EP1322612A1 - Substituted amino-aza-cycloalkanes useful against malaria - Google Patents

Abstract

The invention relates to novel compounds which are substituted amino-aza-cycloalkane derivatives of the general formula I. The invention also concerns related aspects including processes for the preparation of the compounds, pharmaceutical compositions containing one or more compounds of general formula I and especially their use as inhibitors of the plasmodium falciparum protease plasmepsin II or related aspartic proteases.

Description

SUBSTITUTED

AMINO-AZA-CYCLOALKANES

USEFUL AGAINST MALARIA

The invention relates to novel compounds which are substituted amino-aza- cycloalkane derivatives of the general formula I. The invention also concerns related aspects including processes for the preparation of the compounds, pharmaceutical compositions containing one or more compounds of general formula I and especially their use as inhibitors of the plasmodium falciparum protease plasmepsin II or related aspartic proteases.

Background of the invention:

Malaria is one of the most serious and complex health problems affecting humanity in the 21^st century. The disease affects about 300 million people worldwide, killing 1 to 1.5 million people every year. Malaria is an infectious disease caused by four species of the protozoan parasite Plasmodium, P. falciparum being the most severe of the four. All attempts to develop vaccines against P. falciparum have failed so far. Therefore, therapies and preventive measures against malaria are confined to drugs. However, resistance to many of the currently available antimalarial drugs is spreading rapidly and new drugs are needed.

P. Falciparum enters the human body by way of bites of the female anophelino mosquito. The plasmodium parasite initially populates the liver, and during later stages of the infectious cycle reproduces in red blood cells. During this stage, the parasite degrades hemoglobin and uses the degradation products as nutrients for growth [1]. Hemoglobin degradation is mediated by serine proteases, and aspartic proteases. Aspartic proteases have been shown to be indispensable to parasite growth.. A non-selective inhibitor of aspartic proteases, Pepstatin, inhibits the growth of P. falciparum in red blood cells in vitro. The same results have been obtained with analogs of pepstatin [2], [3]. These results show that inhibition of parasite aspartic proteases interferes with the life cycle of P. falciparum. Consequently, aspartic proteases are targets for antimalarial drug development. The present invention relates to the identification of novel low molecular weight, non-peptidic inhibitors of the plasmodium falciparum protease plasmepsin II or other related aspartic proteases to treat and/or prevent malaria.

The compounds of general formula I were tested against plasmepsin II, HIV- protease, human cathepsin D, human cathepsin E and human renin in order to determine their biological activity and their selectivity profile.

In vitro Assays:

The fluorescence resonance energy transfer (FRET) assay for HIV, plasmepsin II, human cathepsin D and human cathepsin E.

The assay conditions were selected according to reports in the literature [4 - 7]. The FRET assay was performed in white polysorp plates (Fluoronunc, cat n° 437842 A). The assay buffer consisted of 50 mM Na acetate pH 5, 12,5% glycerol, 0.1 % BSA + 392 mM NaCl (for HIV-protease). The incubates per well were composed of: - 160 μl buffer - 10 μl inhibitor (in DMSO)

- 10 μl of the corresponding substrate in DMSO (see table A) to a final concentration of 1 μM

- 20 μl of enzyme to a final amount of x ng per assay tube (x = 10 ng/assay tube plasmepsin II, x = 100 ng/assay tube HIV-protease, x = 10 ng/assay tube human cathepsin E and x = 20 ng/assay tube human cathepsin D)

The reactions were initiated by addition of the enzyme. The assay was incubated at 37°C for 30 min (for human cathepsin E), 40 min (for plasmepsin II and HIV- protease) or 120 min (for human cathepsin D). The reactions were stopped by adding 10% (v/v) of a 1 M solution of Tris-base. Product-accumulation was monitored by measuring the fluorescence at 460 nm. Auto-fluorescence of all the test substances is determined in assay buffer in the absence of substrate and enzyme and this value was subtracted from the final signal.

Table A: Summary of the conditions used for the aspartyl proteases fluorescent assays, (at = assay tube)

Enzymatic in vitro assay for renin:

The enzymatic in vitro assay was performed in polypropylene plates (Nunc, Cat No 4-42587A). The assay buffer consisted of 100 mM sodium phosphate, pH 7.4, including 0.1% BSA. The incubates were composed of 190 μL per well of an enzyme mix and 10 μL of renin inhibitors in DMSO. The enzyme mix was premixed at 4°C and composed as follows:

• human recombinant renin (0.16 ng/mL)

• synthetic human tetradecapeptide renin substrate (0.5 μM) • hydroxyquinoline sulfate (0.1 mM)

The mixtures were then incubated at 37°C for 3 h.

To determine the enzymatic activity and its inhibition, the accumulated

Angiotensin I was detected by an enzyme immunoassay (EIA). 10 μL of the incubates or standards were transferred to immuno plates which were previously coated with a covalent complex of Angiotensin I and bovine serum albumin (Ang I - BSA). 190 μL of Angiotensin l-antibodies were added and a primary incubation made at 4°C over night. The plates were washed 3 times and then incubated for one hour at room temperature with a biotinylated anti-rabbit antibody. Thereafter, the plates were washed and incubated at room temperature for 30 min with a streptavidin-peroxidase complex. After washing the plates, the peroxidase substrate ABTS (2.2'-Azino-di-(3-ethyl- benzthiazolinsulfonate), was added and the plates incubated for 10-30 min at room temperature. After stopping the reaction with 0.1 M citric acid pH 4.3 the plate is evaluated in a microplate reader at 405 nm.

Table 1: IC₅₀ values (nM) for selected compounds on plasmepsin II:

References:

1. Goldberg, D. E., Slater, A. F., Beavis, R., Chait, B., Cerami, A., Henderson, G. B., Hemoglobin degradation in the human malaria pathogen Plasmodium falciparum: a catabolic pathway initiated by a specific aspartic protease; J. Exp. Med., 1991 , 173, 961 - 969.

