Abstract:
A library of macrocyclic compounds of the formula (I) 
     
       
                 
         
             
             
         
       
       
         
           
             where part (A) is a 
           
         
       
     
     
       
                 
         
             
             
         
       
       
         
           
              bivalent radical, a —(CH 2 ) y — bivalent radical or a covalent bond; 
             where part (B) is a 
           
         
       
     
     
       
                 
         
             
             
         
       
       
         
           
              bivalent radical, a —(CH 2 ) z — bivalent radical, or a covalent bond; 
             where part (C) is a 
           
         
       
     
     
       
                 
         
             
             
         
       
       
         
           
              bivalent radical, a —(CH 2 ) t — bivalent radical, or a covalent bond; and
           where part (T) is a —Y—L—Z— radical wherein number Y is CH 2  or CO, Z is NH or O and L is a bivalent radical. These compounds are useful for carrying out screening assay or as intermediates for the synthesis of other compound of pharmaceutical interest. A process for their preparation of these compounds in a combinatorial manner, is also disclosed.

Description:
FIELD OF THE INVENTION 
     The present invention relates to new macrocyclic compounds of biologically interesting structures. 
     The invention also relates to a process for preparing these new compounds by lactam or Mitsunobu cyclization. 
     BACKGROUND OF THE INVENTION 
     As everybody knows, medicinal chemistry research has been dramatically transformed by biotechnology. Previously synthetic chemistry and natural products screening dominated drug research, but now molecular biology has become a driving force behind screening and the establishment of molecular targets. 
     Over the last years, most of the research in biotech companies has been directed to peptide and protein therapeutics in spite of problems associated with their low bioavailability, rapid metabolism, and lack of oral activity. Because of these limitations, research groups continue to rely upon chemical synthesis of nonpeptide substances for drug discovery, recognizing that small molecules are likely to remain the most viable avenue for the identification and optimization of potential drugs. 
     As is also known, combinatorial chemistry is a technique by which large chemical libraries can be generated by connecting together appropriate chemical building blocks in a systematic way. The ability to generate such large, chemically diverse libraries, either in a combinational fashion or by any other high throughput parallel synthetic methods, combined with high throughput screening techniques, provides an immensely powerful tool for drug lead discovery and optimization. 
     Drug companies are increasingly interested in harnessing the ability of combinatorial synthesis to produce large collections (or libraries) of molecules to augment their existing sources of molecular diversity, and to fully exploit their capacity to capture millions of biological assay data points annually using high throughput robotic screening instrumentation. 
     This new science is still in its infancy, and to date most successful scaffold are derived from small heterocycles which are usually synthesized in very few steps. Thus several focused libraries have been built around bioactive cores such as benzodiazepines. However, this approach cannot be considered as a true method to generate innovative lead structures. Rather, it is a mean to optimize existing leads and is usually applied in drug development schemes. 
     Random libraries destined to search for innovative leads are very few today. As one example, a library based on diketopiperazine yielded a new submicromolar lead for a neurokinin-2 receptor after screening of this library on a variety of targets. 
     It is obvious that not all scaffolds may lead to potent drug candidates. Some very simple molecules requiring only one or two chemical steps may seem very attractive due to the huge size of the libraries that can be generated from them. Nonetheless, too simple molecules do not usually provide useful leads since they tend to lack target specificity, a prerequisite for a molecule to become a drug. 
     A class of organic structures with outstanding pharmaceutical activity has been termed as “macrocycle family”. Compounds like Taxol, Epothilone, Erythromycin, Neocarzinostatin, Rifampin and Amphotericin are either under clinical study or already marketed drugs and belong to this important family. Most of these products are of natural origin, since they are not usually tackled by medicinal chemists due to lack of knowledge associated with their synthesis. 
     Over the last years, the present inventors have developed expertise in the field of macrocycles synthesis. With such an expertise, they have developed a method of synthesis and evaluation of libraries of partially peptidic macrocycles which mimic β-turns, thereby making it possible to quickly explore huge quantities of conformationally restricted structures. 
     OBJECTS AND SUMMARY OF THE INVENTION 
     A first object of the present invention is to provide a process for preparing macrocyclic compounds, which process can be carried out with a large variety of functional groups and in the presence of a large variety of solvent systems and resins, and thus can be used for preparing a large variety of macrocyclic compounds of biologically interesting structures that could be used for carrying out screening assays or as instrumental for the synthesis of other macrocyclic compounds. Libraries of such synthetic compounds should actually be as attractive as the libraries of extracted natural products which are presently used in high throughput biological screening assays. 
     Another object of the invention is to provide libraries of macrocyclic compounds incorporating two to five building units: one to four amino-acids and a tether chain for controlling the overall shape of the molecule. 
     More specifically, the macrocyclic compounds of the invention have the general formula (I): 
     
       
                 
         
             
             
         
      
         
         
           
             where part (A) is a — 
           
         
       
    
     
       
                 
         
             
             
         
      
         
         
           
              —(CH 2 ) y —NH— bivalent radical having its —NH— group linked to the carbonyl group of part 
           
         
       
    
     
       
                 
         
             
             
         
      
         
         
           
              a —(CH 2 ) y — bivalent radical, or a covalent bonds; 
             where part (B) is a — 
           
         
       
    
     
       
                 
         
             
             
         
      
         
         
           
              —(CH 2 ) z — bivalent radical having its —NH— group linked to part (A), a —(CH 2 ) z — bivalent radical, or a covalent bond; 
             where part (C) is a — 
           
         
       
    
     
       
                 
         
             
             
         
      
         
         
           
              —(CH 2 ) t —NH— bivalent radical having its —NH— group linked to part (B), a —(CH 2 ) t — bivalent radical, or a covalent bond; 
             where part (T) is a —Y—L—Z— radical; and 
             where X is a monovalent group selected from the group consisting of: —SO 2 —Ar, —SO 2 —CH 3 , —SO 2 —CF 3 , —H, —COH, —CO—CH 3 , —CO—Ar, —CO—R, —CO—NHR, —CO—NHAr, —CO—O-tBu, —CO—O—CH 2 —Ar 
           
         
       
    
     
       
                 
         
             
             
         
      
         
         
           
             
               
                 Ar being an aromatic group or substituted aromatic group, 
                 a being an integer selected from the group consisting of 0, 1 and 2, 
                 R being a monovalent group —(CH 2 ) n —CH 3  or —(CH 2 ) n —Ar with n being an integer from 1 to 16, 
                 R 0 , R 1 , R 2 , R 3  and R 4  being independently selected from the group consisting of: 
               
             
           
         
       
    
     
       
                 
         
             
             
         
      
         
         
           
             R 5 , R 6     1    and R 6     2    each being a monovalent radical independantly selected from the group consisting of: —H, —SO 2 —CH 3 , —SO 2 —CF 3 , —COH, —COCH 3 , —CO—Ar, —CO—R or —CO—NHR wherein R is defined as above, —CONHAr, —COO-tBu and —COO—CH 2 —Ar, said radical being or not substituted by at least one monovalent group selected in the group consisting of:
           —O—CH 3 , —CH 3 , —NO 2 , —NH 2 , —NH—CH 3 , —N(CH 3 ) 2 , —CO—OH, —CO—O—CH 3 , —CO—CH 3 , —CO—NH 2 , OH, F, Cl, Br and I;   
         
             R 7  being a monovalent radical selected from the group consisting of:
           —H, —COH, —CO—CH 3 , —CO—R wherein R is defined as above, —CO—Ar and —CO—tBu, said radical being substituted or not by at least one substituent selected from the group consisting of:
               —O—CH 3 , —CH 3 , —NO 2 , —NH 2 , —NH—CH 3 , —N(CH 3 ) 2 , —CO—OH, —CO—O—CH 3 , —CO—CH 3 , —CO—NH 2 , OH, F, Cl, Br and I;   
               
         
             R 8  being a monovalent radical selected from the group consisting of: —OH, —NH 2 , —OCH 3 , —NHCH 3 , —O—tBu and —O—CH 2 —Ar, said radical substituted or not by at least one group selected in the group consisting of:
           —O—CH 3 , —CH 3 , —NO 2 , —NH—CH 3 , —N(CH 3 ) 2 , —CO—OH, —CO—O—CH 3 , —CO—CH 3 , —CO—NH 2 , OH, F, Cl, Br, I;   
         
             R 9  being a monovalent radical selected in the group consisting of: —H, —tBu, —CO—CH 3 , —COAr, —CO—R wherein R is defined as above and —COH, said radicals substituted or not by at least one a monovalent group selected from the group consisting of:
           —O—CH 3 , —CH 3 , —NO 2 , —NH—CH 3 , —N(CH 3 ) 2 , —CO—OH, —CO—O—CH 3 , —CO—CH 3 , —CO—NH 2 , OH, F, Cl, Br and, I;   
         
             where Y is a bivalent group —CH 2 — or —CO—; 
             where Z is a bivalent group —NH— or —O—; 
             wherein x, y, z and t are integers each independently selected from the group consisting of 0,1 and 2; 
             wherein L is a bivalent radical selected from the group consisting of:
           —(CH 2 ) d —A—(CH 2 ) j —B—(CH 2 ) e —, d and e being independently an integer from 1 to 5, j being an integer from 0 to 5,   with A and B being independently selected from the group consisting of:   —O—, —NH—, —NR— wherein R is defined as above, —S—, —CO—, —SO—, —CO—O—, —O—CO—, —CO—NH—, —NH—CO—, —SO 2 —NH—, —NH—SO 2 —,   
         
           
         
       
    
     
       
                 
         
             
             
         
      
     
     
       
                 
         
             
             
         
      
         
         
           
             
               
                  —CH═CH— with the configuration Z or E, —C═C—, 
               
             
           
         
       
    
     
       
                 
         
             
             
         
      
         
         
           
             
               
                  G 2  and 
               
             
           
         
       
    
     
       
                 
         
             
             
         
      
         
         
           
             
               
                  with the substituent —G 2 — in a 1, 2, 1, 3 or 1,4 position, 
                  G 1  being selected from the group consisting of:
               —O—, —NH—, —NR— wherein R is defined as above, —S—, —CH═CH— with a Z configuration, and —CH═N—; and   
             
                 G 2  being selected from the group consisting of:
               —O—, —NH—, —CO—, —NR— wherein R is defined as above, —CO—O—, —O—CO—, —CO—NH—, —NH—CO—, —SO 2 —NH— and —NH—SO 2 —.   
             
