contributes to the survival of tumour cells after chemotherapy and radiotherapy, emphasizing the potential synergy of KDR inhibitors with other agents (Lee et al. Cancer Research, 2000, vol. 60, p. 5565–5570).
Experimental Section
Method A: Analysis by LC/MS
The LC/MS analyses were carried out on a Micromass model LCT device connected to an HP 1100 device. The abundance of the products was measured using an HP G1315A diode array detector over a wavelength of 200–600 nm and a Sedex 65 light scattering detector. The mass spectra were acquired over a range of from 180 to 800. The data were analysed using the Micromass MassLynx software. Separation was carried out on a Hypersil BDS C18, 3 μm (50×4.6 mm) column, eluting with a linear gradient of from 5 to 90% of acetonitrile comprising 0.05% (v/v) of trifluoroacetic acid (TFA) in water comprising 0.05% (v/v) TFA, over 3.5 min at a flow rate of 1 ml/min. The total analysis time, including the period for re-equilibrating the column, is 7 min.
Method B: Purification by LC/MS:
The products were purified by LC/MS using a Waters FractionsLynx system composed of a Waters model 600 gradient pump, a Waters model 515 regeneration pump, a Waters Reagent Manager dilution pump, a Waters model 2700 auto-injector, two Rheodyne model LabPro valves, a Waters model 996 diode array detector, a Waters model ZMD mass spectrometer and a Gilson model 204 fraction collector. The system was controlled by the Waters FractionLynx software. Separation was carried out alternately on two Waters Symmetry columns (C18, 5 μM, 19×50 mm, catalogue reference 186000210), one column undergoing regeneration with a 95/5 (v/v) water/acetonitrile mixture comprising 0.07% (v/v) of trifluoroacetic acid, while the other column was being used for separation. The columns were eluted using a linear gradient of from 5 to 95% of acetonitrile comprising 0.07% (v/v) of trifluoroacetic acid in water comprising 0.07% (v/v) of trifluoroacetic acid, at a flow rate of 10 ml/min. At the outlet of the separation column, one-thousandth of the effluent is separated by means of an LC Packing Accurate, diluted with methyl alcohol at a flow rate of 0.5 ml/min and sent to the detectors, in a proportion of 75% to the diode array detector and the remaining 25% to the mass spectrometer. The rest of the effluent (999/1000) is sent to the fraction collector, where the flow is discarded for as long as the mass of the expected product is not detected by the FractionLynx software. The molecular formulae of the expected products are supplied to the FractionLynx software, which actuates the collection of the product when the mass signal detected corresponds to the ion [M+H]+ and/or to [M+Na]+. In certain cases, depending on the results of the analytical LC/MS, when an intense ion corresponding to [M+2H]++ was detected, the value corresponding to half the calculated molecular mass (MW/2) is also supplied to the FractionLynx software. Under these conditions, the collection is also actuated when the mass signal of the ion [M+2H]++ and/or [M+Na+H]++ are detected.
Method C: EI Analysis
The mass spectra were produced by electron impact (70 eV) on a Finnigan SSQ 7000 spectrometer.
Method D: NMR Analysis
The NMR spectra were produced on a Bruker Avance 300 spectrometer and a Bruker Avance DRX 400 spectrometer.
tert-Butyl 4-(diazoethoxycarbonylmethyl)-4-hydroxypiperidine-1-carboxylate























 








 
















CAS
Name
d
MM
eq
mmol
g
ml



















1
79099-
N-Boc-

199.25
1.00
50.19
10.00




07-3
piper-




idinone


2
623-73-
Ethyl
1.085
114.1
1.05
52.70
6.01
5.54



4
diazo-




acetate


3
109-72-
1.6 M


1.60
80.30

50.19



8
BuLi




hexane


4
108-18-
Diiso-
0.720
101.19
1.60
80.30
8.13
11.29



9
propyl-




amine


5
109-99-
THf on


10


500



9
4′ mole-


vol.




cular




sieve


6
64-19-7
100%
1.050
60.05
5.00
250.94
15.07
14.35




AcOH









A freshly prepared solution of LDA (prepared by the dropwise addition, under an inert atmosphere at −78° C., of 50.19 ml of 1.6 M of BuLi in hexane to a solution of 11.29 ml of diisopropylamine in 200 ml of dry THF) is added dropwise, under an inert atmosphere at −78° C., onto 10.0 g of N-Boc-piperidinone in suspension and 5.54 ml of ethyl diazoacetate in 300 ml of dry THF. The mixture is stirred at −78° C. for 4 hours and is then decomposed at −78° C. with 14.35 ml of concentrated AcOH. The mixture obtained is left at ambient temperature overnight, and the solvent is then evaporated off under reduced pressure, to 1/10 of its initial volume, diluted in diisopropyl ether, and washed 4 times with a saturated NaHCO3 solution. The organic phase is dried over MgSO4. The hydrated salt is removed by filtration and the dry filtrate is concentrated under reduced pressure so as to give 15.12 g of a viscous yellow oil. LC/MS: RT=2.84; [M+1]+=304.33. The product is used as it is for the subsequent step.
tert-Butyl 4-(diazoethoxycarbonylmethyl)-3,6-dihydro-2H-pyridine-1-carboxylate























 








 
















GAS
Name
d
MM
eq
mmol
g
ml



















1

P-31391-

313.35
 1
48.25
15.12





106-4


2
10025-
POCl3
1.67
153.33
 2
96.51
14.80
8.86



87-3


3
110-86-
Pyridine
0.983
79.1
20
965.06
76.34
77.66



1
(4Å m.s.)


4
108-20-
iPr2O


 5


250



3



vol


5
1310-
0.1 M

40
 1
48.25

483



73-2
NaOH









78.0 ml of dry pyridine are added to a solution of 15.12 g of tert-butyl 4-(diazoethoxycarbonylmethyl)-4-hydroxypiperidine-1-carboxylate into 250 ml of iPr2O. The mixture is cooled to −10° C. and 8.86 ml of POCl3 are added slowly with vigorous stirring. The mixture is then left to return to ambient temperature for 12 hours with stirring. The reaction mixture is decomposed with 500 ml of a 0.1M NaOH solution, and is then extracted 3 times with EtOAc. The organic phase is washed with a saturated NaCl solution and dried over MgSO4. The hydrated salt is removed by filtration and the dry filtrate is concentrated under reduced pressure to 1/10 of its initial volume. LC/MS:RT=4.57; [M+1]+=296.31. The product is used as it is for the subsequent step.
6-tert-Butyl 3-ethyl 2,4,5,7-tetrahydropyrazolo[3,4-c]pyridyl-3,6-dicarboxylate























 








 
















CAS
Name
d
MM
eq
mmol
mg
ml



















1

P-31391-120-4

285.35
1.0
48.25




2
108-83-3
PhMe





150









The solution of tert-butyl 4-(diazoethoxycarbonylmethyl)-3,6-dihydro-2H-pyridyl-1-carboxylate in Py/EtOAc obtained in the preceding step is added dropwise to 150 ml of toluene reflux. The azeotrope Py/PhMe is distilled at a rate equivalent to the rate of addition. One hour after the end of addition, the solution is left to cool to ambient temperature, the solvent is evaporated off under reduced pressure and the crude product obtained (15.05 g) is purified by flash chromatography (SiO2, CH2Cl2/MeOH 1% NH37M(MeOH) 40:1 then 30:1 then 20:1). The solvent is evaporated off and 10.05 g (71% over 3 steps) of a black solid are obtained: LC/MS: RT=3.88; [M+1]+=296.27.
(tert-Butyl 2,4,5,7-tetrahydropyrazolo[3,4-c]pyridyl-6-carboxylate)-3-carboxylic acid























 








 
















Cas
Name
d
MM
eq
mmol
g
ml



















1

P-31391-

295.34
1.0
34.03
10.05





123-1


2
1310-66-
LiOH.H2O

41.96
1.1
37.43
1.57



3


3
67-56-1
MeOH


10


375







vol.


4
7732-18-
H2O


1


38



5



vol









1.57 g of LiOH and 38 ml of water are added to a solution of 10.05 g of 6-tert-butyl butyl 3-ethyl 2,4,5,7-tetrahydropyrazolo[3,4-c]pyridyl-3,6-dicarboxylate in 375 ml of MeOH. The mixture obtained is refluxed overnight. The solution is cooled to ambient temperature and is then acidified to pH=2 with 50 ml of a 1M HCl solution. The solution is then extracted 4 times with EtOAc. The organic phase is washed with a saturated NaCl solution and then dried over Na2SO4. The salt obtained is removed by filtration and the solvent is evaporated off under reduced pressure so as to produce 8.90 g (98%) of a white solid. LC/MS: RT=3.21; [M+1]+=268.23.
Preparation of a Library of Products:
tert-Butyl 3-(alkylcarbamoyl, arylcarbamoyl, heteroarylcarbamoyl, etc.)-2,4,5,7-tetrahydropyrazolo[3,4-c]pyridine-6-carboxylate























 








 
















CAS
Name
d
MM

mmol
g
ml



















1

P-31391-031-5

267.28
1.0
3.741
1.00





amine


2.0
6.744


2
538-75-0
DCC

206.33
1.0
3.741
0.772


3
2592-95-2
HOBt.H2O

153.13
1.5
5.612
0.859


4
68-12-2
DMF


5  


19







vol.









General Method:

DCC and HOBT.H2O in solution in DMF with 2 eq of amine (R, Ar, or Het)-NH2 are added to a solution of 1 eq of tert-butyl (2,4,5,7-tetrahydro-pyrazolo[3,4-c]pyridyl-6-carboxylate)-3-carboxylic acid in DMF and the mixture is stirred at ambient temperature for 3 h. The solvent is evaporated off under reduced pressure at 35° C. overnight. The crude product obtained is purified by flash chromatography (SiO2, CH2Cl2/MeOH 1% NH37M(MeOH) 20:1 then 10:1 then 5:1, according to the products).
List of amines R1-NH2 used (Table 1) [Comment: R1-NH2═(R, Ar, or Het)—NH2]:








TABLE 1





Reference number of the amine
Structure
















1






 


2






 


3






 


4






 


5






 


6






 


7






 


8






 


9






 


10






 


11






 


12






 


13






 


14






 


15













3-(Alkylcarbamoyl, arylcarbamoyl, heteroarylcarbamoyl, etc.)-4,5,6,7-tetrahydro -2H-pyrazolo[3,4-c]pyridin-6-ium trifluoroacetate




General Method:

16 eq of tert-butyl 3-(alkylcarbamoyl, arylcarbamoyl, heteroarylcarbamoyl, etc.)-2,4,5,7-tetrahydropyrazolo[3,4-c]pyridine-6-carboxylate in a 1:1 THF/water solution, and the solution is refluxed for 2 h. The solvent is evaporated off under reduced pressure and the viscous oil collected is dried under vacuum overnight. The product thus obtained is used without purification, in the subsequent step.
6-(Alkyl, aryl, heteroaryl, etc.)carbonyl-4,5,6,7-tetrahydro-2H-pyrazolo[3,4c]pyridiene-3-(alkyl, aryl, hetyl, etc.)amide




General Method:

A solution of 2.5 M HOBt.H2O (2 eq) in DMF, a solution of 0.833M HBTU in DMF (2 eq), a solution of 2.5M DIPEA (4 eq) in DMF and a suspension or a solution at an appropriate concentration of an R2COOH (2 eq) in DMF are added, in order, to a solution of 1 eq of 3-(alkylcarbamoyl, arylcarbamoyl, heteroarylcarbamoyl, etc.)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridin-6-ium trifluoroacetate in DMF. The solutions are stirred overnight at ambient temperature and are then acidified with 100 μl of 100% AcOH, filtered and purified by preparative LC/MS.
List of the acids R2COOH used (Table 2):








TABLE 2





Reference number of the



acid
Nomenclature
















1
1-Phenyl-1-cyclopropylcarboxylic acid


2
Acetic acid


3
Propiolic acid


4
Crotonic acid


5
Vinylacetic acid


6
Pyruvic acid


7
Sarcosine


8
Methoxyacetic acid


9
Lactic acid


10
3,3-Dimethylacrylic acid


11
Cyclopropylacetic acid


12
Valeric acid


13
N,N-dimethylglycine


14
3-Mercaptopropionic acid


15
(Methylthio)acetic acid


16
Pyrrole-2-carboxylic acid


17
1-Cyanocyclopropanecarboxylic acid


18
2-Furoic acid


19
4-Pyrazolecarboxylic acid


20
Imidazole-4-carboxylic acid


21
1-Cyclopentenecarboxylic acid


22
Acid


23
Acetoxyacetic acid


24
Hydantoic acid


25
Benzoic acid


26
Nicotinic acid


27
2-Pyrazinecarboxylic acid


28
o-Toluic acid


29
Phenylacetic acid


30
Salicylic acid


31
2-Fluorobenzoic acid


32
3-Cyanobenzoic acid


33
4-Vinylbenzoic acid


34
2-Phenylpropionic acid


35
N-Methylanthranilic acid


36
2-Methoxybenzoic acid


37
2-Hydroxyphenylacetic acid


38
4-Hydroxymethylbenzoic acid


39
2-Fluorophenylbenzoic acid


40
2,6-Difluorobenzoic acid


41
Indole-3-carboxylic acid


42
3,5-Dimethylphenylacetic acid


43
3-(Dimethylamino)benzoic acid


44
Piperonylic acid


45
DL-tropic acid


46
3-Methoxyphenylacetic acid


47
3-Methoxysalicylic acid


48
4-(Methylthio)benzoic acid


49
2-Chlorophenylacetic acid


50
2-Naphthoic acid


51
2-Chloro-6-fluorobenzoic acid


52
1-Methylindole-3-carboxylic acid


53
3-Acetamidobenzoic acid


54
4-(Dimethylamino)salicylic acid


55
2,3-Dimethoxybenzoic acid


56
4-Chlorophenylpropionic acid


57
2-Chloromandelic acid


58
2-Chloro-6-fluorophenylacetic acid


59
1-Phenyl-1-cyclopentanecarboxylic acid


60
2,6-Dichlorobenzoic acid


61
3-Methyl-2-phenylvaleric acid


62
4-Phenylbenzoic acid


63
2-Chloro-4-nitrobenzoic acid


64
2-Benzylbenzoic acid


65
2-Phenoxybenzoic acid


66
2-Ethoxy-1-naphthoic acid


67
4-(4-N-Propylphenyl)benzoic acid


68
3,5-Dibromosalicylic acid


69
2,6-Dichlorophenylacetic acid


70
Cyanoacetic acid









Results

The following products are prepared according to the process described above.




