ASPARTIC PROTEASE INHIBITORS

Field of invention

The present invention relates to medicine and in particular to the treatment of malaria, more particularly to inhibitors of malaria aspartic proteases known as plasmepsins. Even more particularly, the invention relates to novel substituted aminoalkylazoles and pharmaceutical compositions thereof and their use as inhibitors of malarial aspartic protease plasmepsin II or related malarial aspartic proteases.

Background of invention

Malaria is a life threatening infectious disease caused by the Plasmodium parasite carried by mosquitoes. In 2010, World Health Organization has estimated 219 million cases of malaria infection, resulting in 660,000 deaths (WHO. World Malaria Report 2012; World Health Organization: Geneva, 2012; p 59). Widespread resistance to practically all currently used drugs has activated the search for antimalarials with novel mechanisms of action (Hyde, J. E. Drug-resistant malaria - an insight. FEBS J. 2007, 274, 4688-4698; Choi, S. R.; Mukherjee, P.; Avery, M. A. The fight against drug-resistant malaria: novel plasmodial targets and antimalarial drugs. Curr. Med. Chem. 2008, 15, 161-171; Wells, T. K; Alonso, P. L.; Gutteridge, W. E. New medicines to improve control and contribute to the eradication of malaria. Nat. Rev. Drug Discov. 2009, 8, 879-891). Resistance to current anti-malaria agents in regions threatened by malaria continues to spread, and it means that current therapeutic agents will be practically ineffective in the near future. A precondition for the development of a new resistance free malaria agent is inhibition of malaria parasite life cycle by a mechanism that differs from the mode of action of currently used therapeutic agents (N. K. Sahu, S. Sahu and D. V. Kohli, Novel Molecular Targets for Antimalarial Drug. Chem. Biol. Drug. Des. 2008, 71, 287-297). Such product would be resistance free for a particular period of time, and it would be effective in all malaria endemic regions. One group of promising biological targets for the development of new anti-malaria agents are plasmepsins (I, II, IV and HAP) - aspartic proteases of the parasite Plasmodium, which are used by the parasite in the first stage of hemoglobin digestion, which is the main nutrition source during its erythrocytic cycle (K. Ersmark, B. Samuelsson, A. Hallberg, Plasmepsins as Potential Targets for New Antimalarial Therapy. Med. Res. Rev., 2006, 26, 626-666; G. H. Coombs, D. E. Goldberg, M. Klemba, C. Berry, J. Kay and J. C. Mottram, Aspartic proteases of Plasmodium falciparum and other parasitic protozoa as drug targets. Trends in Parasitology, 2001, 17, 532-537). Therapeutic potential of plasmepsins in the treatment of malaria is still unused as there is no marketed anti-malarial drug acting on this target. Most of known plasmepsin inhibitors are based on N-acylated amino alcohols as transition state analogues that are peptidic or peptide-like which suffer from problems in bioavailability and pharmacokinetic profile (K. Ersmark, B. Samuelsson, A. Hallberg, Plasmepsins as Potential Targets for New Antimalarial Therapy. Med. Res. Rev., 2006, 26, 626-666). In order to find more drug-like compound there is an increased interest for non-peptidic plasmepsin inhibitors and several such inhibitors have been reported (O. Corminboeuf, G. Dunet, M. Hafsi, J. Grimont, C. Grisostomi, S. Meyer, C. Binkert, D. Bur, A. Jones, L. Prade, R. Bran, C. Boss, Inhibitors of Plasmepsin II— potential antimalarial agents. Bioorganic & Medicinal Chemistry Letters 2006, 16, 6194-6199; F. Hof, A. Schutz, C. Fah, S. Meyer, D.Bur, J. Liu, D. E. Goldberg, and F. Diederich, Starving the Malaria Parasite: Inhibitors Active against the Aspartic Proteases Plasmepsins I, II, IV Angew. Chem. Int. Ed. 2006, 45, 2138 -2141; T. Luksch, A. Blum, N. Klee, W. E. Diederich, C. A. Sotriffer, G. Klebe, Pyrrolidine Derivatives as Plasmepsin Inhibitors: Binding Mode Analysis Assisted by Molecular Dynamics Simulations of a Highly Flexible Protein. ChemMedChem., 2010, 5, 443-454). Summary of the invention

In a first aspect, the invention features a method of treating malaria in humans, comprising administering to a human in need thereof a therapeutically effective amount of a compound or prodrug thereof, or pharmaceutically acceptable salt, hydrate, solvate, or polymorph of said compound or prodrug, wherein the compound is an inhibitor of malarial aspartic protease plasmepsin II or related malarial aspartic proteases.

In another aspect, the invention features a pharmaceutical composition for treatment of malaria comprising a therapeutically effective amount of a composition comprising (i) a compound or prodrug thereof, or pharmaceutically acceptable salt, hydrate, solvate, or polymorph of said compound or prodrug; and (ii) a pharmaceutically acceptable carrier, wherein the compound is an inhibitor of malarial aspartic protease plasmepsin II or related malarial aspartic proteases. In another aspect, the invention features the use of a compound or prodrug thereof, or pharmaceutically acceptable salt, hydrate, solvate, or polymorph of said compound or prodrug, wherein the compound is an inhibitor of malarial aspartic protease plasmepsin II or related malarial aspartic proteases, in the manufacture of a medicament for treatment or prevention of malaria.

In another aspect, the invention features a compound or prodrug thereof, or pharmaceutically acceptable salt or ester of said compound or prodrug for use in treating or preventing malaria, wherein the compound is an inhibitor of a malarial aspartic protease plasmepsin II or related malarial aspartic proteases.

In one embodiment the inhibitor of a malarial aspartic protease plasmepsin II or related malarial aspartic proteases is a compound of Formula I, generally referred herein as aminoalkylazole derivative:

Formula I

wherein: R ¹ R ² are optional amino group substituents; R ³ R ⁴ are optional phenyl group susbtituents; A-D-E-G-J constitutes saturated or unsaturated azole ring; Q is a covalent bond or a linker.

Compound with general formula I wherein:

R' R ² , R ³ , R ⁴ , R ⁴ ,R ⁵ is independently -H, -F, -CI, -Br, -I, -CF ₃ , -CH ₂ CF ₃ , -CF ₂ CF ₂ H, -OH, -L-OH,-0-L-OH, -OR ⁶ , -O L-NH ₂ , -O-L-NHR ⁶ , -O L-NR ⁶ ₂ , -0-L-NR ⁶ R ⁷ , -L-OR ⁶ ,-0-L-OR ⁶ ,-OCF ₃ , -OCH ₂ CF ₃ , -OCF ₂ CF ₂ H, -L-OR ⁶ ,-0-L-OR ⁶ ,-OCF ₃ , -OCH ₂ CF ₃ , -OCF ₂ CF ₂ H, SR ⁶ , SCF ₃ , CN, -N0 ₂ , -N0 ₂ , -NH ₂ , - HR ⁶ , -NR ⁶ ₂ , -NR ⁶ R ⁷ ,-L-NH ₂ , -L-NHR ⁶ , -L-NR ⁶ 2, -L-NR ⁶ R ⁷ ,

-NH-L-NH ₂ , -NH-L-NHR ⁶ , -NH-L-NR ⁶ ₂ , -NH-L-NR ⁶ R ⁷ ,-NR ⁶ ~L-NH ₂ , -NR ⁶ -L-NHR ^fi , -NR ⁶ -L-NR ⁶ 2, -NR ⁶ -L-NR ⁶ R ⁷ ,L-NR ⁶ R ⁷ ,-C(=0)OH, -C(=0)OR ⁶ , -C(=0)NH ₂ , -C(=0)NHR ⁶ , -C(=0) R ⁶ 2, -C(=0)NR ⁶ R ⁷ , -NHC(=0)R ⁶ , -NR ⁶ C(=0)R ⁷ , -NHC(=0)OR ⁶ , -NR ⁶ C(=0)OR ⁷ , -OC(=0)NH ₂ , -OC(=0)NHR ⁶ , -OC(=0)NR ⁶ ₂ , -OC(=0)NR ⁶ R ⁷ ,-OC(=0)R ⁶ , -C(=0)R ⁶ ,-NHC(=0)NH ₂ , -NHC(=0)NHR ⁶ , -NHC(=0)NR ⁶ ₂ , -NHC(=0)NR ⁶ R ⁷ , -NR ⁶ C(=0)NH ₂ , -NR ⁶ C(=0)NHR ⁷ , -NR ⁶ C(=0)NR ⁷ ₂ , - R ⁶ C(=0)N, -NHS(=0) ₂ R ⁶ , -NR ⁶ S(=0) ₂ R ⁷ ,-S(=0) ₂ NH ₂ , -S(=0) ₂ NHR ⁶ , -S(=0) ₂ NR ⁶ ₂ , -S(=0) ₂ NR ⁶ R ⁷ ,-S(=0)R ⁶ , -S(=0) ₂ R ⁶ ,-OS(=0) ₂ R ⁶ ,-S(=0) ₂ OR ⁶ ,

C _1-6 alkyl, cycloC _3-12 alkyl, cycloC _3-12 alkyl-C _1-6 alkyl, C ₂ - ₆ alkenyl, C ₂ . ₆ alkynyl, aryl, biaryl, arylC _1-6 alkyl, arylC ₂ _ ₆ alkenyl, arylC ₂ . ₆ alkynyl, heteroaryl, heteroarylC _1-6 alkyl, heteroarylC ₂ _ ₆ alkenyl, heteroarylthio, 2,3-dihydro-lH-indenyl, 2-indanylamino, tetrahydrofuryl, pyrrolidino, piperidino, 4-arylpiperidino, 4-heteroarylpiperidino, morpholino, piperazino, 4- Ci-ealkylpiperazino, 4-arylpiperazino, hexamethyleneimino, benzazepinyl, l,3-dihydro-2H- isoindol-2-yl,

wherein: L represents -W-X-Y-Z-, wherein:

W represents a single bond, oxygen, sulfur, -NR ⁶ or -CR ⁶ R ⁷ , X represents oxygen, sulfur, -NR ⁶ or -CR ⁶ R ⁷ , Y represents oxygen, sulfur, -NR ⁶ or -CR ⁶ R ⁷ and Z represents oxygen, sulfur, -NR ⁶ or -CR ⁶ R ⁷ ;

wherein: or R ¹ and R ² together represent -W-X-Y-Z-, wherein:

W represents a single bond, oxygen, sulfur, ~NR ⁶ or -CR ⁶ R ⁷ ,

X represents oxygen, sulfur, -NR ⁶ or -CR ⁶ R ⁷ ,

Y represents oxygen, sulfur, -NR or -CR R and

Z represents oxygen, sulfur, -NR or -CR R ; wherein:

R ⁶ and R ⁷ is independently H, C _1-6 alkyl, cycloC ₃ _ ₁₂ alkyl, cycloCs-^alkyl-Q-ealkyl,

C ₂ - ₆ alkenyl, C ₂ _ ₆ alkynyl, aryl, biaryl, arylC^alkyl, arylC ₂ - ₆ alkenyl, arylC ₂ - ₆ alkynyl, heteroaryl, heteroarylQ.ealkyl, heteroarylC ₂ - ₆ alkenyl, heteroarylthio, 2,3-dihydro-lH-indenyl, C _1- alkoxyC _1-6 alkyl, aryloxyarylQ-ealkoxy, C _1-6 alkylthio, C _4-6 alkenylthio, cycloC _3-12 alkylthio, cycloCs-^alkyl-Q.ealkylthio, cycloC _3-12 alkyl-C _3-6 alkenylthio, Q-ealkoxyQ-ealkylthio, C _\ . ₆ alkoxyC ₃ . ₆ alkenylthio, arylC ₃ _ ₆ alkenylthio, heteroarylC _1-6 alkylthio, Q-ealkylsulfonyl, cycloCs.^alkyl-Ci-ealkylsulfonyl, arylQ-ealkylsulfonyl, C _1-6 alkylamino, di-Ci^alkylamino, cycloC _3-12 alkylamino, Ci-Cealkoxy-cycloCs-C^alkylamino, cycloC ₃ _ ₁₂ alkyl-C ₁ . ₆ alkylamino, di-C _1-6 alkylaminoC _1-6 alkyl, C _1-6 alkoxy-C ₂ _ ₆ alkylamino, arylamino, arylC _1-6 alkylamino, N- cycloCs-nalkyl-N-Q.ealkylamino, N-aryl-N-Q-ealkylamino, N-arylC^alkyl-N-Q, ₆ alkylamino, 2-indanylamino, tetrahydrofuryl, pyrrolidino, piperidino, 4-arylpiperidino, 4- heteroarylpiperidino, morpholino, piperazino, 4-C _1-6 alkylpiperazino, 4-arylpiperazino, hexamethyleneimino, benzazepinyl, l,3-dihydro-2H-isoindol-2-yl, heteroarylC _1-6 alkoxy, heteroarylamino, heteroarylC ] _-6 alkyamino