2. Francis, S. E., Gluzman, I. Y., Oksman, A., Knickerbocker, A., Mueller, R., Bryant, M. L, Sherman, D. R., Russell, D. G., Goldberg, D. E., Molecular characterization and inhibition of a Plasmodium falciparum aspartic hemoglobinase; Embo. J., 1994, 13, 306 - 317.

3. Moon, R. P., Tyas, L., Certa, U., Rupp, K., Bur, D., Jaquet, H., Matile, H., Loetscher, H., Grueninger-Leitch, F., Kay, J., Dunn, B. M., Berry, C, Ridley, R. G., Expression and characterization of plasmepsin I from Plasmodium falciparum, Eur. J. Biochem., 1997, 244, 552 - 560.

4. Carroll, C. D., Johnson, T. O., Tao, S., Lauri, G., Orlowski, M., Gluzman, I.Y., Goldberg, D. E., Dolle, R. E., (1998). "Evaluation of a structure- based statine cyclic diamino amide encoded combinatorial library against plasmepsin II and cathepsin D". Bioorg Med Chem Lett ; 8(22), 3203 - 3206.

5. Peranteau, A. G., Kuzmic, P., Angell, Y., Garcia-Echeverria, C, Rich, D. H., (1995). "Increase in fluorescence upon the hydrolysis of tyrosine peptides: application to proteinase assays". Anal Biochem; 227(1 ):242 - 245. 6. Gulnik, S. V., Suvorov, L. I., Majer, P., Collins, J., Kane, B. P., Johnson,

D. G., Erickson, J. W., (1997). "Design of sensitive fluorogenic substrates for human cathepsin D". FEBS Lett; 413(2), 379 - 384.

7. Robinson, P. S., Lees, W. E., Kay, J., Cook, N. D., (1992). "Kinetic parameters for the generation of endothelins-1 , -2 and -3 by human cathepsin E". Biochem J 284 (Pt 2): 407 - 409.

8. J. March, Advanced Organic Chemistry, pp 918-919, and refs. cited therein; 4^thEd., John Wiley & Sons, 1992. 9. A. Kubo, N. Saito, N. Kawakami, Y. Matsuyama, T. Miwa, Synthesis, 1987, 824-827.

10. R. K. Castellano, D. M. Rudkevich, J. Rebek, Jr., J. Am. Chem. Soc, 1996, 118, 10002-10003. 11. U. Schollkopf, Pure Appl. Chem., 1983, 55, 1799-1806 and refs. cited therein; U. Schollkopf, Top. Curr. Chem., 1983, 109, 65-84 and refs. cited therein; T. Wirth, Angew. Chem. Int. Ed. Engl., 1997, 36, 225-227 and refs. cited therein.

12. T. W. Greene, P. G. M. Wutts, Protective groups in organic synthesis; Wiley-lnterscience, 1991.

13. P. J. Kocienski, Protecting Groups, Thieme, 1994.

14. J. A. Radding, Development of Anti-Malarial Inhibitors of Hemoglobinases, Annual Reports in Medicinal Chemistry, ZA, 1999, 159 - 168. 15. D. F. Wirth, Malaria: A Third World Disease in Need of First World Drug Development, Annual Reports in Medicinal Chemistry, 34, 1999, 349 - 358.

The present invention relates to novel, low molecular weight organic compounds, which are substituted amino-aza-cycloalkanes of the general formula I:

General Formula I

wherein

Q represents -S0₂-R¹; -CO-R¹; -CO-NH-R¹; -CO-N(R )(R²); -CO-OR¹;

-(CH₂)_p-R¹; -(CH₂)_P-CH(R¹)(R²);

X represents -S0₂-R¹; -CO-R¹; -CO-NH-R¹; -CO-N(R¹)(R²); -CO-OR¹; -(CH₂)p-R¹; -(CH₂)_P-CH(R¹)(R²); hydrogen;

R¹, R² and R³ represent lower alkyl; lower alkenyl; aryl; heteroaryl; cycloalkyl; heterocyclyl; aryl-lower alkyl; heteroaryl-Iower alkyl; cycloalkyl-lower alkyl; heterocyclyl-lower alkyl; aryl-lower alkenyl; heteroaryl-Iower alkenyl; cycloalkyl- lower alkenyl; heterocyclyl-lower alkenyl;

R⁴ represents hydrogen; -CH₂-OR⁵; -CO-OR⁵; R⁵ represents hydrogen, lower alkyl; cycloalkyl; aryl; heteroaryl; heterocyclyl; cycloalkyl-lower alkyl; aryl-lower alkyl; heteroaryl-Iower alkyl; heterocyclyl-lower alkyl;

t represents the whole numbers 0 (zero) or 1 and in case t represents the whole number 0 (zero), R⁴ is absent;

m represents the whole numbers 2, 3 or 4;

n represents the whole numbers 1 or 2;

p represents the whole numbers 0 (zero), 1 or 2;

and pure enantiomers, mixtures of enantiomers, pure diastereomers, mixtures of diastereomers, diastereomeric racemates, mixtures of diastereomeric racemates and pharmaceutically acceptable salts thereof

In the definitions of the general formula I - if not otherwise stated - the expression lower means straight and branched chain groups with one to seven carbon atoms, preferably 1 to 4 carbon atoms which may optionally be substituted with hydroxy or lower alkoxy. Examples of lower alkyl groups are methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl. Examples of lower alkoxy groups are methoxy, ethoxy, propoxy, iso-butoxy, sec.-butoxy and tert.-butoxy etc. Lower alkylendioxy-groups as substituents of aromatic rings onto two adjacent carbon atoms are preferably methylen-dioxy and ethylen-dioxy. Lower alkylen-oxy groups as substituents of aromatic rings onto two adjacent carbon atoms are preferably ethylen-oxy and propylen-oxy. Examples of lower alkanoyl-groups are acetyl, propanoyl and butanoyl. Lower alkenylen means e.g. vinylen, propenylen and butenylen.