               
             
           
         
       
    
     Salts of said compounds are also within the scope of the invention. 
     As may be understood, the new macrocyclic compounds according to the invention incorporate two to five building units, one to four amino-acids units and a tether chain which controls the shape of the molecule. 
     These compounds display enhanced stability towards peptidases and exhibit facilitated cell penetration as compared to the corresponding open chain linear equivalents. 
     Some of the compounds according to the invention includes a β-turn motif within their rings: 
     
       
                 
         
             
             
         
      
     
     It is known that β-turn is one of the three major motifs of peptide and protein secondary structure. β-turn plays a key role in many biological molecular recognition events including interactions between antigens and antibodies, peptide hormones and their receptors, and regulatory enzymes and their corresponding substrates. In order to attain high affinity and selective binding to a targeted receptor, a β-turn mimetic must reproduce both the functionality and the orientation of the side chains of the receptor-bound peptide ligand. 
     The inherent diversity in β-turn structure compounded with difficulties in identifying the key residues responsible for binding, make the design of β-turn mimetics quite challenging. 
     As may be appreciated, the present invention permits to circumvent the aforementioned difficulties by providing compounds which, thanks to their structure which incorporate numerous side chain combinations as well as multiple different side chain orientations, can be used as β-turn mimetics and evaluated accordingly. 
     Obviously, the preparation of libraries of β-turn mimetics represent a goal of the present invention. However, the latter is not exclusively restricted to such compounds. There are numerous other compounds according to the invention which include other interesting di- or tri-peptide motif with their structure whose active conformation need be probed by our conformation restrictive approach. 
     As is known many adhesive proteins present in extracellular matrices and in the blood contain the tripeptide arginine-glycine-aspartic acid (RTGD) as their cell recognition site. These proteins include fibronectin, vitronectin, osteopontin, callagens, thrombospondin, fibrinogen adn von Willebrand factor. The RGD sequence of each of the adhesive proteins are recognized by at least one member of a family of structurally related receptors, integrins, which are heterodimeric proteins with two membrane-spanning sub-units. Some of these receptors bind to the RGD sequence of a single adhesion protein only, wheras others recognize groups of them. The conformation of the RGD sequence in the individual proteins may be critical to this recognition specificity. On the cytoplasmic side of the plasma membrane the receptors connect the extracellular matrix to the cytoskeleton. 
     More than ten proved or suspected RGD-containing adhesion-promoting proteins have already been identified, and the integrin family includes at least as many receptors recognizing these proteins. Together the adhesion proteins and their receptors constitute a versatile recognition system providing cells with anchorage, traction for migration, and signals for polarity, position, differentiation and possibly growth. Compounds according to the invention containing the sequence Arg-Gly-Asp in a controled topology could inhibit cell to cell adhesion processes. Such compounds could be important in the areas of antithrombotic and cancer research. 
     Also included within the scope of the invention are compounds of the above mentioned formula (I) containing a biaryl bridge. 
     As can be appreciated, the compounds according to the invention have much flexibility and can adopt structures very different from conventional β-turns, according to the nature of their spacer parts. This means that the scope of the invention is broad and molecular modeling design allows the design of β and non-β-turns. 
     A main advantage of the invention is that the compounds of the formula (I) are neither too tight like small rings nor too loose like aliphatic chains. 
     The process according to the invention for preparing the above mentioned compounds basically comprises the following steps:
         a) preparing by coupling a first building block deriving from natural or synthetic amino-acids, said first building block being of the formula:       

     
       
                 
         
             
             
         
      
         
         
           
             
               
                 wherein X, R 1 , A, B, C are defined as hereinabove, 
               
             
           
         
       
    
     
       
                 
         
             
             
         
      
         
         
           
             
               
                 P is —CH 3  or —CH 2 —Ph where the coupling is carried out in liquid phase and Sp is 
               
             
           
         
       
    
     
       
                 
         
             
             
         
      
         
         
           
             
               
                 and P is polystyrene when the coupling is carried out in solid phase; 
               
             
             b) coupling the first building block prepared in step a) with a second building block hereafter called “tether”, of the formula:
 
H—Y—L—Z—PG z  
 
              wherein Y, L and Z are defined as PG z  is a protective group; and 
             d) carrying out a macrocyclization of the unprotected product obtained in step c) and a cleavage if the preparing and coupling steps (a), (b) were carried out in the solid phase in order to obtain the requested compound of the formula (I). 
           
         
       
    
     As can be noted, this process uses lactam or Mitsunob cyclization to prepare the libraries of compounds according to the invention. It is very versatile and can be carried out in a combinatorial manner, either in solid phase or in solution phase. 
     The invention and its advantages will be better understood upon reading the following non restrictive detailed description and examples made with reference to the accompanying drawings. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a Table detailing the structure that may have the compounds of the formula (I) according to the invention. 
         FIGS. 2   a  and  2   b  are schematic illustrations of the sequence of steps that must be carried out to obtain the library of compounds of formula (8), which is part of the “family 2” libraries shown in  FIG. 1  for which Y is a methylene group (—CH 2 —), and the amino-acids at positions 1, 2 and 3 are α-amino-acids (x, y, z=0). 
         FIG. 3  is a schematic illustration of the steps that must be carried out to obtain other libraries of compounds of formulae (9) to (12), which are also parts of the “family 2” libraries shown in  FIG. 1 , for which Y is a methylene group (—CH 2 —), and the amino-acids at positions 1, 2 and 3 are α-amino-acids (x, y, z 0). 
         FIGS. 4   a  and  4   b  are schematic illustrations of the sequence of steps that must be carried out to obtain libraries of compounds of the formulae (17) and (18) which are parts of the “family 2” libraries shown in  FIG. 1 , for which Y is a carbonyl group (—CO—), and the amino-acids at positions 1, 2 and 3 are α-amino-acids (x, y, z=0). 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     As aforesaid, the process according to the invention is versatile enough to prepare a large number macrocyclic compounds, the main “families” of which are illustrated in  FIG. 1 . This process comprise the following basic steps:
         a) preparing by coupling a first building block deriving from natural or synthetic amino-acids,   b) coupling the first building block prepared in step a) with a second building block called “tether”,   c) removing the protective group from the compound obtained in step b), and   d) carrying out a macrocyclization of the unprotected product obtained in step c) to obtain the requested compound.       