Compound with general formula I

wherein:

A is independently nitrogen or carbon;

D is independently nitrogen or carbon;

E is independently nitrogen or carbon;

G is independently oxygen or sulphur or -N= or -NR ⁸ - -CR ⁹ = or -CR ⁹ R ¹⁰

J is is independently oxygen or sulphur or -N= or -NR ⁸ - -CR ⁹ = or -CR ^y R ¹⁰ ; and at least one of A-D-E-G-J is nitrogen wherein:

R ⁸ ,R ⁹ ,R ¹⁰ is independently H, C _1-6 alkyl, cycloC ₃ .i ₂ alkyl, cycloC ₃ . laalkyl-Ci _6alkyl, C ₂ _ ₆ alkenyl, C _2-6 alkynyl, aryl, biaryl, arylC _1-6 alkyl, arylC ₂ _6alkenyl, arylC2- ₆ alkynyl, heteroaryl, heteroarylC ] -ealkyl, heteroarylC2- ₆ alkenyl, heteroarylthio, 2,3-dihydro-l H-indenyl, C _\ . ₆ alkoxyC _1-6 alkyl, aryloxyarylQ-ealkoxy, Q-ealkylthio, C ₄ . ₆ alkenylthio, cycloC _3-12 alkylthio, cycloCs-^alkyl-Ct-ealkylthio, cycloC3_ ₁₂ alkyl-C ₃ -6alkenylthio, Q-ealkoxyCi-ealkylthio, C _\ . ₆ alkoxyC ₃ .6alkenylthio, arylC3-6alkenylthio, heteroarylQ-ealkylthio, Q.ealkylsulfonyl, cycloC ₃ . ₁₂ alkyl-C _1-6 alkylsulfonyl, arylC _1-6 alkylsulfonyl, Q-ealkylamino, di-C _1-6 alkylamino, cycloC ₃ . ₁₂ alkylamino, Q-Cealkoxy-cycloCs-C^alkylamino, cycloC ₃ _ ₁₂ alkyl-C _1-6 alkylamino, di-Q-ealkylaminoC^alkyl, C _1- alkoxy-C2-6alkylamino, arylamino, arylC _1-6 alkylamino, N- cycloCs-^alkyl-N-Cj-ealkylamino, N-aryl-N-Ci_6alkylamino, N-arylCi-ealkyl-N-Q. ₆ alkylamino, 2-indanylamino, tetrahydrofuryl, pyrrolidino, piperidino, 4-arylpiperidino, 4- heteroarylpiperidino, morpholino, piperazino, 4-C ₁ _ ₆ alkylpiperazino, 4-arylpiperazino, hexamethyleneimino, benzazepinyl, l,3-dihydro-2H-isoindol-2-yl, heteroarylC ].6alkoxy, heteroarylamino, heteroarylC _1-6 alkylamino.

In one preferred embodiment, A-D-E-G-J constitutes 1,2,5-substituted imidazole group of the formula with optional substitiuent R ⁹ :

In one preferred embodiment, A-D-E-G-J constitutes 1,4,5-substituted triazole group formula:

In one preferred embodiment, A-D-E-G-J constitutes 3,4,5-substituted isoxazole group of the formula:

In one referred embodiment, A-D-E-G-J constitutes 1,2,5-pyroline group of the formula:

wherein n is an integer from 0 to 3, and each R ^A is independently a substituent R ⁹ or R ¹⁰ defined herein.

In one preferred embodiment, A-D-E-G-J constitutes 1,2,5-pyrolidine group of the formula:

wherein n is an integer from 0 to 4, and each R ^A is independently a substituent R ⁹ or R ¹⁰ as defined herein.

In one preferred embodiment, A-D-E-G-J constitutes 1,2,5-pyrole group of the formula:

wherein n is an integer from 0 to 2, and each R ^A is independently a substituent R ⁹ or R ¹⁰ as defined herein Compound with general formula I wherein, Q represents a linker group: in some embodiments, Q is a covalent bond; in some embodiments, Q is a linker group.

In one preferred embodiment, Q is a covalent bond.

In one preferred embodiment, Q is a Q^alkylene group and is optionally substituted. In one preferred embodiment, Q is a methylene group and is optionally substituted.

Optical isomers, pharmaceutically acceptable salts, hydrates, solvates, and polymorphs of compound with general formula I

The use of compounds with general formula I; their optical isomers, pharmaceutically acceptable salts, hydrates, solvates, and polymorphs for the treatement of malaria infection.

In one embodiment, the treatment is treatment of a disease or disorder that is mediated by plasmepsins (malarial aspartic protease) or human aspartic proteases.

In one embodiment, the treatment is treatment of a disease or disorder that is ameliorated by the inhibition of plasmepsins (malarial aspartic protease) or human aspartic proteases. In one embodiment, the treatment is treatment of a disease or disorder that is treated by a plasmepsins (malarial aspartic protease) or human aspartic proteases inhibitor.

The use of compounds with general formula I their optical isomers, pharmaceutically acceptable salts, hydrates, solvates, and polymorphs for malaria prevention. In another aspect, the invention features a kit comprising a substituted aminoalkylazole with general formula I, preferably provided as a pharmaceutical composition and in a suitable container and/or with suitable packaging.

In another aspect, the invention features compounds with general formula I obtainable by a method of synthesis as described herein, or a method comprising a method of synthesis as described herein.

In another aspect, the invention features compounds with general formula I obtained by a method of synthesis as described herein, or a method comprising a method of synthesis as described herein. In another aspect, the invention features novel intermediates for the compound with general formula I, as described herein, which are suitable for use in the methods of synthesis described herein.

In another aspect, the invention features the use of such novel intermediates, for the compound with general formula I according to the claim 1 as described herein, in the methods of synthesis described herein.

As will be appreciated by one of skill in the art, features and preferred embodiments of one aspect of the invention will also pertain to other aspects of the invention.

Description of invention Plasmepsins are malarial aspartic proteases have been identified as a group of promising biological targets for the development of new anti-malaria agents. For example, plasmepsin II is used by the parasite in the first stage of hemoglobin digestion, which is the main nutrition source during its erythrocytic cycle (K. Ersmark, B. Samuelsson, A. Hallberg, Plasmepsins as Potential Targets for New Antimalarial Therapy. Med. Res. Rev., 2006, 26, 626-666; G. H. Coombs, D. E. Goldberg, M. Klemba, C. Berry, J. Kay and J. C. Mottram, Aspartic proteases of Plasmodium falciparum and other parasitic protozoa as drug targets. Trends in Parasitology, 2001, 17, 532-537).

When testing the novel substituted aminoalkylazoles derivatives for their ability to inhibit plasmepsin II we have unexpectedly discovered, that said derivatives exhibit pronounced inhibitor properties toward said plasmepsin and thus are useful in treatment of malaria.

According to this invention, the results from plasmepsin inhibition studies demonstrate that substituted are novel class of non-peptidic inhibitors of malarial aspartic proteases - plasmepsins. Several example compounds from the present invention display high nanomolar to low micromolecular inhibitory potency that is comparable to known non-peptidic inhibitors (e.g. F. Hof et. al., Starving the malaria parasite: Inhibitors active against the aspartic proteases plasmepsins I, II, and IV. Angew. Chem. Int. Ed. 2006, 45, 2138-2141; T. Luksch et.al., Computer-Aided Design and Synthesis of Nonpeptidic Plasmepsin II and IV Inhibitors. ChemMedChem., 2008, 3, 1323-1336.; C. Boss et.al., Inhibitors of the Plasmodium falciparum parasite aspartic protease plasmepsin II as potential antimalarial agents. Curr Med Chem. 2003, 10, 883-907; C. Fah et. al., Enantiomerically Pure and Highly Substituted Alicyclic α,α-Difluoro Ketones: Potential Inhibitors for Malarial Aspartic Proteases, the Plasmepsins. Eur. J. Chem., 2010, 4617-4629.; T. Luksch et.al., Pyrrolidine Derivatives as Plasmepsin Inhibitors: Binding Mode Analysis Assisted by Molecular Dynamics Simulations of a Highly Flexible Protein. ChemMedChem., 2010, 5, 443-454)

Stereochemistry

Many of the chemical structures shown herein indicate one or more specific stereoisomeric configurations. Similarly, many of the chemical structures shown herein are silent in this respect, and do not indicate any stereoisomeric configuration. Similarly, many of the chemical structures shown herein indicate the specific stereoisomeric configurations at one or more positions, but are silent with respect to one or more other positions. Where a chemical structure herein is silent with respect to the stereoisomeric configuration at a position, that structure is intended to depict all possible stereoisomeric configurations at that position, both individually, as if each possible stereoisomeric configuration was individually recited, and also as a mixture (e.g., a racemic mixture) of stereoisomers.

Combinations

Each and every compatible combination of the embodiments described above is explicitly disclosed herein, as if each and every combination was individually and explicitly recited.

Examples of Specific Embodiments

The following examples further illustrate the invention, but should not be construed to limit the scope of the invention in any way.

The following aminoalkylazoles were prepared as an examples of the current invention:

General Synthesis

The synthesis of aminoalkylimidazoles 8.1-9 is shown in scheme 1. Commercially available acyl chloride 1 was transformed to amide the intermediate amide was dehydrated to give cyano benzene derivative 2. This was subjected to the reaction with 4-aminomethyl (bromobenzene) hydrochloride (3) in the presence of AlMe ₃ providing amidine derivative 4. This was transformed to 5-formylimidazole 6 in the reaction with unsaturated aldehyde 5 [Shilcrat, Susan C; Mokhallalati, Mohamed K.; Fortunak, Joseph M. D.; Pridgen, Lendon N. A New Regioselective Synthesis of 1 ,2,5-Trisubstituted IH-Imidazoles and Its Application to the Development of Eprosartan. J.Org. Chem. 1997, 62, 8449-8454]. Next, the Suzuki- Miyaura reaction of aldehyde 6 with boronic acid provided intermediate 7 which were subjected to reductive animation with range of amines providing series of compounds 8.1-9.

Scheme 1

The synthesis of aminomethylimidazoles 8.10,11 is shown in scheme 2. Formylimidazole 7 (scheme 1) was reduced to hydoxymethylimidazole 9. This was subjected to azidation with diphenylphosforyl azide (DPP A) followed by catalytic reduction of intermediate azide to aminomethylimidazole 8.10. Reductive alkylation of amine 8.10 with isovaleraldehyde provided N-substituted aminomethylimidazole 8.11.

Scheme 2

The synthesis of 2-aminoprop-2-ylimidazole 8.12 is shown in scheme 3. Aldehyde 7 was subjected to the reaction with Grignard reagent providing alcohol 10. This was oxidized and the intermediate ketone was subjected to the Grignard reagent providing alcohol 11. The Ritter reaction of alcohol 11 with trimethylsilylcyanide (TMSCN) gave amine 8.12.

Scheme 3 Synthesis of intermediate 2

Chloroanhydride 1 (2.1 g, 10 mmol) was dissolved in THF (50 mL) and to this; concentrated aqueous ammonia (15 mL) was added at room temperature. The mixture was stirred for 2 h, then diluted with saturated aqueous NaCl and extracted with EtOAc. The combined organic extract was dried over Na ₂ S0 ₄ and evaporated. The crude amide was suspended in toluene (150 mL) and to this SOCl ₂ (14.6 mL, 200 mmoL) was added. The mixture was refluxed for 48 h and then evaporated. The residue was purified by column chromatography on silica gel (eluent Petroleum ether: EtOAc = 1 : 0 to 20 : 1) to give nitrile 2 (1.16 g, 67 %) as an oil.

Synthesis of intermediate 4 4-(Bromophenyl)methanamine hydrochloride (3) (445 mg, 2 mmol) was suspended in toluene (5 mL) and the mixture was cooled in an ice bath. To this, 2 M AlMe3 in Hexane (1 mL, 2 mmol) was added dropwise. The mixture was warmed to room temperature and stirred for 30 min. Then, a solution of 4-pentylbenzonitrile (2) in toluene (12 mL) was added and the mixture was heated to 110°C for 4 h in a sealed reactor. After cooling to room temperature, 1 M aqueous NaOH (20 mL) was added and the mixture was extracted with EtOAc (3 x 20 mL). The combined organic phase was washed with brine and dried over Na ₂ S0 ₄ . Solvent was removed in vacuo and the residue was treated with Et ₂ 0. The precipitate was collected on a filter and dried to give product 4 (270 mg, 38 %).