The expression cycloalkyl, alone or in combination, means a saturated cyclic hydrocarbon ring system with 3 to 6 carbon atoms , e.g. cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl which may be substituted with lower alkyl groups. The expression heterocyclyl, alone or in combination, means saturated or unsaturated (but not aromatic) five-, six- or seven-membered rings containing one or two nitrogen, oxygen or sulfur atoms which may be the same or different and which rings may be substituted with lower alkyl, lower alkenyl, aryl, aryl- lower alkyloxy, aryl-oxy, amino, bis-(lower alkyi)-amino, alkanoyl-amino, halogen, nitro, hydroxy, lower alkoxy, phenoxy; examples of such rings are morpholinyl, piperazinyl, tetrahydropyranyl, dihydropyranyl, 1 ,4-dioxanyl, pyrrolidinyl, tetrahydrofuranyl, dihydropyrrolyl, imidazolidinyl, dihydropyrazolyl, pyrazolidinyl etc. and substituted derivatives of such type rings with substituents as outlined hereinbefore.

The expression heteroaryl, alone or in combination, means six-membered aromatic rings containing one to four nitrogen atoms; benzofused six-membered aromatic rings containing one to three nitrogen atoms; five-membered aromatic rings containing one oxygen, one nitrogen or one sulfur atom; benzo-fused five- membred aromatic rings containing one oxygen, one nitrogen or one sulfur atom; five membered aromatic rings containig one oxygen and one nitrogen atom and benzo fused derivatives thereof; five membred aromatic rings containing a sulfur and nitrogen or oxygen atom and benzo fused derivatives thereof; five membered aromatic rings containing three nitrogen atoms and benzo fused derivatives thereof or the tetrazolyl ring; examples of such rings are furanyl, thienyl, pyrrolyl, pyridinyl, indolyl, quinolinyl, isoquinolinyl, dihydroquinolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, imidazolyl, triazinyl, thiazinyl, pyridazinyl, oxazolyl, etc. whereby such ring systems may be mono-, di- or tri- substituted with aryl; aryloxy, aryl-lower alkyl-oxy, lower alkyl; lower alkenyl; lower alkyl-carbonyl; amino; lower alkyl-amino; bis-(lower-alkyl)-amino; lower alkanoyl-amino; ω-amino-lower alkyl; halogen; hydroxy; carboxyl; lower alkoxy; vinyloxy; allyloxy; ω-hydroxy-lower alkyl; nitro; cyano; amidino; trifluoromethyl; lower alkyl-sulfonyl etc.

The expression aryl, alone or in combination, means six membered aromatic rings and condensed systems like naphthyl or indenyl etc. whereby such ring systems may be mono-, di- or tri-substituted with aryl, aryloxy, aryl-lower alkyloxy, lower alkyl, lower alkenylen, lower alkyl-carbonyl, aryl-carbonyl, amino, lower alkyl-amino, aryl-amino, bis-(lower-alkyl)-amino, lower alkanoyl-amino, ω- amino-lower alkyl, halogen, hydroxy, carboxyl, lower alkoxy, vinyloxy, allyloxy, ω- hydroxy-lower alkyl, ω-hydroxy-lower alkoxy, nitro, cyano, amidino, trifluoromethyl, lower alkyl-sulfonyl etc.

It is understood that the substituents outlined relative to the expressions cycloalkyl, heterocyclyl, heteroaryl and aryl have been omitted in the definitions of the general formulae I to V and in claims 1 to 5 for clarity reasons but the definitions in formulae I to V and in claims 1 to 5 should be read as if they are included therein.

The expression pharmaceutically acceptable salts encompasses either salts with inorganic acids or organic acids like hydrochloric or hydrobromic acid; sulfuric acid, phosphoric acid, nitric acid, citric acid, formic acid, acetic acid, maleic acid, tartaric acid, methylsulfonic acid, p- toluolsulfonic acid and the like or in case the compound of formula I is acidic in nature with an inorganic base like an alkali or earth alkali base, e.g. sodium hydroxide, potassium hydroxide, calcium hydroxide etc.

The compounds of the general formula I can contain one or more asymmetric carbon atoms and may be prepared in form of optically pure enantiomers, diastereomers, mixtures of diastereomers, diastereomeric racemates and mixtures of diastereomeric racemates. The present invention encompasses all these forms. Mixtures may be separated in a manner known per se, i.e. by column chromatography, thin layer chromatography, HPLC, crystallization etc.

The compounds of the general formula I and their pharmaceutically acceptable salts may be used as therapeutics e.g. in form of pharmaceutical compositions. They may especially be used to in prevention or treatment of malaria. These compositions may be administered in enteral or oral form e.g. as tablets, dragees, gelatine capsules, emulsions, solutions or suspensions, in nasal form like sprays or rectally in form of suppositories. These compounds may also be administered in intramuscular, parenteral or intraveneous form, e.g. in form of injectable solutions.

These pharmaceutical compositions may contain the compounds of formula I as well as their pharmaceutically acceptable salts in combination with inorganic and/or organic excipients which are usual in the pharmaceutical industry like lactose, maize or derivatives thereof, talcum, stearinic acid or salts of these materials.

For gelatine capsules vegetable oils, waxes, fats, liquid or half-liquid polyols etc. may be used. For the preparation of solutions and sirups e.g. water, polyols saccharose, glucose etc. are used. Injectables are prepared by using e.g. water, polyols, alcohols, glycerin, vegetable oils, lecithin, liposomes etc. Suppositories are prepared by using natural or hydrogenated oils, waxes, fatty acids (fats), liquid or half-liquid polyols etc.

The compositions may contain in addition preservatives, stability improving substances, viscosity improving or regulating substances, solubility improving substances, sweeteners, dyes, taste improving compounds, salts to change the osmotic pressure, buffer, anti-oxidants etc.

