Patent Abstract:
The present invention provides antineoplastic peptides of formula I, 
                         R 1 R 2 N-CHX-CO-A-B-D-E-(G) S -K I               
wherein R 1 , R 2 , X, A, B, D, G, K and s have the meanings stated in the description. The compounds have antineoplastic activity.

Full Description:
RELATED APPLICATIONS 
       [0001]    This application is a continuation of U.S. patent application Ser. No. 12/079,980, filed Mar. 31, 2008, which is a is a divisional of U.S. patent application Ser. No. 11/179,074, filed Jul. 11, 2005 which issued as U.S. Pat. No. 7,368,528 on May 6, 2008, which is a divisional of U.S. patent application Ser. No. 09/757,142, filed Jan. 9, 2001, now abandoned, which is a continuation of U.S. patent application Ser. No. 09/097,184, filed Jun. 12, 1998, now abandoned, which is a continuation-in-part of International Application Serial No. PCT/EP96/05518, filed Dec. 11, 1996, which designated the United States, published in English, which claims priority to U.S. Provisional Patent Application Ser. No. 60/059,062, entitled “Antineoplastic Peptides”, which resulted from the conversion of U.S. patent application Ser. No. 08/573,422, filed Dec. 15, 1995, now abandoned. 
         [0002]    The entire teachings of the above applications are incorporated herein by reference. 
     
    
     FIELD OF THE INVENTION 
       [0003]    The invention described herein provides novel peptides and derivatives thereof which offer potentially improved therapeutic utilities for the treatment of neoplastic diseases as compared to dolastatin-10 and -15 (U.S. Pat. Nos. 4,879,276 and 4,816,444) and the compounds described in WO 93/23424. 
       SUMMARY OF THE INVENTION 
       [0004]    Compounds of this invention include novel peptides of the formula i 
         [0000]                            R 1 R 2 N-CHX-CO-A-B-D-E-(G) S -K I            
where
   R 1  is hydrogen, methyl, or ethyl;   R 2  is methyl; or ethyl; or   R 1 —N—R 2  together are a pyrrolidine ring;   A is a valyl, isoleucyl, allo-isoleucyl, 2-tert-butylglycyl, 2-ethylglycyl, norleucyl or norvalyl residue;   B is a N-methyl-valyl, N-methyl-norvalyl, N-methyl-leucyl, N-methyl-isoleucyl, N-methyl-2-tert-butylglycyl, N-methyl-2-ethylglycyl, or N-methyl-norleucyl residue;   D is a prolyl, homoprolyl, hydroxyprolyl, or thiazolidine-4-carbonyl residue;   E is a prolyl, homoprolyl, hydroxyprolyl, or thiazolidine-4-carbonyl, trans-4-fluoro-L-prolyl, cis-4-fluoro-L-prolyl, trans-4-chloro-L-prolyl or cis-4-chloro-L-prolyl residue;   X is ethyl, propyl, butyl, isopropyl, sec. butyl, tert.-butyl, cyclopropyl, or cyclopentyl;   G is a L-2-tert.butylglvcyl, D-2-tert.butylglycyl, D-valyl, D-isoleucyl, D-leucyl, D-norvalyl, 1-aminopentyl-1-carbonyl, or 2,2-dimethylglycyl residue;   S is 0 or 1;   K is —NH—C 1-8 -alkyl, —NH—C 3-8 -alkenyl, —NH—C 3-8  alkinyl, —NH—C 6-8  cycloalkyl, —NH—C 1-4 -alkene-C 3-8 cycloalkyl, C 1-4 -alkyl-N—C 1-6 -alkyl, in which residues one CH 2  group may be replaced by O or S, one H by phenyl or cyano, or 1, 2 or 3H by F, except the N-methoxy-N-methylamino, N-benzylamino, or N-methyl-N-benzylamion residue, or K is   
 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    and the salts thereof with physiologically tolerated acids 
     
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0016]    In specific embodiments of the compounds of formula I, K may be —NHCH 3 , —NHCH 2 CH 3 , —NH(CH 2 ) 2 CH 3 , —NH(CH 2 ) 3 CH 3 , —NH(CH 2 ) 4 —CH 3 , —NH(CH 2 ) 5 CH 3 , —NH(CH 2 ) 6 CH 3 , —NHCH(CH 2 ) 7 CH 3 , —NHCH(CH 3 ) 2 , —NHCH(CH 3 )CH 2 CH 3 , —NHCH(CH 2 CH 3 ) 2 , —NHCH(CH 2 CH 2 CH 3 ) 2 , NHC(CH 3 ) 3 , NHCH(CH 2 CH 3 )CH 2 CH 2 CH 3 , —NHCH(CH 3 )CH(CH 3 ) 2 , —NHCH(CH 2 CH 3 )CH(CH 3 ) 2 , —NHCH(CH 3 )C(CH 3 ) 3 , —NH-cyclohexyl, —NH-cycloheptyl, —NH-cyclooctyl, —N(CH 3 )OCH 2 CH 3 , N(CH 3 )OCH 2 CH 2 CH 3 , —N(CH 3 )OCH(CH 3 ) 2 , —N(CH 3 )O(CH 2 ) 3 , —N(CH 3 )OCH 2 C 6 H 5 , —NH(CH 2 ) 2 C 6 H 5 , —NH(CH 2 ) 3 C 6 H 5 , —NHCH(CH 3 )C 6 H 5 , —NHC(CH 3 ) 2 C 6 H 5 , —NHC(CH 3 ) 2 CH 2 CH 3 , —NHC(CH 3 )(CH 2 CH 3 ) 2 , —NHCH[CH(CH 3 ) 2 ] 2 ), —NHC(CH 3 ) 2 CN, —NHCH(CH 3 )CH(OH)C 6 H 5 , —NHCH 2 -cyclohexyl, NHCH 2 C(CH 3 ) 3 , —NHCH 2 CH(CH 3 ) 2 , —N(CH 3 ) 2 , —N(CH 2 CH 3 ) 2 , —N(CH 2 CH 2 CH 3 ) 2 , —NHCH 2 CF 3 , —NHCH(CH 2 F) 2 , —NHCH 2 CH 2 F, —NHCH 2 CH 2 , OCH 3 , —NHCH 2 CH 2 SCH 3 , —NHCH 2 CHCH 2 , —NH—C(CH 3 ) 2 CH═CH 2 , —NHC(CH 3 ) 2 , C≡CH, —NHC(CH 2 CH 3 ) 2 C≡CH, —NHC(CH 3 ) 2 CH 2 CH 2 OH, —NH(CH 2 CH 2 O) 2 CH 2 CH 3 , —NHC(CH 3 ) 2 CH(CH 3 ) 2 , —NHC(CH 3 ) 2 CH 2 CH 2 CH 3 , —NHC(CH 3 ) 2 CH 2 —C 6 H 5 , —N(OCH 3 )CH(CH 3 ) 2 —N(OCH 3 )CH 2 CH 3 , —N(OCH 3 )CH 2 CH 2 CH 3 , —N(OCH 3 )CH 2 C 6 H 5 , —N(OCH 3 )C 6 H 5 , —N(CH 3 )OC 6 H 5 , —NHCH[CH(CH 3 ) 2 ] 2 , —N(OCH 3 )CH 2 CH 2 CH 2 , CH 3 , or the special ring systems mentioned above. 
         [0017]    In one embodiment of the compounds of formula I described above, s is 0 and E is homoprolyl or hydroxyprolyl. 
         [0018]    Preferred are compounds of the formula I where the substituents R 1 , R 2 , A, B, D, E, X, G and s have the following meanings:
   R 1  hydrogen, methyl, or ethyl, especially methyl;   R 2 , methyl or ethyl, especially methyl;   A valyl, valyl, isoleucyl, 2-tert-butylglycyl, 2-ethylglycyl, norleucyl or norvalyl, especially valyl, isoleucyl, 2-tert-butylglycyl, 2-ethiyigivcyl,   B N-methyl-valyl, N-methyl-norvalyl, N-methyl-isoleucyl, N-methyl-2-tert-butylglycyl, N-methyl-2-ethylglycyl, or N-methyl-norleucyl, especially N-methyl-valyl, N-methyl-2-ethylglycyl, N-methyl-norleucyl, N-methyl-isoleucyl, or N-methyl-2-tert.butyl-glycyl;   D prolyl, homoprolyl or thiazolidine-4-carbonyl, espiecally prolyl or thiazolidine-4-carhonyl;   E prolyl, homoprolyl, thiazolidine-4-carbonyl, trans-4-fluoro-L-prolyl, cis-4-fluoro-L-prolyl, trans-4-chloro-L-prolyl or cis-4-chloro-L-prolyl, espiecally prolyl, trans-4-fluoro-prolyl, cis-4-fluoro-prolyl, trans-4-chloro-prolyl, or cis-4-chloro-prolyl;   X ethyl, propyl, isopropyl, sec.butyl, tert.butyl or cyclo-propyl, especially ethyl, isopropyl, sec.butyl or tert.butyl;   G L-2-tert.butylglycyl, D-2-Tert.butylglycyl, D-valyl, D-isoleucyl, D-leucyl or 2,2-dimethylglycyl residue;   s 0 or 1.   
 