In order to simplify the representation of the products in Table 3 which follows, the pyrazolopiperidine ring presented in Scheme A is symbolized by the letter H, the amines R1-NH2 which are linked to H are symbolized by the letter B followed by a number ranging from 1 to 15, corresponding to the products listed in Table 1, and the acids R2-COOH which are linked to H are symbolized by the letter A followed by a number from 1 to 70, corresponding to the products listed in Table 2.
Thus, a product referred to as A1-H-B1 corresponds to the following structure:







TABLE 3






























A1-H-B1
A2-H-B1
A3-H-B1
A4-H-B1
A5-H-B1
A6-H-B1
A7-H-B1


A1-H-B2
A2-H-B2
A3-H-B2
A4-H-B2
A5-H-B2
A6-H-B2
A7-H-B2


A1-H-B3
A2-H-B3
A3-H-B3
A4-H-B3
A5-H-B3
A6-H-B3
A7-H-B3


A1-H-B4
A2-H-B4
A3-H-B4
A4-H-B4
A5-H-B4
A6-H-B4
A7-H-B4


A1-H-B5
A2-H-B5
A3-H-B5
A4-H-B5
A5-H-B5
A6-H-B5
A7-H-B5


A1-H-B6
A2-H-B6
A3-H-B6
A4-H-B6
A5-H-B6
A6-H-B6
A7-H-B6


A1-H-B7
A2-H-B7
A3-H-B7
A4-H-B7
A5-H-B7
A6-H-B7
A7-H-B7


A1-H-B8
A2-H-B8
A3-H-B8
A4-H-B8
A5-H-B8
A6-H-B8
A7-H-B8


A1-H-B9
A2-H-B9
A3-H-B9
A4-H-B9
A5-H-B9
A6-H-B9
A7-H-B9


A1-H-B10
A2-H-B10
A3-H-B10
A4-H-B10
A5-H-B10
A6-H-B10
A7-H-B10


A1-H-B11
A2-H-B11
A3-H-B11
A4-H-B11
A5-H-B11
A6-H-B11
A7-H-B11


A1-H-B12
A2-H-B12
A3-H-B12
A4-H-B12
A5-H-B12
A6-H-B12
A7-H-B12


A1-H-B13
A2-H-B13
A3-H-B13
A4-H-B13
A5-H-B13
A6-H-B13
A7-H-B13


A1-H-B14
A2-H-B14
A3-H-B14
A4-H-B14
A5-H-B14
A6-H-B14
A7-H-B14


A1-H-B15
A2-H-B15
A3-H-B15
A4-H-B15
A5-H-B15
A6-H-B15
A7-H-B15


A8-H-B1
A9-H-B1
A10-H-B1
A11-H-B1
A12-H-B1
A13-H-B1
A14-H-B1


A8-H-B2
A9-H-B2
A10-H-B2
A11-H-B2
A12-H-B2
A13-H-B2
A14-H-B2


A8-H-B3
A9-H-B3
A10-H-B3
A11-H-B3
A12-H-B3
A13-H-B3
A14-H-B3


A8-H-B4
A9-H-B4
A10-H-B4
A11-H-B4
A12-H-B4
A13-H-B4
A14-H-B4


A8-H-B5
A9-H-B5
A10-H-B5
A11-H-B5
A12-H-B5
A13-H-B5
A14-H-B5


A8-H-B6
A9-H-B6
A10-H-B6
A11-H-B6
A12-H-B6
A13-H-B6
A14-H-B6


A8-H-B7
A9-H-B7
A10-H-B7
A11-H-B7
A12-H-B7
A13-H-B7
A14-H-B7


A8-H-B8
A9-H-B8
A10-H-B8
A11-H-B8
A12-H-B8
A13-H-B8
A14-H-B8


A8-H-B9
A9-H-B9
A10-H-B9
A11-H-B9
A12-H-B9
A13-H-B9
A14-H-B9


A8-H-B10
A9-H-B10
A10-H-B10
A11-H-B10
A12-H-B10
A13-H-B10
A14-H-B10


A8-H-B11
A9-H-B11
A10-H-B11
A11-H-B11
A12-H-B11
A13-H-B11
A14-H-B11


A8-H-B12
A9-H-B12
A10-H-B12
A11-H-B12
A12-H-B12
A13-H-B12
A14-H-B12


A8-H-B13
A9-H-B13
A10-H-B13
A11-H-B13
A12-H-B13
A13-H-B13
A14-H-B13


A8-H-B14
A9-H-B14
A10-H-B14
A11-H-B14
A12-H-B14
A13-H-B14
A14-H-B14


A8-H-B15
A9-H-B15
A10-H-B15
A11-H-B15
A12-H-B15
A13-H-B15
A14-H-B15


A15-H-B1
A16-H-B1
A17-H-B1
A18-H-B1
A19-H-B1
A20-H-B1
A21-H-B1


A15-H-B2
A16-H-B2
A17-H-B2
A18-H-B2
A19-H-B2
A20-H-B2
A21-H-B2


A15-H-B3
A16-H-B3
A17-H-B3
A18-H-B3
A19-H-B3
A20-H-B3
A21-H-B3


A15-H-B4
A16-H-B4
A17-H-B4
A18-H-B4
A19-H-B4
A20-H-B4
A21-H-B4


A15-H-B5
A16-H-B5
A17-H-B5
A18-H-B5
A19-H-B5
A20-H-B5
A21-H-B5


A15-H-B6
A16-H-B6
A17-H-B6
A18-H-B6
A19-H-B6
A20-H-B6
A21-H-B6


A15-H-B7
A16-H-B7
A17-H-B7
A18-H-B7
A19-H-B7
A20-H-B7
A21-H-B7


A15-H-B8
A16-H-B8
A17-H-B8
A18-H-B8
A19-H-B8
A20-H-B8
A21-H-B8


A15-H-B9
A16-H-B9
A17-H-B9
A18-H-B9
A19-H-B9
A20-H-B9
A21-H-B9


A15-H-B10
A16-H-B10
A17-H-B10
A18-H-B10
A19-H-B10
A20-H-B10
A21-H-B10


A15-H-B11
A16-H-B11
A17-H-B11
A18-H-B11
A19-H-B11
A20-H-B11
A21-H-B11


A15-H-B12
A16-H-B12
A17-H-B12
A18-H-B12
A19-H-B12
A20-H-B12
A21-H-B12


A15-H-B13
A16-H-B13
A17-H-B13
A18-H-B13
A19-H-B13
A20-H-B13
A21-H-B13


A15-H-B14
A16-H-B14
A17-H-B14
A18-H-B14
A19-H-B14
A20-H-B14
A21-H-B14


A15-H-B15
A16-H-B15
A17-H-B15
A18-H-B15
A19-H-B15
A20-H-B15
A21-H-B15


A22-H-B1
A23-H-B1
A24-H-B1
A25-H-B1
A26-H-B1
A27-H-B1
A28-H-B1


A22-H-B2
A23-H-B2
A24-H-B2
A25-H-B2
A26-H-B2
A27-H-B2
A28-H-B2


A22-H-B3
A23-H-B3
A24-H-B3
A25-H-B3
A26-H-B3
A27-H-B3
A28-H-B3


A22-H-B4
A23-H-B4
A24-H-B4
A25-H-B4
A26-H-B4
A27-H-B4
A28-H-B4


A22-H-B5
A23-H-B5
A24-H-B5
A25-H-B5
A26-H-B5
A27-H-B5
A28-H-B5


A22-H-B6
A23-H-B6
A24-H-B6
A25-H-B6
A26-H-B6
A27-H-B6
A28-H-B6


A22-H-B7
A23-H-B7
A24-H-B7
A25-H-B7
A26-H-B7
A27-H-B7
A28-H-B7


A22-H-B8
A23-H-B8
A24-H-B8
A25-H-B8
A26-H-B8
A27-H-B8
A28-H-B8


A22-H-B9
A23-H-B9
A24-H-B9
A25-H-B9
A26-H-B9
A27-H-B9
A28-H-B9


A22-H-B10
A23-H-B10
A24-H-B10
A25-H-B10
A26-H-B10
A27-H-B10
A28-H-B10


A22-H-B11
A23-H-B11
A24-H-B11
A25-H-B11
A26-H-B11
A27-H-B11
A28-H-B11


A22-H-B12
A23-H-B12
A24-H-B12
A25-H-B12
A26-H-B12
A27-H-B12
A28-H-B12


A22-H-B13
A23-H-B13
A24-H-B13
A25-H-B13
A26-H-B13
A27-H-B13
A28-H-B13


A22-H-B14
A23-H-B14
A24-H-B14
A25-H-B14
A26-H-B14
A27-H-B14
A28-H-B14


A22-H-B15
A23-H-B15
A24-H-B15
A25-H-B15
A26-H-B15
A27-H-B15
A28-H-B15


A29-H-B1
A30-H-B1
A31-H-B1
A32-H-B1
A33-H-B1
A34-H-B1
A35-H-B1


A29-H-B2
A30-H-B2
A31-H-B2
A32-H-B2
A33-H-B2
A34-H-B2
A35-H-B2


A29-H-B3
A30-H-B3
A31-H-B3
A32-H-B3
A33-H-B3
A34-H-B3
A35-H-B3


A29-H-B4
A30-H-B4
A31-H-B4
A32-H-B4
A33-H-B4
A34-H-B4
A35-H-B4


A29-H-B5
A30-H-B5
A31-H-B5
A32-H-B5
A33-H-B5
A34-H-B5
A35-H-B5


A29-H-B6
A30-H-B6
A31-H-B6
A32-H-B6
A33-H-B6
A34-H-B6
A35-H-B6


A29-H-B7
A30-H-B7
A31-H-B7
A32-H-B7
A33-H-B7
A34-H-B7
A35-H-B7


A29-H-B8
A30-H-B8
A31-H-B8
A32-H-B8
A33-H-B8
A34-H-B8
A35-H-B8


A29-H-B9
A30-H-B9
A31-H-B9
A32-H-B9
A33-H-B9
A34-H-B9
A35-H-B9


A29-H-B10
A30-H-B10
A31-H-B10
A32-H-B10
A33-H-B10
A34-H-B10
A35-H-B10


A29-H-B11
A30-H-B11
A31-H-B11
A32-H-B11
A33-H-B11
A34-H-B11
A35-H-B11


A29-H-B12
A30-H-B12
A31-H-B12
A32-H-B12
A33-H-B12
A34-H-B12
A35-H-B12


A29-H-B13
A30-H-B13
A31-H-B13
A32-H-B13
A33-H-B13
A34-H-B13
A35-H-B13


A29-H-B14
A30-H-B14
A31-H-B14
A32-H-B14
A33-H-B14
A34-H-B14
A35-H-B14


A29-H-B15
A30-H-B15
A31-H-B15
A32-H-B15
A33-H-B15
A34-H-B15
A35-H-B15


A36-H-B1
A37-H-B1
A38-H-B1
A39-H-B1
A40-H-B1
A41-H-B1
A42-H-B1


A36-H-B2
A37-H-B2
A38-H-B2
A39-H-B2
A40-H-B2
A41-H-B2
A42-H-B2


A36-H-B3
A37-H-B3
A38-H-B3
A39-H-B3
A40-H-B3
A41-H-B3
A42-H-B3


A36-H-B4
A37-H-B4
A38-H-B4
A39-H-B4
A40-H-B4
A41-H-B4
A42-H-B4


A36-H-B5
A37-H-B5
A38-H-B5
A39-H-B5
A40-H-B5
A41-H-B5
A42-H-B5


A36-H-B6
A37-H-B6
A38-H-B6
A39-H-B6
A40-H-B6
A41-H-B6
A42-H-B6


A36-H-B7
A37-H-B7
A38-H-B7
A39-H-B7
A40-H-B7
A41-H-B7
A42-H-B7


A36-H-B8
A37-H-B8
A38-H-B8
A39-H-B8
A40-H-B8
A41-H-B8
A42-H-B8


A36-H-B9
A37-H-B9
A38-H-B9
A39-H-B9
A40-H-B9
A41-H-B9
A42-H-B9


A36-H-B10
A37-H-B10
A38-H-B10
A39-H-B10
A40-H-B10
A41-H-B10
A42-H-B10


A36-H-B11
A37-H-B11
A38-H-B11
A39-H-B11
A40-H-B11
A41-H-B11
A42-H-B11


A36-H-B12
A37-H-B12
A38-H-B12
A39-H-B12
A40-H-B12
A41-H-B12
A42-H-B12


A36-H-B13
A37-H-B13
A38-H-B13
A39-H-B13
A40-H-B13
A41-H-B13
A42-H-B13


A36-H-B14
A37-H-B14
A38-H-B14
A39-H-B14
A40-H-B14
A41-H-B14
A42-H-B14


A36-H-B15
A37-H-B15
A38-H-B15
A39-H-B15
A40-H-B15
A41-H-B15
A42-H-B15


A43-H-B1
A44-H-B1
A45-H-B1
A46-H-B1
A47-H-B1
A48-H-B1
A49-H-B1


A43-H-B2
A44-H-B2
A45-H-B2
A46-H-B2
A47-H-B2
A48-H-B2
A49-H-B2


A43-H-B3
A44-H-B3
A45-H-B3
A46-H-B3
A47-H-B3
A48-H-B3
A49-H-B3


A43-H-B4
A44-H-B4
A45-H-B4
A46-H-B4
A47-H-B4
A48-H-B4
A49-H-B4


A43-H-B5
A44-H-B5
A45-H-B5
A46-H-B5
A47-H-B5
A48-H-B5