Synthesis of intermediate 6 Exemplified by the synthesis of 6: A mixture of amidine 4 (270 mg, 0.75 mmol) and (E)-2- bromo-3-isopropoxyacrylaldehyde (5) (174 mg, 0.9 mmol), 6 M aqueous K ₂ C0 ₃ (0.15 mL) and CHC1 ₃ (7 mL) was stirred at room temperature for 48 h. The mixture was diluted with saturated aqueus NaCl and extracted with EtOAc. The organic phase was dried over Na ₂ S0 ₄ and evaporated. The residue was treated with dichloromethane to give crystalline product 6 (54 mg, 18%) which was collected by filtration. An additional amount of product 6 (130 mg, 42%) was isolated from the mother liquid by radial chromatography on silica gel (CH ₂ C1 ₂ : MeOH = 1 : 0 to 1 : 1).

Synthesis of intermediate 7

A mixture of intermediate 6 (130 mg, 0.32 mmol), 3 -pyridine boronic acid (39 mg, 0.32 mmol), Pd(PPh ₃ ) ₄ (3 mg, 5 mol%), 2 M aqueous NaHC0 ₃ (5 mL), isopropylalcohol (5 mL) and toluene (5 mL) was heated at 90°C for 4 h. The mixture was cooled to room temperature 7 and solvents evaporated. The residue was purified by radial chromatography on silica gel (eluent, petroleum ether : EtOAc = 1 : 1 to 0 : 1) to yield 7 (90 mg, 69% yield) as colorless solid.

Synthesis of aminoalkyl imidazoles 8.1-8.9. General method A. Exemplified by the synthesis of 8.1 (GL-1547): To the solution of aldehyde 7. (60 mg, 0.15 mmol) and diethylamine (17 uL, 0.165 mmol) in THF (6 mL) 4A molecular sieves were added and the mixture was cooled in an ice bath. To this, NaBH(OAc) ₄ (48 mg, 0.225 mmol) and AcOH (34 uL, 0.6 mmol) was added and the mixture was stirred at room temperature for 24 h. Saturated aqueous NaHC0 ₃ was added and the mixture was extracted with EtOAc. The organic phase was separated and dried over Na ₂ S0 ₄ . The solvent was then removed in vacuo and the residue was purified by column chromatography (EtOAc : MeOH = 1 : 0 to 1 : 4) to yield 8.1 (21 mg, 30% yield) as colorless solid.

By a method analogous to Method A using compound 7 as a precursor, the following compounds were obtained:

Synthesis of intermediate 9

A solution of aldehyde 7 (1.02 g, 2.5 mmol) in DCM (100 mL) was cooled to -78°C and to this a solution of DIBAL-H in toluene (4.2 mL, 5.0 mmol) was added dropwise. The mixture was stirred at -78°C for 2 h and then quenched by the addition of MeOH (5 mL). Saturated Segnet's salt solution was added and the product was extracted with DCM. The organic phase was dried over Na ₂ S0 ₄ and evaporated to give alcohol 9 (940 mg, 91%).

Synthesis of aminomethylimidazole 8.10

The suspension of alcohol 9 (940 mg, 2.28 mmol) in toluene (23 mL) was cooled in an ice bath. To this mixture, diphenylphosphorylazide (DPP A) (0.64 mL, 2.96 mmol) and DBU (0.44 mL, 2.96 mmol) was added. The mixture was stirred at room temperature for 4 h and diluted with a saturated aqueous NaCl. The product was extracted into EtOAc and the organic phase was dried over Na ₂ S0 ₄ . The solvent was removed in vacuo and the crude product was purified by flash chromatography on silica gel eluting with EtOAc to give intermediate azide (760 mg, 76 %). Azide (140 mg, 0.32 mmol) was dissolved in MeOH (14 mL) and to this solution, 10% Pd on C was added (4 mg). The reaction mixture was stirred in the hydrogen atmosphere (1 atm) for 1 h 30 min and filtered through the short Celite® column. The solvent was evaporated to give product 8.10 (130 mg, 100%). Synthesis of aminoalkylimidazole 8.11

To the solution of amine 8.10 (12 mg, 0.029 mmol) and isovaleraldehyde (5 uM, 0.044 mmol) in MeOH (2 raL) 4 A molecular sieves were added and the mixture was cooled in an ice bath. To this, NaBH(OAc) ₄ (4 mg, 0.066 mmol) and AcOH (7 uL, 0.116 mmol) was added and the mixture was stirred at room temperature for 24 h. Saturated aqueous NaHC0 ₃ was added and the mixture was extracted with EtOAc. The organic phase was separated and dried over Na ₂ S0 ₄ . The solvent was then removed in vacuo and the residue was purified by column chromatography (eluent, EtOAc : MeOH = 1 : 0 to 1 : 4) to yield 8.11 (4 mg, 25% yield) as colorless solid. Synthesis of intermediate 10

A solution of aldehyde 7 (1.7 g, 4.15 mmol) in THF (100 mL) was cooled to -78°C. To this, 2.4 M MeMgBr solution in Et ₂ 0 (3.46 mmol, 8.30 mmol) was added dropwise. The reaction mixture was warmed to room temperature and stirred overnight. It was cooled in an ice bath and quenched by the addition of water, and then aqueous phase was saturated with NaCl and extracted with EtOAc. The organic extract was dried over Na ₂ S0 ₄ and the solvent evaporated in vacuo. The residue was treated with Et ₂ 0 and the precipitate was collected on the filter to give secondary alcohol 10 as crystalline solid 1.36 g (77%).

Synthesis of intermediate 11

The intermediate alcohol 10 (426 mg, 1.0 mmol) was dissolved in DCM (10 mL) and added dropwise to a solution of pyridinium dichromate (752 mg, 2.0 mmol) in DCM (16 mL). The resulting mixture was stirred at room temperature for 4 h and then diluted with Et ₂ 0. The suspension was filtered through the Celite® and the solvent evaporated. The crude residue (170 mg) was dissolved in THF (20 mL) and the solution was cooled to 0°C. Then 2.4 M MeMgBr solution in Et ₂ 0 was added dropwise, the solution was warmed to room temperature and stirred for 2 h. The mixture was cooled in an ice bath and quenched by the addition of water. The aqueous phase was saturated with NaCl and extracted with EtOAc. The organic extract was dried over Na ₂ S0 ₄ and evaporated. The residue was purified by flash chromatography on silica gel (eluent, petroleum ether: EtOAc, 1 : 1 - 0 : 1) to give intermediate 10 (40 mg, 9 %) Synthesis of aminomethylimidazole 8.12

Alcohol 10 (10 mg, 0.023 mmol) was dissolved in AcOH (0.2 mL) and TMSCN (6.1 uL; 0.046 mmol) was added to the solution. The solution was cooled to -15°C and to this concentrated sulfuric acid (0.24 mL) was added dropwise. The mixture was warmed to room temperature and stirred for 16 h, then it was diluted with aqueous saturated NaHC0 ₃ and extracted with EtOAc. The organic phase was concentrated in vacuo and the residue was dissolved in dioxane. To this concentrated aqueous HCl was added and the resulting mixture was heated at 80°C for 2 h. After cooling to room temperature, 1 M aqueous KOH was added and the product was extracted into EtOAc. The organic extract was dried over Na ₂ S0 ₄ and evaporated. Chromatography on preparative TLC provided 8.12 (3 mg, 30%).

Synthesis of aminoalkyltriazoles 33, 34.1-6, 35.1-2 is shown in scheme 4

24: n=0 27: n=0 30: n=0 33: n=0 25: n=1 28: n=1 31.1-6: n=1 34.1-6: n- 26: n=2 29: n=2 32.1-2: n=2 36.1-2: n=2

Scheme 4 Synthesis of intermediate 14

An oven dried pressure tube was charged with 4-methoxycarbonylphenylboronic acid (12) (2.00 g, 11.10 mmol, 1.0 equiv.), 4-bromoanisole (13) (2.08 g, 11.10 mmol, 1.0 eqiuv.) and Pd(PPh ₃ ) ₄ (40 mg, 0.035 mmol, 0.003 eqiuv.). To the starting materials /-PrOH (20 mL), toluene (20 mL) and 2M Na ₂ C0 ₃ aq. solution (25 mL) were added and the mixture was heated at 90°C for 9h. The mixture was extracted with CH2CI2 (4 x 30 mL). Combined organic layers were dried over Na ₂ S0 ₄ and evaporated under reduced pressure. The residue was purified by column chromatography (100% C¾C1 ₂ ) to yield 14 (2.00 g, 74% yield).

Synthesis of intermediate 15 The carboxylic acid methyl ester derivative 14 (2.00 g, 8.25 mmol, 1.0 eqiuv.) was dissolved in THF (dry) (20 mL) and the solution was cooled to 0°C. To the solution 2.4M LiAliL/THF (4.5 mL, 10.70 mmol, 1.3 equiv.) was added dropwise and stirring was continued at r.t. for 2h. The reaction mixture was cooled to 0°C and H ₂ 0 (0.4 mL) was added and stirred for 5 min, then 4N NaOH sol. (0.8 mL) was added and stirred for 10 min, after that H ₂ 0 (1.2 mL) was added and stirred for 10 min. Precipitate was filtered off and filtrate was evaporated, giving the desired product 15 (1.76 g, 99%).

Synthesis of intermediate 16

Alcohol 15 (1.76 g, 8.21 mmol, 1.0 equiv.) was dissolved in CH ₂ C1 ₂ and PBr ₃ (4.22 g, 1.5 mL, 15.60 mmol, 1.9 equiv.) was added dropwise. The mixture was stirred at r.t. for 16h. To the reaction mixture H ₂ 0 (20 mL) was added and stirring was continued for 20 min. Organic layer was separated and washed with H ₂ 0 (2 x 40 mL). Organic layer was dried over Na ₂ S0 ₄ and evaporated under reduced pressure to yield product 16 (2.26 g, 99%).

Synthesis of intermediate 18

Solution of trimethylsilylacetylene (17) (1.42 g; 2.0 mL; 14.40 mmol, 4.0 eqiuv.) in THF (dry) (10 mL) was cooled to 0°C and 2M -PrMgCl/THF (5.4 mL; 10.80 mmol, 3.0 equiv.) was added dropwise. The mixture was stirred at 0°C for 30 min, then at r.t. for lh. Then CuBr (0.31 g, 2.16 mmol, 0.6 equiv.) was added to the reaction mixture and stirred at r.t. for 30 min, after that solution of benzylbromide 16 (1.00 g, 3.60 mmol, 1.0 equiv.) in THF (5 mL) was added. Reaction mixture was heated at reflux for 3h. The solvent was then removed in vacuo and H 0 (10 mL) and EtOAc (10 mL) were added to the residue. Precipitate was filtered off and filtrate was extracted with EtOAc (2 x 30 mL). Combined organic layers were dried over Na ₂ S0 ₄ and evaporated under reduced pressure. Residue was purified by column chromatography (PE EtOAc=95:5) to give product 18 (0.94 g, 89%).

Synthesis of intermediate 19

To the solution of (3~(4'-methoxy-[l,l'-biphenyl]-4-yl)prop-l-yn-l-yl)trimethyl silane (18) (0.90 g, 3.06 mmol, 1.0 eqiuv.) in EtOH (16 niL) AgN0 ₃ (0.78 g, 1.5 eqiuv.) solution in H ₂ 0/EtOH (4 mL, 1:3) was added dropwise. Mixture was stirred at r.t. for 30 min, after that KCN (1.97 g, 9.9 equiv.) solution in H ₂ 0 was added. Stirring was continued for 2h. Reaction mixture was partitioned between EtOAc and H ₂ 0. Organic layer was washed with H ₂ 0 (2 x 20 mL), brine, dried over Na ₂ S0 ₄ and evaporated under reduced pressure to give product 19 (0.65 g, 96%).

Synthesis of intermediate 20

An oven dried pressure tube was charged with 4-(bromomethyl)-4'-methoxy-l,r-biphenyl (16) (1.78 g, 6.40 mmol, 1.0 equiv.), Cul (1.22 g, 6.40 mmol, 1.0 equiv.), K ₂ C0 ₃ (887 mg, 6.40 mmol, 1.0 equiv.) and BU ₄ (2.37 g, 6.40 mmol, 1.0 equiv.). MeCN (15 mL) was added followed by dropwise addition of ethyl propiolate (1.26 g, 12.84 mmol, 2.0 equiv.) was added dropwise. The mixture was stirred at 40°C for 16h. To the reaction mixture sat. NH4CI sol. (30 mL) was added and the resulting suspension was extracted with EtOAc (3 x 25 mL). Combined organic layers were dried over Na ₂ S0 ₄ and evaporated under reduced pressure. The residue was purified by column chromatography (eluent system PE EtOAc=99:l to 85:15). Yield: 1.52 g (80%).