         [0028]    Preferred meanings for K are:
       —NH—C 1-8 -alkyl, —NH—C 6-8 -cycloalkyl, —NH—CH 2 -cyclohexyl, C 1-4 -alkyl-N—C 1-6 -alkyl, in which residues one CH 2  group may be replaced by O, one H by phenyl or 1 or 2H by F, except the N-methoxy-N-methylamino, N-benzylamino, or N-methyl-N-beuzylamino residue, or K is       
 
         [0000]    
       
                 
         
             
             
         
       
     
         [0030]    More preferred K is
       —NHCH 3 , —NHCH 2 CH 3 , —NH(CH 2 ) 2 CH 3 , —NH(CH 2 ) 3 CH 3 , —NH(CH 2 ) 4 —CH 3 , NH(CH 2 ) 5 CH 3 , —NH(CH 2 ) 6 CH 3 , —NH(CH 2 ) 7 CH 3 , —NHCH(CH 3 ) 2 , —NHCH(CH 3 )CH 2 CH 3 , —NHCH(CH 2 CH 3 ) 2 , —NHCH(CH 2 CH 2 CH 3 ) 2 , —NHC(CH 3 ) 3 , —NHCH(CH 2 CH 3 )CH 2 CH 2 CH 3 , —NHCH(CH 3 )CH(CH 3 ) 2 , —NHCH(CH 2 CH 3 )CH(CH 3 ) 2 , —NHCH(CH 3 )C(CH 3 ) 3 , —NH-cyclohexyl, —NH-cycloheptyl, —NH-cyclooctyl, —N(CH 3 )OCH 2 CH 3 , —N(CH 3 )OCH 2 CH 2 CH 3 , —N(CH 3 )OCH(CH 3 ) 2 , —N(OCH 3 )CH(CH 3 ) 2 , —N(CH 3 )OCH 2 C 6 H 5 , —NH(CH 2 ) 2 C 6 H 5 , —NH(CH 2 ) 3 C 6 H 5 , —NHCH(CH 3 )C 6 H 5 , —NHC(CH 3 ) 2 C 6 H 5 , —NHC(CH 3 ) 2 CH 2 CH 3 , —NHC(CH 3 )(CH 2 CH 3 ) 2 , —NHCH(CH 3 )CH(OH)C 6 H 5 , —NHCH 2 -cyclohexyl, —N(CH 3 ) 2 , —N(CH 2 CH 3 ) 2 , —N(CH 2 CH 2 CH 3 ) 2 , —NHCH(CH 2 F) 2 , —NHC(CH 3 )CH═CH 2 , —NHC(CH 3 ) 2 CN, —NHC(CH 3 ) 2 C≡CH, —NHC(CH 3 ) 2 CONH 2 , —NHCH[CH(CH 3 ) 2 ] 2 , —N(OCH 3 )CH 2 C 6 H 5 , —N(OCH 3 )CH 2 CH 3 , —N(OCH 3 )CH 2 CH 2 CH 3 , —N(OCH 3 )CH 2 CH 2 CH 2 CH 3 ,       
 
         [0000]    
       
                 
         
             
             
         
       