A49-H-B5


A43-H-B6
A44-H-B6
A45-H-B6
A46-H-B6
A47-H-B6
A48-H-B6
A49-H-B6


A43-H-B7
A44-H-B7
A45-H-B7
A46-H-B7
A47-H-B7
A48-H-B7
A49-H-B7


A43-H-B8
A44-H-B8
A45-H-B8
A46-H-B8
A47-H-B8
A48-H-B8
A49-H-B8


A43-H-B9
A44-H-B9
A45-H-B9
A46-H-B9
A47-H-B9
A48-H-B9
A49-H-B9


A43-H-B10
A44-H-B10
A45-H-B10
A46-H-B10
A47-H-B10
A48-H-B10
A49-H-B10


A43-H-B11
A44-H-B11
A45-H-B11
A46-H-B11
A47-H-B11
A48-H-B11
A49-H-B11


A43-H-B12
A44-H-B12
A45-H-B12
A46-H-B12
A47-H-B12
A48-H-B12
A49-H-B12


A43-H-B13
A44-H-B13
A45-H-B13
A46-H-B13
A47-H-B13
A48-H-B13
A49-H-B13


A43-H-B14
A44-H-B14
A45-H-B14
A46-H-B14
A47-H-B14
A48-H-B14
A49-H-B14


A43-H-B15
A44-H-B15
A45-H-B15
A46-H-B15
A47-H-B15
A48-H-B15
A49-H-B15


A50-H-B1
A51-H-B1
A52-H-B1
A53-H-B1
A54-H-B1
A55-H-B1
A56-H-B1


A50-H-B2
A51-H-B2
A52-H-B2
A53-H-B2
A54-H-B2
A55-H-B2
A56-H-B2


A50-H-B3
A51-H-B3
A52-H-B3
A53-H-B3
A54-H-B3
A55-H-B3
A56-H-B3


A50-H-B4
A51-H-B4
A52-H-B4
A53-H-B4
A54-H-B4
A55-H-B4
A56-H-B4


A50-H-B5
A51-H-B5
A52-H-B5
A53-H-B5
A54-H-B5
A55-H-B5
A56-H-B5


A50-H-B6
A51-H-B6
A52-H-B6
A53-H-B6
A54-H-B6
A55-H-B6
A56-H-B6


A50-H-B7
A51-H-B7
A52-H-B7
A53-H-B7
A54-H-B7
A55-H-B7
A56-H-B7


A50-H-B8
A51-H-B8
A52-H-B8
A53-H-B8
A54-H-B8
A55-H-B8
A56-H-B8


A50-H-B9
A51-H-B9
A52-H-B9
A53-H-B9
A54-H-B9
A55-H-B9
A56-H-B9


A50-H-B10
A51-H-B10
A52-H-B10
A53-H-B10
A54-H-B10
A55-H-B10
A56-H-B10


A50-H-B11
A51-H-B11
A52-H-B11
A53-H-B11
A54-H-B11
A55-H-B11
A56-H-B11


A50-H-B12
A51-H-B12
A52-H-B12
A53-H-B12
A54-H-B12
A55-H-B12
A56-H-B12


A50-H-B13
A51-H-B13
A52-H-B13
A53-H-B13
A54-H-B13
A55-H-B13
A56-H-B13


A50-H-B14
A51-H-B14
A52-H-B14
A53-H-B14
A54-H-B14
A55-H-B14
A56-H-B14


A50-H-B15
A51-H-B15
A52-H-B15
A53-H-B15
A54-H-B15
A55-H-B15
A56-H-B15


A57-H-B1
A58-H-B1
A59-H-B1
A60-H-B1
A61-H-B1
A62-H-B1
A63-H-B1


A57-H-B2
A58-H-B2
A59-H-B2
A60-H-B2
A61-H-B2
A62-H-B2
A63-H-B2


A57-H-B3
A58-H-B3
A59-H-B3
A60-H-B3
A61-H-B3
A62-H-B3
A63-H-B3


A57-H-B4
A58-H-B4
A59-H-B4
A60-H-B4
A61-H-B4
A62-H-B4
A63-H-B4


A57-H-B5
A58-H-B5
A59-H-B5
A60-H-B5
A61-H-B5
A62-H-B5
A63-H-B5


A57-H-B6
A58-H-B6
A59-H-B6
A60-H-B6
A61-H-B6
A62-H-B6
A63-H-B6


A57-H-B7
A58-H-B7
A59-H-B7
A60-H-B7
A61-H-B7
A62-H-B7
A63-H-B7


A57-H-B8
A58-H-B8
A59-H-B8
A60-H-B8
A61-H-B8
A62-H-B8
A63-H-B8


A57-H-B9
A58-H-B9
A59-H-B9
A60-H-B9
A61-H-B9
A62-H-B9
A63-H-B9


A57-H-B10
A58-H-B10
A59-H-B10
A60-H-B10
A61-H-B10
A62-H-B10
A63-H-B10


A57-H-B11
A58-H-B11
A59-H-B11
A60-H-B11
A61-H-B11
A62-H-B11
A63-H-B11


A57-H-B12
A58-H-B12
A59-H-B12
A60-H-B12
A61-H-B12
A62-H-B12
A63-H-B12


A57-H-B13
A58-H-B13
A59-H-B13
A60-H-B13
A61-H-B13
A62-H-B13
A63-H-B13


A57-H-B14
A58-H-B14
A59-H-B14
A60-H-B14
A61-H-B14
A62-H-B14
A63-H-B14


A57-H-B15
A58-H-B15
A59-H-B15
A60-H-B15
A61-H-B15
A62-H-B15
A63-H-B15


A64-H-B1
A65-H-B1
A66-H-B1
A67-H-B1
A68-H-B1
A69-H-B1
A70-H-B1


A64-H-B2
A65-H-B2
A66-H-B2
A67-H-B2
A68-H-B2
A69-H-B2
A70-H-B2


A64-H-B3
A65-H-B3
A66-H-B3
A67-H-B3
A68-H-B3
A69-H-B3
A70-H-B3


A64-H-B4
A65-H-B4
A66-H-B4
A67-H-B4
A68-H-B4
A69-H-B4
A70-H-B4


A64-H-B5
A65-H-B5
A66-H-B5
A67-H-B5
A68-H-B5
A69-H-B5
A70-H-B5


A64-H-B6
A65-H-B6
A66-H-B6
A67-H-B6
A68-H-B6
A69-H-B6
A70-H-B6


A64-H-B7
A65-H-B7
A66-H-B7
A67-H-B7
A68-H-B7
A69-H-B7
A70-H-B7


A64-H-B8
A65-H-B8
A66-H-B8
A67-H-B8
A68-H-B8
A69-H-B8
A70-H-B8


A64-H-B9
A65-H-B9
A66-H-B9
A67-H-B9
A68-H-B9
A69-H-B9
A70-H-B9


A64-H-B10
A65-H-B10
A66-H-B10
A67-H-B10
A68-H-B10
A69-H-B10
A70-H-B10


A64-H-B11
A65-H-B11
A66-H-B11
A67-H-B11
A68-H-B11
A69-H-B11
A70-H-B11


A64-H-B12
A65-H-B12
A66-H-B12
A67-H-B12
A68-H-B12
A69-H-B12
A70-H-B12


A64-H-B13
A65-H-B13
A66-H-B13
A67-H-B13
A68-H-B13
A69-H-B13
A70-H-B13


A64-H-B14
A65-H-B14
A66-H-B14
A67-H-B14
A68-H-B14
A69-H-B14
A70-H-B14


A64-H-B15
A65-H-B15
A66-H-B15
A67-H-B15
A68-H-B15
A69-H-B15
A70-H-B15











EXAMPLE 1
3-(5,6-Dimethyl-1H-benzoimidazol-2-yl)-2,4,5,7-tetrahydro-pyrazolo[3,4-c]pyridine-6-carboxylic acid 2-phenylethylamide




3-(5,6-Dimethyl-1H-benzoimidazol-2-yl)-2,4,5,7-tetrahydropyrazolo-[3,4-c]pyridine-6-carboxylic acid 2-phenylethylamide can be prepared in the following way:
10 mg of 3-(5,6-dimethyl-1H-benzimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine are suspended in 0.3 ml of tetrahydrofuran. 7.8 μl of 2-phenylethyl isocyanate are added and the reaction mixture is stirred at ambient temperature for 20 hours and then concentrated under reduced pressure.
The evaporation residue is purified by LC/MS (method B). After purification by LC/MS, the fractions containing the 3-(5,6-dimethyl-1H-benzoimidazol-2-yl)-2,4,5,7-tetrahydropyrazolo[3,4-c]pyridine-6-carboxylic acid 2-phenyl-ethylamide are combined and loaded onto SCX phase (500 mg of CUBCX1-HL phase). The SCX phase is subsequently washed with methanol and then extracted with a solution of 2M ammonia in methanol. The extraction solution obtained is then concentrated under reduced pressure. 1.2 mg of 3-(5,6-dimethyl-1H-benzoimidazol-2-yl)-2,4,5,7-tetrahydropyrazolo[3,4-c]-pyridine-6-carboxylic acid 2-phenylethylamide are thus obtained in the form of a white powder, the characteristics of which are as follows:
LC/MS (method A): molecular ion detected: 415.29; retention time=3.48 minutes
EXAMPLE 2
3-(5,6-Dimethyl-1H-benzoimidazol-2-yl)-6-methanesulphonyl-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine




3-(5,6-Dimethyl-1H-benzoimidazol-2-yl)-6-methanesulphonyl-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine can be prepared in the following way:
10 mg of 3-(5,6-dimethyl-1H-benzimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine are suspended in 0.3 ml of dichloromethane. 15.8 μl of triethylamine are added, along with 4.5 μl of methanesulphonyl chloride. The reaction mixture is stirred at ambient temperature for 20 hours and is then concentrated under reduced pressure.
The evaporation residue is purified by LC/MS (method B). After purification by LC/MS, the fractions containing 3-(5,6-dimethyl-1H-benzoimidazol-2-yl)-6-methanesulphonyl-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine are combined and loaded onto SCX phase (500 mg of CUBCX1-HL phase). The SCX phase is subsequently washed with methanol and then extracted with a solution of 2M ammonia in methanol. The extraction solution obtained is then concentrated under reduced pressure. 4.2 mg of 3-(5,6-dimethyl-1H-benzoimidazol-2-yl)-6-methanesulphonyl-4,5,6,7-tetrahydro-2H-pyrazolo-[3,4-c]pyridine are thus obtained in the form of a white powder, the characteristics of which are as follows:
LC/MS (method A): molecular ion detected: 346.30; retention time=3.08 minutes.
1H NMR (300 MHz, (CD3)2SO, δ in ppm): 2.32 (broad s: 6H); 3.02 (s: 3H); 3.03 (mt: 2H); 3.52 (broad t, J=5 Hz: 2H); 4.45 (broad s: 2H); 7.24 (broad s: 1H); 7.42 (broad s: 1H); 12.45 (unresolved peak: 1H); 13.07 (unresolved peak: 1H).
EXAMPLE 3
[3-(5,6-Dimethyl-1H-benzoimidazol-2-yl)-2,4,5,7-tetrahydro-pyrazolo[3,4-c]pyridin-6-yl]-3-pyridinylmethanone