     As aforesaid, the process permits to prepare a large number of compounds either in a solution phase or on a solid support using an IRORI combinatorial chemistry set up or an other set up like an Argonault apparatus. 
     In the synthesis shown in  FIG. 2   a , a first suitably protected amino-acid identified as “A” is activated as a thioester (solution phase or solid phase) or as an oxime ester (Kaiser resin solid phase support) to give a compound of formula (1). The amine protection (PGα in the case of amino-acids PGβ in the case of β-amino-acids and PGγ in the case of γ-amino-acids) is removed to give the compound of formula (2). A second amino-acid identified as “B” is then added in the same way to give a compound of formula (3), followed again by removal of the amine protection to give a compound of formula (4). A third acid of formula (C) ( FIG. 2   b ) is coupled to the amine of formula (4) to yield a sulfonamide of the formula (5), which is immediately coupled with an alcohol of the formula (D) under Mitsunobu conditions to give a compound of formula (6). This compound of formula (6) can also be obtained directly from the compound of formula (4) by peptide coupling with an acid of formula (E). The terminal alcohol (Z═O) or amine (Z═NH) protecting group (PGz) is then cleaved to give the corresponding alcohol or amine of formula (7), which can undergo cyclization and cleavage all at once to give requisted compound according to the invention of formula (8). 
     As shown in  FIG. 3 , the orthogonal protections (PG1, PG2, PG3 and PG4 when a fourth amino-acid is introduced) of the compound of formula (8) can be removed to yield the compound according to the invention of formula (9). Another compound according to the invention of formula (10) can be obtained from the compound of formula (8) by cleaving the sulfonamide portion of the molecules. The resulting free amine can be coupled with various acids (see X groups in  FIG. 1 ) to yield a compound of formula (11) according to the invention. Subsequent cleavage of the orthogonal protecting groups (PG0 when X is an amino-acid, PG1, PG2, PG3 and PG4 when a fourth amino-acid is introduced) yield a compound of formula (12) according to the invention. 
     As shown in  FIG. 4 , it is also possible from the amine of formula (4) to couple an amino-acid of formula (C′) to yield a compound of formula (13) according to the invention, whose amine protecting group (PGa, PGb or PGg) can be cleaved to give the amine of formula (14). A hydroxy-acid (Z═O) or amino-acid (Z═NH) of formula (D′) is then coupled to yield a compound according to the invention of formula (15). The terminal alcohol (Z═O) or amine (Z═NH) protecting group (PGz) is then cleaved to give the corresponding alcohol or amine of formula (16), which can undergo cyclization and cleavage all at once to yield a compound according to the invention of formula (17). The orthogonal protections (PG1, PG2, PG3 and PG4 when a fourth amino-acid is introduced) of the compound of formula (17) see  FIG. 4   b  can be removed to yield a compound of formula (18). 
     The above example of synthesis deals with the preparation of libraries of the “family 2” type for which positions 1, 2 and 3 are filled. Libraries of “families 1, 3 and 4” type as shown in  FIG. 1  can be prepared exactly in the same way. In “family 1” type libraries, an extra amino-acid is incorporated at position 4. In “family 311 type libraries, positions 3 and 4 are empty and in “family 4” type libraries positions 2, 3 and 4 are empty. 
     Thus, it is possible to develop chemical libraries of dozens, hundreds, and even many thousands of discrete chemical compounds, in an efficient and reliable manner. This being the case, it is possible to use these libraries as chemical intermediates for the preparation of pharmaceutical compounds or for the identification of such pharmaceutical compounds or other useful species. Some of these compounds could also be used without further modification. Accordingly, the ability to prepare such complex libraries in a reliable and predictable fashion is highly desired. 
     The term “pharmaceutical” as used herein means the ability of the compounds to provide some therapeutic or physiological beneficial effect. As used herein, the term includes any physiologically or pharmacologically activity that produces a localized or systemic effect or effects in animals including warm blooded mammals such as humans. Pharmaceutically active agents may act on the peripheral nerves, adrenegic receptors, cholinergic receptors, the skeletal muscles, the cardiovascular system, smooth muscles, the blood circulatory system, synoptic sites, neuroeffector junctional sites, endocrine and hormone systems, the immunological system, the reproductive system, the skeletal system, the autocoid system, the alimentary and excretory systems, the histamine system and central nervous systems as well as other biological systems. Thus, compounds derived from compositions of the present invention may be used as sedatives, psychic energizers, tranquilizers, anticonvulsant, muscle relaxants, anti-Parkinson agents, analgesics, anti-inflammatories, local anesthetics, muscle contractants, antibiotic, antiviral, antiretroviral, antimalarials, diuretics, lipid regulating agents, antiandrogenic agents, antiparasities, neoplastics and chemotherapy agents. These compounds could further be used to treat cardiovascular diseases, central nervous system diseases, cancer metabolic disorders, infections and dermatological diseases as well as other biological disorders and infections. 
     Among the potential uses of the compounds according the present invention are uses in scientific research as research reagents. In accordance with the present invention, it is now possible to prepare pluralities of compounds to create libraries of compounds for research. Such libraries are known to be useful and are important in the discovery of new drugs. In view of the chemical and conformational diversities of such compounds e.g. the large number of functionalizable sites, a very large number of different compounds can be prepared. Moreover, such compounds can be prepared differentially, that is, in such a fashion that a population of known species can be prepared reliably, ensuring that all potential members of a family of chemical species are in fact synthesized. 
     In view of the foregoing, persons of ordinary skilled in the art will know how to synthesize such libraries, comprising chemical compositions within the scope and spirit of this invention and to assay the libraries against etiological agents or in tests or assays, in order to identify compounds having antibacterial, antifungal, antiviral, antineoplastic or other desired pharmaceutical, biological, or chemical activity. 
     SPECIFIC EXAMPLES 
     Preparation of macrocyclic compounds according to the invention by the process outlined above will be illustrated by the following non-limiting specific examples: 
     Example 1 
     Library of 135 Members (Solution Phase) (see  FIG. 2   a ) 
     This library (family 2 type) consists of a linear sequence of three natural L-α-amino- acids linked together by an aliphatic chain with 4 or 5 carbons in a head to tail manner. The first amino acids (AA 1 ) are glycine, leucine and methionine, the second ones (AA 2 ) are glycine, histidine (Doc), leucine, proline and valine, and the third ones (AA 3 ) are gylcine, methionine and phenylalanine. 
     The three third amino acids (Boc-AA3) were converted to their thioesters by coupling with methyl 3-mercapto-propionate. The formed compounds were coupled with the second five amino acids to produce 15 dipeptides which were then converted to 135 linear tripeptides by coupling with nine N-alkylated N-betsyl amino-acids. The end-to-end cyclization of the linear tripeptide thioesters was achieved by silver cation-assisted lactamization. 
     Amino Acid, Thioesters (Boc-AA 3 -S(CH 2 ) 2 CO 2 Me): 
     Chlorotrimethylsilane (276 mL, 2.18 mol) was added slowly to a solution of 3-mercaptopropionic acid (70 g, 0.66 mol) in methanol (267 mL, 6.6 mol) at −10° C. The reaction mixture was then stirred for 1 h at 0° C. and for 1 h at room temperature. The mixture was neutralized with saturated aqueous sodium bicarbonate to pH 8 and extracted with dichloromethane (3×300 mL). The combined organic phase was washed with saturated aqueous sodium bicarbonate (2×200 mL), brine (2×200 mL), dried over magnesium sulfate, filtered and evaporated under reduced pressure. The residue was then distilled (70° C./20 mmHg) to give methyl 3-mercaptopropionate as a colorless oil (61.2 g) in the yield of 77%. 
     1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (42 mmol) was added to a solution of N-Boc amino-acid (33 mmol), methyl 3-mercaptopropionate (30 mmol) and 4-dimethylaminopyridine (3 mmol) in dichloromethane (80 mL) at 0° C. The resulting mixture was stirred for 1 hour at 0° C. and 30 min at room temperature. The reaction mixture was diluted with ethyl acetate (200 mL), washed with 1N hydrochloric acid (2×50 mL), saturated aqueous sodium bicarbonate (2×50 mL), brine (2×50 mL), dried over magnesium sulfate, and evaporated to give the desired thioester (Boc-AA 3 -S(CH 2 ) 2 CO 2 Me) in yields ranging from 95 to 100%. 
     Dipeptides (Boc-AA 2 -AA 3 -S(CH 2 ) 2 CO 2 Me): 
     To a solution of the N-Boc amino acid thioester (Boc-AA 3 -S(CH 2 ) 2 CO 2 Me) (5 mmol) in dichloromethane (2 mL), triethylsilane (10 mmol) was added, followed by trifluoroacetic acid (3 mL). The reaction mixture was stirred for 1 h at room temperature, then diluted with toluene (2×10 mL) and the solvent was evaporated to give the TFA salt of H-AA 3 -S(CH 2 ) 2 CO 2 Me. 
     To a solution of N-Boc amino acid (Boc-AA 2 -OH) (5 mmol) and 1-hydroxybenzotriazole (5 mmol) in tetrahydrofuran (5 mL) and dichloromethane (5 mL) at 0° C., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (7 mmol) was added. The resulting mixture was stirred for 5 min at 0° C. and for 20 min at room temperature, then cooled down to 0° C. 
     A solution of the TFA salt of H-AA 3 -S(CH 2 ) 2 CO 2 Me (5 mmol) in dichloromethane (5 mL) was added to the above reaction mixture at 0° C., followed by diisopropylethylamine (7.5 mmol). The resulting reaction mixture was then stirred at the same temperature for 10 min. The ice-water bath was removed and the reaction was stirred for 2 to 4 h at room temperature. Finally, the reaction mixture was diluted with ethyl acetate (80 mL) and washed with 1N hydrochloric acid (2×15 mL), saturated aqueous sodium bicarbonate (2×15 mL), brine (2×20 mL), dried over magnesium sulfate, and evaporated to give the dipeptide (Boc-AA 2 -AA 3 -S(CH 2 ) 2 CO 2 Me) in yields ranging from of 80 to 100%. 
     Alkylated Tripeptides (N-Bts-(N-alkylated)-AA 1 -AA 2 -AA 3 -S(CH 2 ) 2 CO 2 Me): 
     To a solution of N-Boc dipeptide (0.5 mmol) in dichloromethane (1 mL), triethylsilane (2 mmol) was added, followed by trifluoroacetic acid (1 mL). The reaction mixture was stirred for 1 h at room temperature and then diluted with toluene (2×5 mL) and evaporated to give the dipeptide TFA salt. 
     To a solution of alkylated N-Betsyl amino acid (0.5 mmol) and 1-hydroxybenzotriazole (0.5 mmol) in tetrahydrofuran (2 mL) and dichloromethane (2 mL) at 0° C., 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide hydrochloride (0.7 mmol) was added. The resulting mixture was stirred for 5 min at 0° C. then for 30 min at room temperature, and cooled down to 0° C. 
     A solution of the dipeptide TFA salt (0.5 mmol) in dichloromethane (2 mL) was added to the above reaction mixture at 0° C., followed by diisopropylethylamine (0.8 mmol). The reaction mixture was then stirred for 10 min at the same temperature. The ice-water bath was removed and the reaction was stirred for 2 to 4 h at room temperature. The reaction mixture was diluted with ethyl acetate (50 mL) and washed with 1N hydrochloric acid (2×10 mL), saturated aqueous sodium bicarbonate (2×10 mL), brine (2×10 mL), dried over magnesium sulfate, and evaporated to give the alkylated tripeptides (Bts-(N-alkylated)-AA 1 -AA 2 -AA 3 -S(CH 2 ) 2 CO 2 Me) with yields ranging from 11 to 100%. 
     Preparation of Cyclotripeptides: 
     To a solution of the alkylated tripeptide (Bts-(N-alkylated)-AA 1 -AA 2 -AA 3 -S(CH 2 ) 2 CO 2 Me) (0.5 mmol) in dichloromethane (1 mL), triethylsilane (2 mmol) was added, followed by trifluoroacetic acid (1 mL). The reaction mixture was stirred for 1 h at room temperature and then diluted with toluene (2×5 mL) and the solvent was evaporated to give the TFA salt of alkylated tripeptide. 
     To a solution of the TFA salt of alkylated tripeptide (0.5 mmol) in ethyl acetate (250 mL), diisopropylethylamine (1.0 mmol) and silver trifluoroacetate (1.5 mmol) were added. The reaction mixture was stirred for 1 to 3 h at room temperature (Thin Layer Chromatography monitoring of the reaction). Brine (50 mL) and 1.0 M aqueous sodium thiosulfate (30 mL) was added and stirred for 60 min. The organic phase was washed with saturated EDTA aqueous solution (2×50 mL), 1N hydrochloric acid (50 mL), brine (2×50 mL), dried over magnesium sulfate and evaporated to give the crude product. The crude can be purified by flash column chromatography if necessary. 
     