     
         [0032]    In one embodiment of the preferred compounds of formula I described above, is 0 and E is homoprolyl or hydroxyprolyl. 
         [0033]    Especially preferred are compounds of the formula I where
   R 1  and R 2  are methyl,   A is a valyl, isoleucyl, 2-tert.-butylglycyl residue   B is a N-methylvalyl, N-methyl-isoleucyl, methyl-2tert.-butylglyeyl residue,   D is a prolyl or thiazolidine-4-carbonyl residue   E is a prolyl, cis-4-fluoro-L-prolyl, or cis-4-chloro-L-prolyl residue   X is a isopropyl, sec.-butyl, or tert.-butyl residue,   s is 0 and   K is
       —NHCH(CH 3 ) 2 , —NHCH(CH 3 )CH 2 CH 3 , —NHCH(CH 2 CH 3 ) 2 , —NHCH(CH 2 CH 2 CH 3 ) 2 , —NHC(CH 3 ) 3 , —NHCH(CH 2 CH 3 )CH 2 CH 2 CH 3 , —NHCH(CH 3 )CH(CH 3 ) 2 , —NHCH(CH 2 CH 3 )CH(CH 3 ) 2 , —NHCH(CH 3 )C(CH 3 ) 3 , —NH-cycloheptyl, —NH-cyclooctyl, —N(CH 3 )OCH 2 CH 3 , —N(CH 3 )OCH 2 CH 2 CH 3 , —N(CH 3 )OCH(CH 3 ) 2 , —N(OCH 3 )CH(CH 3 ) 2 , —N(CH 3 )OCH 2 C 6 H 5 , —NH(CH 2 ) 2 C 6 H 5 , —NH(CH 2 ) 3 C 6 H 5 , —NHCH(CH 3 )C 6 H 5 , —NHC(CH 3 ) 2 C 6 H 5 , —NHC(CH 3 ) 2 CH 2 CH 3 , NHC(CH 3 )(CH 2 CH 3 ) 2 , —NHCH(CH 3 )CH(OH)C 6 H 5 , —NHCH(CH 2 F) 2 , —NHC(CH 3 ) 2 CH 2 CH 2 OH, —NH(CH 2 CH 2 O) 2 CH 7 CH 3 , —NHC(CH 3 ) 2 CH—CH 2 , —NHC(CH 3 ) 2 CH(CH 3 ) 2 , —N(OCH 3 )CH 2 CH 3 , —N(OCH 3 )CH 2 CH 2 CH 3 , —N(OCH 3 )CH 2 CH 2 CH 2 CH 3 , —NHC(Ch 3 ) 2 CN, —NHC(CH 3 ) 2 C≡CH, —NHCH[CH(CH 3 ) 2 ] 2 , —NHC(CH 3 ) 2 CONH 2 , —NHC(CH 3 ) 2 CH 2 C 6 H 5 , —N(OCH 3 )C 6 H 5 , —N(OCH 3 )CH 2 C 6 H 5 ,   
       