[3-(5,6-Dimethyl-1H-benzoimidazol-2-yl)-2,4,5,7-tetrahydropyrazolo-[3,4-c]pyridin-6-yl]-3-pyridinylmethanone can be prepared in the following way:
10 mg of 3-(5,6-dimethyl-1H-benzimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine are suspended in 0.3 ml of DMF. 6.9 mg of nicotinic acid are added, followed by 7.6 mg of HOBT and 8.7 μl of diisopropylcarbo-diimide.
The reaction mixture is stirred at ambient temperature for 20 hours and is then concentrated under reduced pressure.
The evaporation residue is purified by LC/MS (method B). After purification by LC/MS, the fractions containing the [3-(5,6-dimethyl-1H-benzoimidazol-2-yl)-2,4,5,7-tetrahydropyrazolo[3,4-c]pyridin-6-yl]-3-pyridinylmethanone are combined and loaded onto SCX phase (500 mg of CUBCX1-HL phase). The SCX phase is subsequently washed with methanol and then extracted with a solution of 2M ammonia in methanol. The extraction solution obtained is then concentrated under reduced pressure. 5.3 mg of [3-(5,6-dimethyl-1H-benzoimidazol-2-yl)-2,4,5,7-tetrahydropyrazolo[3,4-c]pyridin-6-yl]-3-pyridinyl-methanone are thus obtained in the form of a white powder, the characteristics of which are as follows:
LC/MS (method A): molecular ion detected: 373.31; retention time=2.87 minutes
1H NMR (400 MHz, (CD3)2SO, at a temperature of 373K, δ in ppm): 2.35 (s: 6H); from 2.90 to 3.10 (mt: 2H); 3.76 (unresolved peak: 2H); 4.76 (broad s: 2H); 7.27 (unresolved peak: 1H); 7.40 (unresolved peak: 1H); 7.51 (dd, J=8 and 5 Hz: 1H); 7.90 (broad d, J=8 Hz: 1H); 8.70 (mt: 2H); from 11.80 to 12.20 (broad unresolved peak: 1H); from 12.50 to 13.00 (broad unresolved peak: 1H).
EXAMPLE 4
6-(3-Chlorobenzyl)-3-(5,6-dimethyl-1H-benzoimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine




6-(3-Chlorobenzyl)-3-(5,6-dimethyl-1H-benzoimidazol-2-yl)4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine can be prepared in the following way:
10 mg of 3-(5,6-dimethyl-1H-benzimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine are suspended in 0.3 ml of methanol. 12.7 μl of 3-chlorobenzaldehyde are added, followed by 4.7 mg of NaBH3CN. The reaction mixture is stirred at ambient temperature for 20 hours and is then concentrated under reduced pressure.
The evaporation residue is purified by LC/MS (method B). After purification by LC/MS, the fractions containing the 6-(3-chlorobenzyl)-3-(5,6-dimethyl-1H-benzoimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine are combined and loaded onto a SCX phase (500 mg of CUBCX1-HL phase). The SCX phase is subsequently washed with methanol and then extracted with a solution of 2M ammonia in methanol. The extraction solution obtained is then concentrated under reduced pressure. 4 mg of 6-(3-chlorobenzyl)-3-(5,6-dimethyl-1H-benzoimidazol-2-yl)4,5,6,7-tetrahydro-2H-pyrazolo-[3,4-c]pyridine are thus obtained in the form of a white powder, the characteristics of which are as follows:
LC/MS (method A): molecular ion detected: 392.26; retention time 3.18 minutes.
1H NMR (400 MHz, (CD3)2SO, at a temperature of 373K, δ in ppm): 2.35 and 2.36 (2 s: 6H in total); 2.83 (t, J=5.5 Hz: 2H); from 2.90 to 3.00 (mt: 2H); 3.62 (broad s: 2H); 3.78 (s: 2H); from 7.25 to 7.50 (mt: 6H); 11.91 (unresolved peak: 1H); from 12.30 to 12.60 (broad unresolved peak: 1H).
EXAMPLE 5
[3-(1H-Benzoimidazol-2-yl)-2,4,5,7-tetrahydropyrazolo[3,4-c]-pyridin-6-yl]-3-pyridinylmethanone




[3-(1H-Benzoimidazol-2-yl)-2,4,5,7-tetrahydropyrazolo[3,4-c]pyridin-6-yl]-3-pyridinylmethanone can be prepared in the following way:
15 mg of 3-(1H-benzimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]-pyridine hydrochloride are suspended in 0.5 ml of DMF. 24.3 mg of diisopropylethylamine are added, followed by 12.7 mg of HOBT, 11.9 mg of diisopropylcarbodiimide and 11.6 mg of nicotinic acid. The reaction mixture is stirred at ambient temperature for 20 hours and then concentrated under reduced pressure.
The evaporation residue is purified by LC/MS (method B). After purification by LC/MS, the fractions containing the [3-(1H-benzoimidazol-2-yl)-2,4,5,7-tetrahydropyrazolo[3,4-c]pyridin-6-yl]-3-pyridinylmethanone are combined and loaded onto SCX phase (500 mg of CUBCX1-HL phase). The SCX phase is subsequently washed with methanol and then extracted with a solution of 2M ammonia in methanol. The extraction solution obtained is then concentrated under reduced pressure. 7.7 mg of [3-(1H-benzoimidazol-2-yl)-2,4,5,7-tetrahydropyrazolo[3,4-c]pyridin-6-yl]-3-pyridinylmethanone are thus obtained in the form of a white powder, the characteristics of which are as follows:
LC/MS (method A): molecular ion detected: 345.22; retention time=1.95 minutes
EXAMPLE 6
6-(3-Chlorobenzyl)-3-(1H-benzoimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine




6-(3-Chlorobenzyl)-3-(1H-benzoimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine can be prepared in the following way:
15 mg of 3-(1H-benzimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]-pyridine hydrochloride are suspended in 0.5 ml of methanol. 26.5 mg of 3-chlorobenzaldehyde are added, followed by 7.9 mg of NaBH3CN. The reaction mixture is stirred at ambient temperature for 20 hours and is then concentrated under reduced pressure.
The evaporation residue is purified by LC/MS (method B). After purification by LC/MS, the fractions containing the 6-(3-chlorobenzyl)-3-(1H-benzoimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine are combined and loaded onto SCX phase (500 mg of CUBCX1-HL phase). The SCX phase is subsequently washed with methanol and extracted with a solution of 2M ammonia in methanol. The extraction solution obtained is then concentrated under reduced pressure. 6.9 mg of 6-(3-chlorobenzyl)-3-(1H-benzoimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine are thus obtained in the form of a white powder, the characteristics of which are as follows:
LC/MS (method A): molecular ion detected: 364.22; retention time=2.19 minutes
EXAMPLE 7
Preparation of an Amide Library




The amide library can be prepared in the following way:
The 19 acids (Table 4) are weighed and placed in 19 individual test tubes.







TABLE 4







Acids used









Entry
Name
Amount












1
ISOBUTYRIC ACID
3.3 mg


2
BENZOIC ACID
4.6 mg


3
2,3-DICHLOROBENZOIC ACID
7.1 mg


4
PHENYLACETIC ACID
5.1 mg


5
ACETIC ACID
2.2 mg


6
CYCLOPROPANECARBOXYLIC ACID
3.2 mg


7
2-CHLOROBENZOIC ACID
5.9 mg


8
3-CHLOROBENZOIC ACID
5.9 mg


9
4-CHLOROBENZOIC ACID
5.9 mg


10
ISOVALERIC ACID
3.8 mg


11
HYDROCINNAMIC ACID
5.6 mg


12
VINYLACETIC ACID
3.2 mg


13
BUTYRIC ACID
3.3 mg


14
2-FUROIC ACID
4.2 mg


15
PIVALIC ACID
3.8 mg


16
N,N-DIMETHYLGLYCINE
3.9 mg


17
VALERIC ACID
3.8 mg


18
THIOPHENE-2-CARBOXYLIC ACID
4.8 mg


19
4-METHYLSULPHONYLBENZOIC ACID
7.5 mg









152 mg of HOBT and 142 mg of diisopropylcarbodiimide are solubilized in 12 ml of DMF and the solution obtained is distributed in each of the 19 test tubes, at a rate of 600 μl per tube.
200 mg of 3-(5,6-dimethyl-1H-benzimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine hydrochloride are suspended in 4 ml of DMF in the presence of 290 mg of N,N-diisopropylethylamine, and the suspension obtained is distributed into each of the 19 test tubes, at a rate of 200 μl per tube.
The 19 reaction mixtures are shaken by means of orbital shaking at ambient temperature for 20 hours.
For each reaction mixture, a 10 μl sample is taken and diluted in 40 μl of DMSO (Gilson Liquid Handler Quad-Z 215). Each sample in solution in DMSO thus obtained is analysed by LC/MS (method A).
The 19 reaction mixtures are then evaporated to dryness and the evaporation residues are each solubilized in 500 μl of DMSO, and the solutions obtained are then purified by LC/MS (method B).
After purification by LC/MS, the fractions containing the desired compounds are (optionally combined) loaded onto SCX phase (500 mg of CUBCX1-HL phase). The SCX phases are subsequently washed with methanol and then extracted with a solution of 2M ammonia in methanol. The extraction solutions are collected in tared glass tubes, evaporated to dryness (Savant AES 2000 or Genevac HT8 centrifugal evaporator), weighed (Mettler Toledo Automated Workstation LA200) and diluted to 10 mM in DMSO (Gilson Liquid Handler Quad-Z 215). Each solution obtained is analysed by LC/MS (method A).
The following compounds (Table 5) were isolated and characterized by means of their retention time and molecular peak in mass spectrometry (method A).







TABLE 5







Amide library obtained













Amount of
Retention
Molecular




product
time
ion


Entry
Name
obtained
(minutes)
detected















1
1-[3-(5,6-Dimethyl-1H-benzoimidazol-
5.8
mg
3.08
338.23



2-yl)-2,4,5,7-tetrahydropyrazolo-



[3,4-c]pyridin-6-yl]-2-methylpropan-1-



one


2
[3-(5,6-Dimethyl-1H-benzoimidazol-2-
6.8
mg
2.68
372.21



yl)-2,4,5,7-tetrahydropyrazolo[3,4-c]-



pyridin-6-yl]phenylmethanone


3
(2,3-Dichlorophenyl)-[3-(5,6-dimethyl-
12
mg
3.05
440.13



1H-benzoimidazol-2-yl)-2,4,5,7-



tetrahydropyrazolo[3,4-c]pyridin-6-yl]-



methanone


4
1-[3-(5,6-Dimethyl-1H-benzoimidazol-
7.9
mg
2.99
386.23



2-yl)-2,4,5,7-tetrahydropyrazolo-



[3,4-c]pyridin-6-yl]-2-phenylethanone


5
1-[3-(5,6-Dimethyl-1H-benzoimidazol-
2.7
mg
2.4
310.19



2-yl)-2,4,5,7-tetrahydropyrazolo-



[3,4-c]pyridin-6-yl]ethanone


6
Cyclopropyl-[3-(5,6-dimethyl-1H-
3.4
mg
2.57
336.21



benzoimidazol-2-yl)-2,4,5,7-



tetrahydropyrazolo[3,4-c]pyridin-6-yl]-



methanone


7
(2-Chlorophenyl)-[3-(5,6-dimethyl-1H-
11.2
mg
2.97
406.18



benzoimidazol-2-yl)-2,4,5,7-



tetrahydropyrazolo[3,4-c]pyridin-6-yl]-



methanone


8
(3-Chlorophenyl)-[3-(5,6-dimethyl-1H-
12.1
mg
3.31
406.16



benzoimidazol-2-yl)-2,4,5,7-



tetrahydropyrazolo[3,4-c]pyridin-6-yl]-



methanone


9
(4-Chlorophenyl)-[3-(5,6-dimethyl-1H-
11.5
mg
3.51
406.17



benzoimidazol-2-yl)-2,4,5,7-



tetrahydropyrazolo[3,4-c]pyridin-6-yl]-



methanone


10
1-[3-(5,6-Dimethyl-1H-benzoimidazol-
4.8
mg
2.72
352.24



2-yl)-2,4,5,7-tetrahydropyrazolo[3,4-c]-



pyridin-6-yl]-3-methylbutan-1-one


11
1-[3-(5,6-Dimethyl-1H-benzoimidazol-
11.9
mg
2.95
400.24



2-yl)-2,4,5,7-tetrahydropyrazolo[3,4-c]-



pyridin-6-yl]-3-phenylpropan-1-one


12
1-[3-(5,6-Dimethyl-1H-benzoimidazol-
10.1
mg
2.72
336.22



2-yl)-2,4,5,7-tetrahydropyrazolo-



[3,4-c]pyridin-6-yl]but-3-en-1-one


13
1-[3-(5,6-Dimethyl-1H-benzoimidazol-
7
mg
2.66
338.23



2-yl)-2,4,5,7-tetrahydropyrazolo-



[3,4-c]pyridin-6-yl]butan-1-one


14
[3-(5,6-Dimethyl-1H-benzoimidazol-2-
9.5
mg
2.67
362.19



yl)-2,4,5,7-tetrahydropyrazolo[3,4-c]-



pyridin-6-yl]furan-2-yl-methanone


15
1-[3-(5,6-Dimethyl-1H-benzoimidazol-
9.3
mg
2.8
352.24



2-yl)-2,4,5,7-tetrahydropyrazolo[3,4-c]-



pyridin-6-yl]-2,2-dimethylpropan-1-one


16
2-Dimethylamino-1-[3-(5,6-dimethyl-
4.7
mg
2.55
353.23



1H-benzoimidazol-2-yl)-2,4,5,7-



tetrahydro-pyrazolo[3,4-c]pyridin-6-



yl]ethanone


17
1-[3-(5,6-Dimethyl-1H-benzoimidazol-
5.4
mg
2.78
352.24



2-yl)-2,4,5,7-tetrahydropyrazolo[3,4-c]-



pyridin-6-yl]pentan-1-one


18
[3-(5,6-Dimethyl-1H-benzoimidazol-2-
7.2
mg
2.75
378.17



yl)-2,4,5,7-tetrahydropyrazolo[3,4-c]-



pyridin-6-yl]thiophen-2-yl-methanone


19
[3-(5,6-Dimethyl-1H-benzoimidazol-2-
14.3
mg
2.79
450.19



yl)-2,4,5,7-tetrahydropyrazolo[3,4-c]-



pyridin-6-yl]-(4-methanesulphonyl-



phenyl)methanone









EXAMPLE 8
Preparation of a Sulphonamide Library




The sulphonamide library can be prepared in the following way:
190 mg of 3-(5,6-dimethyl-1H-benzimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine hydrochloride are suspended in 2 ml of dichloromethane in the presence of 150 μl of triethylamine, and the suspension obtained is distributed into 17 test tubes, at a rate of 500 μl per tube. The 17 sulphonyl chlorides (Table 6) are weighed and added to each of the 17 test tubes.