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Results for the cyclic peptides with the E-alkene linker. 
               
             
          
           
               
                   
                   
                   
                   
                   
                 Purity 
               
               
                   
                   
                   
                 Quantity 
                 Yield 
                 Level 
               
               
                   
                 Name 
                 MW 
                 (mg) 
                 (%) 
                 (%) 
               
               
                   
                   
               
             
          
           
               
                   
                 c-B-GGG-1 
                 451 
                 25 
                 14 
                 100 
               
               
                   
                 c-B-GGM-1 
                 525 
                 170 
                 77 
                 94 
               
               
                   
                 c-B-GGF-1 
                 541 
                 60 
                 44 
                 96 
               
               
                   
                 c-B-LGG-1 
                 507 
                 180 
                 87 
                 92 
               
               
                   
                 c-B-LGM-1 
                 581 
                 95 
                 40 
                 95 
               
               
                   
                 c-B-LGF-1 
                 597 
                 93 
                 83 
                 93 
               
               
                   
                 c-B-MGG-1 
                 525 
                 80 
                 35 
                 96 
               
               
                   
                 c-B-MGM-1 
                 599 
                 66 
                 28 
                 94 
               
               
                   
                 c-B-MGM-1 
                 599 
                 31 
                 10 
                 77 
               
               
                   
                 c-B-MGF-1 
                 615 
                 82 
                 27 
                 97 
               
               
                   
                 c-B-MGF-1 
                 615 
                 25 
                  6 
                 69 
               
               
                   
                 c-B-GHG-1 
                 673 
                 107 
                 53 
                 91 
               
               
                   
                 c-B-GHM-1 
                 747 
                 97 
                 59 
                 98 
               
               
                   
                 c-B-GHF-1 
                 763 
                 177 
                 68 
                 91 
               
               
                   
                 c-B-LHG-1 
                 729 
                 82 
                 61 
                 85 
               
               
                   
                 c-B-LHM-1 
                 803 
                 133 
                 65 
                 80 
               
               
                   
                 c-B-LHF-1 
                 819 
                 162 
                 74 
                 97 
               
               
                   
                 c-B-MHG-1 
                 747 
                 177 
                 63 
                 97 
               
               
                   
                 c-B-MHM-1 
                 821 
                 160 
                 44 
                 90 
               
               
                   
                 c-B-MHF-1 
                 837 
                 201 
                 51 
                 91 
               
               
                   
                 c-B-GLG-1 
                 507 
                 229 
                 90 
                 97 
               
               
                   
                 c-B-GLM-1 
                 581 
                 146 
                 68 
                 99 
               
               
                   
                 c-B-GLF-1 
                 597 
                 130 
                 81 
                 80 
               
               
                   
                 c-B-LLG-1 
                 563 
                 152 
                 88 
                 92 
               
               
                   
                 c-B-MLG-1 
                 581 
                 200 
                 93 
                 91 
               
               
                   
                 c-B-MLM-1 
                 655 
                 162 
                 61 
                 99 
               
               
                   
                 c-B-MLF-1 
                 671 
                 150 
                 89 
                 97 
               
               
                   
                 c-B-GPG-1 
                 491 
                 26 
                 21 
                 85 
               
               
                   
                 c-B-GPM-1 
                 565 
                 121 
                 nd 
                 87 
               
               
                   
                 c-B-GPF-1 
                 581 
                 80 
                 62 
                 89 
               
               
                   
                 c-B-LPG-1 
                 547 
                 73 
                 63 
                 97 
               
               
                   
                 c-B-LPM-1 
                 621 
                 28 
                 31 
                 55 
               
               
                   
                 c-B-LPF-1 
                 637 
                 25 
                  9 
                 85 
               
               
                   
                 c-B-LPF-1 
                 637 
                 135 
                 64 
                 92 
               
               
                   
                 c-B-MPG-1 
                 565 
                 102 
                 58 
                 91 
               
               
                   
                 c-B-MPM-1 
                 639 
                 10 
                 98 
                 99 
               
               
                   
                 c-B-MPF-1 
                 655 
                 140 
                 61 
                 76 
               
               
                   
                 c-B-GVG-1 
                 493 
                 100 
                 56 
                 100 
               
               
                   
                 c-B-GVM-1 
                 567 
                 13 
                  9 
                 59 
               
               
                   
                 c-B-GVF-1 
                 583 
                 142 
                 75 
                 80 
               
               
                   
                 c-B-LVG-1 
                 549 
                 142 
                 77 
                 96 
               
               
                   
                 c-B-LVM-1 
                 623 
                 78 
                 95 
                 90 
               
               
                   
                 c-B-LVF-1 
                 639 
                 290 
                 85 
                 89 
               
               
                   
                 c-B-MVG-1 
                 567 
                 303 
                 95 
                 90 
               
               
                   
                 c-B-MVM-1 
                 641 
                 26 
                 66 
                 96 
               
               
                   
                 c-B-MVF-1 
                 657 
                 222 
                 64 
                 85 
               
               
                   
                 c-B-LLM-1 
                 637 
                 123 
                 69 
                 95 
               
               
                   
                 c-B-LLF-1 
                 653 
                 221 
                 97 
                 96 
               
               
                   
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Results for the cyclic peptides with the Z-alkene linker. 
               
             
          
           
               
                   
                   
                   
                 Quantity 
                 Yield 
                 Purity 
               
               
                   
                 Name 
                 MW 
                 (mg) 
                 (%) 
                 (%) 
               
               
                   
                   
               
             
          
           
               
                   
                 c-B-GGG-2 
                 437 
                 2 
                 1 
                 89 
               
               
                   
                 c-B-GGM-2 
                 511 
                 250 
                 77 
                 86 
               
               
                   
                 c-B-GGF-2 
                 527 
                 46 
                 46 
                 96 
               
               
                   
                 c-B-LGG-2 
                 493 
                 142 
                 76 
                 99 
               
               
                   
                 c-B-LGM-2 
                 567 
                 112 
                 23 
                 55 
               
               
                   
                 c-B-LGF-2 
                 583 
                 64 
                 35 
                 67 
               
               
                   
                 c-B-MGG-2 
                 511 
                 56 
                 49 
                 96 
               
               
                   
                 c-B-MGM-2 
                 585 
                 28 
                 10 
                 87 
               
               
                   
                 c-B-MGM-2 
                 585 
                 40 
                 14 
                 77 
               
               
                   
                 c-B-MGF-2 
                 601 
                 75 
                 20 
                 94 
               
               
                   
                 c-B-MGF-2 
                 601 
                 32 
                 9 
                 88 
               
               
                   
                 c-B-GHG-2 
                 659 
                 80 
                 49 
                 93 
               
               
                   
                 c-B-GHM-2 
                 733 
                 52 
                 22 
                 63 
               
               
                   
                 c-B-GHF-2 
                 749 
                 159 
                 39 
                 66 
               
               
                   
                 c-B-LHG-2 
                 715 
                 72 
                 47 
                 94 
               
               
                   
                 c-B-LHM-2 
                 789 
                 111 
                 31 
                 51 
               
               
                   
                 c-B-LHF-2 
                 805 
                 169 
                 46 
                 67 
               
               
                   
                 c-B-MHG-2 
                 733 
                 30 
                 9 
                 98 
               
               
                   
                 c-B-MHM-2 
                 807 
                 90 
                 6 
                 42 
               
               
                   
                 c-B-MHF-2 
                 823 
                 159 
                 30 
                 69 
               
               
                   
                 c-B-GLG-2 
                 493 
                 166 
                 97 
                 100 
               
               
                   
                 c-B-GLM-2 
                 567 
                 143 
                 85 
                 97 
               
               
                   
                 c-B-GLF-2 
                 583 
                 90 
                 32 
                 57 
               
