US 2006O154325A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2006/0154325 A1 Bogyo et al. (43) Pub. Date: Jul. 13, 2006 (54) SYNTHESIS OF EPOXIDE BASED Publication Classification INHIBITORS OF CYSTEINE PROTEASES (51) Int. Cl. CI2O I/37 (2006.01) (76) Inventors: Matthew Bogyo, Redwood City, CA C07K 7/08 (2006.01) (US); Steven H.L. Verhelst, Stanford, C07D 303/08 (2006.01) CA (US) (52) U.S. Cl. ............................. 435/23: 530/402; 549/551 Correspondence Address: (57) ABSTRACT PETERS VERNY JONES & SCHMITT, L.L.P. 425 SHERMANAVENUE SUTE 230 Epoxide based cysteine protease inhibitors containing pep PALO ALTO, CA 94.306 (US) tide derivatives and methods for synthesizing them in sold phase are disclosed. Preferably, an epoxy succinyl “war head' (for binding to the enzyme) is prepared, having two (21) Appl. No.: 11/329,818 amino acid residues or residue-like structures on either side. Natural and non-natural amino acids may be used. The present process may be carried out entirely on a solid (22) Filed: Jan. 10, 2006 Support, with the proviso that a dipeptide like group is prepared prior to coupling to one side of the Supported Related U.S. Application Data complex. The method lends itself to more efficient inhibitor synthesis, and may be employed with mixtures of peptide (60) Provisional application No. 60/642,891, filed on Jan. compounds, and various modifications of epoxides to create 10, 2005. diverse libraries of inhibitors. Fmoc-NH-AA-OH 1) 20%diorari Piperdine all- AA A 1 0 2 O N-Fmoc di H 1 AA if m RNK Tentage Resin Step 1 2)) Fmoc-NH-AA-OH2 2 1) 20% Piperdine Step 2 Step 3 2) epoxide-NP ester NaOH (): N AA 1 N o al- S H AA O H O O S1NÓ H A A. () OH Step 4 is us N ---N-(i- O Et O A Ao NHR HO-R Step 6 AA o H A A 1 H o 1N N 1R N - NN O 1 NR Ó H A A. () H Ö A.A. () Step 7 Step 8 TFAIHOITIS TFAIHOITIS AA H o AA 1 o "2N Ó - H A A.N () -si-'-RH "',O '-s'A AO or Patent Application Publication Jul. 13, 2006 Sheet 1 of 5 US 2006/0154325 A1 Fig. 1 Fmoc-NH-AA-OH 1) 20%of Piperdineorarr; d) H d H AA RNK Tentage Resin 2) Fmoc-NH-AA-OH 2 1) 20% Piperdine Step 2 Step 3 2) epoxide-NP ester A A O O NaOH 1sloe AAO AA 10 H o N S. SNN 1s-orr1a. A AO Step 7 TFAIHOITIS TFAIHOITIS AAC H o AA H o HN - N ana HN S N-N-si-'o ~ a'sO A AO stra O 1. A A2 O O 1 R Patent Application Publication Jul. 13, 2006 Sheet 2 of 5 US 2006/0154325 A1 Fig. 2 20% Piperadine Patent Application Publication Jul. 13, 2006 Sheet 4 of 5 US 2006/0154325 A1 Fig. 4 Probe JPM ACSV4ACSV5 ACSW4ACSW6 TRSV4 TRSV5 OE Conc (M) 04 2 10 04 2 to Patent Application Publication Jul. 13, 2006 Sheet 5 of 5 US 2006/0154325 A1 Fig. 5 lane Cat Z Cat B catcCath US 2006/0154325 A1 Jul. 13, 2006 SYNTHESIS OF EPOXDE BASED INHIBITORS OF 0.011) Joyce et al. (Cancer Cell, 2004, 5, 443-453) have CYSTEINE PROTEASES shown the importance of the cysteine cathepsin proteases in a number of processes related to cancer progression. These CROSS-REFERENCE TO RELATED processes include tumor growth, angiogenesis, and metasta APPLICATIONS sis. This article discloses that a general, covalent inhibitor, 0001. This application claims priority to provisional which specifically targets the cathepsin protease family can patent application Ser. No. 60/642,891, filed Jan. 10.2005, be administered systemically to transgenic mice that develop entitled “Synthesis of Epoxide Based Inhibitors of Cysteine a multi-stage form of pancreatic cancer and lead to reduced Protease,” and hereby incorporated by reference. tumor burden, metastasis and angiogenesis. STATEMENT OF GOVERNMENTAL SUPPORT 0012 Specifically, Joyce et al. disclose the inhibition of six cathepsin proteases by compounds DCG-04, 530/550 0002 This invention was made during work supported by Biodipy DCG-04, and JPM-OEt. These general covalent U.S. National Institutes of Health under grant U54 epoxide based inhibitors showed little or no general toxicity RR020843. The government has certain rights in this inven when administered to normal mice over a period of several tion. weeks. This work (including work by the present inventors) Suggests that cathepsin cysteine proteases represent a valid REFERENCE TO SEQUENCE LISTING OR COMPACT DISK target for anti-cancer therapeutics. 0003) None. 0013 The general epoxide scaffold for JPM-OEt is based on the natural product E-64, which was discovered to be a BACKGROUND OF THE INVENTION natural product inhibitor of cysteine protease in 1978 (Hanada, K. et al. Agric. Biol. Chem. 1978 42, 523-528 and 0004) 1. Field of the Invention 529-536). 