TABLE 6







Sulphonyl chlorides used









Entry
Name
Quantity













1
BENZENESULPHONYL CHLORIDE
9.9
mg


2
ALPHA-TOLUENESULPHONYL
10.7
mg



CHLORIDE


3
2,3-DICHLOROBENZENE-
13.8
mg



SULPHONYL CHLORIDE


4
4-CHLOROBENZENESULPHONYL
11.9
mg



CHLORIDE


5
2,2,2-TRIFLUOROETHANE-
10.2
mg



SULPHONYL CHLORIDE


6
ETHANESULPHONYL CHLORIDE
7.2
mg


7
1-PROPANESULPHONYL CHLORIDE
8
mg


8
1-BUTANESULPHONYL CHLORIDE
8.8
mg


9
2-CHLOROBENZENESULPHONYL
11.9
mg



CHLORIDE


10
3-CHLOROBENZENESULPHONYL
11.9
mg



CHLORIDE


11
[(4-FLUOROPHENYL)METHYL]-
11.7
mg



SULPHONYL CHLORIDE


12
4-METHOXYBENZENESULPHONYL
11.6
mg



CHLORIDE


13
P-TOLUENESULPHONYL CHLORIDE
10.7
mg


14
O-TOLUENESULPHONYL CHLORIDE
10.7
mg


15
3-METHYLBENZENESULPHONYL
10.7
mg



CHLORIDE


16
3-METHOXYBENZENESULPHONYL
11.6
mg



CHLORIDE


17
2-METHOXY-4-METHYLBENZENE-
12.4
mg



SULPHONYL CHLORIDE









The 17 reaction mixtures are shaken by means of orbital shaking at ambient temperature for 20 h.
For each reaction mixture, a 10 μl sample is taken and diluted in 40 μl of DMSO (Gilson Liquid Handler Quad-Z 215). Each sample in solution in DMSO thus obtained is analysed by LC/MS (method A).
The 17 reaction mixtures are then evaporated to dryness and the evaporation residues are each solubilized in 1 ml of DMSO in the presence of a drop of an aqueous 5N hydrochloric acid solution, and the solutions obtained are purified by LC/MS (method B). After purification by LC/MS, the fractions containing the desired compounds are (optionally combined) loaded onto SCX phase (500 mg of CUBCX1-HL phase). The SCX phases are subsequently washed with methanol and then extracted with a solution of 2M ammonia in methanol. The extraction solutions are collected in tared glass tubes, evaporated to dryness (Savant AES 2000 or Genevac HT8 centrifugal evaporator), weighed (Mettler Toledo Automated Workstation LA200) and diluted to 10 mM in DMSO (Gilson Liquid Handler Quad-Z 215). Each solution obtained is analysed by LC/MS (method A).
The following compounds (Table 7) were isolated and characterized by means of their retention time and molecular peak in mass spectrometry (method A).







TABLE 7







Sulphonamide library obtained













Amount
Retention
Molecular




of product
time
ion


Entry
Name
obtained
(minutes)
detected














1
6-Benzenesulphonyl-3-(5,6-dimethyl-
1.4 mg
3.41
408.18



1H-benzoimidazol-2-yl)-4,5,6,7-



tetrahydro-2H-pyrazolo[3,4-c]pyridine


2
3-(5,6-Dimethyl-1H-benzoimidazol-2-yl)-
0.7 mg
3.51
422.2



6-phenylmethanesulphonyl-4,5,6,7-



tetrahydro-2H-pyrazolo[3,4-c]pyridine


3
6-(2,3-Dichlorobenzenesulphonyl)-3-
6.4 mg
3.25
476.1



(5,6-dimethyl-1H-benzoimidazol-2-yl)-



4,5,6,7-tetrahydro-2H-pyrazolo-



[3,4-c]pyridine


4
6-(4-Chlorobenzenesulphonyl)-3-(5,6-
5.9 mg
3.19
442.12



dimethyl-1H-benzoimidazol-2-yl)-



4,5,6,7-tetrahydro-2H-pyrazolo-



[3,4-c]pyridine


5
3-(5,6-Dimethyl-1H-benzoimidazol-2-yl)-
1.7 mg
3.06
414.14



6-(2,2,2-trifluoroethanesulphonyl)-



4,5,6,7-tetrahydro-2H-pyrazolo-



[3,4-c]pyridine


6
3-(5,6-Dimethyl-1H-benzoimidazol-2-yl)-
5.2 mg
2.63
360.17



6-ethanesulphonyl-4,5,6,7-tetrahydro-



2H-pyrazolo[3,4-c]pyridine


7
3-(5,6-Dimethyl-1H-benzoimidazol-2-yl)-
4.3 mg
2.8
374.19



6-(propane-1-sulphonyl)-4,5,6,7-



tetrahydro-2H-pyrazolo[3,4-c]pyridine


8
6-(Butane-1-sulphonyl)-3-(5,6-dimethyl-
5.6 mg
2.94
388.2



1H-benzoimidazol-2-yl)-4,5,6,7-



tetrahydro-2H-pyrazolo[3,4-c]pyridine


9
6-(2-Chlorobenzenesulphonyl)-3-(5,6-
5.6 mg
3.38
442.13



dimethyl-1H-benzoimidazol-2-yl)-



4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]-



pyridine


10
6-(3-Chlorobenzenesulphonyl)-3-(5,6-
6.9 mg
3.71
442.13



dimethyl-1H-benzoimidazol-2-yl)-



4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]-



pyridine


11
3-(5,6-Dimethyl-1H-benzoimidazol-2-yl)-
0.7 mg
3.05
440.18



6-(4-fluorophenylmethanesulphonyl)-



4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]-



pyridine


12
3-(5,6-Dimethyl-1H-benzoimidazol-2-yl)-
7.5 mg
2.99
438.19



6-(4-methoxybenzenesulphonyl)-



4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]-



pyridine


13
3-(5,6-Dimethyl-1H-benzoimidazol-2-yl)-
7.3 mg
3.22
422.2



6-(toluene-4-sulphonyl)-4,5,6,7-



tetrahydro-2H-pyrazolo[3,4-c]pyridine


14
3-(5,6-Dimethyl-1H-benzoimidazol-2-yl)-
4.8 mg
3.16
422.19



6-(toluene-2-sulphonyl)-4,5,6,7-



tetrahydro-2H-pyrazolo[3,4-c]pyridine


15
3-(5,6-Dimethyl-1H-benzoimidazol-2-yl)-
5.1 mg
3.13
422.19



6-(toluene-3-sulphonyl)-4,5,6,7-



tetrahydro-2H-pyrazolo[3,4-c]pyridine


16
3-(5,6-Dimethyl-1H-benzoimidazol-2-yl)-
6.9 mg
3.07
438.18



6-(3-methoxybenzenesulphonyl)-



4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]-



pyridine


17
3-(5,6-Dimethyl-1H-benzoimidazol-2-yl)-
0.8 mg
3.34
452.19



6-(2-methoxy-4-methyl-benzene-



sulphonyl)-4,5,6,7-tetrahydro-2H-



pyrazolo[3,4-c]pyridine









EXAMPLE 9
Preparation of an Amine Library




The amine library can be prepared in the following way:
180 mg of 3-(5,6-dimethyl-1H-benzimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine hydrochloride are suspended in 2.7 ml of methanol and the suspension obtained is distributed into 16 test tubes, at a rate of 150 μl per tube.
The 16 aldehyde (Table 8) are weighed and added to each of the 16 test tubes.







TABLE 8







Aldehydes used









Entry
Name
Quantity













1
ISOBUTYRALDEHYDE
8.1
mg


2
FORMALDEHYDE
3.4
mg


3
BENZALDEHYDE
11.9
mg


4
PHENYLACETALDEHYDE
13.5
mg


5
2,3-DICHLOROBENZALDEHYDE
19.6
mg


6
FURFURAL
10.8
mg


7
4-CHLOROBENZALDEHYDE
15.8
mg


8
2-THIOPHENECARBOXALDEHYDE
12.6
mg


9
NICOTINALDEHYDE
12
mg


10
TRIMETHYLACETALDEHYDE
9.7
mg


11
ACETALDEHYDE
4.9
mg


12
ISOVALERALDEHYDE
9.7
mg


13
PROPIONALDEHYDE
6.5
mg


14
3-PHENYLPROPIONALDEHYDE
15.1
mg


15
BUTYRALDEHYDE
8.1
mg


16
CYCLOPROPANECARBOXALDEHYDE
7.9
mg









A solution of 85 mg of NaBH3CN in 2.7 ml of methanol is then also distributed into the 16 test tubes, at a rate of 150 μl per tube. The 16 reaction mixtures are shaken by means of orbital shaking at ambient temperature for 20 h. 100 μl of methanol are then added to each of the 16 tubes.
For each reaction mixture, a 10 μl sample is taken and diluted in 40 μl of DMSO (Gilson Liquid Handler Quad-Z 215). Each sample in solution in DMSO thus obtained is analysed by LC/MS (method A).
The 16 reaction mixtures are then evaporated to dryness and the evaporation residues are each solubilized in 500 μl of DMSO and filtered through sintered glass, and the residual solutions are then purified by LC/MS (method B). After purification by LC/MS, the fractions containing the desired compounds are (optionally combined) loaded onto SCX phase (500 mg of CUBCX1-HL phase). The SCX phases are subsequently washed with methanol and then extracted with a solution of 2M ammonia in methanol. The extraction solutions are collected in tared glass tubes, evaporated to dryness (Savant AES 2000 or Genevac HT8 centrifugal evaporator), weighed (Mettler Toledo Automated Workstation LA200) and diluted to 10 mM in DMSO (Gilson Liquid Handler Quad-Z 215). Each solution obtained is analysed by LC/MS (method A).
The following compounds (Table 9) were isolated and characterized by means of their retention time and molecular peak in mass spectrometry (method A).