The compounds of formula I may also be used in combination with one or more other therapeutically useful substances e. g. with other antimalarials like quinolines (quinine, chloroquine, amodiaquine, mefloquine, primaquine, tafenoquine etc), peroxide antimalarials (artemisinin derivatives), pyrimethamine- sulfadoxine antimalarials (e.g. Fansidar etc), hydroxynaphtoquinones (e.g. atovaquone etc.), acroline-type antimalarials (e. g. pyronaridine etc) etc.

The dosage may vary within wide limits but should be adapted to the specific situation. In general the dosage given in oral form should daily be between about 3 mg and about 3 g, peferably between about 10 mg and about 1 g, especially preferred between 5 mg and 300 mg, per adult with a body weight of about 70 kg. The dosage should be administered preferably in 1 to 3 doses per day which are of equal weight. As usual, children should receive lower doses which are adapted to body weight and age.

Preferred compounds are compounds of the formula II

Formula II

wherein X, Q, t, R³ and R⁴ are as defined in general formula I above

and pure enantiomers, mixtures of enantiomers, pure diastereomers, mixtures of diastereomers, diastereomeric racemates, mixtures of diastereomeric racemates and pharmaceutically acceptable salts thereof.

Also preferred compounds are compounds of formula III

Formula III

wherein

Q, t, R³ and R⁴ are as defined in general formula I above

and pure enantiomers, mixtures of enantiomers, pure diastereomers, mixtures of diastereomers, diastereomeric racemates, mixtures of diastereomeric racemates and pharmaceutically acceptable salts thereof.

Especially preferred are also compounds of the formula IV

Formula IV

aryl

wherein

Q is as defined in general formula I above

and pure enantiomers, mixtures of enantiomers, pure diastereomers, mixtures of diastereomers, diastereomeric racemates, mixtures of diastereomeric racemates and pharmaceutically acceptable salts thereof. Especially preferred are compounds of the formula V

and pure enantiomers, mixtures of enantiomers, pure diastereomers, mixtures of diastereomers, diastereomeric racemates, mixtures of diastereomeric racemates and pharmaceutically acceptable salts thereof.

The compounds of the general formula I of the present invention may be prepared according to the general sequences of reactions outlined below, wherein R¹, R², R³, R⁴, R⁵, Q, X, t, m, n and p are as defined in general formula I above (for simplicity and clarity reasons, only parts of the synthetic possibilities which lead to compounds of formulae I to V are described). For general methods of certain steps see also pages 19 - 23. Scheme 1 : Preparation of substituted 4-amino-N-benzyl-piperidines:

Typical procedure for the reductive amination (Synthesis of compounds 2):

The amine (1) and the aldehyde {R³-CHO} (1.5 eq.) are mixed in anhydrous methanol and stirred for 6 h. The mixture is then treated with sodium borohydride (1.5 eq.) and stirred for 2 h. Purified Amberiyst 15 or another suitable scavenger is added and the suspension is shaken for 12 h. The resin is then separated by filtration and washed with methanol. The secondary amine 2 is removed from the resin by adding a 2 M methanolic ammonia solution. The resin is drained after 30 min and washed with methanol. The filtrate is evaporated to yield the pure secondary amine 2. Typical procedure for the acylation (Synthesis of compounds 3):

To a solution of the amine 2 in anhydrous ethyl acetate is added vacuum dried Amberiyst 21 or another suitable scavenger followed by the addition of the carboxylic acid chloride {R¹-(CO)-CI} (1.5 eq.). After shaking the suspension for 2 h, an aliquot of water is added in order to hydrolyze the excess of the carboxylic acid chloride and shaking is continued for 1 h. The resin is then removed by filtration, washed with ethyl acetate and the solution is evaporated to yield the pure amide 3.

The carboxylic acid chlorides {R (CO)-CI} may be obtained in situ from the corresponding carboxylic acid as described in the literature (i. e.: Devos, A.;

Remion, J.; Frisque-Hesbain, A.-M.; Colens,A.; Ghosez, L., J. Chem. Soa, Chem. Commun. 1979, 1180).

The synthesis of the sulfonamide derivatives 5 from the amine 2 is performed in analogy to the above described procedure.

The urea derivatives 4 are obtained by reaction of the amines 2 in dichloromethane, with one equivalent isocyanate.

Typical procedure for the second reductive amination (Synthesis of compound 6):

The amine (2) and the aldehyde or the ketone {RιR₂CO} (1.5 eq.) are mixed in anhydrous dichloromethane and sodium triacetoxyborohydride (1.3 eq.) is added. After stirring the solution for 48h, methanol is added and the reaction mixture is treated in the same manner as described for amines 2. Scheme 2: Preparation of substituted 4-amino-N-(lower alkyl-aryl)-piperidines:

The N-Boc protected 4-amino-piperidine 7 (Scheme 2) can be prepared in a two step procedure starting by reacting 4-hydroxy-N-Boc-piperidine with methanesulfonylchloride in an inert solvent like DCM in the presence of a base like TEA to generate 4-mesyloxy-N-Boc-piperidine. The mesyloxy group is substituted with sodium azide followed by reduction of the azide functionality to the amino group to give 7. The amine 7 is transformed to the secondary amine 8 via the typical procedure for the reductive amination described above. The synthesis of compounds 9, 10, 11 and 12 can also be performed via the typical procedures described above. Boc-deprotection is achieved either with hydrochloric acid in a solvent like diethylether or dioxane or with TFA in DCM. The second reductive amination step of the derivatives 13, 14, 15 and 16 to the fully derivatized final compounds 17, 18, 19 and 20 can be performed according to the typical procedure described above. Compounds 13, 14, 15 and 16 could also be transformed with acylating reagents like isocyanates, acid chlorides or sulfonyl chlorides to yield products with an urea-, amide- or sulfonamide functionality instead of the amine functionality at the ring nitrogen atom.

Compounds based on the 3-amino-piperidine template (see Scheme 3) can be prepared by using 3-amino-N-Boc-piperidine as starting material, which can be prepared as described for 7. All other chemical transformations can be performed as described above in Scheme 2.