0005 The present invention relates to the synthesis of organic molecules useful in the labeling or inhibition of cysteine proteases. HO O H O 0006 2. Related Art H N 0007 Cysteine proteases are proteolytic enzymes, which N w OEt utilize a cysteine residue for their catalytic activity. They can H be grouped in at least 30 protein families. Each family O H contains proteins with similar amino acid sequences and evolutionarily conserved sequence motifs, which are reflected in the family members similar 3D structures. JPM-OEt 0008 Proteases in Family C1 (the papain family) include O H O mammalian enzymes such as cathepsins B and L, which are H H O thought to be involved in cancer growth and metastasis. HN NS-1a1n N N w '' OH Cathepsin K is considered to be involved in bone degrada H 2. tion an osteoporosis. Family C1 also includes parasitic NH O H enzymes being essential for the parasite-host interaction (e.g. cruzipain from Trypanosoma cruzi—ausing Chagas disease, and falcipain from Plasmodium falciparum-caus E-64 ing malaria). 0009 Enzymes belonging to Family C13 (the legumain family) have been shown to play key roles in antigen 0014) The related compound JPM-565 is identical to presentation. Interleukin converting enzyme (ICE) and other JPM-OEt except that the ethyl ester is converted to the free enzymes belonging to Family C14 (the caspase family) have carboxylic acid. gained much interest recently, as key mediators of apoptosis. Many proteases of pathogenic bacteria are virulence factors 0015 These classes of compounds (i.e. having a succinyl and cause severe problems for the host at infections, such as epoxy component) have been used as cysteine protease gingipains (Family C25) of Porhyromonas gingivalis in inhibitors since 1978. A number of research groups have periodontitis and streptopain (Family C10) from Streptococ synthesized analogs of the general epoxide structure over the CuS pyogenes. past 20+ years and the crystal structure of E-64 bound to 0010 Proteolysis controls a number of essential biologi various cysteine proteases in the cathepsin family were cal processes ranging from cell division to cell death. reported as early as 1989. Amongst the cysteine proteases, the lysosomal cathepsins play important roles in a number of human diseases. Ref. 6 0016. Meara and Rich, J. Med. Chem. 1996, 39, 3357 However, this family displays similar substrate preferences 3366 outline some basic SAR data on the epoxysuccinyl making the development of tools to study individual cathe inhibitors of cysteine cathepsins. They worked with the psins a challenging task. basic epoxy Succinyl structure US 2006/0154325 A1 Jul. 13, 2006 bine tags for visualization or purification with warheads that covalently attach to the active sites of enzymatic targets in OR an activity-dependant manner. Using this approach, a spe O w cific protein or protein family can be readily monitored in S- O complex protein mixtures, intact cells, and even in vivo. O Ref. 3 Furthermore, enzyme class specific probes can be R used to develop screens for small molecule inhibitors that can be used functional studies. Ref. 4.5 Therefore, methods that will facilitate the development of novel ABPs have great 0017. They prepared molecules where OR was replaced value for advancing the use of this technology. The natural by a substituted carboxamides, e.g. where the OR was product E-64 Ref. 1 inhibits cysteine proteases via cova replaced with NH-CH(CH)—C(O) NH-CH. lent attachment to the active site sulfhydryl nucleophile. 0018. The use of JPM-565, which is the free acid version This reagent contains a leucine residue that mimics the of JPM-OEt, is disclosed in Shi, et al. J. Biol. Chem. 1992, critical P2 residue of a substrate and therefore binds effi 267, 7258-7262. This paper discloses the molecular cloning ciently in the S2 specificity pocket of virtually all cysteine and expression of human alveolar macrophage cathepsin S. cathepsins. As a result this reagent is a broad-spectrum and its active site labeling with JPM-565. Other inhibitors, inhibitor and activity probe. such as E-64, cystatin c. Cbz-TYr-Tyrco-butyl)-CHN are also disclosed. 0023. One of the first ABPs reported using the epoxide warhead, DCG-04 Ref. 2), has found widespread use for a 0019 Greenbaum et al. Chem. Biol. 2002, 9, 1085-1094 number of functional studies of the cysteine cathepsins. also describe cysteine protease inhibitors. It is noted there However, this general probe makes use of a single peptide that the epoxide class of cysteine protease binding com piece that makes contacts with only one side of the protease pounds has a chiral structure, e.g.