TABLE 9







Amine library obtained













Amount of
Retention
Molecular




product
time
ion


Entry
Name
obtained
(minutes)
detected















1
3-(5,6-Dimethyl-1H-benzoimidazol-2-
5.9
mg
2.62
324.32



yl)-6-isobutyl-4,5,6,7-tetrahydro-2H-



pyrazolo[3,4-c]pyridine


2
3-(5,6-Dimethyl-1H-benzoimidazol-2-
3.5
mg
2.49
282.29



yl)-6-methyl-4,5,6,7-tetrahydro-2H-



pyrazolo[3,4-c]pyridine


3
6-Benzyl-3-(5,6-dimethyl-1H-benzo-
8.2
mg
2.74
358.3



imidazol-2-yl)-4,5,6,7-tetrahydro-2H-



pyrazolo[3,4-c]pyridine


4
3-(5,6-Dimethyl-1H-benzoimidazol-2-
6.4
mg
2.84
372.32



yl)-6-phenethyl-4,5,6,7-tetrahydro-2H-



pyrazolo[3,4-c]pyridine


5
6-(2,3-Dichlorobenzyl)-3-(5,6-dimethyl-
8.6
mg
2.95
426.23



1H-benzoimidazol-2-yl)-4,5,6,7-



tetrahydro-2H-pyrazolo[3,4-c]pyridine


6
3-(5,6-Dimethyl-1H-benzoimidazol-2-
5.9
mg
2.64
348.27



yl)-6-furan-2-ylmethyl-4,5,6,7-



tetrahydro-2H-pyrazolo[3,4-c]pyridine


7
6-(4-Chlorobenzyl)-3-(5,6-dimethyl-1H-
4.7
mg
2.9
392.26



benzoimidazol-2-yl)-4,5,6,7-tetrahydro-



2H-pyrazolo[3,4-c]pyridine


8
3-(5,6-Dimethyl-1H-benzoimidazol-2-
8.4
mg
2.71
364.24



yl)-6-thiophen-2-ylmethyl-4,5,6,7-



tetrahydro-2H-pyrazolo[3,4-c]pyridine


9
3-(5,6-Dimethyl-1H-benzoimidazol-2-
11.7
mg
2.55
359.29



yl)-6-pyridin-3-ylmethyl-4,5,6,7-



tetrahydro-2H-pyrazolo[3,4-c]pyridine


10
3-(5,6-Dimethyl-1H-benzoimidazol-2-
3.7
mg
2.72
338.32



yl)-6-(2,2-dimethylpropyl)-4,5,6,7-



tetrahydro-2H-pyrazolo[3,4-c]pyridine


11
3-(5,6-Dimethyl-1H-benzoimidazol-2-
5
mg
2.55
296.27



yl)-6-ethyl-4,5,6,7-tetrahydro-2H-



pyrazolo[3,4-c]pyridine


12
3-(5,6-Dimethyl-1H-benzoimidazol-2-
5.9
mg
2.76
338.3



yl)-6-(3-methylbutyl)-4,5,6,7-tetrahydro-



2H-pyrazolo[3,4-c]pyridine


13
3-(5,6-Dimethyl-1H-benzoimidazol-2-
6.4
mg
2.62
310.29



yl)-6-propyl-4,5,6,7-tetrahydro-2H-



pyrazolo[3,4-c]pyridine


14
3-(5,6-Dimethyl-1H-benzoimidazol-2-
4.2
mg
2.97
386.31



yl)-6-(3-phenylpropyl)-4,5,6,7-



tetrahydro-2H-pyrazolo[3,4-c]pyridine


15
6-Butyl-3-(5,6-dimethyl-1H-benzo-
4.5
mg
2.68
324.28



imidazol-2-yl)-4,5,6,7-tetrahydro-2H-



pyrazolo[3,4-c]pyridine


16
6-Cyclopropylmethyl-3-(5,6-dimethyl-
3.9
mg
2.62
322.27



1H-benzoimidazol-2-yl)-4,5,6,7-



tetrahydro-2H-pyrazolo[3,4-c]pyridine









EXAMPLE 10
Preparation of a Urea Library




The urea library can be prepared in the following way:
120 mg of 3-(5,6-dimethyl-1H-benzimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine hydrochloride are suspended in 3.6 ml of tetrahydrofuran in the presence of 190 μl of triethylamine, and the suspension obtained is distributed into each of the 9 test tubes, at a rate of 300 μl per tube.
The 9 isocyanates (Table 10) are weighed and added to each of the 9 test tubes.







TABLE 10







Isocyanates used









Entry
Name
Amount





1
PHENYL ISOCYANATE
6.7 mg


2
BENZYL ISOCYANATE
7.5 mg


3
2-CHLOROPHENYL ISOCYANATE
8.6 mg


4
3-CHLOROPHENYL ISOCYANATE
8.6 mg


5
4-CHLOROPHENYL ISOCYANATE
8.6 mg


6
N-BUTYL ISOCYANATE
5.6 mg


7
2-THIENYL ISOCYANATE
  7 mg


8
2-METHOXYPHENYL ISOCYANATE
8.4 mg


9
O-TOLYL ISOCYANATE
7.5 mg









The 9 reaction mixtures are shaken by means of orbital shaking at ambient temperature for 2 hours, and are then evaporated to dryness.
Evaporation residues are each solubilized in 1 ml of DMSO and, for each solution obtained, a 10 μl sample is taken and diluted in 40 μl of DMSO (Gilson Liquid Handler Quad-X 215). Each sample in solution in DMSO thus obtained is analysed by LC/MS (method A).
The residual solutions are purified by LC/MS (method B). After purification by LC/MS, the fractions containing the desired compounds are (optionally combined) either evaporated to dryness (entries 1, 3, 6, 8 and 9) or loaded onto SCX phase (500 mg of CUBCX1-HL phase; entries 2, 4, 5 and 7). The SCX phases are subsequently washed with methanol and then extracted with a solution of 2M ammonia in methanol. The extraction solutions are collected in tared glass tubes, evaporated to dryness (Savant AES 2000 or Genevac HT8 centrifugal evaporator), weighed (Mettler Toledo Automated Workstation LA200) and diluted to 10 mM in DMSO (Gilson Liquid Handler Quad-Z 215). Each solution obtained is analysed by LC/MS (method A).
The following compounds (Table 11) were isolated and characterized by means of their retention time and molecular peak in mass spectrometry (method A).







TABLE 11







Urea library obtained













Amount of
Retention
Molecular




product
time
ion


Entry
Name
obtained
(minutes)
detected















1
3-(5,6-Dimethyl-1H-benzoimidazol-2-
14.6
mg
3.04
387.28



yl)-2,4,5,7-tetrahydropyrazolo[3,4-c]-



pyridine-6-carboxylic acid phenyl-



amide bistrifluoroacetate


2
3-(5,6-Dimethyl-1H-benzoimidazol-2-
1.8
mg
2.78
401.29



yl)-2,4,5,7-tetrahydropyrazolo-



[3,4-c]pyridine-6-carboxylic acid



benzylamide


3
3-(5,6-Dimethyl-1H-benzoimidazol-2-
16
mg
2.92
421.25



yl)-2,4,5,7-tetrahydropyrazolo-



[3,4-c]pyridine-6-carboxylic acid



(2-chlorophenyl)amide bistrifluoro-



acetate


4
3-(5,6-Dimethyl-1H-benzoimidazol-2-
7.9
mg
3.89
421.24



yl)-2,4,5,7-tetrahydropyrazolo-



[3,4-c]pyridine-6-carboxylic acid



(3-chlorophenyl)amide


5
3-(5,6-Dimethyl-1H-benzoimidazol-2-
9.8
mg
3.36
421.25



yl)-2,4,5,7-tetrahydropyrazolo-



[3,4-c]pyridine-6-carboxylic acid



(4-chlorophenyl)amide


6
3-(5,6-Dimethyl-1H-benzoimidazol-2-
2.4
mg
2.8
367.31



yl)-2,4,5,7-tetrahydropyrazolo-



[3,4-c]pyridine-6-carboxylic acid



butylamide bistrifluoroacetate


7
3-(5,6-Dimethyl-1H-benzoimidazol-2-
3.8
mg
2.77
393.24



yl)-2,4,5,7-tetrahydropyrazolo-



[3,4-c]pyridine-6-carboxylic acid



thiophen-2-ylamide


8
3-(5,6-Dimethyl-1H-benzoimidazol-2-
14.4
mg
3.14
417.28



yl)-2,4,5,7-tetrahydropyrazolo-



[3,4-c]pyridine-6-carboxylic acid



(2-methoxyphenyl)amide



bistrifluoroacetate


9
3-(5,6-Dimethyl-1H-benzoimidazol-2-
16.2
mg
2.68
401.29



yl)-2,4,5,7-tetrahydropyrazolo-



[3,4-c]pyridine-6-carboxylic acid



o-tolylamide bistrifluoroacetate









EXAMPLE 11
3-(5,6-Dimethyl-1H-benzimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine




3-(5,6-Dimethyl-1H-benzimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo-[3,4-c]pyridine can be prepared in the following way:
9 ml of water and 2.8 ml of trifluoroacetic acid are added to a solution of 670 mg of tert-butyl 3-(2-amino-4,5-dimethylphenylcarbamoyl)-2,4,5,7-tetrahydropyrazolo[3,4-c]pyridine-6-carboxylate in 9 ml of THF. After stirring for 2 hours at 80° C., the reaction medium is concentrated under reduced pressure. It is then taken up in water and the precipitate formed is recovered by filtration through sintered glass, washed with an aqueous 1N sodium hydroxide solution and dried. The aqueous phase obtained is subsequently extracted with dichloromethane and the organic phase is then dried over magnesium sulphate and concentrated under reduced pressure. The residue obtained and the precipitate are combined and then solubilized in methanol with a few drops of DMF. This solution is then loaded onto MEGA BE-SCX phase. The SCX phase is subsequently washed with methanol and extracted with a solution of 2M ammonia in methanol. The extraction solution obtained is then concentrated under reduced pressure.
46 mg of 3-(5,6-dimethyl-1H-benzimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine are thus obtained in the form of a beige powder, the characteristics of which are as follows:


















EI:
m/z = 267
M+.
base peak




m/z = 238
[M − NHCH2]+




m/z = 209
[M − C3H8N]+.










1H NMR (300 MHz, (CD3)2SO, δ in ppm): 2.31 and 2.32 (2 s: 6H in total); 2.81 (broad t, J=5 Hz: 2H); 2.92 (broad t, J=5 Hz: 2H); 3.83 (broad s: 2H); 7.22 (broad s: 1H); 7.40 (broad s: 1H); 12.28 (unresolved peak: 1H); 12.73 (unresolved peak: 1H).
EXAMPLE 12
3-(5,6-Dimethyl-1H-benzimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine hydrochloride




3-(5,6-Dimethyl-1H-benzimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine can be prepared in the following way:
9 ml of an aqueous 5N hydrochloric acid solution are added to a solution of 1.7 g of tert-butyl 3-(2-amino-4,5-dimethylphenylcarbamoyl)-2,4,5,7-tetrahydropyrazolo[3,4-c]pyridine-6-carboxylate in 40 ml of ethanol. After stirring at 80° C. for 60 hours, the reaction medium is brought back to ambient temperature. The precipitate formed is recovered by filtration through sintered glass and dried. 1.04 g of 3-(5,6-dimethyl-1H-benzimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine hydrochloride are thus obtained in the form of a beige powder, the characteristics of which are as follows:


















EI:
m/z = 267
M+.
base peak




m/z = 238
[M − CH2NH]+




m/z = 209
[M − C3H8N]+.




m/z = 36
[HCl]+










1H NMR (300 MHz, (CD3)2SO with addition of a few drops of CD3COOD, δ in ppm): 2.40 (s: 6H); 3.23 (broad t, J=5.5 Hz: 2H); 3.45 (t, J=5.5 Hz: 2H); 4.45 (s: 2H); 7.54 (s: 2H).
tert-Butyl 3-(2-amino-4,5-dimethylphenylcarbamoyl)-2,4,5,7-tetrahydro-pyrazolo[3,4-c]pyridine-6-carboxylate can be prepared in the following way: 8.5 g of HBTU and also 2.9 g of diisopropylethylamine are added, at ambient temperature, to a solution of 3 g of (tert-butyl 2,4,5,7-tetrahydropyrazolo-[3,4-c]pyridine-6-carboxylate)-3-carboxylic acid in 50 ml of anhydrous DMF. After twenty minutes' stirring at ambient temperature, 3.06 g of 4,5-diamino-o-xylene are added. After stirring at ambient temperature for 60 hours, the reaction medium is diluted in 3 l of an aqueous NaHCO3 solution at pH greater than 7, containing 20 g of NaCl. The aqueous phase is extracted three times with 1 l of ethyl acetate, and the combined organic phases are then dried over magnesium sulphate and concentrated under reduced pressure. The crude residue obtained is taken up in 150 ml of dichloromethane and the insoluble material is removed by filtration through sintered glass. The filtrate is then concentrated under reduced pressure and purified by chromatography on silica (20–45 μm Amicon) with a gradient of from 50 to 100% of ethyl acetate in cyclohexane. The fractions containing the desired product are combined and concentrated under reduced pressure. 4.37 g of tert-butyl 3-(2-aminophenylcarbamoyl)-2,4,5,7-tetrahydro-pyrazolo[3,4-c]pyridine-6-carboxylate are thus obtained in the form of a beige powder, the characteristics of which are as follows:


















EI:
m/z = 385
M+.
base peak




m/z = 329
[M − C4H8]+.




m/z = 312
[M − C4H9O]+




m/z = 57
[C4H9]+










1H NMR (300 MHz, (CD3)2SO, δ in ppm): 1.46 (s: 9H); 2.10 and 2.12 (2 s: 3each); 2.77 (mt: 2H); 3.58 (t, J=5.5 Hz: 2H); 4.53 (s: 2H); 4.57 (unresolved peak: 2H); 6.60 (s: 1H); 7.14 (broad s: 1H); 9.10 (unresolved peak: 1H); 13.08 (unresolved peak: 1H).
EXAMPLE 13
3-(1H-Benzimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo-[3,4-c]pyridine Hydrochloride