Synthesis of intermediate 21

To the solution of 4-methoxy-4'-(prop-2-yn-l-yl)-l,l'-biphenyl (19) (0.55 g, 2.50 mmol, 1.0 eqiuv.) in MeCN (dry) (10 mL) Cul (24 mg, 0.12 mmol, 0.048 equiv.) and ethyldiazoacetate (0.31 g, 2.75 mmol, 1.1 eqiuv.) was added. The mixture was stirred at r.t. for 16h. Solvent was removed in vacuo and the residue was purified by direct phase ARMEN chromatograph, eluent system PE/EtOAc (95:5 to 0:100), 25 g silica gel column, flow rate 18 mL/min.

Obtained product 21 (0.63 g, 82%).

Synthesis of intermediate 22

An oven dried pressure tube was charged with 4-(biOmomethyl)-4'-methoxy- 1 , 1 '-biphenyl (16) (1.22 g, 4.40 mmol, 1.0 equiv.), Cul (838 mg, 4.40 mmol, 1.0 equiv.), K ₂ C0 ₃ (669 mg, 4.84 mmol, 1.1 equiv.) and Bu ₄ NT (1.63 g, 4.40 mmol, 1.0 equiv.). MeCN (15 mL) was added followed by dropwise addition of ethyl pent-4-ynoate (610 mg, 4.84 mmol, 1.1 equiv.). The reaction mixture was stirred at r.t. for 16h. To the reaction mixture sat. NH ₄ C1 sol. (30 mL) was added and resulting suspension was extracted with EtOAc (3 x 25 mL) was performed. Combined organic layers were dried over Na ₂ S0 ₄ and evaporated under reduced pressure. The residue was purified by column chromatography (eluent system

PL7EtOAc=99:l to 85:15). Yield: 749 mg (70%).

Synthesis of azide 23

An oven dried flask was charged with l-bromo-4-pentylbenzene (2.00 g, 8.80 mmol, 1.0 equiv.) and THF (dry) (15 mL) was added. The solution was cooled to -78°C and 2.5M n- BuLi/hexanes (3.9 mL, 9.68 mmol, 1.1 equiv.) was added dropwise. Reaction mixture was stirred at -78°C lh, then 4-methylbenzenesulfonyl azide (2.60 g, 13.20 mmol, 1.5 eqiuv.) solution in THF (8 mL) was added. Reaction mixture was warmed up to -45°C and 20 mL of sat. NH4CI solution was added and resulting mixture was extracted with EtOAc (3 x 35 mL).

Combined organic extracts were washed with brine, dried over Na ₂ S0 ₄ and evaporated under reduced pressure. The residue was purified by column chromatography, eluent system

(PE/EtOAc=98:2). Yield: 1.29 g (77 %).

Synthesis of aminoalkyl triazoles 33, 34.1-6, 35.1-2. General method B. Exemplified by the synthesis of 34.1.

Synthesis of intermediate 25 An oven dried flask was charged with azide 23 (799 mg, 4.22 mmol, 1.2 equiv.), alkyne 21 (1.08 g, 3.52 mmol, 1.0 equiv.) and Cp RuCl(COD) (160 mg, 0.42 mmol, 12 mol%). 1,4- Dioxane (dry) (10 mL) was added and reaction mixture was stirred at r.t. for 16h. The solvent was removed in vacuo and the residue was purified by column chromatography (eluent system PE/EtOAc=9:l to 1:1). Yield: 485 mg (28%). Synthesis of intermediate 28

To the triazole 25 (0.47 g, 0.94 mmol, 1.0 equiv.) in 1,4-dioxane (15 mL) IN NaOH sol. (8 mL) was added. Reaction mixture was stirred at r.t. for 16 h. H ₂ 0 was added and resulting suspesion was extracted with EtOAc (3 x 25 mL). Combined organic layers were washed with brine, dried over Na ₂ S0 ₄ and evaporated under reduced pressure. Product was used in the next step without purification. Yield: 0.26 g (97%). Synthesis of intermediate 31.1

An oven dried MW vial was charged with acid 28 ( 100 mg, 0.21 mmol, 1 .0 equiv.) and HOBt (37 mg, 0.27 mmol, 1.3 eqiuv.), DMF (1.5 mL) and EDC (52 mg, 0.27 mmol, 1.3 equiv.) was added. The mixture was stirred at 0°C for Ih, then N-methylpiperazine (32 mg, 35 μί, 1 .5 equiv.) was added. Stirring continued at r.t. for 16h. To the reaction mixture H ₂ 0 was added and suspension was extracted with EtOAc (3 x 10 mL). Combined organic layers were washed with brine, dried over Na ₂ S0 ₄ and evaporated under reduced pressure. Residue was purified by direct phase ARMEN chromatograph (eluent system CH ₂ Cl ₂ MeOH=l:0 to 4: 1), 12 g silicagel column, flow rate 12 mL/min. Yield: 65 mg (56%). Synthesis of product 34.1

To the solution of amide 31.1 (56 mg, 0.11 mmol) in THF (dry) (1 mL) was added 1M

BH3 THF/THF (0.5 mL). Reaction mixture was stirred at 80°C for 16, the 4N HCl sol. (1 mL) was added and stirring was continued for lh. Reaction mixture was cooled to r.t. and basified with sat. NaHCC»3 sol. to pH=8 and extracted with EtOAc (3 x 10 mL). Combined organic layers were washed with brine, dried over Na ₂ S0 ₄ and evaporated under reduced pressure. The residue was purified by column chromatography (eluent system CH ₂ Cl ₂ /MeOH=l:0 to 4: 1). Yield: 19 mg (36%).

By a method analogous to Method B using compounds 20, 21, 22 as a precursors, the following compounds were obtainded:

* -o taine rom compoun s 3 .5 an 3 .

Synthesis of product 36.1

An oven dried MW vial was charged with NaH (60% mineral oil) (10 mg, 0.24 mmol, 4.0 equiv.), washed with Et ₂ 0 (dry) (3 1 mL). DMF was added (1 mL), mixture was cooled to 0°C and n-DodSH (49 mg, 58 μΐ-, 0.24 mmol, 4.0 equiv.) was added. The mixture was stirred was continued at r.t. for 10 min, then solution of 2-(2-(5-((4'-memoxy-[l,l'-biphenyl]-4- yl)methyl)- 1 -(4-pentylphenyl)- 1H- 1 ,2,3 -triazol-4-yl)ethyl)- 1 ,2,3 ,4-tetrahydroisoquinoline (36.1) (35 mg, 0.06 mmol, 1.0 equiv.) in DMF was added. Reaction mixture was stirred at 130°C for 2h. Solvent was removed in vacuo and H ₂ 0 and sat. NH CI sol. was added, mixture was extracted with EtOAc (3 x 10 mL). Combined organic layers were washed with H ₂ 0, brine, dried over Na ₂ S0 ₄ and evaporated under reduced pressure. The residue was purified by direct phase ARMEN chromatograph, eluent system CH ₂ Cl ₂ /MeOH= 100:0 to 90:10, 12 g silicagel column, flow rate 12 rnL/min. HC1 salt was made and precipitate was washed with petroleum ether. Yield: 13 mg (36%). Product 36.2 was synthesized according to the procedure provided for product 36.1

Synthesis of aminoalkyltriazoles 44, 47, 48.1-5 is shown in scheme 5

Scheme 5

Synthesis of intermediate 38

An oven dried pressure tube was charged with 1 -bromo-4-(prop-2-yn- 1 -yl)benzene (1.24 g, 6.36 mmol, 1.0 equiv.) and THF (dry) (7 mL) was added. Then 1M MeMgBr/THF (7.6 mL, 7.63 mmol, 1.2 equiv.) was added dropwise. Reaction mixture was stirred at 65°C for 3h, then cooled to r.t. and methylchloroformate (2.40 g, 2.0 mL, 25.40 mmol, 4.0 equiv.) was added quickly. The reaction mixture was stirred at 65°C for 18h. Reaction mixture was cooled to r.t. and EtOAc was added. The mixture was washed with IN HC1 sol., H ₂ 0 and brine, dried over Na ₂ S0 ₄ and evaporated under reduced pressure. The residue was purified by direct phase ARMEN chromatograph, eluent system PE EtOAc=98:2 to 90:10, 25 g silicagel column, flow rate 18 niL/min. Yield: 1.14 g (71 ).

Synthesis of intermediate 39

Intermediate 39 was synthesized according to the procedures provided for compound 21.

Synthesis of intermediates 42, 43 Intermediates 42, 43 were synthesized according to the procedures provided for compounds 30-32.

Synthesis of intermediates 45, 46.1-5. General method C. Exemplified by the synthesis of 46.1.

An oven dried pressure tube was charged with phenylboronic acid (26 mg, 0.21 mmol, 1.05 equiv.), amide 43 (100 mg, 0.20 mmol, 1.0 equiv.) and Pd(PPh ₃ ) ₄ (7 mg, 0.006 mmol, 0.03 equiv.). To the starting materials -PrOH (1 mL), toluene (1 mL) and 2M Na ₂ C0 ₃ aq. solution (300 μί) were added and the mixture was heated at 90°C for 16h. Reaction mixture was extracted with EtOAc (3 x 10 mL). Combined organic layers were dried over Na ₂ S0 ₄ and evaporated under reduced pressure. The residue was purified by column chromatography (PE/EtOAc=4: 1 to EtOAc/MeOH=95:5) to yield 46.1 (86 mg, 87% yield).

By a method analogous to Method C using compound 42 and 43 as a precursors, the following compounds were obtainded:

Synthesis of products 47, 48.1-5. General method D. Exemplified by the synthesis of 48.1.

To the solution of amide 46.1 (70 mg, 0.11 mmol, 1.0 equiv.) in THF (dry) (1 mL) was added 1M BH ₃ -THF/THF (0.5 mL). Reaction mixture was stirred at 80°C for 16, the 4N HCl sol. (1 mL) was added and stirring was continued for lh. Reaction mixture was cooled to r.t. and basified with sat. NaHC0 ₃ sol. to pH=8. Extraction with EtOAc (3 x 10 mL) was performed. Combined organic layers were washed with brine, dried over Na ₂ S0 ₄ and evaporated under reduced pressure. The residue was purified by column chromatography (eluent system CH ₂ Cl ₂ /MeOH=l:0 to 4: 1). Yield: 49 mg (68%). By a method analogous to Method D using amides 45, 46.1-5 as a precursors, the following compounds were obtainded:

Obtained from 44 according to general method C. Synthesis of aminoalkylisoxazoles 56.1-2 is shown in scheme 6

54 55

Scheme 6

Synthesis of intermediate 50 Solution of 1 -bromo-4-pentylbenzene (49) (2.00 g, 8.8 mmol, 1.0 equiv.) in THF (10 mL) was cooled to -78°C and 2.5M n-BuLi/hexanes (3.9 mL, 9.7 mmol, 1.1 equiv.) was added dropwise. The mixture was stirred at -78°C for lh. Then DMF (dry) (1.0 mL, 13.2 mmol, 1.5 equiv.) was added dropwise and the reaction mixture was warmed up to r.t. To the reaction mixture sat. NH ₄ C1 sol. was added and mixture was extracted with EtOAc (3 x 30 mL). Combined organic layers were washed with brine, dried over Na ₂ S0 ₄ and evaporated under reduced pressure. The residue was purified by column chromatography (eluent system PE/EtOAc=95:5). Yield: 1.55 g (99%).

Synthesis of intermediate 51 To the solution of 4-pentylbenzaldehyde (50) (1.56 g, 8.85 mmol) in H ₂ 0 (14 mL) and Et ₂ 0 (14 mL) NH ₂ OH HCl (1.53 g, 22.1 mmol, 2.5 equiv.) and NaHCOs (2.23 g, 26.6 mmol, 3.0 equiv.) were added. Stirred at r.t. for 16h. The reaction mixture was transferred to separating funnel and organic layer was separated off. Water layer was extracted with CH ₂ C1 ₂ (2 x 30 mL). Combined extracts were dried over Na ₂ S0 ₄ and evaporated under reduced pressure (bath temperature 26°C). The residue was purified by column chromatography (eluent system PE/EtOAc=98:2 to 90:10). Yield: 1.43 g (85%).

Synthesis of intermediate 53

To the solution of 4-pentylbenzaldehyde oxime (51) (1.30 g, 6.8 mmol, 1.0 equiv.) in DMF (13 mL) solution of NCS 52 (908 mg, 6.8 mmol, 1.0 equiv.) in DMF (3 mL) was added dropwise. Stirred at r.t. for 40 min. To reaction mixture H ₂ 0 was added and extracted with Et ₂ 0 (3 x 20 mL). Combined extracts were washed with H ₂ 0 and brine, dried over Na ₂ S0 ₄ and evaporated under reduced pressure. Yield: 1.53 g (99%).