 
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         [0043]    This invention also provides methods for preparing the compounds of formula I, pharmaceutical compositions containing such compounds together with a pharmaceutically acceptable carrier and methods for using same for treating cancer in mammals. 
         [0044]    The new compounds may be present as salts with physiologically tolerated acids such as: hydrochloric acid, citric acid, tartaric acid, lactic acid, phosphoric acid, methanesulfonic acid, acetic acid, formic acid, fumaric acid, malic acid, succinic acid, malonic acid, sulfuric acid, L-glutamic acid, L-aspartic acid, pyruvic acid, mucic acid, benzoic acid, glucuronic acid, oxalic acid, ascorbic acid and acetylglycine. 
         [0045]    The novel compounds can be prepared by known methods of peptide chemistry. Thus, the peptides can be assembled sequentially from amino acids or by linking suitable small peptide fragments. In the sequential assemblage, starting at the C terminus the peptide chain is extended stepwise by one amino acid each time. In fragment coupuling it is possible to link together fragments of different lengths, and the fragments in turn can be obtained by sequential assemblage from amino acids or themselves by fragment-coupling. 
         [0046]    Both in the sequential assemblage and in the fragment coupling it is necessary to link the units by forming an amide linkage. Enzymatic and chemical methods are suitable for this. 
         [0047]    Chemical methods for forming the amide linkage are described in detail by Mueller, Methoden der organischen Chemie Vol. XV/2, pp 1 to 264, Thieme Verlag, Stuttgart, 1974; Stewart, Young, Solid Phase Peptide Synthesis, pp 31 to 34, 71 to 82, Pierce Chemical Company, Rockford, 1984; Bodanszky, Klausner, Ondetti, Peptide Synthesis, pp 85 to 128, John Wiley &amp; Sons, New York, 1976; The Practice of Peptide Synthesis, M. Bodanszky, A. Bodanszky, Springer-Verlag, 1994, and other standard works on peptide chemistry. Particular preference is given to the azide method, the symmetric and mixed anhydride method, in situ generated or performed active esters, the use of urethane protected N-carboxy anhydrides of amino acids and the formation of the amide linkage using coupling reagents, especially dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), pivaloylchloride, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), n-propanephosphonic anhydride (PPA), N,N-bis(2-oxo-3-oxazolodinyl)-amidophosphoryl chloride (BOP—Cl), bromo-tris-pyrrolidino-phosphonium hexafluororophosphate (PyBrop), diphenylphosphoryl azide (DPPA), Castro&#39;s reagent (BOP, PyBop), O-benzotriazolyl-N,N,N′,N′-tetramethyluronium salts (HBTU), O-azabenzotriazolyl-N,N,N′,N′-tetramethyluronium salts (HATU), diethylphosphoryl cyanide (DEPCN), 2,5-diphenyl-2,3-dihydro-3-oxo-4-hydroxythiophene dioxide (Steglich&#39;s reagent; HOTDO) and 1,1′-carbonyldiimidazole (CDI). The coupling reagents can be employed alone or in combination with additives such as N,N-dimethyl-4-aminopyridine (DMAP), N-hydroxy-benzotriazole (HOBO, N-hydroxybenzotriazine (HOOBt), Azabenzotriazole, N-hydroxysuccinimide (HOSu) or 2-hydroxypyridine. 
         [0048]    Whereas it is normally possible to dispense with protective groups in enzymatic peptide synthesis, reversible protection of reactive groups not involved in formation of the amide linkage is necessary for both reactants in chemical synthesis. Three conventional protective group techniques are preferred for the chemical peptide synthesis: the benzyloxycarbonyl (Z), the t-butoxycarbonyl (Boc) and the 9-fluorenylmethoxycarbonyl (Fmoc) techniques. 
         [0049]    Identified in each case is the protective group on the alpha-amino group of the chain-extending unit. A detailed review of amino-acid Protective groups is given by Mueller, Methoden der organischem Chemie vol. XV/1, pp 20 to 906, Thieme Verlag, Stuttgart, 1974. The units employed for assembling the peptide chain can be reacted in solution, in suspension or by a method similar to that described by Merrifield in J. Amer. Chem. Soc. 85 (1963) 2149. 
         [0050]    Suitable for peptide synthesis in solution are all solvents which are inert under the reaction conditions, especially water, N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), acetonitrile, dichloromethane (DCM), ethyl acetate, 1,4-dioxane, tetrahydrofuran (THF), N-methyl-2-pyrrolidone (NMP) and mixtures of the said solvents. 
         [0051]    Peptide synthesis on the polymeric support can be carried out in all inert organic solvents in which the amino-acid derivatives used are soluble. However, preferred solvents additionally have resin-swelling properties, such as DMF, DCM, NMP, acetonitrile and DMSO, and immixtures of these solvents. After synthesis is complete, the peptide is cleaved off the polymeric support. The conditions under which cleavage off the various resin types is possible are disclosed in the literature. The cleavage reactions most commonly used are acid- and palladium-catalyzed, especially cleavage in liquid anhydrous hydrogen fluoride, in anhydrous trifluoromethanesulfonic acid, in dilute or concentrated trifluoroacetic acid, palladium-catalyzed cleavage in THF or THF-DCM mixturers in the presence of a weak base such as morpholine or cleavage in acetic acid/dichloromethane/trifluoroethanol mixtures. Depending on the chosen protective groups, these may be retained or likewise cleaved off under the cleavage conditions. 
         [0052]    Partial deprotection of the peptide may also be worthwhile when certain derivatization reactions are to be carried out. 
         [0053]    Peptides dialkylated at the N-terminus can be prepared either by coupling on the appropriate N,N-di-alkylamino acids in solution or on the polymeric support, by reductive alkylation of the resin-bound peptide in DMF/1% acetic acid with NaCNBH 3  and the appropriate aldehydes, by hydrogenation of the peptide in solution in the presence of aldehyde or ketone and Pd/C. 
         [0054]    The various non-naturally occurring amino acids as well as the various non-amino acid moieties disclosed herein may be obtained from commercial sources or synthesized from commercially available materials using methods known in the art. For example, amino acids building blocks with R 1  and R 2  moieties can be prepared according to E. Wuensch, Houben Weyl, Meth. d. Org. Chemie, Bd. XV, 1, p. 306 following, Thieme Verlag Stuttgart 1974 and Literature cited therein. 
         [0055]    The compounds of this invention may be used to inhibit or otherwise treat solid tumors (e.g. tumors of the lung, breast, colon, prostate, bladder, rectum, or endometrial tumors) or hematological malignancies (e.g. leukemias, lymphomas) by administration of the compound to the mammal. 
         [0056]    It is a special advantage of the new compounds that they are very resistant to enzymatic degradation and can also be administered orally. 
         [0057]    Administration may be by any of the means which are conventional for pharmaceutical, preferably oncological, agents, including oral and parenteral means such as subcutaneously, intravenously, intramuscularly and intraperitoneally. 
         [0058]    The compounds may be administered alone or in the form of pharmaceutical compositions containing a compound of formula I together with a pharmaceutically accepted carrier appropriate for the desired route of administration. Such pharmaceutical compositions may be combination products, i.e., may also contain other therapeutically active ingredients. 
         [0059]    The dosage to be administered to the mammal will contain an effective tumor-inhibiting amount of active ingredient which will depend upon conventional factors including the biological activity of the particular compound employed; the means of administration; the age, health and body weight of the recipient; the nature and extent of the symptoms; the frequency of treatment; the administration of other therapies; and the effect desired. A typical daily dose will be about 0.05 to 50 milligrams per kilogram of body weight on oral administration and about 0.01 to 20 milligrams per kilogram of body weight on parenteral administration. 
         [0060]    The novel compounds can be administered in conventional solid or liquid pharmaceutical administration forms, e.g. uncoated or (film-)coated tablets, capsules, powders, granules, suppositories or solutions. These are produced in a conventional manner. The active substances can for this purpose be processed with conventional pharmaceutical aids such as tablet binders, fillers preservatives, tablet disintegrants, flow regulators, plasticizers, wetting agents, dispersants, emulsifiers, solvents, sustained release compositions, antioxidants and/or propellant gases (cf. H. Sucker et al.: Pharmazeutische Technologic, Thieme-Verlag, Stuttgart, 1978). The administration forms obtained in this way normally contain 1-90% by weight of the active substance. The following examples are intended to illustrate the invention. The proteinogenous amino acids are abbreviated in the examples using the known three-letter code. Other abbreviations used: Me 2 Val=N,N-dimethylvaline, MeVal=N-methylvaline. 
       Examples 
     A. General Procedures 
       [0061]    I. The peptides of the present invention are either synthesized by classical solution synthesis using standard Z- and Boc-methodology as described above or by standard methods of solid-phase synthesis using Boc and Fmoc protective group techniques. 
         [0062]    In the case of solid phase synthesis, the N,N-dialkylpenta- or hexapeptide acids are liberated from the solid support and further coupled with the corresponding C-terminal amines in solution. BOP—Cl and PyBrop were used as reagents for coupling of the amino acid following the N-methylamino acids. The reaction times were correspondingly increased. For reductive alkylation of the N-terminus, the peptide-resin was deprotected at the N terminus and then reacted with a 3-fold molar excess of aldehyde or ketone in DMF/1% acetic acid with addition of 3 equivalents of NaCNBH 3 . After the reaction was complete (negative Kaisertest) the resin was washed several times with water, isopropanol, DMF and dichloromethane. 
         [0063]    In solution synthesis, the use of either Boc-protected amino acid NCAs (N-tert.-butyloxycarbonyl-amino acid-N-carboxy-anhydrides), Z-protected amino acid NCAs (N-benzyloxycarbonyl-amino acid-N-carboxy-anhydrides), or the use of pivaloylchloride as condensing agent respectively is most advantageous for coupling of the amino acid following the N-methylamino acids. Reductive alkylation of the N terminus can e.g. be achieved by reaction of the N-terminally deprotected peptides or amino acids with the corresponding aldehydes or ketones using NaCNBH 3  or hydrogen, Pd/C. 
         [0064]    II. Purification and Characterization of the Peptides 
         [0065]    Purification was carried out by gel chromatography (SEPHADEX G-10, G-15/10% HOAc, SEPHADEX LH20/MeOH), medium pressure chromatography (stationary phase: HD-SIL C-18, 20-45 mikron, 100 Angstrom; mobile phase: gradient with A=0.1% TFA/MeOH, B=0.1% TFA/water), or preparative HPLC (stationary phase: Waters Delta-Pak C-18, 15 mikron, 100 Angstrom; mobile phase: gradient with A=0.1% TFA/MeOFI, 3=0.1% TFA/water). 
         [0066]    The purity of the resulting products was determined by analytical HPLC (stationary phase: 100 2.1 mm VYDAC C-18, 5 1, 300 A; mobile phase: acetonitrile-water gradient, buffered with 0.1% TFA, 40.degree. C.). 
         [0067]    Characterization was by amino-acid analysis and fast atom bombardment mass pectroscopy. 
       B. Specific Procedures 
       [0068]      
         [0000]    
       
         
               
               
             
           
               
                   
                 Example 1 
               
               
                   