Compounds based on a 5- or 7-membered ring template (see Scheme 4) can be prepared according to the procedures described above. The 7-membered ring 35 can be prepared by ring extension of 1-benzyl-4- piperidone with ethyl diazoacetate in presence of boron trifluoride etherate. Subsequent hydrolysis followed by decarboxylation upon heating a solution in 10% HCl gives the template 35. Amine 36 is then obtained following the typical procedure for the second reductive amination. Scheme 3:

28 29 30

Scheme 4:

Scheme 5: Synthesis of "Hydroxymethyl-Analogues":

According to the synthesis of the example shown in Scheme 5, other derivatives can be prepared by variation of the starting materials.

All chemical transformations can be performed according to well known standard methodologies as described in the literature or as described in the typical procedures above. The following examples illustrate the invention but do not limit the scope thereof. All temperatures are stated in °C.

List of abbreviations:

Boc or boc tert.-butyloxycarbonyl

Cbz benzyloxycarbonyl

DBU 1 ,8-diazabicyclo[5.4.0]undec-7-ene(1 ,5-5)

DCM dichloromethane DMF dimethylformamide

DMSO dimethylsulfoxide

EtOAc ethyl acetate

TEA triethylamine

TFA trifluoroacetic acid THF tetrahydrofuran

TLC thin layer chromatography^

General Procedures and Examples:

The following compounds were prepared according to the procedures described for the synthesis of compounds encompassed by the general formulae hereinbefore. All compounds were characterized by ¹H-NMR (300MHz) and occasionally by ¹³C-NMR (75MHz) (Varian Oxford, 300MHz; chemical shifts are given in ppm relative to the solvent used; multiplicities: s = singlet, d = doublet, t = triplet; m = multiplet), by LC-MS (Waters Micromass; ZMD-platform with ESl- probe with Alliance 2790 HT; Column: 2x30mm, Gromsil ODS4, 3μm, 120A; Gradient: 0 - 100% acetonitrile in water, 6 min, with 0.05% formic acid, flow: 0.45ml/min; t_r is given in minutes, or Finnigan AQA/HP 1100; Column: Develosil C30 Aqua, 50x4.6mm, 5μm; Gradient: 5-95% acetonitrile in water, 1 min, with 0.03% TFA, flow:4.5 ml/min.), by TLC (TLC-plates from Merck, Silica gel 60 F₂₅₄) and occasionally by melting point.

a) General Procedures:

Typical procedure A) for the reductive amination:

The amine and the aldehyde (1.5 eq.) (which are used as starting materials, are known compounds or the synthesis is described above or below, respectively), are mixed in anhydrous methanol and stirred for 6 h. The mixture is then treated with sodium borohydride (1.5 eq.) and stirred for 2 h. Purified Amberiyst 15 or another suitable scavenger is added and the suspension is shaken for 12 h. The resin is then separated by filtration and washed with methanol. The secondary amine is removed from the resin by adding a 2 M methanolic ammonia solution. The resin is drained after 30 min and washed with methanol. The filtrate is evaporated to yield the pure secondary amine. Typical procedure B) for the acylation:

To a solution of the amine in anhydrous ethyl acetate is added vacuum dried Amberiyst 21 or another suitable scavenger followed by the addition of the carboxylic acid chloride (1.5 eq.). After shaking the suspension for two hours, an aliquot of water is added in order to hydrolyze the excess of the carboxylic acid chloride and shaking is continued for 1 h. The resin is then removed by filtration, washed with ethyl acetate and the solution is evaporated to yield the pure amide.

Typical procedure C) for the second reductive amination:

The amine and the aldehyde (1.5 eq.) are mixed in anhydrous dichloromethane and sodium triacetoxyborohydride (1.3 eq.) is added. After stirring the solution for 48 h, methanol is added and the reaction mixture is treated in the same manner as described in procedure A).

Typical procedure D) for the Suzuki coupling:

To a solution of bromide in toluene is added the boronic acid (1.1 eq.) in isopropanol and a 2M aqueous solution of potassium carbonate (5 eq.). The mixture is purged with nitrogen for 10 min and tetrakis (triphenylphosphine) palladium (0.03 eq.) is added. After heating under reflux for 6 h, water is added to the cooled reaction mixture and the product is extracted with ethyl acetate. The organic phase is washed with brine and dried over sodium sulfate. The solvent is evaporated to give the crude aldehyde, which is purified by flash chromatography (ethyl acetate/heptane gradient). b) Examples:

Example 1 :

According to typical procedure B), the secondary amine a), obtained via typical procedure A), is reacted with 4-pentylbenzoyl chloride to give

Λ/-(4-Benzyloxybenzyl)-/V-(1-benzyIpiperidin-4-yl)-4-pentylbenzamide LC-MS: t_R = 4.95; ES+: 561.7

Example 2:

According to typical procedure B), the secondary amine b), obtained via typical procedure A), is reacted with 4-pentylbenzoyl chloride to give

Λ/-(1-Benzylpiperidin-4-yl)-4-pentyl-Λ/-(3-phenylpropyl) benzamide LC-MS: t_R = 4.82; ES+: 483.5 Example 3:

According to typical procedure B), the secondary amine c), obtained via typical procedure A), is reacted with 4-butoxybenzoyl chloride to give

/V-(4-Benzyloxybenzyl)-Λ/-(1-benzylpiperidin-4-yl)-4-butoxybenzamide LC-MS: t_R = 4.57; ES+:563.44

Example 4:

According to typical procedure B), the secondary amine c), obtained via typical procedure A), is reacted with 4-ethylbenzoyl chloride to give

Λ/-(4-Benzyloxybenzyl)-/V-(1-benzylpiperidin-4-yl)-4-ethylbenzamide LC-MS: t_R = 4.32; ES+:519.41 Example 5:

According to typical procedure B), the secondary amine c), obtained via typical procedure A), is reacted with heptanoyl chloride to give