Synthesis of intermediate 54

An oven dried pressure tube was charged with N-hydroxy-4-pentylbenzimidoyl chloride (53) (530 mg, 2.35 mmol, 1.1 equiv.), alkyne 38 (628 mg, 2.13 mmol, 1.0 equiv.), Cp RuCl(COD) (80 mg, 0.21 mmol, 10 mol%) and DCE (dry) (4 mL) was added. To the reaction mixture TEA (431 mg, 592 μί, 4.26 mmol, 2.0 equiv.) solution in DCE (1 mL) was slowly dropwise (during lh) added. Stirred at r.t. for 16h. Solvent was removed in vacuo and the residue was purified by direct phase ARMEN chromatograph, eluent system PE/EtOAc=90: 10 to 75:25, 25 g silicagel column, flow rate 18 mL/min. Yield: 427 mg (37%). Synthesis of intermediate 55

Intermediate 55 was synthesized according to the procedures provided for compounds 30-32.

By a method analogous to Method C andD using compound 55 as a precursor, the following compounds were obtainded:

Synthesis of aminoalkylpyrole 63 is shown in scheme 7

61 62 63

Scheme 7 Synthesis of intermediate 58

An oven dried pressure tube was charged with l-bromo-4-pentylbenzene (500 mg, 2.20 mmol, 1.0 equiv.), bis(pinacolato)diborane (670 mg, 2.64 mmol, 1.2 equiv.), KOAc (1.08 g, 1 1.00 mmol, 5.0 equiv.), Pd(dppf)Cl ₂ -CH ₂ Cl ₂ (18 mg, 0.022 mmol, 0.01 equiv.) and 1 ,4- dioxane (dry) (5 mL) was added. Reaction mixture was heated at 100°C for 18h. To the reaction mixture H ₂ 0 was added and suspension was extracted with EtOAc (3 x 20 mL). Combined organic layers were washed with brine, dried over Na2S04 and evaporated under reduced pressure. The residue was purified by direct phase ARMEN chromatograph, eluent system PE/EtOAc=98:2 to 4:1, 25 g silicagel column, flow rate 25 mL/min. Yield: 480 mg (80%).

Synthesis of intermediate 59

Product 59 was synthesized according to general method C. Synthesis of intermediate 60

An oven dried flask was charged with NaH (60% mineral oil) (33 mg, 0.81 mmol, 1.5 equiv.), washed with Et ₂ 0 (dry) (3 x 2 mL). DMF was added and the suspension was cooled to 0°C.

Then solution of ethyl 5-(4-pentylphenyl)-lH-pyrrole-2-carboxylate (59) (155 mg, 0.54 mmol, 1.0 equiv.) in DMF was added dropwise. Stirred at 0°C for 0.5h, then solution of 1- bromo-4-(bromomethyl)benzene (37) (135 mg, 0.54 mmol, 1.0 equiv.) in DMF was added.

Stirred at r.t. for 16h. H ₂ 0 was added and resulting suspension was extracted with EtOAc (3 x 20 mL). Combined extracts were washed with brine, dried over Na ₂ S0 ₄ and evaporated under reduced pressure. Purified by column chromatography (eluent system PE EtOAc=9:l to 1: 1).

Yield: 165 mg (67%).

Synthesis of intermediate 61

Intermediate 61 was synthesized according to general method C. Synthesis of product 63. General method E.

The carboxylic acid ethyl ester 61 (48 mg, 0.10 mmol, 1.0 equiv.) was dissolved in THF (dry) (2 mL) and the solution was cooled to 0°C. 1M LiAlHJTHF (100 μί, 0.10 mmol, 1.0 equiv.) was added dropwise and the mixture was stirred at r.t. for Ih. The reaction mixture was cooled to 0°C, H ₂ 0 (8 μί) was added and stirred for 5 min, then 4N NaOH sol. ( 16 μί) was added and stirred for 10 min, after that H ₂ 0 (24 μΐ,) was added and stirred for 10 min. The filtrate was evaporated, giving the desired product 62 (40 mg, 91%).

Synthesis of product 63

Solution of alcohol 62 (34 mg, 0.08 mmol, 1.0 equiv.) in CH ₂ C1 ₂ was cooled to 0°C and TEA (15 mg, 20 μί,, 0.15 mmol, 1.9 equiv.) and MsCl (14 mg, 9 \L, 0.12 mmol, 1.5 equiv.) were added. Stirred at 0°C for lh, then diethylamine (29 mg, 41 μΐ., 0.40 mmol, 5.0 equiv.) was added and strirring was continued for 16h at r.t. H ₂ 0 was added and suspension was extracted with EtOAc (3 x 7 mL). Combined organic layers were washed with brine, dried over Na ₂ S0 ₄ and evaporated. The residue was purified by column chromatography (eluent sytem

PE/EtOAc=85: 15 to 0: 100). Yield: 20 mg (50%).

Synthesis of aminoalkyltriazoles 71-73 is shown in scheme 8

70: X=C=0 72: X=C=0

73: X=CH-OH*-obtained from ketone 72

Scheme 8 Synthesis of intermediate 66

Solution of l-(azidomethyl)-4-pentylbenzene (64) (1.3 g, 6.39 mmol) and but-3-yn-l-ol (65) (583 mg, 629 μί, 8.31 mmol, 1.3 equiv.) in CH ₂ C1 ₂ (20 mL) was stirred at r.t. for 15 min, then H ₂ 0 (5 mL), CuS0 ₄ -5H ₂ 0 (209 mg) and sodium ascorbate (587 mg) were added. Stirring was continued for 16h. To the reaction mixture additional H ₂ 0 was added and suspension was extracted with EtOAc (3 x 20 mL). Combined organic layers were washed with H ₂ 0, brine, dried over Na ₂ S0 ₄ and evaporated under reduced pressure. Yield: 1.38 g (79%). Synthesis of intermediate 67

An oven dried pressure tube was charged with 4-methoxyphenol (500 mg, 4.03 mmol, 1.1 equiv.), l-bromo-4-iodobenzene (1.04 g, 3.66 mmol, 1.0 equiv.), NN-dimethylglycine (28 mg, 0.27 mmol, 0.075 equiv.), Cs ₂ C0 ₃ (2.34 g, 7.32 mmol, 2.0 equiv.), Cul (13.9 mg, 0.073 mmol, 0.02 equiv.) and 1,4-dioxane (dry) (8 mL) was added. Reaction mixture was stirred at 90°C for 18h. To the reaction mixture sat. NH ₄ C1 sol. was added and suspension was extracted with EtOAc (3x 50 mL). Combined organic layers were washed with brine, dried over Na ₂ S0 ₄ and evaporated. The residue was purified by column chromatography (eluent system PE/EtOAc=95:5 to 1:1). Yield: 584 mg (57%). Synthesis of intermediate 68

The solution of 1,4-dibromobenzene (1.0 g, 4.24 mmol, 1.0 equiv.) in THF (15 mL) was cooled to -78°C and 2.5M n-BuLi/hexanes (1.7 mL, 4.24 mmol, 1.0 equiv.) was added dropwise. Stirred at -78°C for 30 min, then solution of 4-methoxybenzaldehyde (520 mg, 3.82 mmol, 0.9 equiv.) in THF (10 mL) was added. Stirred for 16h. To the reaction mixture sat. NH4CI sol. was added and suspension was extracted with EtOAc (3 x 35 mL). Combined organic layers were washed with brine, dried over Na ₂ S0 ₄ and evaporated. Product was purified by column chromatography (eluent system PE/EtOAc=9:l to 1:1). Yield: 960 mg (77%).

Synthesis of intermediate 69, general method F. An oven dried MW vial was charged with triazol 66 (300 mg, 1.1 mmol), Pd(OAc) ₂ (12 mg, 0.055 mmol, 0.05 equiv.), tetrabutylammonium acetate (663 mg, 2.2 mmol, 2.0 equiv.) and 1- bromo-4-(4-methoxyphenoxy)benzene (67) (460 mg, 1.65 mmol, 1.5 equiv.) and NMP was added (4 mL). Reaction mixture was stirred at 100°C for 18h. Then H ₂ 0 and brine was added and resulting suspension was extracted with EtOAc (4 x 20 mL). Combined extracts were washed with H ₂ 0 (2 x 60 mL), brine, dried over Na ₂ S0 ₄ and evaporated under reduced pressure. The residue was purified by column chromatography (eluent system PE/EtOAc=9: 1 to 0:100). Yield: 254 mg (49%).

By a method analogous to Methods F, C and D using compound 66 as a precursor, the following compounds were obtainded:

Compound ID

Structure

No

Synthesis of product 73

Solution of (4-(4-(2-(diethylamino)ethyl)- 1 -(4-pentylbenzyl)- 1H- 1 ,2,3 -triazol-5-yl)phenyl)(4- methoxyphenyl)methanone (72) (50 mg, 0.093 mmol, 1.0 equiv.) in MeOH (1.8 mL) was cooled to 0°C and NaBFL; (7 mg, 0.19 mmol, 2.0 equiv.) was added portionwise. Stirred at r.t. for 2h. Reaction mixture was diluted with Et ₂ 0, sat. NaHC0 ₃ sol. was added and the mixture was extracted with EtOAc (3 x 15 mL). Combined organic layers were washed with sat. NaHC0 ₃ sol., brine, dried over Na ₂ S0 ₄ and evaporated under reduced pressure. The residue was purified by column chromatography (eluent system CH ₂ Cl ₂ /MeOH=100:0 to 4:1). Yield: 35 mg (70%).

Synthesis of intermediate 76

An oven dried flask was charged with AICI3 (3.46 g, 26.0 mmol, 1.2 equiv.) and CS ₂ (7 mL) was added. Then solution of pentylbenzene (74) (3.21 g, 21.6 mmol, 1.0 equiv.) in CS ₂ (7 mL) and 3-chloropropionylchloride (75) (3.02 g, 2.3 mL, 23.8 mmol, 1.1 equiv.) solution in CS ₂ (5 mL) was added dropwise. Stirred at r.t. for 15 min. To the reaction mixture H ₂ 0 (25 mL) was added carefully, extracted with EtOAc (3 x 40 mL). Combined organic layers were washed with brine, dried over Na ₂ S0 ₄ and evaporated under reduced pressure. Yield: 4.65 g (90%).

Synthesis of intermediate 78 An oven dried pressure tube was charged with diethylacetamidomalonate (77) (4.23 g, 19.5 mmol, 1.0 equiv.) and NaOEt (1.33 g, 19.48 mmol, 1.0 equiv.), and EtOH (dry) was added (25 mL). Then solution of chloride 76 (4.65 g, 19.48 mmol) in EtOH (15 mL) was added dropwise. Reaction mixture was heated at 85°C for 18h. Solvent was removed in vacuo. The residue was suspended in H ₂ 0 and extracted with EtOAc (3 x 40 mL). Combined organic layers were washed with brine, dried over Na ₂ S0 ₄ and evaporated under reduced pressure. Product 78 was purified by column chromatography (eluent system PE/EtOAc=9:l to 1:1). Yield: 3.12 g (38%).

Synthesis of intermediate 79 To the diethyl 2-acetamido-2-(3-oxo-3-(4-pentylphenyl)propyl)malonate (78) (3.12 g, 7.44 mmol) HCl (cone.) (30 mL) and CH ₃ COOH (10 mL) was added. Reaction mixture was heated at 80°C for 16h. Solvent was removed in vacuo. To the residue toluene was added (2 x 40 mL) and the mixture was evaporated under reduced pressure. Precipitate was washed with mixture of CH ₂ C1 ₂ and Et ₂ 0 (1:1). Yield: 1.77 g (80%). Synthesis of intermediate 80

To the solution of 5-(4-pentylphenyl)-2,3-dihydro-lH-pyrrole-2-carboxylic acid

hydrochloride (79) (600 mg, 2.31 mmol) in EtOH (dry) (15 mL) SOCl ₂ (824 mg, 503 μΕ, 6.93 mmol, 3.0 equiv.) was added. Reaction mixture stirred at 50°C for 24h. Solvent was removed in vacuo and toluene (2 x 20 mL) was added and evaporated under reduced pressure. Yield: 664 mg (100%).