                 (SEQ ID NO: 1) 
               
               
                   
                 Me. 2 Val-Val-MeVal-Pro-Pro-NHCH(CH 3 ) 2   
               
               
                   
                   
               
               
                   
                 a) Z-MeVal-Pro-OME 
               
             
          
         
       
     
         [0069]    66.25 g (250 mmol) Z-MeVal-OH were dissolved in 250 ml dry dichloromethane. After addition of 36.41 ml (262.5 mmol) triethylamine, the reaction mixture was cooled to −25° C. and 32.27 ml (262.5 mmol) pivaloyl chloride were added. After stirring for 2.5 h, 41.89 g (250 mmol) H-Pro-OMe×Ch1 in 250 ml dichloromethane, neutralized with 36.41 ml (262.5 mmol) triethylamine at 0.degree. C., were added to the reaction mixture. Stirring continued for 2 h at −25.degree. C. and overnight at room temperature. The reaction mixture was diluted with dichloromethane and thoroughly washed with saturated aqueous NaHCO 3  solution (3×.), water (1×), 5% citric acid (3×) and saturated NaCl Solution. The organic phase was dried over sodium sulfate and evaporated to dryness. The residue (91.24 g) was stirred with petroleum ether overnight and filtered. 62.3 g of product were obtained. 
         [0000]    
       
         
               
               
             
           
               
                   
                 b) H-MeVal-Pro-OMe 
               
             
          
         
       
     
         [0070]    48.9 g (130 mmol) Z-MeVal-Pro-OMe were dissolved in 490 ml methanol. After addition of 10.9 ml (130 mmol) concentrated hydrochloric acid and 2.32 g 10% Palladium/charcoal, the reaction mixture was hydrogenated. Filtration and evaporation to dryness yielded 36.32 g of the product. 
         [0000]    
       
         
               
               
             
           
               
                   
                 c) Z-Val-MeVal-Pro-OMe 
               
             
          
         
       
     
         [0071]    18.1 g (65 mmol) H-MeVal-Pro-OMe, 21.6 g (78 mmol) Z-Val-N-carboxyanhydride and 22.8 ml (130 mmol) diisopropylethylamine were stirred in 110 ml DMF at 40° C. for 2 d. After evaporation of DMF, dichloromethane was added and the organic phase washed with saturated aqueous NaHCO 3  solution (3×), water (1×), 4% citric acid (3×) and saturated NaCl solution. The organic phase was dried over sodium sulfate and evaporated to dryness. The product (29.3 g) was obtained as a viscous oil. 
         [0000]    
       
         
               
               
             
           
               
                   
                 d) H-Val-MeVal-Pro-OMe 
               
             
          
         
       
     
         [0072]    29.3 g (61.6 mmol) of Z-Val-MeVal-Pro-OMe were dissolved in 230 ml methanol. After addition of 1.15 g 10% Palladium/charcoal, the reaction mixture was hydrogenated. Filtration and evaporation to dryness yielded 21.96 g of the product. 
         [0000]    
       
         
               
               
               
             
           
               
                   
                 e) Z-Val-Val-MeVal-Pro-Ome 
                 (SEQ ID NO: 2) 
               
             
          
         
       
     
         [0073]    15.29 g (61 mmol) Z-Val-OH and 21.96 g (61 mmol) H-Val-MeVal-Pro-OMe were dissolved in 610 ml dichloromethane and cooled to 0.degree. C. After addition of 8.16 ml (73.2 mmol) N-Methylmorpholine, 2.77 g (20.3 mmol) HOBt and 11.73 g (61 mmol) EDCI, the reaction mixture was stirred overnight at room temperature, diluted with dichloromethane and thoroughly washed with saturated aqueous NaHCO 3  solution (3×), water (1×), 5% citric acid (3×) and saturated NaCl solution. The organic phase was dried over sodium sulfate and evaporated to dryness to yield 31.96 g of the product. 
         [0000]    
       
         
               
               
               
             
           
               
                   
                 f) Z-Val-Val-MeVal-Pro-OH 
                 (SEQ ID NO: 2) 
               
             
          
         
       
     
         [0074]    31.96 g (57 mmol) Z-Val-Val-MeVal-Pro-OMe (SEQ ID NO: 2) were dissolved in 250 ml methanol. 102.6 ml of a 1 N LiOH solution was added and the mixture stirred overnight at room temperature. After addition of 500 ml water, the aqueous phase was washed three times with ethyl acetate, adjusted to pH 2 at 0° C. and extracted three times with ethyl acetate. The organic phase was dried over sodium sulfate and evaporated to dryness yielding 30.62 g of the desired product as a white solid. 
         [0000]    
       
         
               
               
             
               
               
               
             
           
               
                   
                 (SEQ ID NO: 1) 
               
             
          
           
               
                   
                 g) 
                 Z-Val-Val-MeVal-Pro-Pro-NHCH(CH 3 ) 2   
               
             
          
         
       
     
         [0075]    2 g (3.35 mmol) Z-Val-Val-MeVal-Pro-OH (SEQ ID NO: 2) and 0.664 g (3.35 mmol) H-Pro-NHCH(CH 3 ) 2  were dissolved in 34 ml of dry dichloromethane. After cooling to 0° C., 1.35 ml (12.1 mmol) N-methylmorpholine, 0.114 g (0.84 mmol) HOBt and 0.645 g (3.35 mmol) EDCI were added and the reaction mixture stirred overnight at room temperature. 80 ml dichloromethane were added and the organic phase thoroughly washed with saturated aqueous NaHCO 3  solution (3×), water (1×), 5% citric acid (3×) and saturated NaCl solution (1×). The organic phase was dried over sodium sulfate and evaporated to dryness to yield 1.96 g of the product which was used in the next reaction without further purification. 
         [0000]    
       
         
               
               
             
               
               
               
             
           
               
                   
                 (SEQ ID NO: 1) 
               
             
          
           
               
                   
                 h) 
                 Me 2  Val-Val-MeVal-Pro-Pro-NHCH(CH 3 ) 2   
               
             
          
         
       