Heptanoic acid (4-benzyloxybenzyl)-(1-benzylpiperidin-4-yl) amide LC-MS: t_R = 4.42; ES+: 499.39

Example 6:

According to typical procedure B), the secondary amine c), obtained via typical procedure A), is reacted with dodecanoyl chloride to give

Dodecanoic acid (4-benzyloxybenzyl)-(1-benzylpiperidin-4-yl) amide LC-MS: t_R = 5.22; ES+: 569.56 Example 7:

According to typical procedure B), the secondary amine d), obtained via typical procedure A), is reacted with 4-pentylbenzoyl chloride to give

Λ/-(1-Benzylpiperidin-4-yl)-4-pentyl-Λ/-(4-phenoxybenzyl) benzamide LC-MS: t_R = 4.80; ES+: 547.46

Example 8:

According to typical procedure B), the secondary amine d), obtained via typical procedure A), is reacted with 4-butoxybenzoyl chloride to give

Λ/-(1-Benzylpiperidin-4-yl)-4-butoxy-Λ/-(4-phenoxybenzyl) benzamide LC-MS: t_R = 4.60; ES+: 549.47 Example 9:

According to typical procedure B), the secondary amine d), obtained via typical procedure A), is reacted with dodecanoyl chloride to give

Dodecanoic acid (1-benzylpiperidin-4-yl)-(4-phenoxybenzyl) amide LC-MS: t_R = 5.16; ES+: 555.50

Example 10:

According to typical procedure B), the secondary amine e), obtained via typical procedure A), is reacted with 4-pentylbenzoyl chloride to give

Λ/-(1-Benzylpiperidin-4-yl)-Λ/-(3,4-bis-benzyloxybenzyl)-4-pentylbenzamide

LC-MS: t_R = 5.05; ES+: 667.55 Example 11 :

According to typical procedure B), the secondary amine e), obtained via typical procedure A), is reacted with 4-butoxybenzoyl chloride to give

Λ/-(1-Benzylpiperidin-4-yl)-Λ/-(3,4-bis-benzyloxybenzyl)-4-butoxybenzamide

LC-MS: t_R = 4.83; ES+: 669.49

Example 12:

According to typical procedure B), the secondary amine e), obtained via typical procedure A), is reacted with 4-ethylbenzoyl chloride to give

Λ/-(1-Benzylpiperidin-4-yl)-/\/-(3,4-bis-benzyloxybenzyl)-4-ethyIbenzamide

LC-MS: t_R = 4.59; ES+: 625.61 Example 13:

According to typical procedure B), the secondary amine e), obtained via typical procedure A), is reacted with dodecanoyl chloride to give

Dodecanoic acid (1-benzylpiperidin-4-yl)-(3,4-bis-benzyloxybenzyl) amide LC-MS: t_R = 5.49; ES+: 675.74

Example 14:

According to typical procedure B), the secondary amine f), obtained via typical procedure A), is reacted with 4-pentylbenzoyl chloride to give

Λ/-(1-Benzylpiperidin-4-yl)-/V-biphenyl-4-ylmethyl-4-pentylbenzamide LC-MS: t_R = 4.82; ES+: 531.46 Example 15:

According to typical procedure B), the secondary amine f), obtained via typical procedure A), is reacted with 4-butoxybenzoyl chloride to give

/V-(1-Benzylpiperidin-4-yl)-/V-biphenyl-4-ylmethyl-4-butoxybenzamide LC-MS: t_R = 4.49; ES+: 533.43

Example 16:

According to typical procedure B), the secondary amine f), obtained via typical procedure A), is reacted with dodecanoyl chloride to give

Dodecanoic acid (1 -benzylpiperidin-4-yl)-biphenyl-4-ylmethylamide LC-MS: t_R = 5.22; ES+: 539.51 Example 17:

According to typical procedure B), the secondary amine g), obtained via typical procedure A), is reacted with 4-te/τ-butylbenzoyl chloride to give

Λ/-(1-Benzylpiperidin-4-yl)-4-tet -butyl-Λ/-(2-pentyl-3-phenylallyl) benzamide

LC-MS: t_R = 4.93; ES+: 537.48

Example 18:

According to typical procedure B), the secondary amine h), obtained via typical procedure A), is reacted with 4-pentylbenzoyl chloride to give

/V-(1-Benzylpiperidin-4-yl)-4-pentyl-Λ/-(4-trifluoromethylbenzyl) benzamide

LC-MS: t_R = 4.58; ES+: 523.43 Example 19:

According to typical procedure B), the secondary amine h), obtained via typical procedure A), is reacted with 4-butoxybenzoyl chloride to give

/V-(1-Benzylpiperidin-4-yl)-4-butoxy-Λ/-(4-trifluoromethylbenzyl) benzamide

LC-MS: t_R = 4.34; ES+: 525.48

Example 20:

According to typical procedure B), the secondary amine h), obtained via typical procedure A), is reacted with dodecanoyl chloride to give

Dodecanoic acid (1-benzylpiperidin-4-yl)-(4-trifluoromethylbenzyl) amide

LC-MS: t_R = 5.03; ES+: 531.43 Example 21

According to typical procedure B), the secondary amine i), obtained via typical procedure A), is reacted with 4-pentylbenzoyl chloride to give

Λ/-(3-Benzyloxy-4-methoxybenzyl)-Λ/-(1-benzylpiperidin-4-yl)-

4-pentylbenzamide LC-MS: t_R = 4.62; ES+: 591.43

Example 22:

According to typical procedure B), the secondary amine j), obtained via typical procedure A), is reacted with 4-pentylbenzoyl chloride to give

Λ/-(4-Benzyloxy-3-methoxybenzyl)-Λ/-(1-benzylpiperidin-4-yl)-

4-pentyIbenzamide

LC-MS: t_R = 4.70; ES+: 591.46 Example 23:

According to typical procedure B), the secondary amine j), obtained via typical procedure A), is reacted with dodecanoyl chloride to give