Synthesis of intermediate 81

Solution of 2,3-dihydro-lH-pyrrole 80 (664 mg, 2.05 mmol, 1.0 equiv.) in THF (dry) (10 mL) was cooled to -78°C and 1M KHMDS/THF (4.7 mL, 4.71 mmol, 2.3 equiv.) was added dropwise. Reaction mixture was stirred at -78°C for 45 min, then 4-brombenzylbromide (693 mg, 2.77 mmol, 1.35 mmol) solution in THF (10 mL) was added dropwise. Stirred at -78°C for lh, then warmed up to r.t. To the reaction mixture H ₂ 0 was added and suspension was extracted with EtOAc (3 x 25 mL). Combined extracts were washed with brine, dried over Na ₂ S0 ₄ and evaporated under reduced pressure. Product was purified by column

chromatography (eluent system PE/EtOAc=7: l). Yield: 363 mg (39%). Synthesis of intermediate 82

Intermediate 82 was synthesized according to general method B. Synthesis of intermediate 83

LTMP solution was prepared as follows: a solution of 2.5M n-BuLi/hexanes (544 μί„, 1 .36 mmol, 2.0 equiv.) was added to a stirred, cold (0°C) solution of TMP (211 mg, 252 μΐ., 1.50 mmol, 2.2 equiv.) in THF. In a separate flask solution of ethyl 1 -((4'-methoxy~| 1 , 1 '-biphenyl |- 4-yl)methyl)-5-(4-pentylphenyl)-2,3-dihydro-lH-pyrrole-2-car boxylate (82) (330 mg, 0.68 mmol, 1.0 equiv.) and CH ₂ Br ₂ (237 mg, 96 μΐ., 1.36 mmol, 2.0 equiv.) in THF was cooled to - 78°C and TMP solution was added via cannula. Stirring was continued for 20 min at -78°C. Reaction was quenched with 1.2 M HC1 aq. sol. and extracted with EtOAc (3 x 15 mL). Combined organic layers were dried over Na ₂ S0 ₄ and evaporated under reduced pressure. Obtained intermediate was dissolved in THF and the solution was cooled to -78°C, then 1M LiHMDS/THF (700 μL·, 1.09 equiv.) was added dropwise. Stirred at -78°C for 20 min, then 2.5M n-BuLi/hexanes (563 μί, 202 equiv.) was added dropwise. Stirring was continued at - 78°C for lh. Reaction mixture was warmed up to r.t., and solution of CH ₃ COCl in MeOH (1:5) was added. H ₂ 0 was added and resulting suspension was extracted with EtOAc (3 x 15 mL). Combined extracts were washed with brine, dried over Na ₂ S0 ₄ and evaporated under reduced pressure. Product was purified by column chromatography (eluent system PE/EtOAc=9:l to 5:1). Yield: 100 mg (32%).

Synthesis of product 84 Product 84 was synthesized according to general method E.

Synthesis of product 85

To the solution of N^-diethyl-2-(l-((4'-methoxy-[l,r-biphenyl]-4-yl)methyl)-5-( 4- pentylphenyl)-2,3-dihydro-lH-pyrrol-2-yl)ethan-l-amine (84) (17 mg, 0.033 mmol) in MeOH (dry) (1 mL) NaBH ₃ CN (2.5 mg, 0.04 mmol, 1.2 equiv.) and HC1 (1 drop) was added. Stirred at r.t. for 16h. To the reaction mixture H ₂ 0 was added and extraction with EtOAc (3 5 mL) was performed. Combined organic layers were washed with brine, dried over Na ₂ S0 ₄ and evaporated under reduced pressure. Product was isolated as HC1 salt. Yield: 10 mg (56%).

Synthesis of aminoalkyltriazole 88 is shown in scheme 9

86 87 88

Scheme 9

Synthesis of intermediate 87

Intermediate 87 was synthesized according to general method E. Synthesis of product 88

To the solution of triazole 87 (40 mg, 0.073 mmol) in MeOH (1 niL) acetaldehyde (10 μΐ.) and NaBH ₃ CN (6.3 mg, 0.1 mmol, 1.5 equiv.) were added. Reaction mixture was stirred at r.t. for lh. The solvent was removed in vacuo and residue was purified by column

chromatography (CH ₂ Cl ₂ /MeOH=100:0 to 4:1). Yield: 15 mg (36%). All compounds were characterized by 1H-NMR and occasionally by ¹³ C-NMR spectroscopy performed on Varian Mercury spectrometer (400 MHz) with chemical shifts values (δ) in ppm relative to TMS using the residual chloroform signal as internal standard and by tandem LC/MS spectrometer on Water Acquity UPLC with SQ mass selective detector.

Physicochemical characterization of compounds 8.1-8.12, 33.1, 34.1-4, 35.1-2, 36.1-2, 48.1-6, 44, 47, 56.1-2, 63, 71-73, 84, 85, 88.

1H-NMR (TMS, CDCI3, 400 MHz)□□: 0.80 ft, 7.0 Hz, 3H); 1.1-1.3 (m, 4H); 1.53 (m, 2H); 2.12 (s, IH); 2.53 (t, 7.8 Hz, 2H); 3.56 (s, 2H); 3.68 (s, 211); 5.40 (s, 2H); 6.96 (d, 8.2 Hz, 2H); 7.04 (t, 3.9 Hz, IH); 7.11 (d, 8.2 Hz, 2H); 7.16 (d, 7.0 Hz, 2H); 7.20 (d, 7.0 Hz, 2H);

GL-1556 8.2

7.29 (dd, 4.7 and 7.8 Hz, IH); 7.38 (d, 8.2 Hz, 2H); 7.42 (d, 8.2 Hz, 2H); 7.76 (dt, 2.0 and 7.8 Hz, IH); 8.52 (dd, 1.6 and 4.7 Hz, IH); and 8.74 ppm (d, 2.0 Hz, IH).

UPLC/MS: [M+l] = 501.5

1H-NMR (TMS, CDCI3, 400 MHz)□□: 0.87 (t, 7.0 Hz, 3H); 1.2-1.4 (m, 4H); 1.60 (m, 2H); 2.60 (t, 7.8 Hz, 2H); 3.51 (s, IH); 3.59 (s, 2H); 3.71 (s, 2H); 3.78 (s, 6H); 5.66 (s, 2H); 6.39 (dd, 2.4 and 8.2

GL-1562 Hz, IH); 6.43 (d, 2.4 Hz, IH); 7.04 (dd, 5.1 and 8.2 Hz, 2H); 7.13 (s,

8.3

IH); 7.18 (d, 8.2 Hz, 2H); 7.37 (dd, 5.1 and 7.8 Hz, 2H); 7.43 (d, 8.2 Hz, 2H); 7.51 (d, 8.2 Hz, 2H); 7.85 (dt, 2.0 and 8.2 Hz, IH); 8.59 (dd, 1.6 and 5.5 Hz, IH); and 8.82 ppm (d, 2.0 Hz, IH).

UPLC/MS: [M+l] = 561.7

1H-NMR (TMS, CDC1 ₃ , 400 MHz)□□: 0.81 (t, 7.4 Hz, 3H); 1.2-1.3 (m, 4H); 1.5-1.6 (m, 2H); 2.12 (s, IH); 2.54 (t, 7.4 Hz, 2H); 3.38 (s, 2H); 4.07 (d, 4.3 Hz, 2H); 5.33 (s, 2H); 6.47 (d, 7.8 Hz, 2H); 6.67 (t, 7.4 Hz, IH); 7.02 (d, 8.2 Hz, 2H); 7.07 (d, 7.4 Hz, 2H); 7.09 (d, 7.4

GL-1570 8.4 Hz, 2H); 7.13 (d, 7.4 Hz, 2H); 7.14 (s, IH); 7.31 (ddd, 0.8 and 4.7 and 7.8 Hz, IH); 7.40 (d, 8.2 Hz, 2H); 7.48 (d, 8.2 Hz, 2H); 7.79 (dt, 2.0 and 8.2 Hz, IH); 8.54 (dd, 1.6 and 5.1 Hz, IH); and 8.77 ppm (d, 2.0 Hz, IH).

UPLC/MS: [M+l] = 487.5

1H-NMR (TMS, DMSO-d ₆ , 400 MHz)□□: 0.5-1.0 (m, 2H); 0.83 (t, 7.0 Hz, 3H); 1.0-1.4 (m, 8H); 1.4-1.7 (m, 7H); 2.26 (d, 6.7 Hz, 4H);

GL-1634 2.5-2.7 (m, 2H); 3.53 (s, 2H); 5.48 (s, 2H); 6.97 (s, IH); 7.04 (d, 8.2

8.5

Hz, 2H); 7.22 (d, 8.2 Hz, 2H); 7.42 (d, 8.2 Hz, 2H); 7.45 (dd, 4.7 and 7.8 Hz, IH); 7.69 (d, 8.2 Hz, 2H); 8.03 (dt, 1.8 and 8.2 Hz, IH); 8.55 (dd, 1.6 and 4.7 Hz, IH); and 8.85 ppm (d, 2.3 Hz, I H). UPLC/MS: [M+l] = 507.5

1H-NMR (TMS, CDC1 ₃ , 400 MHz)□□: 0.87 (t, 7.0 Hz, 3H); 1.37 - 1.21 (m, 4H); 1.5-1.7 (m, 2H); 2.60 (t, 7.8 Hz, 2H); 3.63 (s, 2H); 3.73 (s, 2H); 3.76 (s, 3H); 5.46 (s, 2H); 6.7 6.9 (m, 3H); 7.04 (d, 8.4 Hz, 2H); 7.11 (s, IH); 7.18 (d, 8.3 Hz, 3H); 7.36 (ddd, 0.8 and 4.8 and

GL-1643 8.6

7.9 Hz, IH); 7.45 (d, 8.2 Hz, 2H); 7.50 (d, 8.3 Hz, 2H); 7.83 (ddd, 1.7 and 2.3 and 7.9 Hz, IH); 8.59 (dd, 4.8 and 1.6 Hz, IH) and 8.81 ppm (dd, 0.8 and 2.4 Hz, IH).

UPLC/MS: [M+l] = 531.6

1H-NMR (TMS, CDC1 ₃ , 400 MHz)□□: 0.88 (t, 7.0 Hz, 3H); 1.2-1.4 (m, 7H); 1.5-1.7 (m, 2H); 2.61 (t, 7.8 Hz, 2H); 3.48 (dd, 14.1 and 42.3, 2H); 3.73 (q, 6.7 Hz, IH); 5.42 (dd, 3.5 and 16.8, 2H); 6.98 (d,

GL-1686 8.7 8.2 Hz, 2H); 7.09 (s, IH); 7.19 (d, 8.2 Hz, 2H); 7.2-7.3 (m, 4H); 7.37

(dd, 4.7 and 7.8 Hz, IH); 7.47 (t, 8.2 Hz, 4H); 7.83 (dt, 1.6 and 7.8 Hz, IH); 8.60 (s, IH) and 8.82 ppm (s, IH).

UPLC/MS: [M+l] = 515.6

1H-NMR (TMS, CDC1 ₃ , 400 MHz)□□: 0.87 (t, 7.0 Hz, 3H); 1.2-1.4 (m, 4H); 1.5-1.7 (m, 2H); 2.60 (t, 7.8 Hz, 2H); 3.66 (s, 2H); 3.83 (s, 2H); 5.45 (s, 2H); 6.99 (d, 8.2 Hz, 2H); 7.13 (s, IH); 7.17 (d, 8.2 Hz,

GL-1726 8.8

2H); 7.2-7.6 (m, 14H); 7.76 (dt, 2.3 and 7.8 Hz, IH); 8.77 (dd, 1.6 and 4.7 Hz, IH) and 8.77 (dd, 0.8 and 2.3 Hz, IH).

UPLC/MS: [M+l] = 577.6

1H-NMR (TMS, DMSO-d ₆ , 400 MHz)□□: 0.78 (d, 6.7 Hz, 6H); 0.83 (t, 7.0 Hz, 3H); 1.1-1.3 (m, 6H); 1.4-1.6 (m, 3H); 2.43 (t, 7.4 Hz, 2H); 2.56 (t, 7.4 Hz, 2H); 3.54 (s, 2H); 5.47 (s, 2H); 6.97 (s, IH);

GL-1638 8.9 7.04 (d, 8.2 Hz, 2H); 7.21 (d, 8.2 Hz, 2H); 7.42 (d, 8.2 Hz, 2H); 7.46

(dd, 4.7 and 7.8 Hz, IH); 7.69 (d, 8.2 Hz, 2H); 8.04 (dt, 1.6 and 8.2 Hz, IH); 8.55 (dd, 1.6 and 4.7 Hz, IH); and 8.85 ppm (d, 2.3 Hz, IH)

UPLC/MS: [M+l] = 481.3

GL-1566 8.10 Ή-NMR (TMS, CDCI3, 400 MHz)□□: 0.87 (t, 7.4 Hz, 3H); 1.2-1.4 (m, 4H); 1.5-1.7 (m, 2H); 2.60 (t, 7.8 Hz, 2H); 3.73 (s, 2H); 5.42 (s, 2H); 7.01 (s, IH); 7.12 (d, 8.2 Hz, 2H); 7.18 (d, 8.2 Hz, 2H); 7.3-7.4 (m, IH); 7.44 (d, 8.2 Hz, 2H); 7.55 id, 8.2 Hz, 2H); 7.85 (dt, 2.0 and 8.2 Hz, IH); 8.59 (dd, 1.6 and 4.7 Hz, I II); and 8.83 ppm (dd, 0.8 and 2.4 Hz, IH).