     
         [0076]    1.96 g Z-Val-Val-MeVal-Pro-Pro-NHCH(CH 3 ) 2  (SEQ ID NO: 2) were dissolved in 11 ml methanol. 0.054 g 10% Pd/C were added under nitrogen atmosphere and the reaction mixture hydrogenated at room temperature for 4 h. After addition of 0.86 ml (11.24 mmol) of a 37% aqueous formaldehyde solution and 0.281 g 10% Pd/C, hydrogenation was continued for 5 h. Filtration and evaporation of the solvent gave rise to 2.77 g of crude product. Further purification was achieved by dissolving the peptide in water, adjusting the pH to 2 and extracting the aqueous phase three times with ethyl acetate. The aqueous phase was then adjusted to pH 8-9 and extracted four times with dichloromethane. The organic phase was dried over sodium sulfate to yield 1.37 g of purified product as a white foam. The compound was further purified using medium pressure liquid chromatography (10-50% A in 10 min.; 50-90% A in 320 min.). Fractions containing the product were combined, lyophilized, redissolved in water and the pH adjusted to 9 with 1 N LiOH. After extraction with dichloromethane, the organic phase was dried over sodium sulfate and evaporated to dryness. Lyophilization led to 500 mg of pure product, which was characterized by fast atom bombardment mass spectrometry ([M+H]=593). 
         [0000]    
       
         
               
               
             
           
               
                   
                 Example 2 
               
               
                   
                 (SEQ ID NO: 1) 
               
               
                   
                 Me 2  Val-Val-MeVal-Pro-Pro-NHC(CH 3 ) 3   
               
               
                   
                   
               
               
                   
                 (SEQ ID NO: 1) 
               
               
                   
                 i) Z-Val-Val-MeVal-Pro-Pro-NHC(CH 3 ) 3   
               
             
          
         
       
     
         [0077]    2 g (3.35 mmol) Z-Val-Val-MeVal-Pro-OH (SEQ ID NO: 2) and 0.692 g (3.35 mmol) H-Pro-NHC(CH 3 ) 3  were dissolved in 34 ml of dry dichloromethane. After cooling to 0° C., 1.35 ml (12.1 mmol) N-methylmorpholine, 0.114 g (0.84 mmol) HOBt and 0.645 g (3.35 mmol) EDCI were added and the reaction mixture stirred overnight at room temperature. 80 ml dichloromethane were added and the organic phase thoroughly washed with saturated aqueous NaHCO 3  solution (3×), water (1×), 5% citric acid (3×) and saturated NaCl solution (1×). The organic phase was dried over sodium sulfate and evaporated to dryness to yield 1.8 g of the product which was used in the next reaction without further purification. 
         [0000]    
       
         
               
               
             
           
               
                   
                 (SEQ ID NO: 1) 
               
               
                   
                 k) Me 2  Val-Val-MeVal-Pro-Pro-NHC(CH 3 ) 3   
               
             
          
         
       