3-(1H-Benzimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine hydrochloride can be prepared in the following way:
2.2 ml of an aqueous 5N hydrochloric acid solution are added to a solution of 200 mg of tert-butyl 3-(2-aminophenylcarbamoyl)-2,4,5,7-tetrahydro-pyrazolo[3,4-c]pyridine-6-carboxylate in 1 ml of ethanol. After stirring for 20 hours at 80° C., the reaction medium is brought back to ambient temperature. The insoluble material is removed by filtration through sintered glass and the filtrate is concentrated under reduced pressure. 84 mg of 3-(1H-benzimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine hydrochloride are thus obtained in the form of an orange-coloured powder, the characteristics of which are as follows:
LC/MS (method A): molecular ion detected: 240.26; retention time=1.68 minutes
tert-Butyl 3-(2-aminophenylcarbamoyl)-2,4,5,7-tetrahydro-pyrazolo[3,4-c]-pyridine-6-carboxylate can be prepared in the following way:
425 mg of HBTU and also 145 mg of diisopropylethylamine are added, at ambient temperature, to a solution of 150 mg of (tert-butyl 2,4,5,7-tetrahydro-pyrazolo[3,4-c]pyridine-6-carboxylate)-3-carboxylic acid in 1 ml of anhydrous DMF. After stirring for twenty minutes at ambient temperature, 121 mg of orthophenylenediamine are added.
After stirring at ambient temperature for 20 hours, the reaction medium is diluted in 100 ml of water and 50 ml of ethyl acetate. The aqueous phase is extracted three times with 50 ml of ethyl acetate and the combined organic phases are then dried over magnesium sulphate and concentrated under reduced pressure. The crude residue obtained is purified by HPLC (reverse phase C18 Lichroprep 12 μm) with a linear gradient of from 5 to 95% of acetonitrile comprising 0.07% (v/v) of trifluoroacetic acid in water comprising 0.07% (v/v) of trifluoroacetic acid, at a flow rate of 10 ml/min. The fractions containing the desired product are combined and loaded onto MEGA BE-SCX phase. The SCX phase is subsequently washed with methanol and extracted with a solution of 2M ammonia in methanol. The extraction solution obtained is then concentrated under reduced pressure. 200 mg of tert-butyl 3-(2-aminophenylcarbamoyl)-2,4,5,7-tetrahydropyrazolo[3,4-c]pyridine-6-carboxylate are thus obtained in the form of a beige powder, the characteristics of which are as follows:
LC/MS (method A): molecular ion detected: 358.34; retention time=3.19 minutes.
EXAMPLE 14
Preparation of a Sulphonamide Library




The sulphonamide library can be prepared in the following way:
40 mg of 6-[3-(5,6-dimethyl-1H-benzimidazol-2-yl)-2,4,5,7-tetrahydro-pyrazolo[3,4-c]pyridin-6-yl]pyridin-3-ylamine are suspended in 2 ml of dichloromethane and the solution obtained is distributed into 4 test tubes, at a rate of 500 μl per tube.
The 4 sulphonyl chlorides (Table 12) are weighed and added to each of the 4 test tubes, followed by 15.6 μl of triethylamine.







TABLE 12







Sulphonyl chlorides used









Entry
Name
Amount





1
THIOPHENE-2-SULPHONYL CHLORIDE
5.6 mg


2
4-METHOXYBENZENESULPHONYL CHLORIDE
6.3 mg


3
2-CHLOROBENZENESULPHONYL CHLORIDE
6.4 mg


4
2-METHOXY-4-METHYLBENZENESULPHONYL
6.8 mg



CHLORIDE









The 4 reaction mixtures are shaken by means of orbital shaking at 40° C. for 15 h.
For each reaction mixture, a 5 μl sample is taken and diluted in 100 μl of DMSO (Gilson Liquid Handler Quad-Z 215). Each sample in solution in DMSO thus obtained is analysed by LC/MS (method A).
The 4 reaction mixtures are then evaporated to dryness and the evaporation residues are each solubilized in 500 μl of DMSO and the solutions obtained are purified by LC/MS (method B). After purification by LC/MS, the fractions containing the desired compounds are (optionally combined) loaded onto SCX phase (500 mg of CUBCX1-HL phase). The SCX phases are subsequently washed with methanol and then extracted with a solution of 2M ammonia in methanol. The extraction solutions are collected in tared glass tubes, evaporated to dryness (Savant AES 2000 or Genevac HT8 centrifugal evaporator), weighed (Mettler Toledo Automated Workstation LA200) and diluted to 10 mM in DMSO (Gilson Liquid Handler Quad-Z 215). Each solution obtained is analysed by LC/MS (method A).
The following compounds (Table 13) were isolated and characterized by means of their retention time and molecular peak in mass spectrometry (method A)







TABLE 13







Sulphonamide library obtained













Amount
Retention
Molecular




of product
time
ion


Entry
Name
obtained
(minutes)
detected





1
N-{6-[3-(5,6-Dimethyl-1H-
2.9 mg
3.07
506.21



benzimidazol-2-yl)-2,4,5,7-



tetrahydropyrazolo[3,4-c]pyridin-



6-yl]pyridin-3-yl}thiophene-2-



sulphonamide


2
N-{6-[3-(5,6-Dimethyl-1H-
3.0 mg
3.20
530.25



benzimidazol-2-yl)-2,4,5,7-



tetrahydropyrazolo[3,4-c]pyridin-



6-yl]pyridin-3-yl}-4-methoxy-



benzenesulphonamide


3
2-Chloro-N-{6-[3-(5,6-dimethyl-
3.0 mg
3.38
534.21



1H-benzimidazol-2-yl)-2,4,5,7-



tetrahydropyrazolo[3,4-c]pyridin-



6-yl]pyridin-3-yl}benzene-



sulphonamide


4
N-{6-[3-(5,6-Dimethyl-1H-
3.8 mg
3.38
544.26



benzimidazol-2-yl)-2,4,5,7-



tetrahydropyrazolo[3,4-c]pyridin-



6-yl]pyridin-3-yl}-2-methoxy-4-



methylbenzenesulphonamide









EXAMPLE 15
6-[3-(5,6-Dimethyl-1H-benzimidazol-2-yl)-2,4,5,7-tetrahydro-pyrazolo[3,4-c]pyridin-6-yl]pyridin-3-ylamine




6-[3-(5,6-Dimethyl-1H-benzimidazol-2-yl)-2,4,5,7-tetrahydropyrazolo-[3,4-c]pyridin-6-yl]pyridin-3-ylamine can be prepared in the following way:
55 mg of Pd/CaCO3 10% are added to a solution of 545 mg of 3-(5,6-dimethyl-1H-benzimidazol-2-yl)-6-(5-nitropyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine in 60 ml of ethanol. After stirring for 15 hours at 35° C. under 3 bar of hydrogen, the reaction medium is brought back to ambient temperature, filtered through celite and then concentrated under reduced pressure. 300 mg of 6-[3-(5,6-dimethyl-1H-benzimidazol-2-yl)-2,4,5,7-tetrahydropyrazolo [3,4-c]pyridin-6-yl]-pyridin-3-ylamine are thus obtained in the form of a brown powder, the characteristics of which are as follows:


















EI
m/z = 359
M+
base peak




m/z = 266
(M—C5H5N2)+










EXAMPLE 16
3-(5,6-Dimethyl-1H-benzimidazol-2-yl)-6-(5-nitropyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine




3-(5,6-Dimethyl-1H-benzimidazol-2-yl)-6-(5-nitropyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine can be prepared in the following way:
287 mg of 2-chloro-5-nitropyridine and 500 mg of potassium carbonate are added to a solution of 500 mg of 3-(5,6-dimethyl-1H-benzimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine hydrochloride in 5 ml of dimethylformamide. After stirring for 20 hours at ambient temperature, the reaction medium is added to 50 ml of water. The precipitate formed is recovered by filtration through sintered glass, washed with 3 times 15 ml of water and then dried under reduced pressure. 548 mg of 3-(5,6-dimethyl-1H-benzimidazol-2-yl)-6-(5-nitropyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo-[3,4-c]pyridine are thus obtained in the form of a yellow powder, the characteristics of which are as follows:


















EI:
m/z = 389
M+.
base peak




m/z = 266
(M − C5H3N2O2)+










EXAMPLE 17
6-{5-[3-(2-Fluoro-5-trifluoromethylphenyl)ureido]pyridin-2-yl}-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine-3-carboxamide




6-{5-[3-(2-Fluoro-5-trifluoromethylphenyl)ureido]pyridin-2-yl}-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine-3-carboxamide can be prepared in the following way from ethyl 6-(5-tert-butoxycarbonylaminopyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine-3-carboxylate:
The ethyl ester of ethyl 6-(5-tert-butoxycarbonylaminopyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine-3-carboxylate is converted to carboxamide by amidation using a solution of aqueous ammonia, and results in the obtaining of 6-(5-tert-butoxycarbonylaminopyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine-3-carboxamide.
EI: m/z=358
The amine group of the 6-(5-tert-butoxycarbonylaminopyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine-3-carboxamide is deprotected in acid medium (trifluoroacetic acid in dichloromethane) and results in the obtaining of 6-(5-aminopyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine-3-carboxamide.
EI: m/z=258
The urea function is introduced onto the 6-(5-aminopyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine-3-carboxamide according to the method described in Example 1, using 2-fluoro-5-(trifluoromethyl)phenyl isocyanate, and results in the obtaining of 6-{5-[3-(2-fluoro-5-trifluoromethylphenyl)-ureido]pyridin-2-yl}4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine-3-carb-oxamide.
EI: m/z=463
EXAMPLE 18
Ethyl 6-(5-tert-butoxycarbonylaminopyridin-2-yl)-4,5,6,7-tetra-hydro-2H-pyrazolo[3,4-c]pyridine-3-carboxylate




Ethyl 6-(5-tert-butoxycarbonylaminopyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine-3-carboxylate can be prepared in the following way:
5 mg of Pd/C 10% and 38 mg of di-tert-butyl dicarbonate are added to a solution of 50 mg of ethyl 6-(5-nitropyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine-3-carboxylate in 6 ml of methanol. After stirring for 12 hours at ambient temperature under 3 bar of hydrogen, the reaction medium is filtered through celite and then concentrated under reduced pressure. The reaction crude obtained is purified by flash chromatography (SiO2, CH2Cl2/MeOH gradient 75/25 to 25/75). 20 mg of ethyl 6-(5-tert-butoxycarbonylaminopyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine-3-carboxylate are thus obtained in the form of a white powder, the characteristics of which are as follows:


















EI:
m/z = 387
M+.





m/z = 331
(M − C4H8)+.
base peak




m/z = 286
(m/z = 331 − CO2H)+




m/z = 194
C9H12N3O2+




m/z = 57
C4H9+










EXAMPLE 19
Ethyl 6-(5-nitropyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo [3,4-c]pyridine-3-carboxylate




Ethyl 6-(5-nitropyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine-3-carboxylate can be prepared in the following way:
522 mg of 2-chloro-5-nitropyridine are added to a solution of 1 g of 3-(5,6-dimethyl-1H-benzimidazol-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine trifluoroacetate in 10 ml of pyridine. After stirring for 20 hours at ambient temperature, the reaction medium is concentrated under reduced pressure. The precipitate formed is recovered by filtration through sintered glass, washed with 3 times 15 ml of water, and dried under reduced pressure. The reaction crude obtained is purified by flash chromatography (SiO2, cyclohexane/EtOAc gradient 75/25 to 25/75). 450 mg of ethyl 6-(5-nitro-pyridin-2-yl)-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine-3-carboxylate are thus obtained in the form of a yellow powder, the characteristics of which are as follows:


















EI:
m/z = 317
M+.
base peak




m/z = 271
(M − NO2)+.




m/z = 194
(M − C5H3N2O2)+




m/z = 148
(m/z = 194 − C2H6O)+










EXAMPLE 20
Ethyl 4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine-3-carboxylate trifluoroacetate




Ethyl 4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine-3-carboxylate trifluoro-acetate can be prepared in the following way:
50 ml of water followed by 12 ml of trifluoroacetic acid are added to a solution of 3 g of 6-tert-butyl-3-ethyl 2,4,5,7-tetrahydropyrazolo[3,4-c]pyridyl-3,6-dicarboxylate in 50 ml of tetrahydrofuran. After stirring for 2 hours at reflux, the reaction medium is brought back to ambient temperature and a saturated aqueous Na2CO3 solution is added until a basic pH is obtained. The aqueous phase obtained is extracted 3 times with ethyl acetate. The combined organic phases are dried over magnesium sulphate and concentrated under reduced pressure. 1.49 g of ethyl 4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine-3-carboxylate trifluoroacetate are thus obtained, the characteristics of which are as follows:


















EI:
m/z = 195
M+.





m/z = 166
(M − CH3N)+.
base peak




m/z = 138
(M − C3H7N)+.




m/z = 120
(m/z = 166 − C2H6O)+.




m/z = 92
(m/z = 120 − CO)+.