               
                   
                 c-B-LLG-2 
                 549 
                 110 
                 48 
                 94 
               
               
                   
                 c-B-MLG-2 
                 567 
                 308 
                 55 
                 56 
               
               
                   
                 c-B-MLM-2 
                 641 
                 38 
                 5 
                 28 
               
               
                   
                 c-B-MLF-2 
                 657 
                 206 
                 32 
                 44 
               
               
                   
                 c-B-GPG-2 
                 477 
                 31 
                 29 
                 92 
               
               
                   
                 c-B-GPM-2 
                 551 
                 76 
                 nd 
                 82 
               
               
                   
                 c-B-GPF-2 
                 567 
                 55 
                 42 
                 92 
               
               
                   
                 c-B-LPG-2 
                 533 
                 0 
                 nd 
               
               
                   
                 c-B-LPM-2 
                 607 
                 34 
                 54 
                 49 
               
               
                   
                 c-B-LPF-2 
                 623 
                 19 
                 8 
                 96 
               
               
                   
                 c-B-MPG-2 
                 551 
                 46 
                 22 
                 78 
               
               
                   
                 c-B-MPM-2 
                 625 
                 5 
                 1 
                 59 
               
               
                   
                 c-B-MPF-2 
                 641 
                 44 
                 11 
                 49 
               
               
                   
                 c-B-GVG-2 
                 479 
                 61 
                 23 
                 85 
               
               
                   
                 c-B-GVM-2 
                 553 
                 59 
                 80 
                 93 
               
               
                   
                 c-B-GVF-2 
                 569 
                 191 
                 86 
                 76 
               
               
                   
                 c-B-LVG-2 
                 535 
                 92 
                 36 
                 89 
               
               
                   
                 c-B-LVM-2 
                 609 
                 60 
                 57 
                 93 
               
               
                   
                 c-B-LVF-2 
                 625 
                 152 
                 36 
                 87 
               
               
                   
                 c-B-MVG-2 
                 553 
                 112 
                 30 
                 82 
               
               
                   
                 c-B-MVM-2 
                 627 
                 6 
                 7 
                 48 
               
               
                   
                 c-B-MVF-2 
                 643 
                 0 
                 nd 
               
               
                   
                 c-B-LLM-2 
                 623 
                 85 
                 26 
                 61 
               
               
                   
                 c-B-LLF-2 
                 639 
                 193 
                 43 
                 77 
               
               
                   
                   
               
             
          
         
       
     
                                                                         TABLE 3                   Results for the cyclic peptides with the alkyne linker.                        Quantity   Yield   Purity           Name   MW   (mg)   (%)   (%)                            c-B-GGG-3   435   11   6   96           c-B-GGM-3   509   258   58   85           c-B-GGF-3   525   15   9   93           c-B-LGG-3   491   150   69   91           c-B-LGM-3   565   60   22   98           c-B-LGM-3   565   86   27   84           c-B-LGF-3   581   36   34   91           c-B-LGF-3   581   10   8   91           c-B-LGF-3   581   22   18   83           c-B-MGG-3   509   60   38   89           c-B-MGM-3   583   32   12   96           c-B-MGM-3   583   54   17   83           c-B-MGF-3   599   96   31   98           c-B-MGF-3   599   55   13   73           c-B-GHG-3   657   104   54   94           c-B-GHM-3   731   52   26   61           c-B-GHF-3   747   80   24   81           c-B-LHG-3   713   119   70   85           c-B-LHM-3   787   173   66   73           c-B-LHF-3   803   224   64   75           c-B-MHG-3   731   107   29   93           c-B-MHM-3   805   97   13   78           c-B-MHF-3   821   163   38   83           c-B-GLG-3   491   63   38   100           c-B-GLM-3   565   53   15   51           c-B-GLF-3   581   20   7   44           c-B-LLG-3   547   124   46   83           c-B-LLG-3   547   61   11   41           c-B-LLM-3   621   148   62   81           c-B-LLF-3   637   196   55   82           c-B-MLG-3   565   167   24   40           c-B-MLM-3   639   27   9   76           c-B-MLF-3   655   100   22   65           c-B-GPG-3   475   37   46   99           c-B-GPM-3   549   15   16   50           c-B-GPF-3   565   45   83   99           c-B-LPG-3   531   0   1   58           c-B-LPM-3   605   54   29   38           c-B-LPF-3   621   26   10   91           c-B-MPG-3   549   23   15   98           c-B-MPM-3   623   10   41   41           c-B-MPF-3   639   5   nd           c-B-GVG-3   477   39   19   79           c-B-GVM-3   551   33   34   66           c-B-GVF-3   567   36   6   28           c-B-LVG-3   533   158   64   82           c-B-LVM-3   607   75   64   83           c-B-LVF-3   623   356   64   79           c-B-MVG-3   551   285   63   62           c-B-MVM-3   625   8   9   50           c-B-MVF-3   641   123   31   77                        
Spectral Data for Betsylated c-Met-Leu-Phe-Linker Compounds:
 
Trans-Linker (c-B-MLF-1):
 