     
         [0078]    1.8 g Z-Val-Val-MeVal-Pro-Pro-NHC(CH 3 ) 3  (SEQ ID NO: 1) were dissolved in 10 ml methanol. 0.049 g 10% Pd/C were added under nitrogen atmosphere and the reaction mixture hydrogenated at room temperature for 4 h. After addition of 0.86 ml (11.24 mmol) of a 37% aqueous formaldehyde solution and 0.252 g 10% Pd/C, hydrogenation was continued for 5 h. Filtration and evaporation of the solvent gave rise to 1.82 g of crude product. The compound was further purified using medium pressure liquid chromatography 910-50% A in 10 min.; 50-90% A in 320 min.). Fractions containing the product were combined, lyophilized, redissolved in water and the pH adjusted to 9 with 1 N LiOH. After extraction with dichloromethane, the organic phase was dried over sodium sulfate and evaporated to dryness. Lyophilization led to 547 mg of pure product, which was characterized by fast atom bombardment mass spectrometry ([M+H] + =607). 
         [0079]    The following compounds were prepared or can be prepared according to examples 1 and 2: 
         [0000]                                                        3.   Xaa   Val   Xab   Pro   Xac           4.   Xaa   Val   Xab   Pro   Xad       5.   Xaa   Val   Xab   Pro   Xae       6.   Xaa   Val   Xab   Pro   Xaf       7.   Xaa   Val   Xab   Pro   Xag       8.   Xaa   Val   Xab   Pro   Xah       9.   Xaa   Val   Xab   Pro   Xai       10.   Xaa   Val   Xab   Pro   Xak       11.   Xaa   Val   Xab   Pro   Xal       12.   Xaa   Val   Xab   Pro   Xam       13.   Xaa   Val   Xab   Pro   Xan       14.   Xaa   Val   Xab   Pro   Xao       15.   Xaa   Val   Xab   Pro   Xap       16.   Xaa   Val   Xab   Pro   Xaq       17.   Xaa   Val   Xab   Pro   Xar       18.   Xaa   Val   Xab   Pro   Xas       19.   Xaa   Val   Xab   Pro   Xat       20.   Xaa   Val   Xab   Pro   Xau       21.   Xaa   Val   Xab   Pro   Xav       22.   Xaa   Val   Xab   Pro   Xaw       23.   Xaa   Val   Xab   Pro   Xax       24.   Xdd   Val   Xab   Pro   Xay       25.   Xaa   Val   Xab   Pro   Xaz       26.   Xaa   Val   Xab   Pro   Xba       27.   Xaa   Val   Xab   Pro   Xbb       28.   Xaa   Val   Xab   Pro   Xay       29.   Xaa   Val   Xab   Pro   Xbd       30.   Xaa   Val   Xab   Pro   Xbe       31.   Xaa   Val   Xab   Pro   Xbf       32.   Xaa   Val   Xab   Pro   Xbg       33.   Xaa   Val   Xab   Pro   Xbh       34.   Xaa   Val   Xab   Pro   Xbi       35.   Xaa   Val   Xab   Pro   Xbk       36.   Xaa   Val   Xab   Pro   Xbl       37.   Xaa   Val   Xab   Pro   Xbm       38.   Xaa   Val   Xab   Pro   Xbn       39.   Xaa   Val   Xab   Pro   Xb0       40.   Xaa   Val   Xab   Pro   Xbp       41.   Xaa   Val   Xab   Pro   Xbq       42.   Xaa   Val   Xab   Pro   Xbr       43.   Xaa   Val   Xab   Pro   Xbx       44.   Xaa   Val   Xab   Pro   Xbt       45.   Xaa   Val   Xab   Pro   Xbu       46.   Xaa   Val   Xab   Pro   Xbv       47.   Xaa   Val   Xab   Pro   Xbw       48.   Xaa   Val   Xab   Pro   Xbx       49.   Xaa   Val   Xab   Pro   Xby       50.   Xaa   Val   Xab   Pro   Xbz       51.   Xaa   Val   Xab   Pro   Xca       52.   Xaa   Val   Xab   Pro   Xcb       53.   Xaa   Val   Xab   Pro   Xcc       54.   Xaa   Val   Xab   Pro   Xcd       55.   Xaa   Val   Xab   Pro   Xce       56.   Xaa   Val   Xab   Pro   Xcf       57.   Xaa   Xdf   Xab   Pro   Xay       58.   Xaa   Val   Xab   Pro   Xch       59.   Xaa   Val   Xab   Pro   Xci       60.   Xaa   Val   Xab   Pro   Xck       61.   Xaa   Val   Xab   Pro   Xcl       62.   Xaa   Val   Xab   Pro   Xcm       63.   Xaa   Val   Xab   Pro   Xcn       64.   Xaa   Val   Xab   Pro   Xco       65.   Xaa   Val   Xab   Pro   Xcp       66.   Xaa   Val   Xab   Pro   Xcq       67.   Xaa   Val   Xab   Pro   Xcr       68.   Xaa   Val   Xab   Pro   Xcs       69.   Xaa   Val   Xab   Pro   Xct       70.   Xaa   Val   Xab   Pro   Xcu       71.   Xcx   Val   Xab   Pro   Xcv       72.   Xcx   Val   Xab   Pro   Xcv       73.   Xaa   Val   Xab   Pro   Pro   Xcy       74.   Xaa   Val   Xab   Pro   Pro   Xcz       75.   Xaa   Val   Xda   Pro   Xcv       76.   Xaa   Xdb   Xab   Pro   Xcv       77.   Xdc   Val   Xab   Pro   Xcv       78.   Xaa   Ile   Xab   Pro   Xcv       79.   Xdd   Val   Xab   Pro   Xcv       80.   Xde   Val   Xab   Pro   Xcv       81.   Xaa   Xdf   Xab   Pro   Xcv       82.   Xaa   Val   Xab   Pro   Xcg       83.   Xaa   Val   Xab   Pro   Pro   Xdg       84.   Xaa   Val   Xab   Pro   Pro   Xdh       85.   Xaa   Val   Xab   Pro   Pro   Xdi       86.   Xaa   Val   Xab   Pro   Pro   Xdk       87.   Xaa   Val   Xdl   Pro   Xcv       88.   Xde   Val   Xab   Pro   Xay       89.   Xaa   Val   Xdl   Pro   Xay       90.   Xaa   Val   Xab   Pro   Xdm       91.   Xaa   Val   Xab   Pro   Xdn       92.   Xaa   Val   Xab   Pro   Xdo       93.   Xaa   Val   Xab   Pro   Xdp       94.   Xaa   Val   Xab   Pro   Xdq       95.   Xaa   Val   Xab   Pro   Pro   Xdr       96.   Xaa   Val   Xab   Pro   Xds       97.   Xaa   Val   Xbc   Pro   Xcv       98.   Xaa   Ile   Xab   Pro   Xay       99.   Xcw   Val   Xab   Pro   Xay       100.   Xaa   Val   Xbc   Pro   Xal       101.   Xaa   Val   Xdl   Pro   Xal       102.   Xaa   Xdf   Xab   Pro   Xal       103.   Xaa   Ile   Xab   Pro   Xal       104.   Xdd   Val   Xab   Pro   Xal       105.   Xde   Val   Xab   Pro   Xal       106.   XcX   Val   Xab   Pro   Xcy       107.   Xcw   Val   Xab   Pro   Xal       108.   Xcx   Val   Xab   Pro   Xal       109.   Xcw   Val   Xab   Pro   Xav       110.   Xcx   Val   Xab   Pro   Xav       111.   Xcw   Val   Xab   Pro   Xaw       112.   Xcx   Val   Xab   Pro   Xaw       113.   Xab   Val   Xab   Pro   Xay       114.   Xab   Val   Xab   Pro   Xcv       115.   Xab   Val   Xab   Pro   Xal       116.   Xab   Val   Kab   Pro   Xam       117.   Xab   Val   Xab   Pro   Xam       118.   Xab   Val   Xab   Pro   Xao       119.   Xab   Val   Xab   Pro   Xay       120.   Xab   Val   Xab   Pro   Xaw       121.   Xab   Val   Xab   Pro   Xau       122.   Xab   Val   Xab   Pro   Xau       123.   Xab   Val   Xab   Pro   Xbf       124.   Xab   Val   Xab   Pro   Xbm       125.   Xab   Val   Xab   Pro   Xbm       126.   Xab   Val   Xab   Pro   Xbo       127.   Xab   Val   Xab   Pro   Xch       128.   Xaa   Val   Xab   Pro   Xdt       129.   Xaa   Val   Xab   Pro   Xdu       130.   Xaa   Val   Xab   Pro   Xdv       131.   Xaa   Val   Xab   Pro   Xdw       132.   Xaa   Val   Xab   Pro   Xdx       133.   Xaa   Val   Xab   Pro   Xdy       134.   Xaa   Val   Xab   Pro   Xdz       135.   Xaa   Val   Xab   Pro   Xea       136.   Xaa   Val   Xab   Pro   Xeb       137.   Xaa   Val   Xab   Pro   Xec       138.   Xaa   Val   Xab   Pro   Xed       139.   Xaa   Val   Xab   Pro   Xef       140.   Xaa   Val   Xab   Pro   Xeg       141.   Xaa   Val   Xab   Pro   Xeh       142.   Xaa   Val   Xab   Pro   Xei       143.   Xaa   Val   Xab   Pro   Xek       144.   Xaa   Val   Xab   Pro   Xel       145.   Xaa   Val   Xab   Pro   Xem       146.   Xaa   Val   Xab   Pro   Xen       147.   Xaa   Val   Xab   Pro   Xeo       148.   Xaa   Val   Xab   Pro   Xep       149.   Xaa   Val   Xab   Pro   Xeq       150.   Xaa   Val   Xab   Pro   Xer       151.   Xaa   Val   Xab   Pro   Xcg                    
Examples for the MS-characterization of the synthesized novel compounds are given in the following table.
 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE I 
               
             
             
               
                   
               
               
                 Sequence Identification of Compounds Prepared 
               
               
                 According to Examples 1 and 2 
               
             
          
           
               
                   
                 EXAMPLE 
                 Fast atom bombardment MS analysis. 
               