UPLC/MS: [M+l] = 411.6

1H-NMR (TMS, CDC1 ₃ , 400 MHz)□□: 0.79 (d, 6.3 Hz, 12H); 0.87 (t, 7.0 Hz, 3H); 1.1-1.3 (m, 4H); 1.3-1.4 (m, 4H); 1.4-1.5 (m, 2H); 1.5-1.7 (m, 2H); 2.37 (t, 7.8 Hz, 4H); 2.59 (t, 7.8 Hz, 2H); 3.33 (s, 2H); 5.55 (s, 2H); 7.05 (d, 8.2 Hz, 2H); 7.07 (s, IH); 7.17 (d, 8.2 Hz,

GL-1625 8.11

2H); 7.37 (dd, 4.7 and 7.8 Hz, IH); 7.43 (d, 8.2 Hz, 2H); 7.54 (d, 8.2 Hz, 2H); 7.85 (dt, 2.0 and 8.2 Hz, IH); 8.59 (dd, 1.2 and 4.7 Hz, IH); and 8.83 ppm (d, 2.0 Hz, IH).

UPLC/MS: [M+l] = 551.6

1H-NMR (TMS, DMSO-d ₆ , 400 MHz)□□: 0.6-0.9 (m, 3H); 1.0-1.3 (m, 6H); 1.38 (s, 6H); 1.7-2.1 (m, 2H); 5.96 (s, 2H); 6.90 (d, 8.3 Hz,

GL-1639

2H); 6.93 (s, IH); 7.11 (d, 8.2 Hz, 2H); 7.36 (d, 8.2 Hz, 2H); 7.44

8.12

(dd, 8.0 and 4.8 Hz, IH); 7.60 (d, 8.3 Hz, 2H); 8.03-7.98 (m, IH); 8.53 (dd, 4.7 and 1.5 Hz, IH) and 8.83 ppm (d, 2.4 Hz, IH).

UPLC/MS: [M+l] = 439.6

1H-NMR (DMSO-d ₆ ) δ: 8.53-8.41 (3H, m) 7.55-7.49 (2H, m) 7.45- 7.40 (2H, m) 7.38-7.33 (2H, m) 7.32-7.27 (2H, m) 7.02-6.97 (2H, m) 6.97-6.93 (2H, m) 4.24 (2H, s) 4.13 (2H, q, 7=5.6 Hz) 3.78 (3H, s) 2.65 (2H, t, 7=7.5 Hz) 1.59 (2H, quintet, 7=7.3 Hz) 1.36-1.21 (4H, m) 0.85 (3H, t, 7=7.0 Hz)

LV-1136 33.1

¹³ C- MR (100 MHz, CDC1 ₃ ) δ: 159.0, 144.7, 138.9, 136.3, 134.7, 133.6, 133.0, 129.0, 128.6, 127.9, 126.7, 125.6, 114.1, 55.3, 35.5, 31.4, 30.9, 22.4, 14.0

HRMS: Calculated for C28H33N40: 441.2654 Found: 441.2659 [M+H] ⁺

Ή-NMR (CDCI ₃ ) δ: 7.51-7.44 (2H, m) 7.43-7.38 (2H, m) 7.25-7.18

LV-1087 34 _* Ϊ

(4H, m) 7.00-6.93 (4H, m) 4.04 (2H, s) 3.84 (3H, s) 2.91-2.83 (2H, m) 2.79-2.71 (2H, m) 2.64 (2H, t, 7=7.8 Hz) 2.59-2.33 (6H, m) 2.28 (3H, s) 2.10-1.72 (2H, m) 1.62 (2H, quintet, 7=7.4 Hz) 1.39-1.23 (4H, m) 0.89 (3H, t, 7=7.0 Hz)

ESI/MS: [M+l] = 538

Ή-NMR (DMSO-d ₆ ) δ: 11.24-1 1.07 (1H, m) 7.55-7.49 (2H, m) 7.47-7.41 (2H, m) 7.39-7.30 (4H, m) 7.01-6.97 (2H, m) 6.97-6.92 (2H, m) 4.16 (2H, s) 3.97 (2H, dd, 7=12.1, 2.2 Hz) 3.84-3.78 (2H, m)

LV-1154 34.2 3.78 (3H, s) 3.54-3.47 (2H, m) 3.47-3.38 (2H, m) 3.24-3.16 (2H, m)

3.16-3.04 (2H, m) 2.65 (2H, t, 7=7.7 Hz) 1.59 (2H, quintet, 7=7.4 Hz) 1.36-1.20 (4H, m) 0.85 (3H, t, 7=7.0 Hz)

ESI-MS (m/z): 525 [M+H] ⁺ .

1H-NMR (CDC1 ₃ ) δ: 7.49-7.44 (2H, m) 7.42-7.38 (2H, m) 7.24-7.22 (4H, m) 6.98-6.91 (4H, m) 4.03 (2H, s) 3.84 (3H, s) 3.34 (2H, t, 7=6.4 Hz) 2.99 (2H, t, 7=6.4 Hz) 2.64 (2H, t, 7=7.7 Hz) 1.62 (2H, quintet, 7=7.3 Hz) 0.89 (3H, t, 7=7.0 Hz)

LV-1155 34.3 ¹³ C- NMR (100 MHz, CDC1 ₃ ) δ: 159.2, 144.8, 134.9, 133.2, 132.8,

129.3, 128.3, 127.9, 126.9, 125.3, 114.2, 55.3, 47.3, 43.3, 35.5, 31.3, 30.9, 22.5, 14.0

HRMS: Calculated for C31H39N40: 483.3124 Found: 483.3119 [M+H] ⁺

1H-NMR (CDCI ₃ ) δ: 7.49-7.44 (2H, m) 7.42-7.38 (2H, m) 7.24-7.22 (4H, m) 6.98-6.91 (4H, m) 4.03 (2H, s) 3.84 (3H, s) 3.34 (2H, t, 7=6.4 Hz) 2.99 (2H, t, 7=6.4 Hz) 2.64 (2H, t, 7=7.7 Hz) 1.62 (2H, quintet, 7=7.3 Hz) 0.89 (3H, t, 7=7.0 Hz)

LV-1157 34.4 ¹³ C-NMR (CDC1 ₃ ) δ: 159.1, 144.8, 139.2, 134.7, 134.2, 133.7, 132.8,

129.2, 128.4, 127.9, 126.8, 125.4, 114.2, 55.3, 35.5, 31.4, 30.9, 22.4, 14.0

HRMS: Calculated for C29H35N40: 455.2811 Found: 455.2806 [M+H] ⁺

1H-NMR (CDCI3) δ: 7.51-7.46 (2H, m) 7.46-7.41 (2H, m) 7.30-7.26 (4H, m) 7.01-6.92 (4H, m) 4.06 (2H, m) 3.84 (3H, s) 3.06-2.93 (6H,

LV-1 130 m) 2.71 (2H, t, 7=7.0 Hz) 2.66 (2H, t, 7=7.8 Hz) 2.28-2.17 (2H, m)

1.64 (2H, quintet, 7=7.4 Hz) 1.36-1.26 (9H, m) 0.89 (3H, t, 7=7.0 Hz) ESI/MS (m/z): 525 [M+H] ⁺ .

1H-NMR (CDC1 ₃ ) δ: 7.50-7.46 (2H, m) 7.45-7.40 (2H, m) 7.30-7.25 (4H, m, overlapped with CDC1 ₃ ) 7.01-6.93 (4H, m) 4.06 (2H, s) 3.84

LV-1124 (3H, s) 3.06 (2H, t, .7=8.0 Hz) 2.74 (2H, t, 7=7.0 Hz) 2.66 (2H, t,

7=7.8 Hz) 2.30-2.19 (2H, m) 2.13-1.97 (4H, m) 1.64 (2H, quintet, 7=7.6 Hz) 1.40-1.27 (4H, m) 0.89 (3H, t, 7=7.0 Hz)

ESI/MS (m/z): 523 [M+H] ⁺ .

1H-NMR (CD ₃ OD- I4) δ: 7.45-7.40 (2H, m) 7.40-7.33 (3H, m) 7.32- 7.17 (4H, m) 7.13-7.07 (1H, m) 7.04-6.97 (2H, m) 6.84-6.77 (2H, m) 4.57 (1H, d, 7=15.2 Hz) 4.31 (1H, d, 7=15.2 Hz) 4.15 (2H, s) 3.87-

KD-302 36.1 3.72 (1H, m) 3.62-3.46 (2H, m) 3.46-3.34 (1H, m) 3.25-3.15 (2H, m)

3.14-3.01 (2H, m) 2.68 (2H, t, 7=7.6 Hz) 1.64 (2H, quintet, J=7.2 Hz) 1.39-1.29 (4H, m) 0.88 (3H, t, 7=7.0 Hz)

ESI/MS (m/z): 557 [M+H] ⁺ .

1H-NMR (CDCI ₃ ) δ: 7.31-7.26 (4H, m) 7.25-7.19 (4H, m) 4.04 (2H, s) 3.10-2.94 (4H, m) 2.86-2.72 (4H, m) 2.65 (2H, t, 7=7.7 Hz) 1.62 (2H, quintet, 7=7.5 Hz) 1.40-1.27 (4H, m) 1.13 (6H, t, 7=7.1 Hz)

VGP-153 36.2 0.89 (3H, t, 7=7.0 Hz)

¹³ C-NMR (CDCI ₃ ) δ: 156.5, 144.8, 139.5, 134.8, 133.9, 131.8, 129.3, 128.2, 127.9, 126.7, 125.3, 116.0, 51.7, 46.3, 35.5, 31.3, 30.9, 28.2, 22.5, 14.0

ESI/MS (m/z): 497 [M+H] ⁺ .

1H-NMR (CDC13) δ: 7.56-7.51 (2H, m) 7.48-7.43 (2H, m) 7.43-7.39 (2H, m) 7.37-7.31 (1H, m) 7.25-7.21 (4H, m) 7.03-6.98 (2H, m) 4.08 (2H, s) 2.91 (2H, s) 2.71-2.59 (6H, m) 1.63 (2H, quintet, J=7.5 Hz)

KD-304 48.1 1.40-1.26 (4H, m) 1.06 (6H, t, J=7.2 Hz) 0.89 (3H, t, J=7.0 Hz)

13C-NMR (100 MHz, CDC13) δ: 144.7, 140.4, 139.8, 136.0, 134.1, 132.8, 129.3, 128.8, 128.4, 127.4, 127.3, 126.9, 125.3, 52.0, 46.8, 35.5, 31.3, 30.9, 28.3, 22.4, 14.0, 11.2

LC/MS: 481 [M+H]+

1H-NMR (CDCI ₃ ) δ: 12.38-12.22 (1H, m) 7.49-7.42 (6H, m) 7.34-

KD-307 48.2 7.26 (4H, m) 7.05-7.00 (2H, m) 4.22 (2H, s) 3.48-3.32 (4H, m) 3.22- 3.03 (4H, m) 2.68 (2H, t, 7=7.7 Hz) 1.71-1.59 (2H, m) 1.41-1.30 (10H, m) 0.90 (3H, t, 7=7.0 Hz) °C-NMR (100 MHz, CDC1 ₃ ) δ: 150.4, 144.8, 139.6, 137.5, 135.6, 134.0, 129.3, 128.4, 127.2, 126.5, 125.7, 125.3, 62.8, 51.7, 46.8, 35.5, 34.5, 31.3, 30.9, 29.8, 28.3, 22.4, 14.0

ESI/MS: 537 [M+H] ⁺ .

Ή-NMR (CDC1 ₃ ) δ: 7.45-7.40 (2H, m) 7.29-7.22 (4H, m, overlapped with CDCI ₃ ) 7.09 (1H, d, 7=8.3, 2.1 Hz) 7.05 (IH, d, 7=2.1 Hz) 7.02- 6.98 (2H, m) 6.92 (1H, d, 7=8.3 Hz) 4.09 (2H, s) 3.93 (3H, s) 3.91 (3H, s) 3.07-2.95 (4H, m) 2.87-2.70 (4H, m) 2.66 (2H, t, 7=7.7 Hz)

KD-329 48.4 1.63 (2H, quintet, 7=7.5 Hz) 1.39-1.26 (4H, m) 1.14 (6H, t, 7=7.0

Hz) 0.89 (3H, t, 7=7.0 Hz)

¹³ C-NMR (100 MHz, CDC1 ₃ ) δ: 149.2, 148.7, 144.8, 139.6, 135.5, 134.0, 133.4, 129.4, 128.4, 127.1, 125.3, 119.2, 111.5, 110.2, 56.0, 55.9, 46.7, 35.5, 31.3, 30.9, 28.3, 22.5, 14.0

ESI/MS: 541 [M+H] ⁺ .