Measurements of the Inhibitory Potential of the Products with Respect to the Activity of the Tie2 and KDR Kinases:

The inhibitory activity of the products with respect to the Tie2 and KDR kinases is tested according to the experimental protocols described below.
1. Tie2
The coding sequence of human Tie2 corresponding to amino acids 776–1124 of the intracellular domain was generated by PCR using the cDNA isolated from human placenta as model. This sequence was introduced into a baculovirus expression vector pFastBacGT in the form of a GST fusion protein.
The inhibitory effect of the molecules is determined in an assay for phosphorylation of PLC by Tie2 in the presence of GST-Tie2 purified to approximately 80% homogeneity. The substrate is made up of the SH2-SH3 fragments of PLC, the latter being expressed in the form of a GST fusion protein.
The kinase activity of Tie2 is measured in a 20 mM MOPS buffer, pH 7.2, containing 10 mM MgCl2, 10 mM MnCl2, 1 mM DTT and 10 mM of glycerophosphate. A reaction mixture made up of 70 μl of kinase buffer containing 100 ng of GST-Tie2 enzyme per well is placed in a flashplate 96-well plate kept on ice. 10 μl of the molecule to be tested, diluted in DMSO, at a concentration of at most 10% are then added. For a given concentration, each measurement is carried out in quadruplicate. The reaction is initiated by adding 20 μl of solution containing 2 μg of GST-PLC, 2 μM of cold ATP and 1 μCi of 33P[ATP]. After incubation for 1 hour at 37° C., the reaction is stopped by adding 1 volume (100 μl) of 200 mM EDTA. After removal of the incubation buffer, the wells are washed three times with 300 μl of PBS. The radioactivity is measured on a Wallac MicroBeta 1450.
The inhibition of the Tie2 activity is calculated and expressed as percentage inhibition with respect to the control activity determined in the absence of compound.
2. KDR
The inhibitory effect of the compounds is determined in an assay for phosphorylation of substrate by the KDR enzyme in vitro using a scintillation technique (96-well plate, NEN).
The cytoplasmic domain of the human KDR enzyme was cloned in GST fusion form into the baculovirus expression vector pFastBac. The protein was expressed in SF21 cells and purified to approximately 60% homogeneity.
The KDR kinase activity is measured in 20 mM MOPS, 10 mM MgCl2, 10 mM MnCl2, 1 mM DTT, 2.5 mM EGTA, 10 mM b-glycerophosphate, pH=7.2, in the presence of 10 mM MgCl2, 100 μM Na3VO4 and 1 mM NaF. 10 μl of the compound are added to 70 μl of kinase buffer containing 100 ng of KDR enzyme at 4° C. The reaction is initiated by adding 20 μl of solution containing 2 μg of substrate (SH2-SH3 fragment of PLCγ expressed in the form of an GST fusion protein), 2 μCi γ33P[ATP] and 2 μM cold ATP. After incubation for 1 hour at 37° C., the reaction is stopped by adding 1 volume (100 μl) of 200 mM EDTA. The incubation buffer is removed and the wells are washed three times with 300 μl of PBS. The radioactivity is measured in each well using a Top Count NXT (Packard) radioactivity counter.
The background noise is determined by measuring the radioactivity in four different wells containing the radioactive ATP and the substrate alone.
A total activity control is measured in four different wells containing all the reagents (γ33P-[ATP], KDR and PLC-γ substrate) but in the absence of compound.
The inhibition of the KDR activity with the compound of the invention is expressed as percentage inhibition of the control activity determined in the absence of compound.
The compound SU5614 (Calbiochem) (1 μM) is included in each plate as inhibition control.
Results:
















Tie2
KDR



% Inhib at 10 μM (FRX)
% Inhib at 10 μM











Chemistry
Assay 1
Assay 2
Assay 1
Assay 2





P-31378-112-3
78.7
79.1
59.0
56.5








 


P-31378-112-6
92.7
93.8
71.4
68.0








 


P-31378-112-2
92.6
92.6
98.1
97.4








 


P-31378-112-8H
87.8
93.3
86.8
89.0








 


P-31378-112-4
83.1
87.2
56.2
50.1








 


P-31378-112-9
82.6
85.9
53.2
45.1








 


P-31378-112-14
69.7
73.8
19.5
13.0








 


P-31378-112-1
83.1
88.1
96.5
96.7








 


P-31378-112-16
88.7
88.3
90.2
89.1








 


P-31378-112-5
87.1
86.4
94.7
95.4








 


P-31378-112-10
90.6
87.9
42.3
28.7








 


P-31378-112-7
92.7
92.0
68.6
64.3








 


P-31378-112-15
80.1
83.8
75.5
77.4





















KDR % Inhib.
Tie2 % Inhib.



Chemistry
10 μM
10 μM











77.20



Chemistry 2



 







78.10



Chemistry 3



 







25.40



Chemistry 4



 







88.80



Chemistry 5



 







2.10
41.2



Chemistry 6



 







12.15
48.6



Chemistry 7



 







85.45
64.3



Chemistry 8



 







15.45
56.7



Chemistry 9



 







79.55
83.9



Chemistry 10



 







70.35
66.1



Chemistry 11



 







69.05
60.3



Chemistry 12



 







84.50
41.5



Chemistry 13



 







14.85
50.5



Chemistry 14



 







39.30
69.4



Chemistry 15



 







−5.75
36.4



Chemistry 16



 







−2.90
50.7



Chemistry 17



 







−3.70
55.0



Chemistry 18



 







17.90
68.1



Chemistry 19



 







1.65
47.6



Chemistry 20



 







1.85
33.0



Chemistry 21



 







3.65
24.9



Chemistry 22



 







48
92.5



Chemistry 23



 







70.75
91.5



Chemistry 24



 







31.85
89.1



Chemistry 25



 







27.75
89.1



Chemistry 26



 







22.95
65.4



Chemistry 27



 







16.50
78.5



Chemistry 28



 







13.35
47.5



Chemistry 29



 







15.30
44.2



Chemistry 30



 







45.70
93.2



Chemistry 31



 







46.45
91.5



Chemistry 32



 







57.70
95.9



Chemistry 33



 







18.15
84.7



Chemistry 34



 







27.40
88.8



Chemistry 35



 







49.40
90.8



Chemistry 36



 







41.20
88.3



Chemistry 37



 







20.85
84.6



Chemistry 38



 







30.90
97.9



Chemistry 39



 







14.40
83.4



Chemistry 40



 







72.50
86.3



Chemistry 41



 







52.65
97.4



Chemistry 42



 







10.90
84.4



Chemistry 43



 







41.10
90.8



Chemistry 44



 







33.40
94.1



Chemistry 45



 







73.05
96.7



Chemistry 46



 







78.55
87.7



Chemistry 47



 







41.95
83.8



Chemistry 48



 







12.20
84.8



Chemistry 49



 







74.75
82.4



Chemistry 50



 







38.70
72.9



Chemistry 51



 







29.35
89.3



Chemistry 52



 







74.55
92.7



Chemistry 53



 







11.25
81.3



Chemistry 54



 







15.95
65.4



Chemistry 55



 







46.65
92.5



Chemistry 56



 







75.15
94.6



Chemistry 57



 







27.90
82.3



Chemistry 58



 







17.6
33.2



Chemistry 59



 







33.55
64.85



Chemistry 60



 







12.7
3.75



Chemistry 61



 







12.85
21



Chemistry 62



 







−0.3
23.75



Chemistry 63



 







18
38.5



Chemistry 64



 







63.3
85.9



Chemistry 65



 







8.1
18.05



Chemistry 67



 







19.5
19.1



Chemistry 68



 







18.55
59.3



Chemistry 69



 







92.1
65.5



Chemistry 70



 








98



 








100



 








99



 








100

## Claims

1. A compound of formula (I), including its tautomers:




wherein:
L is a bond;
X is chosen from a bond, CH2, CO, SO2, CONH and COO;
R1 is OH or H, or is chosen from alkyl, cycloalkyl, heterocycle, aryl, heteroaryl, chosen from benzimidazolyl, indolyl or pyrrolyl, all of which is optionally substituted, and, when X is a bond, then R1 may also be halogen;
R2 is H or is chosen from alkyl, alkylene, cycloalkyl, heterocycle, aryl, heteroaryl, chosen from pyridyl, furanyl or thienyl, all of which is optionally substituted;

and in that the optional substituents are chosen independently from R3, O—R3, halogen, NO2, SO2—R3, CO—R3, SO2NH—R3, CONH—R3, N—(R3)2, NHCO—R3, NHSO2—R3, NHCONH—R3, NHSO2NH—R3, OCO—R3, COO—R3, OSO2—R3, SO2O—R3, OCONH—R3 and OSO2NH—R3, wherein R3 is H or is chosen independently from alkyl, cycloalkyl, alkenyl, aryl, heteroaryl chosen from benzimidazolyl, indolyl, pyridyl, furanyl, thienyl or pyrrolyl, heterocycle, all of which is optionally substituted with halogen, aryl, heteroaryl chosen from benzimidazolyl, indolyl, pyridyl, furanyl, thienyl or pyrrolyl, R4, OR4 or N(R4)2,
wherein each R4 is chosen independently from H, C1–C4 alkyl and halogenated C1–C4 alkyl, or
a racemate, a stereoisomer, an enantiomer, or a mixture in any combination thereof, or a pharmaceutically acceptable salt thereof;
with the proviso that:
when R1 is OH and X is a bond or COO, then R2 is not hydrogen or methyl;
when R1 is methyl, cyclopropyl or 4-fluorophenyl, and X is a bond, then R2 is not hydrogen.

2. The compound as set forth in claim 1, which is of the formula (II):




and its tautomers, wherein:
X is chosen from a bond, CH2, CO, SO2, CONH and COO;
R1 is chosen from alkyl, cycloalkyl, heterocycle, aryl, heteroaryl chosen from benzimidazolyl, indolyl, or pyrrolyl, all of which is optionally substituted;
R2 is H or is chosen from alkyl, alkylene, cycloalkyl, heterocycle, aryl, heteroaryl chosen from pyridyl, furanyl or thienyl, all of which is optionally substituted;

and in that the optional substituents are chosen independently from R3, O—R3, halogen, NO2, SO2—R3, CO—R3, SO2NH—R3, CONH—R3, N—(R3)2, NHCO—R3, NHSO2—R3, NHCONH—R3, NHSO2NH—R3, OCO—R3, COO—R3, OSO2—R3, SO2O—R3, OCONH—R3 and OSO2NH—R3, wherein R3 is H or is chosen independently from alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, chosen from benzimidazolyl, indolyl, pyridyl, furanyl, thienyl or pyrrolyl, heterocycle, all of which is optionally substituted with halogen, aryl, heteroaryl chosen from benzimidazolyl, indolyl, pyridyl, furanyl, thienyl or pyrrolyl, R4, OR4 or N(R4)2, in which each R4 is chosen independently from H and C1–C4 alkyl, or
a racemate, a stereoisomer, an enantiomer, or a mixture in any combination thereof, or a pharmaceutically acceptable salt thereof.

3. The compound as set forth in claim 1, wherein R1 is heteroaryl chosen from benzimidazolyl, indolyl or pyrrolyl, which is optionally substituted.

4. The compound as set forth in claim 2, wherein R1 is heteroaryl chosen from benzimidazolyl, indolyl or pyrrolyl, which is optionally substituted.

5. The compound as set forth in claim 3, wherein R1 is chosen from benzimidazolyl, indolyl, pyrrolyl, optionally substituted with halogen, R4 or O—R4, wherein R4 is chosen independently from H and C1–C4 alkyl.

6. The compound as set forth in claim 5, wherein R1 is chosen from benzimidazol-2-yl, indol-2-yl, pyrrol-2-yl, optionally substituted with halogen, R4 or O—R4, wherein R4 is chosen independently from H and C1–C4 alkyl.

7. The compound as set forth in claim 1, wherein R2 is chosen from phenyl, pyridyl, thienyl, C1–C4 alkyl, and C3–C7 cycloalkyl, all of which is optionally substituted.

8. The compound as set forth in claim 1, wherein X is chosen from CO and SO2.

9. The compound as set forth in claim 1, wherein R1 is H.

10. The compound as set forth in claim 1, wherein R1 is substituted aryl.

11. The compound as set forth in claim 1, wherein X is a bond, and R2 is chosen from substituted aryl and substituted heteroaryl wherein heteroaryl is chosen from pyridyl or thienyl.

12. The compound as set forth in claim 1, which is:

13. A pharmaceutical composition comprising a compound of formula (I), including its tautomers, in combination with one or more pharmaceutically acceptable excipient, diluent or a carrier:




wherein:
L is a bond;
X is chosen from a bond, CH2, CO, SO2, CONH and COO;
R1 is OH or H, or is chosen from alkyl, cycloalkyl, heterocycle, aryl, heteroaryl, chosen from benzimidazolyl, indolyl or pyrrolyl, all of which is optionally substituted, and, when X is a bond, then R1 may also be halogen;
R2 is H or is chosen from alkyl, alkylene, cycloalkyl, heterocycle, aryl, heteroaryl chosen from pyridyl, furanyl or thienyl, all of which is optionally substituted;

and in that the optional substituents are chosen independently from R3, O—R3, halogen, NO2, SO2—R3, CO—R3, SO2NH—R3, CONH—R3, N—(R3)2, NHCO—R3, NHSO2—R3, NHCONH—R3, NHSO2NH—R3, OCO—R3, COO—R3, OSO2—R3, SO2O—R3, OCONH—R3 and OSO2NH—R3, wherein R3 is H or is chosen independently from alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, chosen from benzimidazolyl, indolyl, pyridyl, furanyl, thienyl or pyrrolyl, heterocycle, all of which is optionally substituted with halogen, aryl, heteroaryl chosen from benzimidazolyl, indolyl, pyridyl, furanyl, thienyl or pyrrolyl, R4, OR4 or N(R4)2,
wherein each R4 is chosen independently from H, C1–C4 alkyl and halogenated C1–C4 alkyl, or
a racemate, a stereoisomer, an enantiomer, or a mixture in any combination thereof, or a pharmaceutically acceptable salt thereof;
with the proviso that:
when R1 is OH and X is a bond or COO, then R2 is not hydrogen or methyl;
when R1 is methyl, cyclopropyl or 4-fluorophenyl, and X is a bond, then R2 is not hydrogen.