               
                   
                 [No.] 
                 [Mol.-Weight (measured)] 
               
               
                   
                   
               
             
          
           
               
                   
                 3. 
                 565 
               
               
                   
                 4. 
                 579 
               
               
                   
                 5. 
                 593 
               
               
                   
                 6. 
                 607 
               
               
                   
                 7. 
                 621 
               
               
                   
                 8. 
                 635 
               
               
                   
                 11. 
                 607 
               
               
                   
                 12. 
                 607 
               
               
                   
                 13. 
                 621 
               
               
                   
                 14. 
                 649 
               
               
                   
                 15. 
                 635 
               
               
                   
                 16. 
                 635 
               
               
                   
                 17. 
                 635 
               
               
                   
                 18. 
                 635 
               
               
                   
                 19. 
                 621 
               
               
                   
                 20. 
                 621 
               
               
                   
                 21. 
                 635 
               
               
                   
                 22. 
                 635 
               
               
                   
                 25. 
                 633 
               
               
                   
                 26. 
                 647 
               
               
                   
                 27. 
                 661 
               
               
                   
                 31. 
                 623 
               
               
                   
                 32. 
                 671 
               
               
                   
                 33. 
                 667 
               
               
                   
                 34. 
                 631 
               
               
                   
                 35. 
                 655 
               
               
                   
                 36. 
                 655 
               
               
                   
                 37. 
                 669 
               
               
                   
                 38. 
                 621 
               
               
                   
                 39. 
                 635 
               
               
                   
                 41. 
                 649 
               
               
                   
                 42. 
                 621 
               
               
                   
                 43. 
                 633 
               
               
                   
                 44. 
                 667 
               
               
                   
                 45. 
                 607 
               
               
                   
                 46. 
                 647 
               
               
                   
                 47. 
                 668 
               
               
                   
                 48. 
                 655 
               
               
                   
                 49. 
                 669 
               
               
                   
                 50. 
                 685 
               
               
                   
                 51. 
                 629 
               
               
                   
                 52. 
                 625 
               
               
                   
                 53. 
                 721 
               
               
                   
                 55. 
                 579 
               
               
                   
                 58. 
                 623 
               
               
                   
                 61. 
                 597 
               
               
                   
                 62. 
                 621 
               
               
                   
                 63. 
                 609 
               
               
                   
                 64. 
                 625 
               
               
                   
                 65. 
                 635 
               
               
                   
                 66. 
                 591 
               
               
                   
                 67. 
                 715 
               
               
                   
                 68. 
                 685 
               
               
                   
                 69. 
                 685 
               
               
                   
                 70. 
                 591 
               
               
                   
                 71. 
                 607 
               
               
                   
                 72. 
                 621 
               
               
                   
                 74. 
                 706 
               
               
                   
                 75. 
                 579 
               
               
                   
                 76. 
                 579 
               
               
                   
                 77. 
                 579 
               
               
                   
                 78. 
                 607 
               
               
                   
                 79. 
                 607 
               
               
                   
                 80. 
                 607 
               
               
                   
                 81. 
                 607 
               
               
                   
                 82. 
                 637 
               
               
                   
                 83. 
                 692 
               
               
                   
                 84. 
                 706 
               
               
                   
                 85. 
                 706 
               
               
                   
                 86. 
                 706 
               
               
                   
                 87. 
                 607 
               
               
                   
                 90. 
                 635 
               
               
                   
                 92. 
                 659 
               
               
                   
                 93. 
                 617 
               
               
                   
                 94. 
                 636 
               
               
                   
                 95. 
                 678 
               
               
                   
                 128. 
                 671 
               
               
                   
                 131. 
                 625 
               
               
                   
                 139. 
                 625 
               
               
                   
                 151. 
                 637 
               
               
                   
                   
               
             
          
         
       
     
       Compound Number(s) 
       [0080]    1-56, 58-72, 75, 77, 79, 80, 82,
 
87-94, 96, 97, 99-101, 104-151
 
73, 74, 83-86, 95,
 
57, 76, 81, 102
 
78, 98, 103
 
The symbols Xaa in the summary have the following meanings:
 
         [0000]    
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
     
         [0081]    Compounds of this invention may be assayed for anti-cancer activity by conventional methods, including for example, the methods described below. 
       A. In Vitro Methodology 
       [0082]    Cytotoxicity was measured using a standard methodology for adherent cell lines such as the microculture tetrazolium assay (MTT). Details of this assay have been published (Alley, M C et al, Cancer Research 48:589-601, 1988). Exponentially growing cultures of tumor cells such as the HT-29 colon carcinoma or LX-1 lung tumor are used to make microtiter plate cultures. Cells are seeded at 3000 cells per well in 96-well plates (in 150.mu.l or media), and [varying from 10 0.4  M to 10 0.10  M. Cells are then incubated for 72 hours. To determine the number of viable cells in each well, the MTT dye is added (50 μl or 3 mg/ml solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in saline). This mixture is incubated at 37° C. for 5 hours, and then 50 μl of 25% SDS, pH2 is added to each well. After an overnight incubation, the absorbance of each well at 550 nm is read using an ELISA reader. The values for the mean+/−SD of data from replicated wells are calculated, using the formula % T/C (% viable cells treated/control). 
         [0000]    
       
         
           
             
               
                 
                   
                     
                       
                         O 
                          
                         
                             
                         
                          
                         D 
                          
                         
                             
                         
                          
                         of 
                          
                         
                             
                         
                          
                         treated 
                          
                         
                             
                         
                          
                         cells 
                       
                       
                         O 
                          
                         
                             
                         
                          
                         D 
                          
                         
                             
                         
                          
                         of 
                          
                         
                             
                         
                          
                         control 
                          
                         
                             
                         
                          
                         cells 
                       
                     
                     × 
                     100 
                   
                   + 
                   
                     % 
                      
                     
                         
                     
                      
                     T 
                      
                     
                       / 
                     
                      
                     C 
                   
                 
               
               
                 
                     
                 
               
             
           
         
       
     
         [0083]    The concentration of test compound which gives a T/C of 50% growth inhibition was designated as the IC 50  value. 
       B. In Vivo Methodology 
       [0084]    Compounds of this invention were further tested in pre-clinical assay for in vivo activity which is indicative of clinical utility. Such assays were conducted with nude mice into which tumor tissue, preferably of human origin, had been transplanted (xenografted), as is well known in this field. Test compounds were evaluated for their anti-tumor efficacy following administration to the xenograft-bearing mice. 
         [0085]    More specifically, human breast tumors (MX-1) which had been grown in athymic nude mice were transplanted into new recipient mice, using tumor fragments which were about 50 mg in size. The day of transplantation was designated as day O, Six to ten days later, mice were treated with the test compounds given as an intravenous injection or orally, in groups of 5-10 mice at each dose. Compounds were given every other day, for 3 weeks, at doses from 1-200 mg/kg body weight. 
         [0086]    Tumor diameters and body weights were measured twice weekly. Tumor volumes were calculated using the diameters measured with Vernier calipers, and the formula 
         [0000]      (Length×width 2 )/2=mm 3  of tumor volume
 
         [0087]    Mean tumor volumes are calculated for each treatment group, and T/C values determined for each group relative to the untreated control tumors. 
         [0088]    The new compounds possess good tumor inhibiting properties.

Technology Classification (CPC): 2