1H-NMR (CDCI ₃ ) δ: 7.50-7.45 (2H, m) 7.44-7.40 (2H, m) 7.29-7.24 (4H, m, overlapped with CDC1 ₃ ) 7.01-6.93 (4H, m) 4.08 (2H, s) 3.90-3.85 (4H, m) 3.24-3.16 (4H, m) 3.10-2.97 (4H, m) 2.87-2.74 (4H, m) 2.66 (2H, t, 7=7.7 Hz) 1.63 (2H, quintet, 7=7.4 Hz) 1.40-

KD-330 48.5 1.28 (4H, m) 1.15 (6H, t, 7=7.0 Hz) 0.89 (3H, t, 7=7.0 Hz)

1 ³ C-NMR (100 MHz, CDC1 ₃ ) δ: 150.6, 144.8, 139.4, 135.0, 134.0, 131.7, 129.3, 128.4, 127.5, 126.7, 125.3, 115.7, 66.8, 49.1, 46.7, 35.5, 31.3, 30.9, 28.3, 22.5, 14.2, 14.0

ESI/MS: 566 [M+H] ⁺ .

1H-NMR (CDC1 ₃ ) δ: 7.36-7.32 (2H, m) 7.27-7.23 (2H, m, overlapped with CDCI ₃ ) 7.20-7.15 (2H, m) 6.84-6.78 (2H, m) 4.02 (2H, s) 3.17-

KD-294 44 2.90 (4H, m) 2.89-2.70 (4H, m) 2.66 (2H, t, 7=7.7 Hz) 1.63 (2H, quintet, 7=7.6 Hz) 1.40-1.27 (4H, m) 1.24-1.07 (6H, m) 0.90 (3H, t, 7=7.0 Hz)

ESI/MS: 484 [M+H] ⁺ .

1H-NMR (CD ₃ OD-d ₄ ) δ: 7.85-7.80 (2H, m) 7.59-7.53 (2H, m) 7.48- 7.42 (2H, m) 7.27-7.22 (2H, m) 7.22-7.18 (2H, m) 6.99-6.93 (2H, m)

KD-315 48.6 4.06 (2H, s) 2.78-2.66 (4H, m) 2.59 (2H, t, 7=7.7 Hz) 2.51 (4H, q,

7=7.2 Hz) 1.55 (2H, t, 7=7.2 Hz) 1.31-1.17 (4H, m) 0.93 (6H, t, 7=7.2 Hz) 0.80 (3H, t, 7=7.0 Hz) °C-NMR (100 MHz, CD3OD- I4) δ: 170.5, 145.2, 143.7, 143.2, 138.5, 136.8, 133.8, 133.8, 132.3, 129.2, 128.5, 127.9, 127.0, 126.4, 125.2, 51.4, 46.3, 35.0, 31.0, 30.8, 27.6, 22.1, 21.2, 12.9, 9.9

ESI/MS: 524 [M+H .

1H-NMR (400 MHz, CDCI3) δ: 8.83-8.76 (IH, m) 8.57 (IH, d, 7=3.5 Hz) 7.86-7.79 (IH, m) 7.43 (2H, d, 7=8.2 Hz) 7.35 (IH, d, 7=5.5, 7.7 Hz) 7.26-7.16 (4H, m) 7.07 (2H, d, 7=8.2 Hz) 4.20 (2H, s) 3.75 (2H,

KD-263 47 s) 2.63 (4H, q, 7=7.0 Hz) 2.40 (3H, s) 1.06 (6H, t, 7=7.0 Hz)

1 ³ C-NMR (100 Hz, CDCI3) δ: 148.4, 148.1, 139.7, 137.2, 136.1, 134.1, 133.9, 129.9, 128.9, 127.2, 125.3, 123.5, 62.7, 48.0, 46.6, 29.9, 28.3, 21.2, 11.4

ESI/MS: 412 [M+H] ⁺ .

1H-NMR (400 MHz, CDC1 ₃ ) δ: 13.05 (s, IH), 7.51-7.41 (m, 6H), 7.23-7.21 (m, IH), 7.15-7.09 (m, 2H), 6.98-6.92 (m, 2H), 4.16 (s, 4H), 3.83 (s, 3H), 3.15-3.05 (m, 4H), 2.62 (t, 2H, 7=7.7 Hz), 1.65- 1.55 (m, 3H), 1.46 (t, 6H, 7=7.2 Hz), 1.34-1.27 (m, 4H), 0.87 (t, 3H,

KD-199 56.1 7= 6.8 Hz).

¹³ C- MR (100 Hz, CDCI3) δ: 163.9, 159.2, 158.0, 145.4, 139.5, 136.0, 132.8, 129.0, 128.5, 128.2, 127.9, 127.1, 125.1, 119.8, 114.2, 55.3, 47.9, 43.7, 35.7, 31.4, 30.8, 27.5, 22.5, 14.0, 9.3 .

ESI/MS (m/z): 497 [M+H] ⁺ .

1H-NMR (400 MHz, CDC1 ₃ ) δ: 7.44-7.40 (m, IH), 7.38-7.34 (m, IH), 7.18-7.16 (m, IH), 7.14-7.08 (m, IH), 7.04-6.98 (m, IH), 3.89

KD-233 56.2 (s, 2H), 3.64 (s, 2H), 2.57-2.44 (m, 6H), 1.54 (quintet, 2H, 7=7.7 Hz),

1.29- 1.21 (m, 4H), 1.18 (m, 2H), 0.99-0.91 (m, 6H), 0.81 (t, 3H, 7= 6.8 Hz).

ESI/MS (m/z): 473 [M+H] ⁺ .

1H-NMR (400 MHz, CDC1 ₃ ) δ: 7.52-7.48 (2H, m) 7.47-7.43 (2H, m) 7.26-7.22 (2H, m) 7.13-7.08 (2H, m) 6.98-6.94 (2H, m) 6.94-6.90 (2H, m) 6.20 (IH, d, 7=3.5 Hz) 6.14 (IH, d, 7=3.5 Hz) 5.44 (2H, s)

LV-1341 63 3.85 (3H, s) 3.35 (2H, s) 2.57 (2H, t, 7=7.8 Hz) 2.48 (2H, q, 7=7.1

Hz) 1.60 (2H, quintet, 7=7.6 Hz) 1.38-1.25 (4H, m) 0.92 (3H, t, 7=7.1 Hz, 0.88 (3H, t, 7=7.0 Hz)

ESI/MS: 495 [M+Hf. Ή-NMR (400 MHz, CDC1 ₃ ) 6: 7.10-6.98 (6H, m) 6.97-6.88 (6H, m) 5.36 (2H, s) 3.83 (3H, s) 2.88-2.72 (4H, m) 2.65-2.45 (6H, m) 1.56

IN- 1402 71 (2H, quintet, 7=7.4 Hz) 1.39-1.17 (4H, m) 1.07-0.91 (6H, m) 0.87

(3H, t, 7=6.9 Hz)

ESI/MS: 527 [M+H] ⁺ .

Ή-NMR (400 MHz, CDC1 ₃ ) δ: 7.86-7.80 (2H, m) 7.80-7.75 (2H, m) 7.31-7.24 (2H, m, overlapped with CDC1 ₃ ) 7.09-7.02 (2H, m) 7.02- 6.95 (2H, m) 6.95-6.89 (2H, m) 5.41 (2H, s) 3.90 (3H, s) 2.89-2.78

LV-1417 72

(4H, m) 2.63-2.47 (6H, m) 1.55 (2H, quintet, 7=7.4 Hz) 1.38-1.18 (4H, m) 0.98 (6H, t, 7=7.1 Hz), 0.86 (3H, t, 7=6.9 Hz)

ESI/MS: 539 [M+H] ⁺ .

1H-NMR (400 MHz, CDC1 ₃ ) δ: 7.45-7.39 (2H, m) 7.31-7.24 (2H, m, overlapped with CDC1 ₃ ) 7.14-7.08 (2H, m) 7.05-6.98 (2H, m) 6.92-

LV-1424 6.85 (4H, m) 5.84 (IH, s) 5.35 (2H, s) 3.81 (3H, s) 2.84-2.72 (4H, m)

73

2.59-2.45 (6H, m) 1.55 (2H, quintet, 7=7.5 Hz) 1.39-1.18 (4H, m) 0.95 (6H, t, 7=7.1 Hz) 0.87 (3H, t, 7=7.0 Hz)

ESI/MS: 541 [M+Hf.

1H-NMR (400 MHz, CDC1 ₃ ) 6: 7.69-7.64 (2H, m) 7.50-7.45 (2H, m) 7.40-7.34 (2H, m) 7.24-7.17 (4H, m) 6.97-6.91 (2H, m) 3.83 (3H, s) 3.07 (IH, d, 7=13.4 Hz) 2.88 (IH, d, 7=13.4 Hz) 2.80-2.70 (IH, m)

LV-1528 84 2.69-2.44 (7H, m) 2.32-2.21 (IH, m) 2.08-1.76 (4H, m) 1.63 (2H, quintet, 7=7.4 Hz) 1.40-1.27 (4H, m) 1.10-0.98 (6H, m) 0.90 (3H, t, 7=6.9 Hz)

ESI/MS: 511 [M+H] ⁺ .

1H-NMR (400 MHz, CDC1 ₃ ) δ: 7.62-7.46 (4H, m) 7.45-7.37 (2H, m) 7.30-7.22 (2H, m, overlapped with CDC1 ₃ ) 7.17-7.09 (2H, m) 7.03- 6.94 (2H, m) 5.18-5.01 (IH, m) 4.24-4.04 (IH, m) 3.86 (3H, s) 3.52-

LV-1532 85 3.14 (4H, m) 3.13-2.92 (IH, m) 2.84-2.65 (IH, m) 2.60-2.38 (4H, m)

2.38-2.16 (IH, m) 2.11-1.87 (IH, m) 1.63-1.47 (2H, m) 1.47-1.19 (10H, m) 0.90-0.83 (3H, m).

ESI/MS: 513 [M+H] ⁺ .

Ή-NMR (400 MHz, CDCI ₃ ) δ: 8.50 (IH, d, 7=1.4 Hz) 8.45 (I H, dd,

LV-1570 88 7=4.8, 1.4 Hz) 7.64-7.58 (IH, m) 7.49-7.44 (2H, m) 7.42-7.37 (2H,

m) 7.25-7.21 (4H, m) 7.21-7.15 (IH, m) 6.99-6.94 (2H, m) 6.94-6.89 (2H, m) 3.97 (2H, s) 3.84 (3H, s) 3.60 (2H, s) 2.87-2.80 (4H, m) 2.64 (2H, t, 7=7.7 Hz) 2.56 (2H, q, 7=7.1 Hz) 1.62 (2H, quintet, 7=7.3 Hz) 1.40-1.23 (4H, m) 1.02 (3H, t, 7=7.1 Hz) 0.89 (3H, t, 7=6.9 Hz) ESI/MS: 574 [M+H] ⁺ .

In vitro Assay

Fluorescence resonance energy transfer (FRET) assay was performed to evaluate ability of compounds to inhibit plasmepsin II Solution of compounds for testing (concentration 0.01- lOOuM) on 96 well plate were added to the enzyme (Plasmepsin II) in buffer (0.1 M NaOAc, pH = 4.5, 10% glycerin). The mixture was incubated for 30 min at 37°C. Substrate (DABCYL-Glu-Arg-Nle-Phe-Leu-Ser-Phe-Pro-EDANS, AnaSpec Inc) was added to reach final concentration 5 uM. Hydrolysis of substrate was detected as an increase in fluorescence (Em 490 nm, Ex 336 nm) at 37°C. Compounds were tested in three repeated triplicate experiments.

IC50 values for selected inhibitors on plasmepsins II:

11. 'GL-1625 8.11 1,3

12. GL-1639 8.12 4,1

13. LV-1136 33.1 2,8

14. LV-1087 34.1 1,8

15. LV-1154 34.2 2,9

16. LV-1155 34.3 3

17. LV-1157 34.4 6,5

18. LV-1170 34.5 2,2

19. LV-1077 34.6 0,6

20. LV-1130 35.1 2,7

21. LV-1124 35.2 2,9

22. KD-302 36.1 0.38

23. VGP-153 36.2 1,2

24. KD-294 44 3,5

25. KD-263 47 i.a

26. KD-304 48.1 1,8

27. KD-307 48.2 2,8

28. KD-309 48.3 1,6

29. KD-329 48.4 1,9

30. KD-330 48.5 1,2

31. KD-315 48.6 0,78

32. KD-199 56.1 10

33. KD-233 56.2 13 34. LV-1341 63 10.0

35. LV-1402 71 8.0

36. LV- 1417 72 15

37. LV-1528 84 4.5

38. LV-1532 85 5.7

39. LV-1570 88 2.2

i.a. - inactive at the concentration below 200 uM; n.d. - not determined