       1 H NMR (CDCl 3 , 300 MHz): 8.32 (1H, br), 8.19 (1H, dd, J=8.0 and 1.7 Hz), 8.01 (1H, dd, J=8.5 and 1.7 Hz), 7.70–7.59 (2H, m), 7.34–7.22 (5H, m), 6.91 (1H, br), 6.66 (1H, br), 5.70–5.60 (1H, m), 5.21–5.16 (1H, m), 4.64–4.59 (2H, m), 3.92–3.85 (3H, m), 3.35–3.20 (2H, m), 3.30 (1 H, dd, J=14.0 and 4.7 Hz), 3.16 (1H, dd, J=13.9 and 9.3 Hz), 2.47–2.32 (3H, m), 2.09–2.04 (3H, m), 1.97 (3H, s), 1.73–1.44 (3H, m), 0.86 (3H, d, J=6.3 Hz), 0.81 (3 H, d, J=6.3 Hz). 
     LC-MS: m/e: 671 (M + ) 
     Cis-Linker (c-B-MLF-2): 
       1 H NMR (CDCl 3 , 300 MHz): 8.65 (1H, J=5.9 Hz), 8.05 (1H, dd, J=8.9 and 1.6 Hz), 8.00 (1H, d, J=8.1 Hz), 7.66–7.57 (2H, m), 7.33–7.22 (3H, m), 7.16 (2H, d, J=7.1 Hz), 6.71–6.68 (2H, m), 5.72–5.56 (2H, m), 5.01 (1H, t, J=7.3 Hz), 4.54 (1H, dt, J=8.7 and 4.9 Hz), 4.08–4.00 (2H, m), 3.94 (1H, dd, J=16.4 and 7.3 Hz), 3.67–3.56 (2H, m), 3.23 (1H, dd, J=14.1 and 4.8 Hz), 3.00 (1H, dd, J=14.1 and 9.0 Hz), 2.67–2.47 (2H, m), 2.36–2.11 (1 H, m), 2.09 (3H, s), 2.07–1.99 (1H, m), 1.92–1.82 (1H, m), 1.61–1.52 (1H, m), 1.49–1.41 (1H, m), 0.93 (3H, d, J=6.5 Hz), 0.87 (3H, d, J=6.3 Hz). 
     LC-MS: m/e: 657 (M + ). 
     Acetylene-Linker (c-B-MLF-3): 
       1 H NMR (CDCl 3 , 300 MHz): 8.32(2H, d, J=8.1 Hz), 8.01 (1 H, d, J=8.0 Hz), 7.72–7.60 (2H, m), 7.49 (1H, br), 7.31–7.20 (5H, m), 6.80 (1H, br), 4.80–4.76 (1H, m), 4.42–4.36 (2H, m), 4.13–3.95 (3H, m), 3.45–3.40 (1H, m), 3.29 (1H, dd, J=14.2 and 5.6 Hz), 3.14 (1H, dd, J=14.1 and 10.1 Hz), 2.54–2.47 (3H, m), 2.12–2.08 (1H, m), 1.97 (3H, s), 1.74 (2H, t, J=7.6 Hz), 1.42–1.33 (1H, m), 0.82 (3H, d, J=6.6 Hz), 0.78 (3H, d, J=6.6 Hz). 
     LC-MS: m/e: 655 (M + ). 
     Example 2 
     Removal of Betsyl Group of Cyclotripeptides 
     1) in solution: 
     Potassium trimethylsilanolate (0.2 mmol) was added to a solution of 2-naphthalenethiol (0.2 mmol) in a deoxygenated mixture of THF and EtOH (1:1, 1 mL) at room temperature and the resulting mixture was stirred for 20 min. N-Bts-cyclotripeptide (0.1 mmol) was then added. The resulting mixture was stirred for 1 h and evaporated to dryness. The residue was purified by column to give the deprotected product with yields ranging from 77 to 86%. 
     2) on solid support: 
     Polystyrene-thiophenol Resin (0.2 mmol) was added to a solution of potassium trimethylsilanolate (0.2 mmol) in a deoxygenated mixture of THF and EtOH (1:1, 1 mL) at room temperature and the resulting mixture stirred for 20 min. 
     The solution was removed by filtration. The resin was washed with a deoxygenated mixture of THF and EtOH (1:1, 3×2 mL) and added to a solution of N-Bts-cyclotripeptide (0.1 mmol) in a deoxygenated mixture of THF and EtOH (1:1, 1 mL). The resulting mixture was stirred for 1 h. The resin was removed by filtration and washed with THF and EtOH (1:1 mixture, 2×2 mL). The filtrate was concentrated to give the crude product in quantitive yield. 
     Data for c-Met-Leu-Phe-Linker Compounds: 
     Trans-Linker (c-MLF-1): 
       1 H NMR (CDCl 3 , 300 MHz): 7.77 (1H, d, J=7.7 Hz), 7.30–7.14 (5H, m), 6.65 (1H, d, J=7.4 Hz), 6.53 (1H, br), 5.56–5.64 (1H, m), 5.32–5.22 (1H, m), 4.03–3.95 (2H, m), 3.79–3.68 (1H, m), 3.57–3.35 (3H, m), 3.12–3.02 (2H, m), 2.96–2.86 (1H, m), 2.61–2.50 (2H, m), 2.24–1.98 (3H, m), 2.10 (3H, s), 1.78 (2H, m), 1.55–1.39 (2H, m), 0.86 (3 H, d, J=6.6 Hz), 0.83 (3H, d, J=6.8 Hz). 
     LC-MS: m/e: 474 (M + ) 
     Cis-Linker (c-MLF-2): 
       1 H NMR (CDCl 3  &amp; CD 3 OD, 300 MHz): 7.27–7.14 (5H, m), 5.80–5.71 (1H, m), 5.68–5.53 (1H, m), 4.21–4.09 (2H, m), 3.88 (1H, dd, J=14.0 and 6.0 Hz), 3.64 (1H, dd, J=14.0 and 7.3 Hz), 3.34–3.15 (5H, m), 2.52 (2H, t, J=7.3 Hz), 2.07 (3H, s), 2.00–1.86 (2H, m), 1.53–1.15 (3H, m), 0.80 (6H, d, J=6.6 Hz). 
     LC-MS: m/e: 460 (M + ). 
     Acetylene-Linker (c-MLF-3): 
     H NMR (CDCl 3 , 300 MHz): 7.92 (1H, d, J=8.8 Hz), 7.34 (1 H, d, J=9.2 Hz), 7.31–7.17 (5H, m), 7.02 (1H, t, J=5.9 Hz), 4.21 (1H, q, J=8.0 Hz), 4.10–3.99 (2H, m), 3.79 (1H, dm, J=15.5 Hz), 3.67 (1H, dm, J=16.0 Hz), 3.49–3.35 (3H, m), 3.25 (1H, dd, J=8.8 and 4.3 Hz), 2.60–2.54 (2H, m), 2.15–1.99 (1H, m), 2.11 (3H, s), 1.80–1.68 (2H, m), 1.57–1.46 (2H, m), 0.86 (6H, d, J=6.3 Hz). 
     LC-MS: m/e: 458 (M + ). 
     Example 3 
     Preparation of N-Formyl-Cyclotripeptides 
     The free amine macrocycle (see example 2) was added to a mixture of formic acid (0.2 mL) and acetic anhydride (0.1 mL) and the reaction mixture was stirred for 15 min at room temperature, then for 5 min at 55° C. and finally for 2 h at room temperature. The reaction mixture was evaporated to dryness and purified by column chromatography to give the desired compounds in yields ranging from 81 to 100% 
     Data for Formylated c-Met-Leu-Phe-Linker Compounds: 
     Trans-Linker (c-f-MLF-1): 
       1 H NMR (CDCl 3 , 300 MHz): 8.72 (1H, s), 7.92 (1H, s), 7.38–7.22 (5H, m), 6.78 (1H, br), 5.83 (1H, d, J=10.0 Hz), 5.68–5.58 (1H, m), 4.96–4.89 (1H, m), 4.02–3.68 (5H, m), 3.34 (1H, dd, J=14.0 and 10.0 Hz), 2.78 (1H, dd, J 13.5 and 5.2 Hz), 2.67–2.51 (3H, m), 2.42–2.31 (1H, m), 2.18–2.04 (1H, m), 2.06 (3H, s), 1.99–1.53 (6H, m), 1.03 (3H, d, J=6.0 Hz), 0.94 (3H, d, J=6.0 Hz). 
     LC-MS: m/e: 502 (M + ) (84.2%). 
     Cis-Linker (c-f-MLF-2): 
       1 H NMR (CDCl 3 , 300 MHz): 8.04 (1H, s), 7.29–7.18 (5H, m), 6.82–6.74 (1H, m), 6.39 (1H, d, J=9.5 Hz), 6.16 (1H, dd, J=18.0 and 7.5 Hz), 6.94–6.85 (1H, m), 4.80–4.72 (1 H, m), 4.24–3.68 (5H, m), 3.50–3.43 (1H, m), 2.87–2.79 (1 H, m), 2.61–2.45 (3H, m), 2.14–2.01 (1H, m), 2.11 (3H, s), 1.74–1.62 (1H, m), 1.43–1.25 (3H, m), 0.93–0.78 (6H, m). 
     LC-MS: m/e: 488 (M + ). 
     Acetylene-Linker (c-f-MLF-3): 
     H NMR (CDCl 3 , 300 MHz): 8.10 (1H, s), 7.83 (1H, br), 7.31–7.16 (5H, m), 6.97 (1H, br), 6.45 (1H, br), 4.354.01 (5H, m), 3.87–3.71 (2H, m) r , 3.38 (1H, dd, J=14.5 and 5.0 Hz), 3.24–3.16 (1H, m), 2.59–2.23 (3 H, m), 2.08 (3H, s), 1.66–1.25 (4H, m), 0.84 (6H, t, J=6.0 Hz). 
     LC-MS: m/e: 486 (M + ). 
     Example 4 
     Solid Phase Synthesis on the Kaiser Resin 
     Anchoring Boc-Amino Acid to the Resin: 
     To the Kaiser resin (2.0 g, 0.95 mmol/g) was added a 0.2M solution of N-Boc amino acid and 4-dimethylaminopyridine (0.25 eq). After shaking for 5 min, diisopropylcarbodiimide (1.5 eq) was added and the reaction mixture was agitated for 16 h. The resin was washed with dichloromethane (3×30 mL), tetrahydrofuran (1×30 mL), methanol (1×30 mL), dichloromethane (1×30 mL), methanol (1×20 mL), tetrahydrofuran (1×30 mL), methanol (1×20 mL), dichloromethane (2×30 mL) and dried by nitrogen flow. The unreacted hydroxy group on the resin was then capped by reacting with acetic anhydride (5 mL) and diisopropylethylamine (1 mL) in dichloromethane (20 mL) at room temperature for 2 h. The resin was washed and dried by the same procedure mentioned above. The substitution level was 0.2–0.3 mmol/g. 
     Formation of Dipeptides (Performed on the Quest 210™): 
     25%. TFA in dichloromethane (20 mL) was added to the above resin (0.4 mmol, 2.0 g, 0.2 mmol/g) and agitated for 30 min. The resin was then washed with dichloromethane (3×30 mL), methanol (1×20 mL), dichloromethane (1×30 mL), methanol (1×20 mL), dichloromethane (1×30 mL), methanol (1×20 mL), dichloromethane (2×30 mL) and dried by nitrogen flow. 
     A 0.2M solution of hydroxybenzotriazole and diisopropy-carbodiimide in 60% dichloromethane/tetrahydrofuran was added to the N-Boc amino acid, followed by diisopropylethylamine (1.5 eq). The resulting mixture was stirred for 30 min at room temperature, and then transfered to the resin (200 mg on Quest 210™) and agitated at room temperature for 30 min. Diisopropylethylamine (2.0 mmol) was then added and agitated until Kaiser test of an aliquot of the resin was negative (2 to 4 h). The resin was washed with dichloromethane (3×4 mL), tetrahydrofuran (1×4 mL), methanol (1×4 mL), dichloromethane (1×4 mL), methanol (1×4 mL), tetrahydrofuran (1×4 mL), methanol (1×4 mL), dichloromethane (2×4 mL) and dried by nitrogen flow. 
     Formation of Tripeptides: 
     25% TFA in dichloromethane (4 mL) was added to the Boc-protected dipeptide resin (0.04 mmol, 200 mg g, 0.2 mmol/g) and agitated for 30 min. The resin was then washed with dichloromethane (3×4 mL), methanol (1×4 mL), dichloromethane (1×4 mL), methanol (1×4 mL), dichloromethane (1×4 mL), methanol (1×4 mL), dichloromethane (2×4 mL) and dried by nitrogen flow. 
     A 0.2M solution of hydroxybenzotriazole and diisopropyl-carbodiimide in 60% dichloromethane/tetrahydrofuran was added to the N-Bts amino acid, followed by diisopropyl-ethylamine (1.5 eq). The resulting mixture was stirred for 30 min at room temperature, and then transferred to the resin (200 mg on Quest 210™) and agitated at room temperature for 30 min. The resin was washed with dichloromethane (3×4 mL), tetrahydrofuran (1×4 mL), methanol (1×4 mL), dichloromethane (1×4 mL), methanol (1×4 mL), tetrahydrofuran (1×4 mL), methanol (1×4 mL), dichloromethane (2×4 mL) and dried by nitrogen flow. 
     Mitsunobu Reaction: 
     A 0.2M solution of 5-(tert-butoxycarbonylamino)-trans-2-penten-1-ol or N-tert-butoxycarbonyl-2-(2-hydroxyethoxy)-cinnamyl amine, and triphenylphosphine was added to the tripeptide resin (0.036 mmol, 180 mg, 0.2 mmol/g on the Quest 210™) in anhydrous tetrahydrofuran, followed by diethyl azodicarboxylated (1.5 eq). The mixture was agitated for 2 h and the resin was washed and dried by the same procedure mentioned above. 
     Cyclization of Alkylated Tripeptides: 
     The N-alkylated linear tripeptide (0.016 mmol, 80 mg) was treated with 25% TFA in dichloromethane (4 mL) for 30 min and washed with dichloromethane, methanol and dried by nitrogen flow. 
     Toluene (2 mL), acetic anhydride (1 mL) and diisopropyl-ethylamine (1 mL) were added to the above resin and agitated for 2 h. The resin was removed by filtration and washed with dichloromethane (3×4 mL). An aliquot of the the filtrate was analysized by LC-MS. The filtrate was concentrated and then diluted with ethyl acetate (15 mL). The solution was washed with 1N hydrochloric acid (2 mL), saturated sodium bicarbonate (2 mL), brine (3 mL), dried and evaporated to give the crude product. 
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Library of Macrocyclic Tripeptides Synthesized on 
               