Dodecanoic acid (4-benzyloxy-3-methoxybenzyl)-(1 -benzylpiperidin-4-yl) amide LC-MS: t_R = 5.12; ES+: 599.71

Example 24:

According to typical procedure B), the secondary amine k), obtained via typical procedure A), is reacted with 4-pentylbenzoyl chloride to give

Λ/-(1-Benzylpiperidin-4-yl)-/V-(4-butylbenzyl)-4-pentylbenzamide LC-MS: t_R = 5.02; ES+: 511.56 Example 25:

According to typical procedure B), the secondary amine I), obtained via typical procedure A), is reacted with 4-pentylbenzoyl chloride to give

Λ/-(1-Benzylpiperidin-4-yl)-Λ/-(4-butoxybenzyl)-4-pentylbenzamide LC-MS: t_R = 4.92; ES+: 527.58

Example 26:

According to typical procedure B), the secondary amine m), obtained via typical procedure A), is reacted with 4-pentylbenzoyl chloride to give

Λ/-(1-Benzylpiperidin-4-yl)-4-pentyl-Λ/-(4-pentyIbenzyl) benzamide LC-MS: t_R - 5.14; ES+: 525.60 Example 27:

According to typical procedure B), the secondary amine a), obtained via typical procedure A), is reacted with 4-butylphenylisocyanate to give

1-(4-Benzyloxybenzyl)-1-(1-benzylpiperidin-4-yl)-3-(4-butylphenyI) urea LC-MS: t_R = 4.70; ES+: 562.53

Example 28:

According to typical procedure B), the secondary amine n), which is prepared as indicated in scheme 4, is reacted with 4-pentylbenzoyl chloride to give

Λ/-[( 7S)-1 -(4-Benzyloxyphenyl)-2-hydroxyethyl]-N-(1 -benzyIpiperidin-4-yl)-

4-pentylbenzamide LC-MS: t_R = 4.47; ES+: 591.61 Example 29:

According to typical procedure B), the secondary amine a), obtained via typical procedure A), is reacted with 4-propylphenylsulfonyl chloride to give

/V-(4-Benzyloxybenzyl)-/V-(1-benzylpiperidin-4-yl)-4-propyl benzenesulfonamide

LC-MS: t_R = 4.63; ES+: 569.56

Example 30:

According to typical procedure C), the secondary amine m), obtained via typical procedure A), is reacted with 4-trifluoromethylbenzaldehyde to give

(1-Benzylpiperidin-4-yl)-(4-pentylbenzyl)-(4-trifluoromethylbenzyl) amine LC-MS: t_R = 4.91; ES+: 509.60 Example 31 :

According to typical procedure C), the secondary amine m), obtained via typical procedure A), is reacted with biphenyl-4-carbaldehyde to give

(1-Benzylpiperidin-4-yl)-biphenyl-4-ylmethyl-(4-pentylbenzyI) amine LC-MS: t_R = 4.84; ES+: 517.55

Example 32:

According to typical procedure C), the secondary amine o), obtained via typical procedures A) and B), is reacted with furan-3-carbaldehyde to give

Λ/-(4'-Cyanobiphenyl-4-ylmethyl)-Λ/-(1-furan-3-ylmethylpiperidin-4-yl)-4-pentylbenzamide

LC-MS: t_R = 1.05 ; ES+: 546.19 Example 33:

According to typical procedure C), the secondary amine p), obtained via typical procedure A), is reacted with 4-pentylbenzaldehyde to give

2-(4'-{[(1-Benzylpiperidin-4-yl)-(4-pentylbenzyl)-amino]methyl}biphenyl-4-yloxy)ethanol

LC-MS: t_R = 4.32 ; ES+:577.49

Example 34:

According to typical procedure C), the secondary amine q), which is prepared as indicated in Scheme 4, is reacted with 4-pentylbenzaldehyde to give

frac -(1-Benzylazepan-4-yl)biphenyl-4-ylmethyl-(4-pentylbenzyl) amine LC-MS: t_R = 4.41 ; ES+:531.53 Example 35:

According to typical procedure B), the secondary amine q), which is prepared as indicated in Scheme 4, is reacted with 4-pentylbenzoyl chloride to give

(?ac.^,-N-(1-Benzylazepan-4-yl)-N-biphenyl-4-ylmethyI-4-pentyl benzamide LC-MS: t_R = 4.94; ES+:545.42

Example 36:

According to typical procedure B), the secondary amine r), obtained via typical procedure A), is reacted with 4-pentylbenzoyl chloride to give

W-((3S)-1-Benzylpyrrolidin-3-yl)-Λ/-biphenyl-4-ylmethyl-4-pentylbenzamide LC-MS: t_R = 5.08 ; ES+:517.44

Example 37:

According to typical procedure B), the secondary amine s), obtained via typical procedure C), is reacted with 4-pentylbenzoyl chloride to give

Λ/-(4-Benzyloxyphenyl)-Λ-(1-benzylpiperidin-4-yl)-4-pentylbenzamide LC-MS: t_R = 4.57 ; ES+: 547.34

Additional Examples:

^! LC-MS measured on the Finningan AQA/HP system.

Further Examples:

c) Referential Examples: (e.g. not commercially available starting materials)

Referential Example 1 :

According to typical procedure D), 4-formylbenzeneboronic acid is coupled with 2-(4-bromophenoxy) ethanol to give

4'-(2-Hydroxy-ethoxy)-biphenyl-4-carbaldehyde

Referential Example 2:

According to typical procedure D), 4-formylbenzeneboronic acid is coupled with 1-bromo-2-fluorobenzene to give

2'-Fluoro-biphenyl-4-carbaldehyde

Referential Example 3:

According to typical procedure D), 4-formylbenzeneboronic acid is coupled with 1-bromo-3-trifluoromethylbenzene to give

3'-Trifluoromethylbiphenyl-4-carbaldehyde

Referential Example 4:

According to typical procedure D), 4-formylbenzeneboronic acid is coupled with 1-bromo-2-chlorobenzene to give

2'-Chlorobiphenyl-4-carbaldehyde

Referential Example 5:

According to typical procedure D), 4-formylbenzeneboronic acid is coupled with 5-bromopyrimidine to give

4-Pyrimidin-5-yl-benzaldehyde Referential Example 6:

According to typical procedure D), 4-formylbenzeneboronic acid is coupled with 1-bromo-3-(trifluoromethoxy)benzene to give

3'-Trifluoromethoxybiphenyl-4-carbaldehyde

Referential Example 7:

According to typical procedure D), 4-formylbenzeneboronic acid is coupled with 1-bromo-3,4-dimethoxybenzene to give

3',4'-Dimethoxybiphenyl-4-carbaldehyde

Referential Example 8:

According to typical procedure D), 4-formylbenzeneboronic acid is coupled with 5-bromo-benzo[1 ,3]dioxole to give

4-Benzo[1 ,3]dioxol-5-yl-benzaldehyde Referential Example 9:

According to typical procedure D), 4-formylbenzeneboronic acid is coupled with 3-bromopyridine to give

4-Pyridin-3-yl-benzaIdehyde

Referential Example 10:

According to typical procedure D), 4-formylbenzeneboronic acid is coupled with 4-bromopyridine to give

4-Pyridin-4-yl-benzaldehyde

Referential Example 11 :

According to typical procedure D), 4-formylbenzeneboronic acid is coupled with 4-bromobenzonitrile to give

4'-Formylbiphenyl-4-carbonitrile Referential Example 12:

According to typical procedure D), 4-formylbenzeneboronic acid is coupled with 3-bromotoluene to give

3'-Methylbiphenyl-4-carbaldehyde

Referential Example 13:

The following biaryl-derivatives could be prepared according to the typical procedure D):

Claims

Claims:

1. Compounds of the general formula I

FT

X

General Formula I

wherein

Q represents -SO₂-R¹; -CO-R¹; -CO-NH-R¹; -CO-N(R¹)(R²); -CO-OR¹;

-(CH₂)p-R¹; -(CH₂)p-CH(R¹)(R²);

X represents -SO2-R¹; -CO-R¹; -CO-NH-R¹; -CO-N(R¹)(R²); -CO-OR¹; -(CH₂)p-R¹; -(CH₂)p-CH(R¹)(R²); hydrogen;

R¹, R² and R³ represent lower alkyl; lower alkenyl; aryl; heteroaryl; cycloalkyl; heterocyclyl; aryl-lower alkyl; heteroaryl-Iower alkyl; cycloalkyl-lower alkyl; heterocyclyl-lower alkyl; aryl-lower alkenyl; heteroaryl-Iower alkenyl; cycloalkyl- lower alkenyl; heterocyclyl-lower alkenyl;

R* represents hydrogen; -CH₂-OR⁰; -CO-OR 5 o.. R⁵ represents hydrogen, lower alkyl; cycloalkyl; aryl; heteroaryl; heterocyclyl; cycloalkyl-lower alkyl; aryl-lower alkyl; heteroaryl-Iower alkyl; heterocyclyl-lower alkyl;

t represents the whole numbers 0 (zero) or 1 , in case t represents the whole number 0 (zero), R⁴ is absent;

m represents the whole numbers 2, 3 or 4;

n represents the whole numbers 1 or 2;

p represents the whole numbers 0 (zero), 1 or 2;

and pure enantiomers, mixtures of enantiomers, pure diastereomers, mixtures of diastereomers, diastereomeric racemates, mixtures of diastereomeric racemates and pharmaceutically acceptable salts 3reof

2. Compounds of formula II

Formula II

wherein

X, Q, t, R³ and R⁴ are as defined in general formula I above and pure enantiomers, mixtures of enantiomers, pure diastereomers, mixtures of diastereomers, diastereomeric racemates, mixtures of diastereomeric racemates and pharmaceutically acceptable salts thereof.

3. Compounds of formula III

Formula III

wherein Q, t, R³ and R are as defined in general formula I above

and pure enantiomers, mixtures of enantiomers, pure diastereomers, mixtures of diastereomers, diastereomeric racemates, mixtures of diastereomeric racemates and pharmaceutically acceptable salts thereof.

4. Compounds of formula IV

Formula IV

wherein

Q is as defined in general formula I above

and pure enantiomers, mixtures of enantiomers, pure diastereomers, mixtures of diastereomers, diastereomeric racemates, mixtures of diastereomeric racemates and pharmaceutically acceptable salts thereof.

5. Compounds of formula V

and pure enantiomers, mixtures of enar.-. mers, pure diastereomers, mixtures of diastereomers, diastereomeric racemates, mixtures of diastereomeric racemates and pharmaceutically acceptable salts thereof.

6. A compound as described as end-product in any of the examples 1 to 140.

7. Pharmaceutical compositions containing one or more compounds as claimed in any one of claims 1 to 6 and inert excipients.

8. Pharmaceutical compositions according to claim 7 for treatment of diseases demanding the inhibition of aspartic proteases.

9. Pharmaceutical compositions according to claim 7 for treatment of disorders associated with the role of plasmepsin II and which require selective inhibition of plasmepsin II.

10. Pharmaceutical compositions according to claim 7 for treatment or prevention of malaria.

11. Pharmaceutical compositions according to claim 7 for treatment or prevention of diseases caused by protozoal infection (e.g. Chagas disease, Sleeping sickness etc).

12. Pharmaceutical compositions according to claim 7, which contain aside of one or more compounds of the general formula I a known plasmepsin II, a known HIV protease or a known cathepsin D or E inhibitor.

13. A process for the preparation of a pharmaceutical composition according to any one of claims 8 to 11 , characterized by mixing one or more active ingredients according to any one of claims 1 to 6 with inert excipients in a manner known per se.

14. Use of at least one of the compounds of the general formula I for the treatment or prevention of diseases.

15. The invention as herein before described.