               
                 the Kaiser Resin Using the Quest 210 ™: 
               
             
          
           
               
                   
                 SAMPLE ID 
                 ISOLATED QUANTITY (MG) 
                 PURITY 
               
               
                   
                   
               
               
                   
                 c-B-MPG-1 
                 5.9 (42%) 
                 Low 
               
               
                   
                 c-B-MVG-1 
                 4.4 (48%) 
                 Medium 
               
               
                   
                 c-B-MGM-1 
                 4.6 (32%) 
                 Low 
               
               
                   
                 c-B-LPM-1 
                 7.5 (50%) 
                 Low 
               
               
                   
                 c-B-LLM-1 
                 7.1 (46%) 
                 Good 
               
               
                   
                 c-B-GVM-1 
                 3.1 (22%) 
                 Low 
               
               
                   
                 c-B-GLF-1 
                 7.8 (81%) 
                 Good 
               
               
                   
                 c-B-MVG-4* 
                 2.4 (23%) 
                 Good 
               
               
                   
                 c-B-MGM-4* 
                 5.7 (34%) 
                 Low 
               
               
                   
                 c-B-LPM-4* 
                 3.3 (19%) 
                 Low 
               
               
                   
                 c-B-LMM-4* 
                 5.0 (29%) 
                 Medium 
               
               
                   
                 c-B-GVM-4* 
                 4.0 (25%) 
                 Low 
               
               
                   
                 c-B-GLF-4* 
                 2.9 (26%) 
                 Low 
               
               
                   
                   
               
               
                   
                 *Nature of linker 4 is shown in Scheme 2. 
               
             
          
         
       
     
     Example 5 
     Synthesis of Betsylated Macrocyclic Tripeptides on Thioester Resin in IRORI MACROKANS™: 
     Macrocyclic tripeptides were synthesized in MACROKANS™ (160 mg of aminomethyl resin) following the same procedure as that given for the synthesis on solid support (KAISER Resin, see example 4): 
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Library of Macrocyclic Tripeptides Synthesized 
               
             
          
           
               
                   
                 SAMPLE ID 
                 Quantity (mg) 
                 PURITY 
               
               
                   
                   
               
               
                   
                 c-B-GHG-1 
                 7.6 (6%) 
                 Good 
               
               
                   
                 c-B-GLF-1 
                 16.3 (14%) 
                 Excellent 
               
               
                   
                 c-B-GVM-1 
                 20 (18%) 
                 Good 
               
               
                   
                 c-B-LHF-1 
                 43 (27%) 
                 Excellent 
               
               
                   
                 c-B-LPM-1 
                 26.7 (22%) 
                 Excellent 
               
               
                   
                 c-B-LLM-1 
                 20.6 (17%) 
                 Excellent 
               
               
                   
                 c-B-MVG-1 
                 10.5 (10%) 
                 Good 
               
               
                   
                 c-B-MPG-1 
                 6.4 (6%) 
                 Low 
               
               
                   
                 c-B-GHG-4 
                 12.4 (11%) 
                 Excellent 
               
               
                   
                 c-B-GLF-4 
                 12.4 (9%) 
                 Excellent 
               
               
                   
                 c-B-GVM-4 
                 9.3 (7%) 
                 Good 
               
               
                   
                 c-B-LHF-4 
                 21.8 (16%) 
                 Excellent 
               
               
                   
                 c-B-LPM-4 
                 29.1 (2.1%) 
                 Excellent 
               
               
                   
                 c-B-LLM-4 
                 25.8 (18%) 
                 Excellent 
               
               
                   
                 c-B-MVG-4 
                 37.5 (30%) 
                 Excellent 
               
               
                   
                 c-B-MPG-4 
                 30.3 (24%) 
                 Excellent 
               
               
                   
                   
               
             
          
         
       
     
     Example 6 
     Synthesis of Mesylated Macrocyclic Tripeptides on Thioester Resin in IRORI MINIKANS™ 
     Macrocyclic tripeptides were synthesized in MINIKANS™(60 mg of aminomethyl resin) following the same procedure as that given for the synthesison solid support (KAISER Resin, see example 4) 
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Library of Macrocyclic Tripeptides Synthesized 
               
             
          
           
               
                   
                 SAMPLE ID 
                 Quantity (mg) 
                 PURITY 
               
               
                   
                   
               
               
                   
                 c-Ms-GLG-4 
                 3 (9%) 
                 Low 
               
               
                   
                 c-Ms-GLG-1 
                 3 (12%) 
                 Low 
               
               
                   
                 c-Ms-E(OMe)LG-4 
                 1.9 (5%) 
                 Good 
               
               
                   
                 c-Ms-E(Ome)LG-1 
                 4 (13%) 
                 Good 
               
               
                   
                 c-Ms-LPM-4 
                 0.1 
                 Good 
               
               
                   
                 c-Ms-LPM-1 
                 0.5 
                 Low 
               
               
                   
                 c-Ms-YLG-4 
                 2 (5%) 
                 Good 
               
               
                   
                 c-Ms-YLG-1 
                 6 (18%) 
                 Good 
               
               
                   
                 c-Ms-GLF-4 
                 6 (16%) 
                 Good 
               
               
                   
                 c-Ms-GLF-1 
                 5 (15%) 
                 Good 
               
               
                   
                 c-Ms-LLG-4 
                 1 (3%) 
                 Low 
               
               
                   
                 c-Ms-LLG-1 
                 — 
                 Low 
               
               
                   
                 c-Ms-LLM-4 
                 — 
                 Low 
               
               
                   
                 c-Ms-SLG-4 
                 — 
                 Good 
               
               
                   
                 c-Ms-E(COOH)LG-4 
                 13.3* 
                 Good 
               
               
                   
                 c-Ms-E(COOH)LG-1 
                 21* 
                 Good 
               
               
                   
                   
               
               
                   
                 *Unpurified 
               
             
          
         
       
     
     SPECIFIC EXAMPLES OF BUILDING UNITS 
     Tether Building Blocks (type D, see  FIG. 2   b ) 
     The tether building blocks have the following formula: HOY—L—Z—PGz. 
     The three following tether building blocks 4-(tert-butoxycarbonylamino)-cis-2-buten-1-ol (Y═CH 2 , L=[Z]alkene, Z=CH 2 ) and 4-(tert-butoxycarbonylamino)-2-butyn-1-ol (Y═CH 2 , L=alkyne, Z=CH 2 ) and 5-(tert-butoxycarbonylamino)-trans-2-penten-1-ol (Y═CH 2 , L=CH 2 -[E]alkene, Z=CH 2 ) were synthesized from commercial available cis-2-butene-1,4-diol, 2-butyne-1,4-diol and 3-amino-1-propanol respectively. 
     Preparation of 4-(tert-butoxycarbonylamino)-2-butyn-1-ol: 
     Dowex 50WX8-100 ion-exchange resin (53 g) was added to a suspension of 2-butyne-1,4-diol (153.9 g, 1.77 mol) and dihydropyran (250 mL, 2.66 mol) in dichloromethane (800 mL) at room temperature. The resulting mixture was stirred for 60 min and quenched with triethylamine (10 mL). The resin was removed by filtration. The filtrate was washed with a saturated aqueous solution of sodium bicarbonate (100 mL), brine (3×300 mL), dried over magnesium sulfate, filtered and evaporated under reduced pressure to give the desired monoprotected 2-butyne-1,4-diol with a yield of 50%. 
     To a solution of monoprotected 2-butyne-1,4-diol (20.4 g, 0.12 mol) and triphenylphosphine (40.9 g, 0.16 mol) in tetrahydrofuran (50 mL), hydrazoic acid (113 mL, 1.6 M in toluene, 0.18 mol) was added at 0° C. Diisopropyl azodi-carboxylate (29.5 mL, 0.15 mol) was added dropwise to the solution whose temperature was around 0° C. The reaction was stirred for 30 min at 0° C. and for 30 min at room temperature. Triphenylphosphine (40.9 g, 0.16 mol) was added at 0° C. and the reaction was stirred overnight at room temperature. After addition of water (50 mL), the mixture was heated at 60° C. for 4 h, 1N hydrochloric acid (140 mL) was added. After stirring for 1 h, brine (140 mL) and dichloromethane (500 mL) were added. The aqueous phase was washed with dichloromethane (3×100 mL). Potassium carbonate (16.5 g, 0.12 mol) was added, followed by a solution of di-tert-butyl dicarbonate (26.2, 0.12 mol) in tetrahydrofuran (100 mL). The reaction mixture was stirred for 18 h, then extracted with dichloromethane (1×300 mL, 2×50 mL). The combined organic extract was washed with brine (50 mL), dried over magnesium sulfate, filtered and evaporated under reduced pressure. The residue was purified by a dry-pack silica gel column to give the titled compound in 50% yield. 
     Preparation of 4-(tert-butoxycarbonylamino)-cis-2-buten-1-ol: 
     The title compound was synthesized from cis-2-butene-1,4-diol in an overall yield of 30% according to the procedure used for 4-tert-butoxycarbonylamino-2-butyn-1-ol. 
     Preparation of 5-(tert-butoxycarbonylamino)-trans-2-penten1-ol: 
     A solution of di-tert-butyl dicarbonate (382.8 g, 1.75 mol) in dichloromethane (1.6 L) was added to 3-amino-1-propanol (263.6 g, 3.51 mol) during 2 h. The reaction mixture was stirred for an additional 40 min and water (1 L) was added. 
     The organic phase was washed with water (3×500 ml),), dried over magnesium sulfate, filtered and evaporated under reduced pressure to give 3-(tert-butoxycarbonylamino)-1-propanol in 96% yield. 
     Dimethyl sulfoxide (321 mL, 4.5 mol) and triethylamine (941 mL, 6.75 mol) were added to a solution of 3-tert-butoxycarbonylamino-1-propanol (262.6 g, 1.5 mol) in dichloromethane (1.2 L) at 0° C. Sulfur trioxide pyridine complex (286.4 g, 1.8 mol) was added in 6 portions. The reaction mixture was then stirred for 30 min at 0° C. and for 30 min at room temperature. The reaction was cooled down to 0° C., quenched with 1N hydrochloric acid, washed with brine (2×500 mL), dried over magnesium sulfate, filtered and evaporated under reduced pressure to give 3-(tert-butoxycarbonylamino)propionaldehyde. 
     To a solution of the above crude aldehyde (1.5 mol) in acetonitrile (1.3 L), trimethyl phosphonoacetate (409.7 g, 2.25 mol) and lithium hydroxide (53.9 g, 2.25 mol) were added and stirred overnight at room temperature. The reaction was quenched with water (50 mL). The acetonitrile solvent was removed by evaporation under reduced pressure. The residue was then diluted with diethyl ether (800 mL), washed with 1N sodium hydroxide (3×300 mL), brine (2×500 mL), dried over magnesium sulfate, filtered and evaporated under reduced pressure to give the desired trans-unsaturated methyl ester. 
     Diisobutylaluminium hydride (1.12 L, 1.0 M in dichloromethane, 1.12 mol) was added dropwise to a solution of this methyl ester (102.75 g, 0.445 mol) in dichloromethane (250 mL) at 0° C. The resulting mixture was stirred for an additional 1 h and then poured slowly into a 1M tartaric acid aqueous solution (1.4 L), extracted with dichloromethane (3×500 mL). The combined organic phase was dried over magnesium sulfate, filtered and evaporated under reduced pressure. The residue was purified by dry-pack silica gel column to give the titled compound with a yield of 40% for the three steps. 
     Amino-Acid Building Block (type A and B, see  FIG. 2   a ) 
     The N-Boc amino acids are commercial available except N ∝ -Boc-N im -Doc-histidine which was preparated by the method described below. 
     Preparation of N ∝ -Boc-N” im -Doc-histidine: 
     A solution of 2,4-dimethyl-3-pentanol (83.2 g, 0.72 mol) and triethylamine (125 mL, 0.90 mol) in toluene (300 mL) was added slowly (30 min) to a solution of phosgene (531 mL, 20% in toluene, 1.07 mol) in toluene (531 mL) at 0° C. The mixture was stirred for an additional 30 min at the same temperature. Ice-cold water (500 mL) was added and the organic phase was washed with ice-cold water (2×100 mL), dried over magnesium sulfate, filtered and evaporated under reduced pressure. The residue was then distilled under reduced pressure (6 cm of Hg, 33–35° C.) to give Doc-Cl as a colorless oil (92 g, 72%). 
     A solution of Doc-Cl (57.9 g, 0.32 mol) in tetrahydrofuran (250 mL) was added slowly to a solution of N ∝ -Boc-histidine (69 g, 0.27 mol) and potassium carbonate (41.1 g, 0.28 mol) in water (350 mL) at 0° C. The resulting mixture (pH=10) was stirred for 2 h at the same temperature and for 1 h at room temperature. Water (150 mL) and hexane (200 mL) were added to the reaction mixture. The separated aqueous phase was washed with a mixture of diethyl ether and hexane (1:1; 3×200 mL), acidified with 20% citric acid aqueous solution to pH 2–3, and then extracted with dichloromethane (3×200 mL). The combined dichloromethane solution was washed with brine (200 mL), dried over magnesium sulfate, filtered and evaporated under reduced pressure to give the crude N ∝ -Boc-N im -Doc-histidine in quantitive yield. 
     Building Blocks of Type C (see  FIG. 2   b ) 
     N-Betsyl protected amino acids (Bts-AA1) were synthesized by the reaction of amino acids with Betsyl chloride (benzothiazole-2-sulfonylchloride) which was obtained from mercaptobenzothiazole and chlorine. 
     Preparation of N-Betsyl Amino Acid (Bts-AA 1 OH): 
     Chloride gas was bubbled into a solution of acetic acid (250 mL) in water (500 mL) at 5° C. until an orange precipitate was formed in good quantity. A solution of mercaptobenzothiazole (0.7 mol) in aqueous acetic acid (750 mL, 33% in water) was added in portions to the above reaction mixture in a period of 3 hours. The reaction mixture was stirred for 1 h, filtered at 0° C. then washed with cold water. The solid was dissolved in cold dichloromethane (500 mL) and washed with cold brine (2×100 mL), cold saturated sodium bicarbonate (100 mL), brine (100 mL), dried over magnesium sulfate, filtered and evaporated at 10° C. under reduced pressure. The solid was then washed with cold diethyl ether (100 mL), cold acetonitrile (100 mL), filtered and pumped to give betsyl chloride (benzothiazole-2-sulfonylchloride). 
     To a solution of amino acid (0.11 mol) in 0.25 N aqueous sodium hydroxide (0.08 mol) at room temperature (initial pH around 9.5), betsyl chloride (0.1 mol) was added. The resulting mixture was stirred vigorously for 18 h. The pH of the reaction was adjusted between 9.5 to 10.0 with 1.0 N aqueous sodium hydroxide during the reaction progress. The reaction mixture was washed with diethyl ether (3×50 mL). The aqueous phase was then cooled to 0° C., acidified to pH 1.5–2.0 with 6 N HCl, and extracted with ethyl acetate (3×100 mL). The combined ethyl acetate solution was dried over magnesium sulfate, filtered and evaporated under reduced pressure to give the desired compound in 74–85% yield. 
     Building Blocks of Type E (see  FIG. 2   b ) 
     These building blocks correspond to building blocks of type C which have been alkylated using Mitsunobu reaction conditions with the building blocks of type D. To be able to carry out this alkylation, the acid functional group of C must be protected. The protecting group used is finally removed to get the desired compounds 
     Preparation of N-alkylated N-Betsyl Amino Acid: 
     Dihydrofuran (90 mmol) and pyridinium p-toluenesulfonate (1.5 mmol) were added to a suspension of N-Betsyl amino acid (30 mmol) in dichloromethane (50 mL) at 0° C. The resulting mixture was stirred for 15 min at 0° C. and for 60 min at room temperature. The reaction mixture was diluted with diethyl ether (150 mL), washed with saturated aqueous sodium bicarbonate (20 mL), brine (20 mL), dried over magnesium sulfate, filtered and evaporated under reduced pressure to give the tetrahydrofuranyl ester of amino acid. A mixture of this ester (30 mmol), an alcohol (type D building block) (i.e. 4-(tert-butoxycarbonylamino)-cis-2-buten-1-ol, or 4-(tert-butoxycarbonylamino)-2-butyn-1-ol, or 5-(tert-butoxycarbonylamino)-trans-2-penten-1-ol)) (43.5 mmol) and triphenylphosphine (49 mmol) were suspended in toluene (20 mL) and azeotropically distilled three times in vacuum. The residue was dissolved in tetrahydrofuran (40 mL). Diisopropyl azodicarboxylate (43.5 mmol) was added at 0° C. After stirring for 15 min at 0° C. and for 30 min at room temperature, 1N hydrochloric acid (30 mL) and methanol (30 mL) was added and stirred for an additional 60 min. After removal of the organic solvents by evaporation, the aqueous phase was diluted with water (30 mL), the pH of the medium was adjusted to 12 with potassium carbonate, washed with diethyl ether (3×60 mL), and then acidified to pH 2–3 with 1N hydrochloric acid, extracted with dichloromethane (3×200 mL). The combined dichloromethane solution was washed with brine (100 mL), dried over magnesium sulfate, filtered and evaporated under reduced pressure to give the desired alkylated N-Betsyl amino acid in the overall yield of 68–89%.